Osteoblast differentiation is a tightly regulated process in which key transcription factors (TFs) and their target genes constitute gene regulatory networks (GRNs) under the control of osteogenic signaling pathways. Among these TFs, Sp7 works as an osteoblast determinant critical for osteoblast differentiation. Following the identification of Sp7 and a large number of its functional studies, recent genome-scale analyses have made a major contribution to the identification of a “non-canonical” mode of Sp7 action as well as “canonical” ones. The analyses have not only confirmed known Sp7 targets but have also uncovered its additional targets and upstream factors. In addition, biochemical analyses have demonstrated that Sp7 actions are regulated by chemical modifications and protein–protein interaction with other transcriptional regulators. Sp7 is also involved in chondrocyte differentiation and osteocyte biology as well as postnatal bone metabolism. The critical role of SP7 in the skeleton is supported by its relevance to human skeletal diseases. This review aims to overview the Sp7 actions in skeletal development and maintenance, particularly focusing on recent advances in our understanding of how Sp7 functions in the skeleton under physiological and pathological conditions. Mammalian skeletons are derived from the ectoderm and mesoderm . The neuTwo distinct modes of ossification underlie skeletal formation from these origins: intramembranous ossification and endochondral ossification. The cranial vault, most of the facial bones, and the body of the clavicle are generated by the former, whereas the cranial base, the mandibular condyle, the vertebral column, the sternum, the ribs, the end of the clavicle, and the appendicular skeleton are generated by the latter ,4. In boRunx2-positive osteoblast precursors and committed to the osteoblast lineage; the Runx2-positive osteoblast precursors then become Runx2–Sp7-double-positive osteoblast precursors [Runx2–Sp7-double-positive precursors differentiate into osteoblasts and osteocytes, contributing to the formation and maintenance of bone tissues and bone marrows [Osteoblast differentiation is a tightly regulated process in which key transcription factors (TFs) and their target genes constitute gene regulatory networks (GRNs) under the control of osteogenic signaling pathways. Those TFs and signaling pathways include runt Runx2 (related transcription factor 2), Sp7/Osterix, Dlx , Msx (msh homeobox), ATF4 (activating transcription factor 4), Wnt/β-catenin signaling, Hedgehog (Hh) signaling, bone morphogenetic protein (BMP) signaling, transforming growth factor-β (TGF-β) signaling, fibroblast growth factor (FGF) signaling, and Notch signaling (extensively reviewed in ). Among ecursors . The Run marrows .For more than a decade after its discovery in 2002, Sp7 had been thought to be an osteoblast determinant, which induced transcription of osteoblastic genes by directly binding to their regulatory regions on the genome. However, a next-generation sequencer (NGS)-based genome-scale search for Sp7-associated regions indicated that a “non-canonical” mode of DNA binding might be the preferred action of Sp7 on the osteoblast genome; emerging evidence further supports the presence of such a “non-canonical” mode, even in other Sp family members, and its relevance to human diseases. In addition, recent studies suggest the potential roles of Sp7 in the terminal differentiation of chondrocytes and osteocyte biology as well as osteoblastogenesis. In this review, we, therefore, aim to summarize and discuss the actions of Sp7 in skeletal tissues, particularly focusing on recent advances in the understanding of its modes of action on the genome, its broader biological functions in skeletal cells, its genomic targets, the regulation of its expression and functions, and its clinical relevance.Sp7 gene is located at chromosome 15, whereas human SP7 is found at chromosome 12q13.13 [Sp7 gene is composed of three exons and two introns. Rapid amplification of 5′ complementary DNA ends (5′ RACE) identified three alternatively spliced variants of Sp7. The organization of the Sp7 gene appears to be conserved between mice and humans [SP7, the type I isoform is generated by removal of both introns and exon 2; the type II isoform by removal of introns 1 and 2; and the type III isoform by removal of only intron 2. The expected protein products are identical between type II and type III because of the absence of an initiation codon in exon 1 and the identical coding sequence between the two isoforms, while the protein from the type I transcript lacks the first 18 N-terminal amino acids [Sp7 was identified by screening for genes that were differentially expressed in BMP-2-treated C2C12 cells compared to untreated ones [d humans ,11. In hno acids . Althougno acids , functioThe gene was initially called Osterix (Osx). Its current official gene symbol is Sp7; it contains three C2H2-type zinc fingers—which have high homology with those in Sp family transcription factors Sp1, Sp3, and Sp4—at its C terminus. Based on the following observations, as well as the presence of C2H2-type zinc fingers, Sp7 was thought to have typical properties of TFs. First, Sp7 bound to the GC-box in an electrophoretic mobility-shift assay (EMSA), as Sp1 did. Second, Sp7 had a proline-rich transactivation domain at its N terminus. Third, its subcellular localization was restricted to the nucleus.Sp7 transcripts first appear in chondrocytes and the surrounding perichondrium of endochondral bones and in the condensed mesenchyme of intramembranous bones at embryonic day (E) 13.5. Strong Sp7 expression is detected in the osteoblast lineage located in primary spongiosa and the perichondrium/bone collars at E15.5 and later. The expression is also detectable in prehypertrophic chondrocytes in the growth plate. At the postnatal stages, Sp7 is expressed in osteoblasts on the surface of trabecular bones, the endosteum, and the periosteum and in osteocytes in the bone matrix [In the developing mouse skeleton, e matrix .Sp7-null mice died immediately after birth due to breathing difficulty, although the mice with heterozygous deletion of Sp7 appeared normal and fertile. Sp7-null embryos completely lacked bone formation in both endochondral and intramembranous bones, although their cartilage development remained normal. Sp7-null mice failed to express osteoblast-related genes, including Sparc , Spp1 , Ibsp , and Bglap  and showed severely reduced expression of Col1a1, the main component of bone matrix, in their skeletal elements. Given that Runx2 is expressed in Sp7-null skeletal elements, it seems likely that osteoblast differentiation is arrested at the Runx2-positive stage in Sp7-null mutants. The absence of Sp7 expression in Runx2-null mice further suggests that Sp7 acts genetically downstream of Runx2 [Sp7-null cells that failed to differentiate into osteoblasts ectopically expressed chondrocyte regulator and cartilage matrix genes, including Sox5 (SRY-box transcription factor 5), Sox9 (SRY-box transcription factor 9), Ihh (Indian hedgehog), and Col2a1, in both endochondral and intramembranous bones. Thus, the phenotypes of Sp7-null mice indicate that Sp7 acts as a critical determinant for bone-forming osteoblasts in skeletal development.In the initial report that identified Sp7, the critical role of Sp7 in osteoblast differentiation was demonstrated by means of a mouse genetic study [Sp7 at postnatal stages caused a reduction of new bone formation; there were few mature osteoblasts, and Col1a1 expression was severely decreased in the long bones of the mutants [Dmp1 (dentin matrix protein 1), Phex (phosphate regulating endopeptidase homolog X-linked), and Sost (sclerostin) was reduced in the mutants [Sp7 using a 2.3-kb Col1a1-Cre driver line rescued the perinatal lethality of Sp7-null mice, and the newborn mutants appeared normal. However, they showed osteopenic phenotypes at growing stages, probably due to a delay of osteoblast maturation [Col1a1-CreERT2 driver line basically yielded phenotypes similar to those of the above mutants [Sp7 in osteoblasts using a transgenic approach resulted in osteopenia with suppression of osteoblast differentiation [Sp7 may be required for the proper execution of the osteoblast program in adults.Several studies also support the postnatal roles of Sp7 in bone homeostasis. Inducible but ubiquitous deletion of  mutants . The mut mutants . Osteoblturation . Inducib mutants . These rntiation , an apprUntil fairly recently, conventional mechanistic studies on the actions of TFs had focused on local gene regulation, i.e., how the TFs regulate transcription of certain sets of target genes, and this approach provided a good model for understanding their modes of action. However, it remained unclear whether the models were relevant to the overall actions of the TFs on the genome and to their cell-type distinct functions. Next-generation sequencer (NGS)-based genome-scale studies have recently addressed these questions, uncovering how TFs are associated with the genome, how they potentially interact with other proteins over the genome, and how the actions potentially elicit biological outcomes.GFP-positive osteoblasts of neonatal Sp7-GFP mouse calvarias [A chromatin immunoprecipitation-sequencing (ChIP-seq) study unveiled an Sp7-DNA association profile in mouse calvaria-derived primary osteoblasts and mouse pre-osteoblastic cell line MC3T3-E1, showing several key features of Sp7 actions on the osteoblast genome . First, alvarias  further The last but most important feature of the actions of Sp7 was found by de novo motif analysis on Sp7-associated regions. The analysis revealed enrichment of an AT-rich sequence under the Sp7-associated regions. This was unexpected because Sp7 belongs to the Sp family; Sp family members bind to the GC-box consensus motif via their zinc finger domains . The modIn a comparative analysis of Sp1 and Sp7 ChIP-seq in the pre-osteoblastic cell line MC3T3-E1, the AT-rich motif was again enriched in Sp7-associated regions, whereas the GC-box was enriched in Sp1-associated ones . The Sp7Dlx knock-down attenuated Sp7 engagements with the osteoblast enhancers on the genome, leading to suppression of the Sp7 target gene expression [How is Sp7 associated with the AT-rich motifs on the osteoblast genome? No direct binding of Sp7 to the AT-rich motifs was detected in EMSA. The AT-rich motif resembled a homeodomain-responsive element, which raised the possibility that Sp7 is indirectly bound to the motif through homeodomain-containing TFs. Several lines of evidence supported the conclusion that homeodomain-containing Dlx factors act with Sp7. Dlx factors have been implicated in osteoblast differentiation ,25,26. Ipression .The above series of data suggests that Sp7 executes the osteoblast program via a “non-canonical” mode of action, in which it acts as a co-factor for Dlx rather than as a TF that directly binds to DNA. Other homeodomain-containing TFs, such as Msx1/2, Satb2 , and Alx4 , expressed in osteoblasts  may bindSp8 and Sp6 are expressed in the limb ectoderm and play necessary roles for proximal–distal (PD) and dorsal–ventral (DV) patterning in limb development. Motif analysis on the Sp8 ChIP-seq in the mouse limb ectoderm showed that a GC-rich motif was the most over-represented one, and an AT-rich motif was the second-most over-represented. This suggests that Sp8 has a dual mode of action, direct DNA binding through the typical Sp consensus motif and indirect binding through homeodomain-containing TFs. Indeed, physical interactions were detected between Sp8 and Dlx5 and between Sp6 and Dlx5. However, the functional outcome of the interaction seems to be complex and likely depends on the availability of the interacting TFs [Recent ChIP-seq studies suggest that the “non-canonical” mode of action is not specific to Sp7 but is present in other Sp family members as well. ting TFs . Sp6 ChIting TFs .As mentioned earlier, amino acid variants in the α-helical domain distinguish Sp7 from other Sp family members, causing loss of the GC-box preference. Interestingly, a sequence comparison among vertebrate and non-vertebrate chordate species revealed that the closest non-boney vertebrates , the cephalochordates , and the ascidians  do not have Sp7-like zinc finger variants, whereas all boney vertebrates and cartilaginous fish that arose from a boney ancestor have Sp7 genes or a gene with Sp7-like zinc finger variants . TherefoFmod (fibromodulin) [Col1a1 [Col1a2 [Col5a1 [Col5a3 [Ibsp [Sost [Bglap [Zbtb16 [Cx43 [Vegf  [Mmp9  [Mmp13  [Zip1 [Ucma (upper zone of growth plate and cartilage matrix associated) [Enpp1 (pyrophosphatase/phosphodiesterase 1) [Sp7 has been shown to upregulate transcription of various osteoblast-related genes, including modulin) , Col1a1  [Col1a1 ,31, Col1 [Col1a2 , Col5a1  [Col5a1 , Col5a3  [Col5a3 , Ibsp [2a3 [Ibsp ,35, Sostsp [Sost ,36, Bglat [Bglap , Zbtb16  [Zbtb16 , Cx43 [316 [Cx43 , Vegf (v factor) , Mmp9 (minase 9) , Mmp13 (nase 13) ,43, Zip13) [Zip1 , Ucma (uociated) , and Enperase 1)  (extensierase 1) ). This sRunx2 [Col1a1 [Runx2 enhancer by ChIP. However, Dlx5 and Msx2, but not Sp7, are directly bound to the core sequence of the enhancer in EMSA. Since a physical interaction between Dlx5 and Sp7 was detected by GST pulldown, they proposed an enhanceosome model of the Runx2 enhancer, in which Dlx5/6 is directly bound to the core-sequence, and Sp7 indirectly did so by forming a complex with Dlx5/6. The Sp7 ChIP-seq study at least partly confirmed the model and, more importantly, further expanded it to a genome scale , and Kremen1 further suggested cross-talk between Sp7 and osteogenic signaling pathways; their signaling activity is likely fine-tuned by Sp7 at the transcriptional level.The Sp7 ChIP-seq study also revealed a set of enhancers as novel genomic targets of Sp7. Site-directed mutations in AT-rich motifs within these targets suppressed their enhancer activities , suggestdrocytes . Sp7 ChISp7-null mouse embryos had no abnormality in cartilage, chondrocyte-specific deletion of Sp7 using a Col2a1-Cre driver line resulted in impaired endochondral ossification with reduced expression of chondrocyte-related genes [Col2a1-Cre driver line showed that the calcification of cartilage matrix was delayed in the mutants [Sp7 in chondrocytes using a Col2a1-CreERT2 driver line leads to reduced trabecular bone mass in mice. This phenotype was accompanied by a delay of chondrocyte hypertrophy and cartilage-to-bone conversion [Although global ed genes . Another mutants . Postnatnversion . Thus, tSp7 deletion in mature osteoblasts using a Dmp1-Cre driver line led to severe defects in osteocyte dendrites in mice. RNA-seq in Sp7-deficient and Sp7-overexpressing Ocy454 cells, a mouse osteocyte-like cell line, revealed that Sp7-dependent genes were enriched in gene ontology (GO) terms linked to cell projection organization and neuronal development. Comparative analysis of Sp7 ChIP-seq between Ocy454 cells and primary osteoblasts revealed Ocy-specific Sp7-associated regions, which were connected with genes associated with axon guidance. These results suggest that the osteocyte network bears similarity to the network of intercellular connections between neurons in terms of both gene expression and morphology [Sp7 also has a crucial role in osteocyte biology ,52, and Sp7 transcription is regulated by signaling pathways, TFs, and miRNAs, as recently reviewed in detail by Liu et al. [Sp7 regulation  by ChIP in E13.5 mouse limb bud cells treated with BMP-2 [Sp7 transcription.u et al. . With regulation . As for gulation . Most oft manner . In the th BMP-2 . This maSp7 transcription through a novel Sp7 enhancer, which is located at approximately 11 kb distal to the Sp7 transcription start site [Sp7 enhancer. EMSA showed the direct binding of Runx2 on the Runx consensus motif in the enhancer; mutagenesis on the Runx consensus motif confirmed the requirement of the Runx2 binding site for the enhancer activity in vitro. Thus, a further analysis focusing on not only promoter regions but also enhancer regions will help to elucidate how Sp7 is regulated in a cell type-specific manner.In addition to these findings, we recently revealed that Runx2 controls art site . An analSp7 transcription through directly binding to 3′ untranslated region (UTR) of Sp7 [Sp7 expression indirectly by regulating a negative factor of osteogenesis [Sp7 transcription by modifying histone marks, including H3K4me3 on the Sp7 promoter [Sp7 expression, possibly through inhibition of H3K27me3 on the promoter [Sp7 transcription is finely tuned through multiple mechanisms, although the whole picture of the regulation has not been revealed yet.MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) were reported to participate in the Sp7 regulation . A numbe) of Sp7 ; some miogenesis . lncRNAspromoter , whereaspromoter . OverallThe activities of Sp7 are regulated post-translationally by the chemical modification of Sp7 and its interactions with other transcriptional regulators . p38 MAPFbw7 E3 ligase complex; Fbw7-targeted Sp7 undergoes ubiquitination and subsequent degradation [Another report showed that phosphorylation of Sp7 at Ser-73 and Ser-77 is necessary for its interaction with Fbw7 (F-box/WD repeat-containing protein 7), which works as a receptor subunit of the Skp1-Cullin1-F-box-protein (SCF)radation . Thus, pSp7 is acetylated at K307 and K312 by p300/CBP, and acetylation, in turn, enhances the stability of the Sp7 protein . Sp7 aceSirt7-null mice showed decreased bone formation, and this phenotype was recapitulated by osteoblast-specific Sirt7 deletion. Biochemical data suggest that Sirt7 increases the transcriptional activity of Sp7 in a DNA binding-independent manner through physical interaction. It is worth noting that Sirt7 did not affect the Sp7 function as a co-factor of Dlx5 and the Sp7 protein stability. From a mechanistic point of view, in vitro data suggest that Sirt7 causes deacylation of K368 in Sp7; the C-terminal deacylation is likely to enhance its N-terminal transactivation activity. K368 deacylation also facilitates Sirt1-mediated depropionylation of Sp7, which enhances its transactivation activity as well.Sirtuin 7 (Sirt7), one of the nicotinamide adenine dinucleotide (NAD)-dependent lysine deacylases, has been reported to regulate the transactivation property of Sp7 . Global Sp7 has been shown to interact with other TFs in the osteoblast program. Physical interactions and synergistic functions between Sp7 and Runx2 have been demonstrated in the context of the transcription of osteoblast-related genes ,63. In ap53 acts as a negative regulator of Sp7 in both its “canonical” and “non-canonical” modes of action . PhysicaZfhx4-null mice demonstrated impaired calcification of cartilage and reduced expression of the late hypertrophic chondrocyte marker Mmp13, which were similar to the cartilage phenotypes of Sp7 mutant mice [Sp7 under the Zfhx4-null background led to a more severe impairment of chondrocyte maturation than was observed in the Zfhx4-null mice, and Zfhx4 physically interacted with Sp7 [A recent study highlighted zinc finger homeodomain 4 (Zfhx4) as a transcriptional partner of Sp7 at the late stage of endochondral ossification . Zfhx4-nant mice . The intwith Sp7 .SP7 have been identified as a rare cause of osteogenesis imperfecta (OI type XII) [SP7 locus and bone mineral density [SP7-mutation-caused diseases have been studied well in OI cases. OI comprises a group of connective tissue disorders characterized by bone fragility and low bone mass [COL1A1 or COL1A2, numerous variants around other genes, including in SP7, have also been identified [SP7 were identified to be associated with recessive OI cases. First, a homozygous single base pair deletion (p.Glu351GlyfsTer19) in SP7 caused a frameshift, resulting in the removal of the last 81 amino acids of the protein in a patient with OI. The clinical features of this patient included recurrent fractures, mild bone deformities, delayed tooth eruption, and white sclera [SP7; the patient presented with osteoporosis, low-trauma fractures, and short stature [SP7 [SP7 variant (p.Glu340Ala) [SP7 mutations in recessive OI. However, the low bone turnover in the patients with dominant OI contrasts with the high turnover state seen in the previously reported patients with recessive OI. Recently, moreover, we and another group independently reported a dominant SP7 mutation (p.Ser309Trp) in two individuals who showed sclerosis, bone fragility, and high turnover [Sp7. Its skeletal phenotype partially recapitulated the patient’s phenotypes [The significance of SP7 in osteogenesis has been confirmed in humans. Mutations in ype XII) ,68 in onype XII) . A genom density . The patone mass . Althougentified ,68. So fe sclera . A secon stature . The thiure [SP7 . In addiu340Ala) . Low corturnover ,75. ThesSP7, and some were associated with the mode of action of SP7. Specifically, we found that the p.Ser309Trp variant increased the binding of SP7 to the GC-box and decreased the binding to an AT-rich motif [SP7 variants on the AT-rich motif through complex formation with DLX5 showed that SP7 carrying either the p.Ser309Trp or p.Glu340Ala variants decreased the activity on the AT-rich motif, compared to the wild type, whereas the p.Arg316Cys and p.Cys275Tyr variants did not significantly change the activity [SP7 variants were associated with different skeletal phenotypes indicates that the pathological phenotypes caused by SP7 variants cannot be simply explained by the gain- or loss-of-function of Sp7. Rather, a complicated mode of SP7 action, possibly including both canonical and non-canonical modes, may underlie the pathological mechanism.Importantly, most of the variants described above were located in the zinc finger domain of ch motif . A crossactivity . In consSP7-related skeletal diseases and the development of new therapeutic approaches for skeletal defects.Mouse genetic studies and a large number of in vitro studies established Sp7 as an osteoblast determinant. As discussed in this review, additional layers of Sp7 actions have been unveiled over the past decade. Identification of the non-canonical mode of Sp7 action has given us a better understanding of the regulatory mechanism underlying Sp7-mediated osteoblast differentiation. It has also provided a new perspective on the pathological mechanisms underlying Sp7-associated bone diseases. However, the overall picture of Sp7-associated GRNs remains to be clarified. The next step could be defining Sp7 from the broader viewpoint of skeletal development and maintenance: How is Sp7 wired to skeletal GRNs depending on cell types and/or distinct phases of differentiation? How does Sp7 select one of the two modes of action in different situations? In the non-canonical mode of action, what are the Sp7 partners in the distinct cell types and differentiations? Recently advancing technologies for single-cell analyses and multiome analysis, including proteomics, will clarify these points, leading to the understanding of the pathological mechanisms underlying


Nicotiana benthamiana, showing the value of this tool in extracting important biologicalinformation such as the determination of the limiting steps in anenzymatic pathway.Programmable transcriptional factors based on the CRISPRarchitectureare becoming commonly used in plants for endogenous gene regulation.In plants, a potent CRISPR tool for gene induction is the so-calleddCasEV2.1 activation system, which has shown remarkable genome-widespecificity combined with a strong activation capacity. To explorethe ability of dCasEV2.1 to act as a transactivator for orthogonalsynthetic promoters, a collection of DNA parts was created (GB_SynP)for combinatorial synthetic promoter building. The collection includes(i) minimal promoter parts with the TATA box and 5′UTR regions,(ii) proximal parts containing single or multiple copies of the targetsequence for the gRNA, thus functioning as regulatory cis boxes, and(iii) sequence-randomized distal parts that ensure the adequate lengthof the resulting promoter. A total of 35 promoters were assembledusing the GB_SynP collection, showing in all cases minimal backgroundand predictable activation levels depending on the proximal partsused. GB_SynP was also employed in a combinatorial expression analysisof an autoluminescence pathway in  Cauliflower mosaic virus (CaMV) 35S promoter, which could easily cause pleiotropic or even detrimentaleffects in the transformed organism due to interferences with essentialprocesses during their development. To reach its full potential, plantgenetic engineering is thus in need of tools for orthogonal and fine-tunedexpression of genes. Synthetic promoters are strong allies, not onlyas tools for gene regulation, but also for designing tailor-made metabolicpathways by controlling multiple genes simultaneously.Plant synthetic biology is evolving fast,as high-throughput omicstools provide us with high-quality and precise knowledge about geneexpression networks, providing clues for successful engineering interventions.However, there is a shortage of tools capable of controlling the expressionof genes in the same precise way as occurs in nature. Many studiesstill rely on conventional genetic manipulation strategies such asgene knockout or overexpression driven by constitutive promoters likethe 2 GoldenBraid,3 Mobius Assembly,4 orLoop5 facilitate combinatorial rearrangementof promoter elements. GoldenBraid (GB) was conceived as an easy andmodular assembly platform based on type IIS restriction enzymes, whichmakes use of the Phytobricks common syntax7 tofacilitate the exchangeability of parts. The GB system also proposeda standard measurement using Luciferase/Renilla transient assay toestimate relative expression levels of promoter elements.8Plantsynthetic promoters typically comprise a minimal promoterand a 5′ regulatory region where cis-regulatory elements areinserted. Regulatory DNA elements are often recruited from the bindingsites of natural transcription factors (TFs). The dual architectureof many TFs allows the generation of synthetic TFs that combine theirDNA-binding domains with the transcriptional regulatory domains ofa different TF and vice versa, creating multiple functional combinations.Moreover, the availability of modular and interchangeable DNA partsgreatly expands the possibilities of promoter design. In this regard,modular cloning methods such as MoClo,12 The main advantage of CRISPRa tools lays in its programmable DNAbinding activity, which is encoded in its custom-designed 20-nucleotideguide RNA (gRNA). Another remarkable feature of these tools is theirmultiplexing capacity, which enables several gRNAs to be directedto the same target gene to ensure higher activation levels, or totarget different genes simultaneously to obtain a cascade of activation.13 CRISPRa tools reported in plants include different protein-fusionstrategies, such as SunTag14 and dCas9-TV,15 and strategies that make use of modified gRNAscaffolds to anchor additional activator domains.17 In this last category falls the recently created dCasEV2.1, whichmakes use of a modified gRNA scaffold  that includestwo aptamer loops at the end of its sequence to allow the attachmentof the viral MS2 protein. The use of this gRNA2.1 thus allows thecombination of two activation domains in dCasEV2.1 system, first theEDLL plant motif fused to the dCas9 protein, and second the VPR  complex fused to MS2 protein. This system showed a strongactivation level for endogenous genes that even surpassed those oftheir natural activation factors.18 Interestingly,the transcriptional activation achieved with dCasEV2.1 in Nicotiana benthamiana results in remarkablegenome-wide specificity. When the promoter region of the endogenousdihydroflavonol-4-reductase (NbDFR) gene was targetedfor activation in N. benthamiana leaves, transcriptomicanalysis showed that only the two NbDFR homologousgenes were significantly activated, with negligible changes in therest of the transcriptome. Similar genome-wide specificity was shownfor another dCas9-based activation system,19 pointing toward dCasEV2.1 as the ideal system for creating orthogonalsynthetic promoters.A limitation of this classical approachlies in the hardwired DNAbinding specificities of natural TFs, which impose cis-regulatoryelements in a fixed DNA sequence, thus precluding free design, reducingcombinatorial power, and comprising full orthogonality. These limitationscould be overcome by employing programmable transcriptional factorsbased on CRISPR/Cas9 architecture. The so-called CRISPR activation(CRISPRa) tools, based on the nuclease-deactivated Cas9 protein (dCas9),are becoming commonly used in plants for endogenous gene regulation.SlDFR genepromoter (pSlDFR) as a “model” promoter,given its remarkable inducibility using dCasEV2.1.18 The strongest activation of pSlDFR occurredwhen targeted at a 20-nucleotides sequence at position −150from its transcriptional start site (TSS). Taking the pSlDFR structure as a prototype, and randomizing most of its sequence,we created a set of synthetic DNA parts comprising distal, proximal,and minimal promoter parts  driven by the 2Kb 5′ regulatory region of the SlDFR promoter (herewith referred to as pSlDFR).18 The responsiveness of pSlDFR was also confirmed in stably transformed reporter plant lines carryingthe pSlDFR:Luc construct, which outperformed other reporter linesemploying other promoters. Here, by performing a nonsaturated scanof possible target sites in different regions of the pSlDFR fragment, we located a 20-nucleotides target box located at position−150 relative to the TSS, named gRNA1, yielding maximum transcriptionalactivation in transient analysis (SlDFR structure as the basis for the design of a new set of dCasEV2.1-regulatedsynthetic promoters (SlDFR gene (named mDFR) as previously reportedby Garcia-Perez et al.21 This element wasassigned a standard A3(−B2) position, according to the Phytobrickssyntax, thus being flanked by TCCC and AATG overhangs . The gRNA1 targetin the synthetic parts was maintained at position −150 relativeto the TSS, mimicking the structure of the native pSlDFR. We hereby defined a series of gRNA target positions or sites, namedwith lower case letters to differentiate them from the capital lettersused in Phytobricks syntax. This target position of −150 fromTSS was therefore named the “a site” as being the firstexplored for this promoter collection. To expand the availabilityof unique A2 parts and avoid repetitions in promoter choice, six differentA2 parts were initially designed (named G1a.1 to G1a.6). These A2parts contain a single gRNA1 target site at this “a site”(position −150) and each one has a different random backgroundsequence. For convenience, the target sequence of a different gRNA(named gRNA2) was also included in all G1a.N parts at position −210from the TSS (referred to as the “b site”). Later, thecollection was further expanded with a second series of five new A2parts (G1ab.1 to G1ab.5 parts), where a repetition of the gRNA1 boxwas inserted at the “b site” (position −210).Finally, a third group of five A2 sequences (G1abc.1 to G1abc.5) wascreated containing the target sequence three times, with the thirdcopy located at position −100 (referred as the “c site”)from the TSS (G1abc.1 to G1abc.5). To finalize the promoter design,an A1 “distal promoter” part (named R1) consisting of1240 nucleotides of random DNA sequence was designed to mimic thelength of the native pSlDFR. All randomly designedA1 and A2 sequences were analyzed with the TSSP software (http://www.softberry.com/berry) to ensure the absence of spurious cis-regulatory elements.Previously, we showed in transient transactivationstudies analysis 1B. Owingromoters 1C. A “miverhangs 1A. Next 35S terminator. All the resulting transcriptionalunits were further combined with Renilla luciferase (RLuc) under CaMV 35S promoter for normalization (as required for the standardLuciferase/Renilla transient assay) and the P19 silencingsuppressor and subsequently assayed in transient transactivation experimentsin N. benthamiana leaves. All promoters showednegligible basal expression levels when cotransformed with a dCasEV2.1loaded with a gRNA (named gRNA3), a target sequence which is not presentin the sequences of the promoters. On the contrary, cotransformationwith gRNA1 led to substantial transcriptional activation in all promotersassayed, yielding a range of activation levels that increased withthe number of copies for the gRNA target present in the A2 element.13 Promoters with the target sequencepresent three times (G1abc.N series) reached activation levels ofaround 50 RPUs on average. The G1ab.N promoter series showed intermediatetranscription levels, similar to those obtained with CaMV 35S promoter, when activated with dCasEV2.1.Promoter parts were next assembled to generate a total of 16 syntheticpromoters, which were subsequently combined with the FLuc coding sequenceand the CaMV  element1C. PromoTheproposed modular GB_SynP structure allows, in principle, a limitlessextension of the gRNA1-responsive promoter collection by the additionof new distal (using A1 syntax) and minimal promoter (with A3–B2syntax) parts. To test this, two new A1 distal elements (R2 and R3)with random DNA sequences different to R1 were designed. These newparts were assayed in combination with A2 proximal promoters describedabove having one (G1a.1), two (G1ab.1), or three (G1abc.1) repetitionsof the target sequence for gRNA1 2A. As obSolanum lycopersicum, Nicotianatabacum, andArabidopsis thaliana. In addition, two minimal promoters based on fungal sequences werealso created. Table S1 summarizes the genomicregions selected as A3 parts. All minimal promoters were assembledupstream with R1 and G1abc.1 (3xgRNA1-target) parts, downstream withthe Luc/Ren reporter, and tested functionally. As shown in N. benthamiana; however,fungal mPAF A3 part promoted high transcriptional levels, similarto other promoter regions obtained from plants.Next, new A3 minimal promoter parts were also addedto the collectionand functionally assayed. Minimal promoter elements were designedbased on the sequences of different strongly regulated and/or tissue-specificgenes from NOS promoter used asreference, reaching values of 0.04 RPUs.Despite theexpression differences found employing different minimalpromoters in the GB_SynP design, the A2 proximal region carrying thedCasEV2.1 cis protospacer elements concentrates most of the regulatoryactivity. Therefore, it was interesting to investigate modificationsin its structure that could accommodate additional regulatory features.Accordingly, we first analyzed the influence of the relative positionof the cis gRNA1 target to the TSS. New A2 proximal parts were thusdesigned, which included the target for gRNA1 at positions −120(named “d site”), −150 (“a site”),−210 (“b site”), and −320 (named “esite”) upstream of the TSS . In both G3a.1 and G1a.7 parts, the target sequencefor the gRNA2 at the “b site” (position −210bp) was also present .This target sequence is found at position 376 upstream of the TSSin the SlDFR promoter and showed low activation inthe native promoter,18 which could be dueto its distance from the TSS. The new A2 parts were then combinedwith R1 and mDFR parts , while double activation using gRNA2+gRNA1for G1aG2b.7 and gRNA2+gRNA3 for G3aG2b.1 resulted in higher activationlevels  when compared to usingeach gRNA individually.Next to the position of the target sequence, weanalyzed the inclusionof new cis-regulatory elements other than gRNA1. For this, we chosethe target sequence of gRNA3 as a new cis element, which is nativelypresent at position −161 in the rget see S1B. In brget see S1B. In b12 asit combines the use of two proteins, dCas9 and MS2, to which two activationdomains are fused . This modular architecturecan be exploited as an additional source of variability in the system,incorporating different activation domains to the dCas9 and MS2 modules, thus expanding the range of trans-activatorsfor GB_SynP promoters. In addition, other dCas9-based transactivationstrategies, such as the SunTag system, can be also incorporated. Inthe SunTag approach, activation domains are fused to a single-chainvariable fragment (ScFv) antibody, which in turn binds to a SunTagmultiepitope peptide fused to dCas9 protein. To explore these additionalexpansions of the system, we assayed the two activation domains, ERF2and EDLL, in four different combinations with dCas9 and MS2 modules,as well as the dCas9:SunTag system with EDLL, ERF2, or VPR fused tothe ScFv antibody. All these dCas9-based TFs were coinfiltrated withthe reporter R1:G1abc.1:mDFR:FLuc and transiently assayed  was the onlycombination that reached expression levels comparable to the CaMV 35S promoter, thus confirming the unique characteristicsof this activation tool in plants.The dCasEV2.1 system is considered tobe a second-generation CRISPRa tool assayed 4. Signif22 was recently adaptedto plants.23 The LUZ pathway has as a majoradvantage that uses the plant’s endogenous caffeic acid asa substrate to produce luciferin, thus avoiding the need for exogenousaddition of luciferin substrate. Moreover, the self-sustainable luminescenceemission implies that nondestructive assays can be performed, allowingfor instance the visualization of time-course kinetics. The pathwaycomprises four genes, named HispS (hispidin synthase), H3H (hispidin-3 hydroxylase), Luz (luciferase),and CPH (caffeylpyruvate hydrolase). HispS encodes for the larger enzyme of the pathway, which catalyzes threeconsecutive reactions to convert caffeic acid into hispidin, whichis then turned into luciferin by a reaction catalyzed by H3H enzyme. Finally, luciferin is used by LUZ enzymeas a substrate to create a high energy intermediate that emits lightupon its degradation to caffeylpyruvic acid. The fourth enzyme ofthe pathway, CPH, is included to recycle this degradationproduct back to caffeic acid, thus closing the cycle , two , or three targets for the gRNA1 . The CPH recycling enzyme was kept underthe constitutive CaMV 35S promoter in all geneticconstructs to reduce the complexity of the analysis. An enhanced GFPprotein (eGFP) under the CaMV 35S promoter was alsoincluded in each construct to serve as an internal reference for normalization.The normalized luminescence values of the resulting 27 pathway combinationscoinfiltrated with dCasEV2.1 and gRNA1 are depicted in 35S promoter,were reached when all three genes in the pathway were regulated by3× gRNA-target promoters, while the lowest transcriptional levelswere found when the three genes were under 1× gRNA-target promoter.Almost no luminescence was observed for any of the 27 combinationswhen coinfiltrated with dCasEV2.1 and an irrelevant gRNA .Adapting the LUZ pathway as a reporter for geneexpression analysisin plants requires identifying which genes in the pathway act as limitingsteps, so that changes in their transcriptional levels are directlytranslated into changes in light intensity. Therefore, to understandthe limiting steps governing the expression of this pathway in HispS was driven by 1× gRNA-target promoters,the resulting normalized luminescence values remained at basal levelsregardless of the synthetic promoters used to regulate the remaininggenes  or the zinc finger proteins, which require re-engineeringof the DNA-binding motifs for each target sequence,29 GB_SynP allows the creation of promoters with completely differentcis-boxes by simply creating new A2-type cis-regulatory parts, andits corresponding gRNA transcriptional unit, both elements being onlya few hundred base pairs long. Here, we also demonstrated that twocompletely different gRNAs (gRNA1 and gRNA3) can reach similar activationlevels when positioned in the same position within the GB_SynP syntheticpromoter, implying that potentially any 20-nucleotide sequence canbe used as a cis-regulatory box. The specificity of the transcriptionalactivation signaling GB_SynP promoters was also demonstrated, sincecoexpression of dCasEV2.1 with an unrelated gRNA led to negligiblebasal expression in all assays. These results position the GB_SynPcollection as a promising tool for the regulation of complex multigenecircuits with different gRNAs present in the cis-regulatory boxesof each promoter, thus creating logic gates that could be useful tofurther explore different metabolic fluxes within biosynthetic pathways.Moreover, other studies reported the successful expression of gRNAsunder pol-II promoters, which in turn could be regulated by differentinducers,27 thus allowing customizable control of eachgRNA with different stimuli to further direct the multigene circuitsin different ways.The synthetic promoters whose expressionis regulated via CRISPRasystems are promising orthogonal tools for Synthetic Biology. CRISPRa-basedsynthetic promoters have been previously reported in bacteria,Table S2, that can be usedto assemble more than 500 different promoters without the need forcreating any new sequence, standing out as one of the CRISPRa-basedsynthetic expression tools currently available for plants with thehighest diversity and combinatorial strength. Moreover, plant syntheticpromoters created so far mostly rely on the well-characterized CaMV 35S minimal promoter,30 which might lead tohigher basal expression in comparison with other minimal promoterslike mDFR.21 To overcome this limitation,GB_SynP includes newly designed minimal promoter parts, for whichnegligible basal expression was shown in all cases, as well as a rangeof activation levels. The total length of the synthetic promoter shouldalso be considered, as short sequences could easily be interferedwith by other nearby promoters once they are introduced into the plantgenome, especially considering the preference for T-DNA to be insertedinto transcriptionally active regions.32 In this regard,different A1 random parts were also included in the GB_SynP collectionto allow easy modulation of the length of the resulting promoter,while adding an extra source of sequence variation.While we reported here the assembly and behaviorof 35 syntheticpromoters, the GB_SynP collection includes to date 32 promoter parts,compiled in 9 where VPR reached the highestfluorescence values out of the 22 different activation domains tested,including the commonly used VP64. VPR is in fact a combination ofthe activation domains VP64, P65, and Rta, of which VP64 is in turncomprised of four tetrameric repetitions of the herpes simplex virusVP16 protein. Depending on the intended application of GB_SynP promoters,concerns may arise from the use of viral proteins for the regulationof GB_SynP promoters. In this regard, as an alternative, we proposethe combination of dCas9 and MS2 proteins with the EDLL and ERF2 activationdomains, respectively, which triggered a considerable activation thatled to a signal comparable to a NOS promoter level. Nevertheless,better-performing CRISPRa tools are continuously being developed,12 which could also be used in combination withdifferent activation domains to increase the expression levels ofGB_SynP promoters developed here.AlthoughdCasEV2.1 remains as the most optimal system to regulateGB_SynP synthetic promoters, here we demonstrated that their activationcan also be triggered by combining dCas9 and MS2 proteins with otheractivation domains, or by using other CRISPRa strategies, such asthose based on dCas9:SunTag fusion. Among the combinations tested,the VPR activation domain showed the highest activation levels forboth systems, which correlates with what was previously observed byChavez et al.23 wherethey reported that the addition of caffeic acid to N. benthamiana leaves expressing the autoluminescence pathway resulted in the developmentof lower and slower luminescence than the addition of hispidin orluciferin.The combinatorial power andthe wide range of expression levelsprovided by the GB_SynP collection were further exploited in the optimizationof a multigene bioluminescence pathway. The new synthetic promoterswere shown to regulate the expression of three genes in the pathwayin a predictable and reliable way, with the lowest pathway outputlevels (luminescence) obtained when all three genes were under theregulation of the weakest promoter, and the highest expression wasreached when the three genes were driven by the strongest promoters.In this case, we showed that the GB_SynP system was also useful tofurther characterize the regulatory requirements of the syntheticpathway. We found that, unlike the rest of the genes, low HispS expressionlimits the flux in the pathway, rendering the regulation of the remainingsteps useless. Such behavior is in line with previous observationsdescribed by Mitiouchkina et al.,33 definedfive fundamentalcharacteristics for efficient and predictable genetic engineering,which are independence, orthogonality, reliability, tunability, andcomposability. Our GB_SynP system described here is a modular andcomposable system that has shown to be highly gRNA-specific and whoseorthogonality is ensured by the negligible basal expression of thesynthetic promoters generated when used in combination with the genome-widespecific dCasEV2.1 system.18 We furtherdemonstrated that the GB_SynP system works in a reliable way for expressingthe bioluminescence pathway and includes a range of expression levelsthat can be further modulated by the use of inducible pol-II drivengRNAs. All in all, the GB_SynP system constitutes a promising toolfor the easy design and optimization of multigenic circuits in thefield of plant genetic engineering.Lucks et al.34 The DNA sequences of the constructs generatedin this work are available at https://gbcloning.upv.es/search/features by entering the IDs provided in Table S3. Random DNA sequences were generated at https://www.bioinformatics.org/sms2/random_dna.html,35 and each promoter part designed wasordered as gBlocks (IDT) and assembled following the GoldenBraid (GB)domestication strategy. Briefly, DNA parts were first cloned intothe pUPD2 entry vector and verified by digestion and sequencing. Transcriptionalunits were then generated via restriction-ligation reactions withthe different DNA parts contained in pUPD2 vectors, and combined withbinary assemblies into multigenic constructs via restriction-ligationwith T4 ligase and BsaI or BsmBI. All constructs were cloned intoEscherichia coliTOP 10 strain using Mix&Go kit(Zymo Research) as indicated by the manufacturer. All assemblies wereconfirmed by digestion.All plasmidsused in this work were assembled using GoldenBraid (GB) cloning.N. benthamiana plants grown at 24 °C/20 °C(light/darkness) with a 16 h:8 h photoperiod. Expression vectors weretransferred to Agrobacterium tumefaciens GV3101 by electroporation. Cultures were grown overnight in liquidLB medium supplemented with rifampicin and the corresponding antibioticfor plasmid selection. Cells were then pelleted and resuspended inagroinfiltration buffer , incubatedfor 2 h in the dark, and adjusted to an OD600 of 0.1. Forcoinfiltration, cultures were mixed at equal volumes, maintaininga final OD of 0.1. The silencing suppressor P19 was included in alltested constructs. Agroinfiltration was carried out using a 1 mLneedleless syringe through the abaxial surface of the three youngestfully expanded leaves of each plant.Transientexpression assays were performed by agroinfiltration of 4–5-week-old g) at 4 °C for10 min. Ten μL of working plant extract (supernatant) was thentransferred to a 96 well plate, where 40 μL LARII buffer wasadded to measure the Fluc signal in a GloMax 96 Microplate Luminometer(Promega) with a 2-s delay and a 10-s measurement. RLuc signal wasmeasured afterward by adding 40 μL Stop&Glow reagent andmeasuring in the same way.Agroinfiltrated sampleswere collected 5 days postinfiltration using a Ø 8 mmcorkborer to extract a disc per each agroinfiltrated leaf, and snapfrozen in liquid nitrogen. Expression of Firefly luciferase (FLuc)and Renilla luciferase (RLuc) were determined with the Dual-Glo LuciferaseAssay System (Promega) following the manufacturer’s instructionswith some modifications. Frozen leaf samples were first homogenizedand extracted with 180 μL Passive Lysis Buffer, followed bya centrifugation (14 000NOS promoter (pNOS) and Renilla is under CaMV 35S promoter (p35S). Reference FLuc/RLuc ratios are arbitrarily set as1.0 relative promoter units (RPUs). Differences between the FLuc/RLucratios were analyzed with one-way ANOVA followed by the post hoc multiplecomparisons Tukey’s test (p ≤ 0.05)using GraphPad Prism 8.0.1 software. As residuals of FLuc/RLuc ratiosdid not follow a normal distribution according to Anderson–Darling,D’Agostino–Pearson omnibus, Shapiro–Wilk, andKolmogorov–Smirnov tests, a logarithmic transformation of thedata (Y = log(Y)) was performedpreviously to the statistical analysis to properly fit the ANOVA assumptions.FLuc/RLuc ratios were determinedas the mean value of three independent agroinfiltrated leaves of thesame plant and were normalized to the FLuc/RLuc ratio obtained froma sample agroinfiltrated with a reference construct (GB1398) whereLuciferase is driven by Agroinfiltrated leafdiscs were collected 24 h postinfiltration using a Ø 6 mmcorkborer to extract a disc per each agroinfiltrated leaf, and placeddirectly in white 96 well plates containing 200 μL/well of solidMS medium . Plates weremeasured once per day for 8 days in a GloMax 96 Microplate Luminometer(Promega), first for luminescence and immediately after for fluorescence.For luminescence, a 2-s delay and 10-s measurement parameters wereused as previously described for in vitro Luciferase/Renilla assays.For eGFP measurement, an optical kit was used with an excitation peakat 490 nm and emission at 510–570 nm.


Finally, we discuss the roles of caRNAs in cancer as well as epigenetic and epitranscriptomic mechanisms contributing to cancer, which could provide insights into the relationship between different caRNAs and cancer, as well as tumor treatment and intervention.Eukaryotic genomes are prevalently transcribed into many types of RNAs that translate into proteins or execute gene regulatory functions. Many RNAs associate with chromatin directly or indirectly and are called chromatin-associated RNAs (caRNAs). To date, caRNAs have been found to be involved in gene and transcriptional regulation through multiple mechanisms and have important roles in different types of cancers. In this review, we first present different categories of caRNAs and the modes of interaction between caRNAs and chromatin. We then detail the mechanisms of chromatin-associated nascent RNAs, chromatin-associated noncoding RNAs and emerging m Xist and other novel ncRNAs containing repeat elements and associated transcription start sites [In human genomes, approximately 74.7% of genomes can be transcribed to form primary RNAs while 62.1% of genomes generating RNAs will be processed [rt sites . These nrt sites .In eukaryotic cells, naturally occurring RNAs can basically be divided into protein-coding RNAs and ncRNAs, and ncRNAs can be further divided into several types, including housekeeping ncRNAs and regulatory ncRNAs , 10. Howcis, trans and cis-trans cooperation [cis, although there are a few nascent RNAs that can interact with chromatin in trans [trans or cis-trans cooperation mode, those RNA-chromatin interactions usually occur in noncoding regions such as promoter and enhancer regions of the genome and most of them involve binding to chromatin together with other proteins, even though some lncRNAs can regulate transcription in cis by binding DNA to form R-loops [In terms of the different locations of caRNAs interacting with chromatin on the genome loci, there are three modes of caRNA-chromatin interactions: peration . Nascentin trans –17. Then R-loops .According to the types of combinations between caRNAs and chromatin, the types of caRNAs-chromatin interactions can be basically classified as direct and indirect binding modes. CaRNAs within specific sequences are able to bind both DNA and some proteins such as transcription factors (TFs), RBPs, histone modifiers and chromatin remodelers. Apart from nascent RNAs that directly bind DNA through base complementary pairing, other caRNAs such as paRNAs and repeat RNAs can also directly interact with chromatin , 12. On cis and trans mechanisms. The R-loop is formed by nascent RNA: DNA hybrids and a displaced single-stranded DNA during the transcription process especially in highly transcribed regions and repeats such as telomeres [cis to regulate transcription [trans [Nascent RNA including nascent precursor mRNA(pre-mRNA) and nascent ncRNA can both act as caRNAs through elomeres , which nelomeres , 20–23. cription . In eukacription –26. A qucription . Additiocription . R-loopscription . These rn [trans . One exan [trans   and eventually increase RNA pol II processing and transcription, which require the H3K27me3 demethylase JMJD3/KDM6B to recruit and interact with DDX21 at the transcription start site (TSS) [cis-acting, and although many chromatin-associated lncRNAs play an essential role in gene regulation as cis- or trans-acting [trans mode and distant action of the nascent RNA-chromatin interaction and its functions should be further discovered. In addition to histone modifications, the chromatin remodelers also associate with R-loops in specific ways, one example is that BRG1, a subunit of chromatin remodeler SWI/SNF complexes, contributes to the resolution of R-loops and transcription-replication conflicts [Another model of nascent RNAs interacting with chromatin involves nascent caRNAs and epigenetic factor cooperation mechanisms. Recent studies have shown a strong relationship between histone modification and transcription. Some histone modifications  as well as histone modification factors such as polycomb repressive complexe 2 (PRC2) have a broad range of dependence on Pol II. In particular, accumulation of H3K27me3 can be observed after blocking transcription . For inste (TSS) . Most ofs-acting , the traThe classification of noncoding RNAs is constantly updating and changing as an increasing number of functions of ncRNAs are discovered. Currently, the most classic category and most studied of ncRNAs are mainly s3mall ncRNAs including microRNAs (miRNAs) and ncRNAs over 200 nt in length including circRNAs and lncRNAs , 38. NcRcis. In mouse ESCs, the TARID antisense transcript acts as a chromatin-associated lncRNA to form an R-loop at the Transcription Factor 21 (TCF21) promoter, and then epigenetic R-loop reader growth arrest and DNA damage inducible alpha (GADD45A) binds the R-loop and recruits DNA demethylase Tet methylcytosine dioxygenase 1 (TET1) to TCF21 promoter CGIs, which leads local DNA demethylation and TCF21 expression [trans. Telomeric-repeat-containing RNA (TERRA) is a chromatin-associated lncRNA that is usually recruited to chromosome ends in trans. Recently, the mechanism of the recruitment of TERRA to chromatin has been revealed, and specific UUAGGG repeats of TERRA and recombinase RAD51 can promote the formation of R-loops by lncRNA TERRA invading into telomere DNA, which contributes to telomere fragility [Arabidopsis is able to target distant genes and form R-loops, which can decoy the histone modification factor PRC1 component LHP1 to regulate local chromatin 3D conformation and subsequent gene expression [There are large numbers of lncRNAs that have the ability to interact with chromatin in eukaryotes, which generally contain specific sequences or structures to bind DNA, chromatin-associate proteins and chromatin remodelers , 40. Naspression  , which refers to the phenomenon in which female embryonic cells randomly inactivate one of the X chromosomes in order to balance gene dosage in males and females during early mammalian embryonic development . The X-ipression . Howeverpression . This prpression . Althougpression . Then, apression   has been the most abundant dynamic modification that has been found in eukaryotes [6A modification on RNAs depends on METTL3/METTL14/WTAP complexes, which consist of the core protein METTL3 with methyltransferase catalytic activity, adaptor protein METTL14 acting as an allosteric activator and guide protein WTAP recruiting METTL3/METTL14 [6A methyltransferases existing in higher eukaryotes [6A including YTH domain-containing proteins, hnRNPs, IGF2BPs (insulin-like growth factor 2 mRNA-binding proteins) and ELAV Like RNA Binding Protein 1 (ELAVL1/HuR) as well as some ‘erasers’ such as AlkB Homolog 5 (ALKBH5) and Fat mass and obesity-associated protein (FTO) [6A modification on different RNAs cooperates with its readers, erasers and writers and has extensive functions in cotranscriptional nascent RNA splicing, mRNA translation, RNA translocation, RNA stability, chromatin state and transcriptional regulation and is related to many diseases especially cancer [6A modification is associated with the translation of mRNAs in acute myeloid leukemia (AML) cells, and the m6A ‘writer’ METTL3 can be recruited by the TF CEBPZ to specific genomic loci promoters, which leads to m6A on a few oncogene mRNAs especially SP1 and promotes translation as well as regulates c-MYC expression and AML cell growth [To date, N6-methyladenosine  , 75. They cancer , 76–79. l growth .6A on chromatin-associated nascent RNAs, chromatin-associated regulatory RNAs (carRNAs) and chromatin-associated lncRNAs all have important roles in transcriptional regulation by interacting with m6A ‘readers’, histone modifiers and transcription factors [6A on nascent mRNAs can impact nascent RNA synthesis. The present study reveals that METTL3/METTL14/WTAP complex-mediated m6A modification of nascent mRNAs, eRNAs and upstream promoter RNAs can protect premature termination by integrator complexes while increasing nascent RNA transcription [6A modification of nascent RNAs and repeat RNAs is able to impact the formation of R-loops thereby influencing transcription termination and heterochromatic RNA synthesis. One example is that the depletion of the METTL3 will dramatically decreases R-loop formation and perturbs transcription termination, and METTL3-deposited m6A on nascent RNAs can increase R-loop formation in gene terminator regions and promote transcription termination [6A on major satellite repeats (MSRs) can also facilitate the R-loop formation, which promotes the MSR transcription and stabilizes the heterochromatin [Many studies have demonstrated that the m factors –84. Firscription , 85  complexes, resulting in carRNA degradation. Conversely, when demethylated carRNAs bind to chromatin, they can recruit histone modifiers to add transcriptional activators of H3K27ac and H3K4me3, then recruit TF YY1 and repel PRC2, ultimately promoting chromatin opening and gene transcription [6A ‘eraser’ FTO can increase m6A abundance in long interspersed nuclear element 1 (LINE1) RNA, and then m6A recruits YTHDC1 to chromatin, further leading the local chromatin closure through the following two mechanisms: (i) YTHDC1 recruits NEXT complexes and causes LINE1 RNA decay inhibiting the deposition of active histone marks H3K4me3/H3K27ac; (ii) YTHDC1 recruits histone modifier SET domain bifurcated histone lysine methyltransferase 1 (SETDB1) and cofactor tripartite motif containing 28/KRAB-associated protein (TRIM28/KAP1), which adds the repressive histone marks H3K9me3 [6A on LINE1 is recognized by YTHDC1, then LINE1 acting as a scaffold recruits TRIM28/KAP1 and NCL to chromatin and deposits the repressive histone mark H3K9me3 on 2C-related retrotransposons, which eventually inhibits retrotransposons transcription [6A -associated chromatin regions by YTHDC1, however, this decreases the H3K9me2 level and promotes gene expression [6A modification in cooperation with histone modifier mechanisms ultimately regulate the repeat RNAs and heterochromatin formation during embryonic development.More importantly, mpression , 88. In cription   and its paralog RBM15B recruit WTAP/METTL3 complexes to specific sites in Xsit which deposits m6A modifications on Xist. This leads YTHDC1 to preferentially recognize m6A modification and is required for Xist-mediated gene silencing [6A can be easily deposited in conserved AUCG tetraloop hairpin regions of the Xsit A-repeats and YTHDC1 recognizes it in a single-stranded conformation [Finally, milencing   . ReplicaSimilarly, R-loop formation causes genomic instability, which can inhibit tumor growth in human cancers. It is well known that one of the major hallmarks of cancer is genome instability . A few sHOTTIP directly interacts with CTCF-binding sites (CBSs) to form R-loops and then recruits CTCF/cohesin complexes and R-loop-associated factors, which reinforces the CTCF boundary and promotes the formation of the β-catenin topologically associated domain (TAD). The above HOTTIP and CTCF/cohesin mediated R-loop formation at TAD boundaries drives canonical Wnt/β-catenin transcription and ultimately facilitates leukemogenesis and AML development [On the other hand, R-loop-associated transcriptional regulation also plays essential roles in tumorigenesis and cancer progression. Aberrant R-loop accumulation localized at the TSS can promote tumorigenesis by affecting associated gene transcription. BRCA1 mutation causes nascent RNA-formed R-loop accumulation at the luminal gene and its enhancer region in luminal progenitor cells, which inhibits the enhancer-promoter interactions and transcriptional activation and subsequent primary luminal progenitor cell differentiation into mature luminal cells, resulting in oncogenic transformation and basal-like tumor formation . Chromatelopment   and histone deacetylase inhibitors (HDACis) are associated with replication stress and R-loops accumulation –116. Hen6A modification can be associated with many disease occurrence mechanisms including cancer by m6A writers-, erasers- and readers-mediated RNA fate decisions and translation regulation [6A reader YTHDF1 specifically binds m6A-modified Eukaryotic Translation Initiation Factor 3 Subunit C (EIF3C) mRNA and facilitates its translation. Then the increased EIF3C, a subunit of the protein translation initiation factor EIF3, can promote ovarian cancer tumorigenesis and metastasis [6A methyltransferase METTL3 in macrophages can decrease the YTHDF1-mediated translation of Sprouty Related EVH1 Domain Containing 2 (SPRED2), impairing the inhibition of ERK by SPRED2, which eventually leads the activation of NF-κB and Signal Transducer and Activator of Transcription 3 (STAT3) as well as facilitates tumor growth and metastasis [6A readers mediate RNA decay, which is associated with tumorigenesis, YTHDF2 is observed to be overexpression in glioblastoma cells and plays an important role in cholesterol metabolism-associated liver X receptor a (LXRa) and HIVEP zinc finger 2 (HIVEP2) mRNA decay in a m6A-dependent manner, which finally causes cholesterol dysregulation and promotes the glioblastoma cell proliferation, invasion and tumorigenesis [Numerous previous studies have shown that cytoplasmic mRNAs and noncoding RNAs with mgulation , 119–122tastasis . Corresptastasis . On the igenesis .6A readers and associated proteins being discovered, especially m6A -associated factors located in the nucleus and chromatin, the functions of caRNAs associated with m6A modification are gradually expanding. Among them, there are a few caRNAs related to m6A modification involved in cancer progression and tumorigenesis . Researchers have found that the m6A demethylase ALKBH5 can specifically recognize the 3′ UTR of nascent Forkhead Box M1(FOXM1) pre-mRNA, resulting in a decrease in m6A level on FOXM1 pre-mRNA, and an increase in HuR-mediated FOXM1 expression, which leads to GSC self-renewal and tumorigenesis. The lncRNA FOXM1 antisense transcript is able to facilitate the interaction between nascent FOXM1 pre-mRNA and ALKBH5 [6A modification level and stability of ARHGAP5 mRNA in the cytoplasm, which ultimately increases the chemoresistance of gastric cancer (GC) [6A writer KIAA1429 to the 3′ UTR of GATA3 pre-mRNA and deposit m6A methylation, which inhibits the interaction between GATA3 pre-mRNA and HuR, leading to the subsequent pre-mRNA degradation [6A-modified transcripts play contrasting roles in mRNA stabilization by interacting with the RBP HuR, hence further mechanisms of m6A and HuR-mediated RNA regulation should be investigated. The above examples show that lncRNAs indirectly associate with m6A modification by cooperating with mRNA and nascent pre-mRNA. Additionally, chromatin-associated lncRNAs can be directly deposited on m6A and subsequently participate in oncogene regulation and cancer progression. For example, the novel lncRNA lung cancer associated transcript 3 (LCAT3), is stabilized and upregulated in lung adenocarcinomas due to METTL3-mediated m6A modification, and then LCAT3 recruits Far Upstream Element Binding Protein 1 (FUBP1) to the MYC far-upstream element sequence, which leads to MYC activation and lung cancer cell proliferation, invasion and metastasis [6A and epigenetic regulation plays an important role in gene regulation, there are still few studies on its function in tumorigenesis and development. The newest study found that the RNA m6A level in esophageal squamous cell carcinoma tissues is significantly higher than that in paracarcinomic tissue. Further studies have shown that RNA m6A can negatively regulate the DNA methylation of esophageal cancer through FMR1 Autosomal Homolog 1 (FXR1) and TET1 and play an important role in the development of cancer [6A modification in cancer. Further studies should provide insight into the elusive and unknown association between m6A-modified caRNAs and cancer.More importantly, with an increasing number of msis Fig. B. A signd ALKBH5 . Likewiscer (GC) . Anotherradation . Interestastasis . Althougf cancer . HoweverNoncoding RNAs, especially lncRNAs, miRNAs and circRNAs, can participate in and modulate many biological processes, including RNA transcription, translation, and alternative splicing, contributing to the regulation of gene expression and cancer , 132. ThMany oncogenic chromatin-associated lncRNAs have the capability to interact with many transcriptional regulators, including TFs, DNA/RNA-binding proteins and epigenetic factors Fig. A. At theApart from the above oncogenic lncRNAs contributing to cancer cell proliferation, invasion and metastasis, other chromatin-associated lncRNAs play a contrary role in cancer progression, and tumor suppressor lncRNAs also mainly function by regulating related gene transcription Fig. B. For inOverall, chromatin-associated ncRNAs have dual roles in tumorigenesis and cancer development and usually act as RNA scaffolds or chaperones to recruit TFs, epigenetic regulators , transcriptional coactivators, DNA topoisomerase and DNA/RNA helicase to chromatin, which is essential for modulating cancer development-associated gene transcription, downstream signal transduction and subsequent physiological processes. Recently, one of the chromatin-associated snoRNA subsets was found to be relevant to DNA damage and cancer genome instability; notably, among them, the orphan snoRNA SNORA73 can combine poly (ADP-ribose) polymerase 1 (PARP1) and the canonical H/ACA proteins DKC1/NHP2 to form a snoRNP at DNA damage genomic loci, which blocks PARP1 autoPARylation and DNA damage repair and leads to genome instability and cell differentiation in AML . This suIn recent years, an increasing number of circulating ncRNAs, including circRNAs, lncRNAs and miRNAs, have been detected as biomarkers in the serum to predict tumor progression –148. SimOn the other hand, the mechanism of caRNAs involved in tumor development may be a target for cancer therapy . HOX traSince caRNAs are widely involved in gene regulation in various cancers, compounds and nucleic acid drugs targeting caRNAs may be used for cancer treatment. Although there are currently a large number of RNA agents in phase II or III clinical development , no caRN6A modification plays a vital role in various human cancers and could be a potential target for cancer therapy [6A, some recent studies suggest that m6A-deposited caRNAs or m6A-associated caRNAs may play an important role in tumors, which also indicates the crosstalk between RNA m6A modification and epigenetic mechanisms. Other RNA modifications might also be associated with caRNAs in gene regulation; for instance, pseudouridylation is the second most abundant RNA modification taking place cotranscriptionally in human pre-mRNA, and the most recent research maps the pseudouridine (Ψ) profiling of human chromatin-associated RNAs, which reveals that pseudouridine modification is abundant in alternatively spliced regions of nascent pre-mRNA and overlaps hundreds of binding sites for RBPs [In conclusion, different types of caRNAs might have potential roles in gene regulation by directly binding to chromatin or specific proteins such as TFs, RBPs and epigenetic factors that can be connected with chromatin. In addition to caRNA-mediated transcriptional activation and repression, caRNAs especially nascent RNAs can impact genome stability and DNA damage repair in cancer progression. Various caRNAs can play a role in promoting or inhibiting cancer progression, even for the same type of caRNAs, such as chromatin-associated lncRNAs or circRNAs, which both have dual roles in cancer depending on which protein is recruited by caRNA to chromatin. Recently, an increasing number of studies have suggested that RNA m therapy , 162. Alfor RBPs . This infor RBPs . With th


We conducted controlled experiments under realistic simulated data from ChIP-seq to confirm spatzie is capable of discovering co-enriched motif interactions even in noisy conditions. We then use spatzie to investigate cell type specific transcription factor cooperativity within recent human ChIA-PET enhancer–promoter interaction data. The method is available online at https://spatzie.mit.edu.Genomic interactions provide important context to our understanding of the state of the genome. One question is whether specific transcription factor interactions give rise to genome organization. We introduce  Genome organization plays an important role in the function of the genome in development  and disespatzie, an R/Bioconductor package named after the German diminutive for sparrow and inspired by the long-range geographical patterns of their songs . Raw data for all experiments is accessible from GEO  uses the presence or absence of a motif within an enhancer or promoter to determine a statistically significant association, thus Iab is the number of interactions that contain a match for motif a in the enhancer and motif b in the promoter, Ea is the number of enhancers that contain motif a  and twos method , and Bens method ), all P-The goal of spatzie is to identify pairs of transcription factor motifs which have a relationship such that the presence of motif A in an enhancer is associated with the presence of motif B in the promoter, indicating these transcription factors may be cooperating to drive enhancer–promoter interactions Figure . Given aWe validate spatzie by simulating a co-enrichment relationship where binding of USF1 at promoters is co-dependent on binding of ELF1 at enhancers. Using ELF1 and USF1 ChIP-seq data from ENCODE, we aligned and called binding events with GPS . We thenN percent of enhancer interactions and randomly permute them such that they are paired with new promoters and then run spatzie using score-based correlation, count-based correlation, and motif presence/absence association . The source code of the R package is hosted on GitHub (https://github.com/gifford-lab/spatzie). The core functionality is also available online at https://spatzie.mit.edu, which includes enhancer–promoter motif co-enrichment analysis with HOCOMOCO or user-defined motifs on interactions data mapped to either hg38, hg19, mm9 or mm10.The functionality of spatzie is bundled as an R/Bioconductor package with the same name (gkac036_Supplemental_FileClick here for additional data file.


Accumulating evidence points out that sperm carry epigenetic instructions to embryo in the form of retained histones marks and RNA cargo that can transmit metabolic and behavioral traits to offspring. However, the mechanisms behind epigenetic inheritance of paternal environment are still poorly understood. Here, we curated male germ cells RNA-seq data and analyzed the expression profile of all known histone lysine writers and erasers enzymes across spermatogenesis, unraveling the developmental windows at which they are upregulated, and the specific activity related to canonical and non-canonical histone marks deposition and removal. We also characterized the epigenetic enzymes signature in the mature sperm RNA cargo, showing most of them positive translation at pre-cleavage zygote, suggesting that paternally-derived enzymes mRNA cooperate with maternal factors to embryo chromatin assembly. Our study shows several histone modifying enzymes not described yet in spermatogenesis and even more, important mechanistic aspects behind transgenerational epigenetics. Epigenetic enzymes not only can respond to environmental stressors, but could function as vectors of epigenetic information and participate in chromatin organization during maternal-to-zygote transition. In the last years, accumulating research has documented that male germ cell maturation presents windows of vulnerability for epigenetic reprogramming by environmental stressors that can affect fertility and even transmit developmental, metabolic and behavioral traits to offspring . This tySpermatogenesis is a finely regulated process where unipotent spermatogonia undergo meiosis and, subsequently, spermiogenesis, to become sperm cells. During the developmental stages of spermatogenesis, male germ cells experience dramatic chromatin reorganization, where approximately 90% (human) to 95% (mouse) of histones are evicted and replaced by protamines to compact the paternal genome . ImportaThe role of epigenetic enzymes comes to focus, as they are responsible for the histones’ PTMs writing and erasing balance. Moreover, histone-modifying enzymes have been shown to respond to the organism physiology and metabolism, several disease conditions, and environmental stressors . Even thFASTQ files were downloaded from NCBI GEO and ArrayExpress databases. Transcriptomic data of male germ cells was obtained from GSE162740, consisting of germ cells isolated from “Stra8-Tom” mice obtained from the cross of Stra8-cre mice with CAG-LSLtdTomato mice . The grahttps://github.com/Gonzalez-Lab/Gonzalez-2022-germ-cells.FASTQ files from germ cells and sperm were processed with the following pipeline: quality control with FASTQC, trimming with fastp, mapping with STAR and GRCm39 (MM10 gencode.vM29.annotation), and counting with featureCounts. Processed data was manipulated using Rstudio (v1.2.1033) and Tidyverse packages Dplyr and ggplot2 . The malTo analyze the expression profiles of writers and erasers families for histone K acetylation and methylation across spermatogenesis, we curated RNA-seq data available in purified male germ cells from adult mice and selected the dataset GSE162740 . This daSmyd2, Kdm8, Hdac2, Mcm3ap), ii) downregulated at PD , and iii) downregulated at RStid   analysis using the DESeq2 algorithm, and performed contrasts on consecutive stages of cell development: SGdiff vs. SGund, PreL vs. SGdiff, LZ vs. PreL, PD vs. LZ and RStid vs. PD . For eact6b) see . To visuEzh1 at the SGund (Kdm6b (mt2a/b (Mll1/2), Setd2, Kmt5b (Suv420h1), Ehmt2 and Kdm1a  to compact the nucleus. at RStid , and alstage see  consistetage see . The RSter Ehmt2 , consister Ehmt2 . Additioogenesis  , preparing germ cells for gene activation at late spermatogenesis and embryonic development  formation at meiotic hot spots , that were found to counteract the enzymatic activity of EHMT2  receptor (RAR), suggesting that these HDACs could mediate chromatin silencing induced by RA, essential for the transition of SGund into SGdiff  at the PD stage, that were recently shown to interact with BEND2 and participate in DSB repair, synapsis and transcriptional repression , misregulation of meiotic and somatic genes, anomalous synapsis and misssegregation of chromosomes and apoptosis at pachytene stage (Ezh1/2 at PreL and LZ stages that were found involved in mammalian X chromosome inactivation (Kdm6a is part of the COMPASS complex with Kmt2b/c/d and Ashl2 that activate genes in response to RA by H3K4me deposition and H3K27me removal (Kdm6a expression induce defects that persisted transgenerationally (Setd6 at PreL stage, which in ESC was found with H3K27me3 close to differentiation marker genes and removed upon RA signal (mt1 at LZ stage, a recently described epigenetic mark found at promoters of genes encoding cell cycle regulators (During prophase I, recombination hotspots are mainly marked by H3K4me3 catalyzed by KMT2 family and PRDM9 methyltransferases . Moreovein vivo . It was ertility . We also onwards . The het onwards . Repressne stage . Interesne stage . Also, Sne stage . A recenne stage . Moreovene stage . In linetivation . Further removal  and moretionally . Additiotionally . We alsoA signal , suggestgulators .Kat2b and Kat8 at RStid stage, suggesting that these acetyltransferases are responsible for histone hyperacetylation prior to the histone-to-protamine transition (Msl3 and Kat8, and also p300/CBP and Kat2b that keep their expression from spermatocyte stage, due to their crucial role to regulate histone eviction (Hat1 expression was found related to the incorporation of H4K5/12ac and H3.3 variant at DSBs sites and the promotion of DNA repair (Chromatin organization dramatically changes during mid-to late-spermiogenesis, due to histone eviction and replacing by PRMs to facilitate the condensation and packaging of the paternal genome. In mice, 1%–10% of histones are retained in sperm chromatin and form a heterogeneous mixture of nucleo-histones and nucleo-protamines . At the ansition . In lineeviction . Moreoveeviction  that coueviction . AdditioA repair . H4K5/8/A repair  and moreA repair .Dotl1, H3K9me writer Ehtm2 and H3K27me writer Ezh2 at RStid stage. It was found that DOTL1 is enriched in post-meiotic stages of mouse germ cells and precedes the histone-to-protamine transition (Ezh2 and Ehmt2 methyltransferases were detected at spermatid stage, suggesting that spermatids have a potential preference for epigenetic transcriptional repression and heterochromatin formation (Multiple histone methylation have been also identified in spermatids, pointing to a balance of “opened” and “closed” chromatin regions during the histone-to-protamine transition . Here, wansition . Moreoveormation . During ormation . Moreoveormation , and proormation .Kmt5a/c, the H3K9me2 writers Ehmt1/2, and the H3K9me3 writers Suv39h2 and Setdb1/2, with established roles in chromatin structure organization. Moreover, some of these enzymes were found increased in the embryo compared to the MII oocyte, as indicative of paternal contribution to their pool levels. Hence, sperm’s epigenetic enzymes contribution to the zygote’s mRNA pool could cooperate to the translation of the machinery necessary for the extensive chromatin remodeling that takes place to allow a new developmental program to start.It is established that mature sperm transport a cargo of miRNAs, tsRNAs, lncRNAs, circRNAs, and protein-coding mRNAs, that carry an epigenetic blueprint involved in early embryo development . Some ofIn summary, the analysis performed here shows important windows during spermatogenesis, where interference with epigenetic enzymes gene expression may have phenotypic consequences in the offspring, even when they are not inherited. Moreover, we show that epigenetic enzymes mRNA could become functional in pre-cleavage zygotes and contribute to early chromatin organization with deep implications in future embryo development. The epigenetic enzyme’s paternal contribution could be another mechanism for epigenetic inheritance that deserves further consideration.


Drosophila melanogaster provide a valuable in vivo model to investigate how the adult stem cell identity is maintained and the differentiation of the daughter cells is regulated. GSCs are embedded into a specialized cellular microenvironment, the so-called stem cell niche. Besides the complex signaling interactions between the germ cells and the niche cells, the germ cell intrinsic mechanisms, such as chromatin regulation and transcriptional control, are also crucial in the decision about self-renewal and differentiation. The key differentiation regulator gene is the bag of marbles (bam), which is transcriptionally repressed in the GSCs and de-repressed in the differentiating daughter cell. Here, we show that the transcription factor MESR4 functions in the germline to promote GSC daughter differentiation. We find that the loss of MESR4 results in the accumulation of GSC daughter cells which fail to transit from the pre-cystoblast (pre-CB) to the differentiated cystoblast (CB) stage. The forced expression of bam can rescue this differentiation defect. By a series of epistasis experiments and a transcriptional analysis, we demonstrate that MESR4 positively regulates the transcription of bam. Our results suggest that lack of repression alone is not sufficient, but MESR4-mediated transcriptional activation is also required for bam expression.Ovarian germline stem cells (GSCs) of  Drosophila female germline stem cells (GSCs) provides an excellent model for studying stem cell maintenance and differentiation processes . W. Wbam anA niches E. Similaess CycB F. The acMESR4 regulates GSC differentiation, we first determined the expression pattern of GFP-tagged MESR4 expressed from a genomic bacmid construct (MESR4:GFP) [MESR4 variant completely rescued the lethal phenotype associated with the 79MESR4 mutation, indicating that the MESR4:GFP fusion protein is fully functional. We detected ubiquitous MESR4 expression in the somatic and germ cells of the ovaries of MESR4:GFP females. In the cells, MESR4 localized to the nuclei, suggesting a role for MESR4 in the transcriptional regulation of bam (To identify the molecular mechanisms by which SR4:GFP) . The tagn of bam A.bam transcription has been shown to be negatively regulated by the dpp signaling pathway through the SE element of the bam regulator region. In the bamPΔSE-Bam:GFP transgenic females, the SE element is removed and the GSCs do not respond to the inhibitory pMad-mediated Dpp signal and initiate bam expression from the transgene. Ectopically expressed bam then leads to the differentiation of the GSCs into CBs and cysts, which in turn results in “empty” niches containing no GSCs    B–E. Furt n = 99) B–E. TakeMESR4 revealed that the MESR4 protein possesses a plant homeodomain (PHD) finger generally involved in chromatin remodeling nine C2H2 type zinc finger (ZF) domains mediating sequence specific DNA–protein interactions [MESR4 can control the bam expression by several, not mutually exclusive, mechanisms. By the PHD finger, it may act as a chromatin remodeling factor and promote bam expression by the formation of a permissive chromatin environment. Alternatively, MESR4 can function as a DNA-binding transcription factor associating directly with cis-regulatory elements of differentiation promoting genes by the ZF domains. To investigate how MESR4 controls the bam expression, we analyzed the activity of the MESR4 PHD finger domain using a CRISPR-based loss-of-function genetic approach.An in silico analysis of ractions . Due to MESR4, we applied CRISPR/Cas9-mediated gene editing to generate MESR4 alleles lacking this domain. This way, we isolated a ΔMESR4PHD allele lacking the C-terminal 106 amino acids of MESR4 covering the entire PHD finger. The homozygous mutant ΔMESRPHD animals were viable, indicating that the PHD finger of MESR4 is not required for viability. The homozygous females were fertile, and wild type fusome containing cysts were detected in their niches. A lack of germ cell differentiation defects indicates that the PHD finger is dispensable for the MESR4 function in bam-mediated pre-CB to CB transition.PHD fingers have been reported to be involved in epigenetic gene regulation by mainly recognizing various unmodified or methylated lysines of H3 histone molecules . To testMESR4 controls the fate of germ cells in a very narrow developmental window by promoting the pre-CB to CB transition, and functions as a positive transcriptional regulator of the differentiation gene bam.The mechanisms behind the decision between self-renewal and differentiation act at multiple levels of gene regulation. Transcriptional control, regulation of protein or RNA turnover and translational regulation of differentiation genes act in concert to ensure the proper temporal and spatial regulation of GSC maintenance and differentiation. Here, we show that bam to prevent the differentiation [bam is released to trigger the transition of the pre-CBs to the differentiated CBs. While the mechanisms mediating the transcriptional suppression of bam in the GSCs are well known, the identity of positive transcriptional regulators that drive bam expression to facilitate the differentiation of the GSC daughter pre-CBs remains elusive. We propose that MESR4 is such a factor which functions in the pre-CB to promote bam transcription.At the transcriptional level, GSC differentiation can be theoretically triggered by the repression of self-renewal factors, by preventing the expression of differentiation inhibitory factors or by stimulating the expression of differentiation factors. In these processes, epigenetic mechanisms, such as the incorporation of histone variants, posttranslational histone modifications and repositioning of nucleosomes by chromatin remodelers play a key role by establishing a global chromatin landscape to control transcription in GSCs and in their differentiating daughter cells . In the ntiation ,44,45,46MESR4 is expressed in the germ cells and in all somatic cell types of the GSC niche, its function is exclusively required cell-autonomously in the germ cells at the pre-CB stage, as demonstrated by the accumulation of undifferentiated pre-CBs upon germline-specific MESR4 silencing. How the activity of MESR4 is spatially and temporally regulated remains elusive. MESR4 may function as a component of a protein complex specifically assembled in the pre-CBs and may bind a yet undetermined factor, the expression of which is restricted to the pre-CBs. Alternatively, MESR4 may be activated by upstream processes specifically taking place in the pre-CBs. In the pre-CBs, the transcriptional repressor Pgc is transiently expressed mediating a pulse of global transcriptional silencing which prevents the transcription of stem cell fate regulators [MESR4 to drive bam expression directly or indirectly.Although gulators . This isMESR4 function has been implicated in various developmental processes as a downstream effector of signaling pathways. It is able to suppress the constitutively activated Ras-MAPK pathway [MESR4 is required for the proper control of EGFR/ERK signaling during embryonic development and wing formation [MESR4 does not function as a component of the EGFR/ERK pathway. Impaired EGFR signaling in the niche results in the accumulation of GSC-like cells induced by the expanded dpp activity, whereas silencing of MESR4 in the germ cells induces the accumulation of pre-CBs. The MESR4 function is dispensable for proper germ cell development in the escort cells where the EGFR pathway is active.The  pathway  or the F pathway . In addiormation . In the ormation . SeveralMESR4 regulates germ cell development is the transcriptional control of bam. Based on its protein domain composition, MESR4 may promote bam transcription as a chromatin remodeling factor via the PHD finger domain [MESR4 function in germ cell differentiation. Consistent with our observation, previous studies failed to detect the direct association of MESR4 with the modified histones [MESR4 is involved in chromatin remodeling, we prefer an alternative hypothesis for the MESR4 function. According to this hypothesis, MESR4 directly interacts with the DNA via its Zn-fingers to specifically control the transcription of key differentiation regulators. An obvious candidate for a target of MESR4 regulation could be bam. This hypothesis is supported by two lines of evidence. We detected decreased bam mRNA levels in MESR4-silenced ovaries. Furthermore, the phenotype caused by the unsilenced bam expression from the bamΔSE transgene can be suppressed by silencing MESR4, thus confirming that MESR4 promotes bam expression. However, the precise mechanisms of bam regulation by MESR4 remain elusive. MESR4 may indirectly affect bam expression through the transcriptional regulation of a yet unknown factor. Alternatively, the transcriptional control of bam by MESR4 may be mediated directly by the association of MESR4 with the bam regulatory sequences. However, further studies are needed to distinguish between these possibilities.Our results suggest that the molecular mechanism by which r domain ,52. PHD r domain ,54,55,56histones . While w


For pregnancy to be established, uterine cells respond to the ovarian hormones, estrogen, and progesterone, via their nuclear receptors, the estrogen receptor (ESR1) and progesterone receptor (PGR). ESR1 and PGR regulate genes by binding chromatin at genes and at distal enhancer regions, which interact via dynamic 3-dimensional chromatin structures. Endometrial epithelial cells are the initial site of embryo attachment and invasion, and thus understanding the processes that yield their receptive state is important. Here, we cultured and treated organoids derived from human epithelial cells, isolated from endometrial biopsies, with estrogen and progesterone and evaluated their transcriptional profiles, their PGR cistrome, and their chromatin conformation. Progesterone attenuated estrogen-dependent gene responses but otherwise minimally impacted the organoid transcriptome. PGR ChIPseq peaks were co-localized with previously described organoid ESR1 peaks, and most PGR and ESR1 peaks were in B (inactive) compartment regions of chromatin. Significantly more ESR1 peaks were assigned to estrogen-regulated genes by considering chromatin loops identified using HiC than were identified using ESR1 peak location relative to closest genes. Overall, the organoids model allowed a definition of the chromatin regulatory components governing hormone responsiveness. The female reproductive system encompasses anatomical structures, physiological and cellular processes, and external environmental signals working in a coordinated manner. In order to prepare the uterus for pregnancy, estrogen and progesterone are released from the ovary to act on the uterine endometrium, which consists of epithelial and stromal cells lining the uterus. A receptive endometrium will allow attachment and implantation of embryos . The endEstrogen and progesterone impact intracellular processes via nuclear hormone receptor family members, the estrogen receptor (ESR) and progesterone receptor (PGR) ,5. NucleIhh) gene promoter was shown to be essential both for basal uterine expression of Ihh and its regulation by progesterone [PGR alters gene transcription by interacting with hormone-responsive enhancer (HRE) motifs in regions where pioneer factors, such as members of the Forkhead or GATA transcription factor families, have increased the accessibility of the chromatin . When PGesterone . HiC is esterone . These Testerone  and of eesterone  has beenMany of the molecular details of progesterone signaling have been derived from mouse models, which allow both genetic manipulations to assess the roles of progesterone response components as well as treatments with exogenous substances. The development of models to similarly interrogate and define mechanisms in human uterine tissue is more challenging due to the invasive method required to obtain endometrial samples. Studies in cultured stromal cells are one key model, as they are amenable to mechanistic studies; however, in vitro models in which to study epithelial cells have proven challenging. Recently, however, innovative organoid culture systems have been developed that provide a platform to study processes intrinsic to endometrial epithelial cells ,24,25.Here, we assessed the progesterone-induced responses of human endometrial epithelial organoids by examining their transcriptome and PGR cistrome. We integrated these findings, and our previously published estrogen-induced transcriptional responses and ESR1 cistrome of human endometrial epithelial organoids , with HiOrganoid culture, isolation, and analysis of RNA and chromatin were described previously . OrganoiRNA was isolated using Trizol  and analyzed by RT-PCR or RNAseq as previously described . For RT-https://broadinstitute.github.io/picard/ (accessed on 22 October 2018); Broad Institute, Cambridge, MA, USA). BAM files were imported into Partek Genomics Suite for analysis. RPKM for RefSeq genes was determined, and the maximum RPKM value from any sample for each gene (MAX) was listed. We noted differences between donor 1 and donor 2 datasets that reflect differences in RNA and library quality. For this reason, we analyzed the datasets from each donor separately and then compared the DEG so that each would be relative to its own V control. Genes for which the MAX < 1% of the mean RPKM for the whole dataset were filtered out . Values of 0 for V samples were replaced with a value of 0.05% of MAX to allow calculation of E2/V or (E2+MPA)/V. After PCA, it was noted that donor 1 V-1 and donor 1 E2-1 samples were outliers, potentially due to low reads, and were excluded from further analysis. Partek ANOVA was used to determine E2 vs. V, E2+MPA vs. V, and E2+MPA vs. E2 fold changes, and the List Manager function was used to find DEG . Partek Venn diagrams were used to construct a list of all DEG in either donor to combine fold changes of the datasets for visualization. Data are deposited in GEO (GSE200807).For RNAseq, RNA  was DNAse treated and cleaned up using the RNeasy Mini kit  or the RNA Clean and Concentrator 5 kit . RNA was submitted to the NIEHS sequencing core for library preparation using Illumina’s Ribo-Zero Gold kit for donor 1 RNA and Illumina Stranded mRNA Prep for donor 2 and paired-end sequencing using the Illumina Novaseq . Raw data were filtered to remove low-quality reads, mapped to hg38 using Tophat  and de-dFor HiC, frozen E2+MPA treated organoid samples were shipped to Active Motif  for HiC and library preparation using the Arima HiC kit . Libraries were sequenced by the NIEHS Genomics Core on the Illumina NovaSeq . The raw reads were mapped to human reference genome (GRCh38/hg38) using HiCUP version 0.7.1 . Juicer For PGR ChIPseq, crosslinked E2+MPA treated organoid cells were sent to Active Motif for Factor Path analysis using anti-PGR antibody sc-7208 . Resulting libraries were sequenced by the NIEHS Genomics Core On the Illumina NextSeq-High Output . Raw ChIP-seq reads were processed and aligned to the human reference genome hg38 using Bowtie . The rea® 680RD Goat anti-Rabbit IgG Secondary Antibody diluted 1:20,000.E2+MPA treated organoid cell pellets were homogenized in 50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 2.5 mg Na Deoxycholate with added protease  and phosphatase  inhibitors. Protein levels were assayed using the BCA kit , and 12 µg per lane was loaded onto a Protean mini gel  run, and transferred to nitrocellulose membrane using the Turbo-Blot transfer system (BioRad) according to manufacturer’s directions. Membrane was blocked with 5% milk  in TBST , 140 mM NaCl (Lonza), 1% TWEEN-20 (Sigma). PGR was detected using a mixture of IMA5 12,658 , hPRa7 (kindly provided by Dr. Dean Edwards), and mAb 1294  each at 1:1000 in milk overnight at 4C. Bands were detected using IRDye 800 CW Goat anti-Mouse IgG Secondary Antibody  diluted 1:20,000 in 5% milk for 45 min, and images generated using a Licor Fc instrument with Image Studio software (Licor). β-ACTIN was similarly detected using Actin (I-19)-R  diluted 1:5000, and IRDyeIHH) transcript in organoids derived from two of three different donors (PGR transcript levels manifested a similar pattern of estrogen induction and MPA inhibition (IHH and PGR (p < 0.05) of E2 vs. V or E2+MPA vs. V of donor one or two derived organoids  yielded 102 genes . This aThe 3-dimensional structure of chromatin facilitates interactions between distal enhancer regions that bind transcription factors, including ESR1 or PGR, and the genes they regulate. Using HiC, we analyzed loops in organoid chromatin and found a comparable number of loops from either donor a. We utiGREB1 gene is in the direct, anchor/anchor, and span anchor/groups  by deteBecause we see evidence that PGR is located more distally from genes, is not co-localizing with whole endometrial PGR sites, and the organoids are insensitive to progesterone response, we investigated the locations of the PGR peaks relative to the A (active chromatin) and B (inactive chromatin) compartments. This analysis reveals that most PGR peaks (68%) are co-localized in the B compartment . For comThe proportion of organoid of mouse uterus PGR or ESR1 peaks that co-localize with A or B compartments identified in HiC from the same sample and treatments are shown.IHH and PGR, are shown as examples. Both genes were increased by estrogen and showed attenuated inductions from estrogen+MPA  treated with estrogen, progesterone, and cAMP were included [ESR1 and PGR peaks and loops in the vicinity of two genes, ogen+MPA a. ESR1 aincluded .PGR. In organoids, the PGR gene is within a 450 kb loop . Since the mouse HiC analysis involves whole uterine tissue, a more complex looping pattern occurs, with multiple interactions in the region, but similar to the human gene, the loops in the region incorporate >400 kb. There is intense PGR and ESR1 binding to multiple sites, especially at the 3′ end of the Pgr transcript  compartment localization of PGR in organoids correlated with progesterone’s limited activity. However, ESR1 is similarly localized in the B compartment but mediates gene responses . PGR is PGR and IHH (PGR and ESR1 often bind DNA sites in enhancers that are not close to the genes they regulate but interact with genes via loops in the 3D structure of the chromatin. One approach often used to “assign” enhancer-gene interaction is to find the closest gene within a maximum kb cutoff. We used HiC to identify chromatin loops and then describe interactions of ESR1 or PGR binding peaks relative to loops . We loca and IHH , suggestIn sum, our study describes transcriptional profiles, the PGR cistrome, and the three-dimensional chromatin structure of hormone-treated endometrial epithelial cells cultured as organoids. Although progesterone primarily led to attenuation of estrogen-regulated genes, future work will investigate whether altered culture conditions might increase progesterone responsiveness. The ability to control hormone responsiveness in this in vitro model will advance our understanding of mechanisms involved in processes intrinsic to endometrial epithelial cells.