In this study, Saccilotto et al. investigated the molecular connection between proangiogenic signals and downstream gene expression during angiogenesis. By characterizing a Dll4 enhancer directing expression to endothelial cells at the angiogenic front, the authors identified MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Dll4 enhancer directing expression to endothelial cells at the angiogenic front, we found that MEF2 factors directly transcriptionally activate the expression of Dll4 and many other key genes up-regulated during sprouting angiogenesis in both physiological and tumor vascularization. Unlike ETS-mediated regulation, MEF2-binding motifs are not ubiquitous to all endothelial gene enhancers and promoters but are instead overrepresented around genes associated with sprouting angiogenesis. MEF2 target gene activation is directly linked to VEGFA-induced release of repressive histone deacetylases and concurrent recruitment of the histone acetyltransferase EP300 to MEF2 target gene regulatory elements, thus establishing MEF2 factors as the transcriptional effectors of VEGFA signaling during angiogenesis.Angiogenesis, the fundamental process by which new blood vessels form from existing ones, depends on precise spatial and temporal gene expression within specific compartments of the endothelium. However, the molecular links between proangiogenic signals and downstream gene expression remain unclear. During sprouting angiogenesis, the specification of endothelial cells into the tip cells that lead new blood vessel sprouts is coordinated by vascular endothelial growth factor A (VEGFA) and Delta-like ligand 4 (Dll4)/Notch signaling and requires high levels of Notch ligand DLL4. Here, we identify MEF2 transcription factors as crucial regulators of sprouting angiogenesis directly downstream from VEGFA. Through the characterization of a  Sprouting angiogenesis, in which new blood vessels form from existing ones, is driven in response to insufficient supplies of nutrients and oxygen. This response involves an exquisitely regulated pattern of gene expression directly downstream from vascular endothelial growth factor A (VEGFA) stimulation . As a coWhile many factors can elicit an angiogenic response, and numerous pathways influence sprouting angiogenesis, the Notch signaling pathway is crucially required to coordinate endothelial cell behavior during vessel patterning downstream from VEGFA signaling. In particular, the specification of endothelial cells into tip and stalk cells is regulated by Delta-like ligand 4 (Dll4)/Notch signaling . Higher Dll4, are precisely controlled at the angiogenic front (cis-regulatory elements (enhancers) that directly regulate differential expression at the angiogenic front in vivo has presented a major challenge when attempting to identify direct gene targets of transcriptional and signaling angiogenic networks. Many studies have focused instead on putative regulatory sequences identified by proximity to the core promoter. For example, multiple studies have focused on the Dll4 promoter region   and in the postnatal retina . The reporter gene expression at the angiogenic front in the retina was specific and persisted throughout sprouting angiogenesis .Previously, two arterial enhancers for the Notch ligand nhancer)  and the nhancer) . Similar factors . HoweverLacZ activity appeared equally robust in arterial endothelial cells at both embryonic and postnatal stages, no transgene activity could be detected in endothelial cells undergoing active angiogenesis . E11 hindbrains from multiple independent transgenic founders were examined to exclude the possibility of influences downstream from transgene insertion . These results demonstrate that the Dll4in3 enhancer contains DNA sequences that convey unique transcriptional information and suggest that gene expression in sprouting angiogenesis is regulated by factors other than, or in addition to, ETS, RBPJ, and SOXF.Dll4in3 and Dll4-12 enhancers both contain the ETS-, RBPJ-, and SOXF-binding motifs essential for arterial expression . ConsequSupplemental Fig. 2A,B). The Dll4in3 MEF2-binding motif bound MEF2A, MEF2C, and MEF2D proteins in electophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) analysis . These results therefore indicate that the MEF2-binding motif within the Dll4in3 enhancer is essential for directing gene expression in endothelial cells undergoing active sprouting angiogenesis.A comparison of the Dll4in3 and Dll4-12 enhancer sequences demonstrated that the angiogenic Dll4in3 enhancer contained consensus MEF2- and Forkhead C (FOXC)-binding motifs that were not present in the Dll4-12 sequence . As with the postnatal day 6 (P6) retina, expression was not detected in every endothelial cell ; instead, the pattern closely resembled that reported in Unc5bLacZ/+ mice, in which LacZ expression correlated with sprouting angiogenesis  despite the maintenance of transgene expression in the vasculature of some adult organs . These results clearly demonstrate the importance of the MEF2-binding motif for Dll4 enhancer expression during both physiological and pathological angiogenesis.Sprouting angiogenesis also occurs in the adult in response to physiological cues that support cycles of remodeling and repair and in pathological conditions, including cancer, where establishment of a vascular system is essential for tumour growth and metastasis . Therefoogenesis . StrikinDll4 gene and the acquisition of arterial fate more generally , we hypothesized that multiple MEF2 factors may regulate Dll4 expression. Supporting this, we detected expression of MEF2A, MEF2C, and MEF2D in endothelial cells from multiple tissues and with expression dynamics correlating to that of Dll4 after VEGFA . Combined siRNA-mediated knockdown of MEF2A,C and MEF2D in human umbilical vein endothelial cells (HUVECs) resulted in significantly reduced Dll4 expression after VEGFA stimulation . Furthermore, these MEF2 knockdown HUVEC cells demonstrated a reduction of tip cell competitiveness compared with control cells in VEGFA-induced mosaic spheroid sprouting assays . The same phenotype was observed in VEGFA-induced sprouting of embryoid bodies after CRISPR/Cas9-mediated ablation of Mef2A and Mef2C in embryonic stem (ES) cells . This reduced competitiveness was similar to previous reports demonstrating that Dll4LacZ/+ heterozygous ES cells with reduced DLL4 levels cannot compete with wild-type cells for the tip cell position during sprouting angiogenesis Cre-ERT2;Mef2Afl/fl;Mef2Cfl/fl, denoted as induced endothelial cell-specific (iEC) Mef2A/C knockout]. Although Mef2D was not targeted in these mice, DLL4 levels were reduced in the highly angiogenic E11 hindbrains 3 d after induction of gene deletion . Pan-genomic analysis of MEF2C-binding locations revealed a significant enrichment of enhancer-associated MEF2C binding near genes known to be up-regulated during sprouting angiogenesis . Loci containing significant MEF2-binding peaks included the transcription factors ETS1, ELK3, and HLX, all of which are also implicated in the regulation of angiogenic behavior   was particularly notable, as HLX/hlx expression is unusually specific to sprouting angiogenesis  , the strongest of which was able to bind MEF2A, MEF2C, and MEF2D in EMSA analysis at levels comparable with the Dll4in3 MEF2 motif . Mutation of these MEF2 sites resulted in total ablation of transgene expression in both transgenic zebrafish and mice . Together, these results suggest that MEF2 factors play a crucial role in the transcriptional activation of multiple genes associated with sprouting angiogenesis.The MEF2C-bound enhancer element 3 kb upstream of the ogenesis . Furtherx-3:GFP) E. This eMef2a and Mef2d in endothelial cells remain unknown, Mef2c transcription is known to be directly activated by ETS factors, including ETS1 .Our data support a role for MEF2 factors in the activation of sprouting angiogenesis downstream from VEGFA signaling in endothelial cells. We therefore next investigated the mechanisms by which MEF2 factors are able to specifically activate gene transcription in sprouting, but not quiescent, endothelial cells. Although the transcriptional regulators of ing ETS1 . TherefoMef2c enhancer itself is not specific to sprouting angiogenesis . Furthermore, ChIP analysis demonstrates that MEF2 factor binding to the DLL4in3 and HLX-3 enhancers occurs independently of VEGFA stimulation , although both HLX expression . To confirm that the repressive activity of HDACs on DLL4 expression occurred directly via MEF2 interaction, we tested the effects of two different small molecule inhibitors of class IIa HDACs. BML-210 works by specifically targeting and disrupting the MEF2:HDAC complex .The activity of MEF2 factors in other cell types is known to be modulated by the mutually exclusive recruitment of EP300 and class II HDACs . Class In HUVECs C and Dlln HUVECs D and ex  complex , whereas complex . Supporteraction . Converspression E. Other pression  but withMef2 genes though a feed-forward loop, enhances the angiogenic transcriptional cascade, and drives the high levels of Dll4 expression required to form a leading tip endothelial cell. This activation of Notch signaling then triggers the lateral inhibition and guided vascular patterning essential for sprouting angiogenesis.In conclusion, these results support a model F in whicDll4 in arteries downstream from VEGFA  are primarily restricted to arterial and angiogenic endothelial cells in mammals . SOXF faIn addition to collaborative binding with other transcription factors, our data also indicate that the specificity of MEF2 activation to sprouting angiogenesis is modulated by the removal of repressive class IIa HDAC binding and recruitment of EP300 downstream from VEGFA. However, MEF2 factors are able to confer signal responsiveness downstream from multiple signaling pathways. For example, multiple calcium-regulated protein kinases are able to modulate HDAC binding to MEF2 factors, and phosphorylation by MAP kinases can activate MEF2 . The latDll4. This may mirror the situation in skeletal muscle regeneration, where an absolute requirement for MEF2 was revealed only after compound deletion of Mef2A, Mef2C, and Mef2D  stable line was created by outcross of adult F0 carriers generated using the tol2 system. X-gal analysis of embryos and postnatal organs was as described  supplemented with protease and phosphatase inhibitor cocktail (Roche). Hindbrains were lysed in Tris buffer  supplemented with protease and phosphatase inhibitor cocktail (Roche), boiled at 100°C, and then incubated at 750 rpm at 80°C. Insoluble material was removed, protein concentration was determined with the BCA protein assay kit (Thermo Scientific), and 10 μg of protein per lane was separated by SDS-PAGE. DLL4 was detected using anti-DLL4  and membranes were reprobed with anti-β-Actin antibody . Band intensities of DLL4 and β-Actin were quantified with ImageLab software (Bio-Rad).Hindbrains and retinas were processed as described , and tum165 . ChIP assays were performed as described  at a final concentration of 100 nM using Oligofectamine (Life Technologies). MEF2A, MEF2C, or MEF2D stable knockdown was performed using lentiviral particles containing shRNAmir for MEF2A, MEF2C, MEF2D, or nonsilencing target (Open Biosystems).HUVECS (pooled) (Lonza) were grown as described previously . For VEGescribed  using anescribed  using prMEF2C and corresponding H3K27ac ChIP-seq data were obtained using publicly available ChIP-seq data  accessibHUVECS were starved for 16 h in EBM-2; treated with 400 nM TSA, 10 µM BML-210 (Abcam), 10 µM MC-1568 (Sigma), or DMSO (incubation times are in the figure legends); and stimulated with EBM-2 supplemented with 25 ng/mL VEGF-A for 1 h before harvesting for RNA extraction. Embryo culture in TSA was as described previously  with modRNA extraction was carried out using the RNAspin minikit  following the manufacturer's instructions from either HUVECs (directly lysed on the cell culture plate in R1 buffer) or whole-lung extracts (homogenized in liquid nitrogen using a pestle and mortar and subsequently lysed in R1 buffer). Total RNA (0.5–1 µg) was then retrotranscribed using random primers and SuperScript III (Life Technologies) as per the manufacturer's instructions, and the cDNA was analyzed by qRT–PCR  using gene-specific TaqMan probes (Applied Biosystems).


In mammals, the unicellular zygote starts the process of embryogenesis and differentiates into all types of somatic cells, including both fetal and extraembryonic lineages—in a highly organized manner to eventually give rise to an entire multicellular organism comprising more than 200 different tissue types. This feature is referred to as totipotency. Upon fertilization, oocyte maternal factors epigenetically reprogram the genomes of the terminally differentiated oocyte and spermatozoon and turn the zygote into a totipotent cell. Today, we still do not fully understand the molecular properties of totipotency. In this review, we discuss recent findings on the molecular signature and mechanism of transcriptional regulation networks in the totipotent mouse embryo. Multicellular organisms typically originate from a single totipotent cell, the zygote. In plants, structurally and functionally specialized cells of leaves, roots, stem, floral parts, and endosperm retain the potential to revert back to the undifferentiated state and form entire new plants, irrespective of their ploidy level . The potential of terminally differentiated cells to regenerate whole plants was referred to as “cellular totipotency” by the remarkable German plant physiologist Göttlieb Haberlandt in his famous address to the German Academy in 1902 , 4. The Following fertilization, maternal factors play a leading role in epigenetically resetting the parental DNA and histones across the genome of the zygote, thereby preparing for whole-genome activation and establishment of totipotency. A burst of transcription—known as zygotic genome activation (ZGA)—begins at the late one-cell stage and peaks at the two-cell stage in the mouse . ZGA is During oogenesis, the volume of oocytes dramatically increases to accommodate the storage of maternal factors  required for establishing totipotency and ZGA, such as nucleoplasmin (NPM) 2 , and theHyperaccessibility of chromatin by transcriptional machinery is a prerequisite for ZGA. Chromatin accessibility is largely determined by histone modifications of its N-terminal tails (“marks”), which acts as a fundamental epigenetic regulator to control the gene expression during embryo development in mammals.There are two major types of histone modifications involved in regulation of gene expression during the ZGA: lysine acetylation and lysine (tri)methylation. H4 acetylation makes pronucleus permissive for active transcription . Loss ofThe opposing marks histone H3 lysine 4 trimethylation (H3K4me3) and histone H3 lysine 27 trimethylation (H3K27me3) at gene promoter regions are associations with active and repressed genes, respectively. Following fertilization, H3K4me3 and H4 acetylation in the paternal genome are responsible for a minor ZGA. They are depleted in late zygotes stage but reestablished on promoter regions during the major ZGA at the late two-cell stage . On the At the time of fertilization, the chromatin molecules of the paternal and maternal genomes exhibit different epigenetic marks and organization. The paternal genome is haploid, and most of it is packaged densely, with protamines rather than histones, while the maternal genome is diploid, as it arrests at metaphase II, and is packaged with histones. After a sperm penetrates the cytoplasm of the oocyte, the paternal genome decondenses, enabling protamine removal and repackaging with the stored maternal histones in the absence of DNA replication, while the maternal genome completes meiosis. These newly integrated histones possess a transcriptionally permissive pattern of modifications, including H4 hyperacetylation  and H3K9During preimplantation development, striking changes in epigenetic modifications in the form of deposition of histone variants, reestablishment of histone marks, and DNA demethylation occur throughout the genome.Both canonical histones  and variant histones  are incorporated into chromatin throughout the first cell cycle of the zygote. Variant histones preferentially are deposited into specific genomic regions to form nucleosomes with unique biophysical characteristics. As one of the three variants of histone H3 in mammals, H3.3 differs from canonical H3 in only four amino acids and incorporates into chromatin in both a replication-independent and a replication-coupled manner. H3.3 interacts with the chaperones HIRA and Daxx/ATRX and is enriched in transcriptionally active regions . H3.3 alThe genome-wide cytosine methylation profile differs among cell types, and it functions as a form of memory of the cell’s identity . 5-methyIn concordance with histone acquisition, the paternal genome undergoes genome-wide loss of DNA methylation via an active mechanism prior to the start of DNA replication , 51.Recent studies have found a new mechanism of active demethylation involving prior modification of methylated cytosine and nucleotide excision and repair. 5-hydroxy-methylcytosine (5hmC), a stable hydroxylated metabolite of 5mC, was first identified in the genome of T-even bacteriophages , and it Tet3 has been shown to prevent both elevation of 5hmC and reduction of 5mC levels in the paternal genome, impair promoter demethylation of Oct4 (Pou5f1) and Nanog, delay the activation of a paternally derived Oct4 transgene, and cause frequent death of the resulting embryos [Although recent sequence data has shown active demethylation in maternal DNA as well , 65, hig embryos . These f embryos , furtherOct4flox/flox/ZP3Cre/+ female mice are capable of completing full-term development after fertilization, indicating that Oct4 is not required for initiating totipotency or pluripotency in embryos [Oct4 activation in early embryos is critical for understanding the molecular regulation network of totipotency and transition from totipotency to pluripotency. There are a few transcriptional factors found to be involved in the regulation of Oct4 expression. In proliferating stem cells, Promyelocytic leukemia (Pml) protein, along with the transcription factors TR2, SF1, and Sp1, and the Brg1-dependent chromatin remodeling complex (BRGC), associates with the Oct4 promoter to maintain a nucleosome-free region for Oct4 gene expression [Oct4 and Nanog in transplanted somatic nuclei in Xenopus oocytes [Tpt1 by small interfering RNA (siRNA) does not reduce Oct4 expression in mouse embryos [Oct4 distal enhancer (DE), and evidence shows that injection of Sall4 siRNA into zygotes knocking down Sall4 mRNA levels by 50% leads to a 70% reduction of Oct4 expression levels, suggesting that Sall4 is a transcriptional activator of Oct4 expression [Sall4 by injection of more efficient Sall4 siRNA into maternal Oct4-deficient zygotes—to avoid any possible effect of maternal Oct4 as a positive autoregulator—does not lead to any Oct4 expression changes at the blastocyst stage [Oct4, but to play no evident role in the self-renewal of ESCs [AID) has also been shown to be required for Oct4 activation during reprogramming [Oct4 expression, and shown that Paf1C overexpression blocks the differentiation of ESCs while Paf1C knockdown causes expression changes similar to those caused by Oct4 or Nanog depletion [Eif4eloo) [Oct4 expression is activated in the embryo.The maternal octamer-binding transcription factor 4 (Oct4), encoded by the gene Pou5f1 hereafter referred to as Oct4, is at the top of the pluripotency regulatory hierarchy in pluripotent cells , 63. How embryos –70. The  embryos , suggest embryos , 72. It  embryos  and is e embryos . Therefopression . Cancer- oocytes , but kno embryos . The matpression . Contradst stage . The nuc of ESCs . However of ESCs , and to  of ESCs . As a cogramming . A genomepletion . Studiesif4eloo)  and a laif4eloo) . To thisUnlike the case for pluripotency, the mechanism underlying the molecular regulation of totipotency remains largely unknown. In mice, only the zygote and two-cell-stage blastomeres can generate an entire organism on their own, and are therefore regarded as totipotent cells . The morRetrotransposon transcripts contribute a significant portion to the transcriptome during ZGA. Retrotransposons can also act as alternative promoters in the activation of protein-coding genes by generating chimeric transcripts with retrotransposon gene junctions . The mosZscan4 is activated during ZGA [Zscan4 by siRNAs delays progression from the two-cell to four-cell stage, and thus leads to the formation of blastocysts that fail to implant or proliferate in blastocyst outgrowth culture [ring ZGA  and can ring ZGA . Knockdo culture . Zscan4  culture . Express culture . Of note culture .Furthermore, another recent study has described a small transient ESC/iPSC population with fluctuating expression of a particular retrotransposon, MERVL and a transcriptome that closely resembles that observed in the blastomeres of the totipotent, two-cell embryos , indicatRecent progress in identifying two-cell marker genes and new players in the genome-wide demethylation process has shed light on the molecular mechanism governing totipotency. Nevertheless, many questions still remain unanswered. To date, no totipotent cell lines have been established in vitro. The upstream “master” signals that trigger the establishment of totipotency have not yet been identified. Given the distinct phases of ZGA in the mouse, it is likely that multiple regulators/cofactors are associated with regulators of ZGA to ensure temporal gene activation for establishing totipotency. It would be interesting to see how the upstream signals relate to histone replacement or modification, genome-wide DNA demethylation, ZGA, and the expression of two-cell-specific genes, particularly those of maternal origin. The identification of two-cell marker genes has moved us closer to solving the fundamental question in developmental biology of how totipotency is established.


Campylobacter jejuni Fur (CjFur) employs the same surface to positively and negatively control gene expression regardless of the presence or absence of metals. Moreover, the crystal structure determination of a CjFur devoid of any regulatory metals shows that subtle reorientation of the transcription factor DNA binding domain negatively impacts DNA binding, gene expression and gut colonization in chickens. Overall, these results highlight the versatility of the CjFur DNA binding domain in mediating all gene regulatory events controlled by the metalloregulator and that the full metalation of CjFur is critical to the Campylobacter jejuni life cycle in vivo.Ferric uptake regulators (Fur) are a family of transcription factors coupling gene regulatory events to metal concentration. Recent evidence has expanded the mechanistic repertoires employed by Fur to activate or repress gene expression in the presence or absence of regulatory metals. However, the mechanistic basis underlying this extended repertoire has remained largely unexplored. In this study, we used an extensive set of mutations to demonstrate that  However, when found in excess, these metal ions are toxic. To prevent toxicity and control their intracellular availability, metal ion concentrations are tightly controlled4. In bacteria, a large group of metal-sensing transcription factors regulates the intracellular homeostasis of metal cofactors. These metalloregulators are divided into seven different families that are important for the detection of the six biologically essential transition metals, MnII, FeII, CoII, NiII, CuI and ZnII, as well as other transition metals such as Ag, Au, CdII and HgII 3. One family, the ferric uptake regulator (Fur) family of metalloregulators, is involved in the regulation of iron, manganese, zinc and nickel homeostasis through the activity of Fur, Mur (manganese uptake regulator), Zur (zinc uptake regulator) and Nur (nickel uptake regulator) proteins, respectively. In addition to these members, the Fur family of metalloregulators also includes the peroxide stress regulator (PerR) and the heme-dependent iron responsive regulator (Irr)11. Depending on the availability of metal cofactors, the Fur superfamily regulates the expression of proteins involved in metal acquisition, storage and consumption in order to maintain metal homeostasis and ensure that the concentration of free metals do not reach toxic levels2. In addition, the Fur family of transcription factors regulates the expression of mRNAs encoding for proteins implicated in energy metabolism, acid and oxidative stress defense, the tricarboxylic acid cycle, protein glycosylation and flagella biogenesis13. Moreover, members of the Fur superfamily play a role in the expression of virulence factors in most pathogens4 and are therefore important factors contributing to bacterial pathogenicity15.Transition metals are crucial components of several metabolic pathways and are critical for DNA, RNA and protein synthesis24 revealed that these transcription factors fold into two distinct domains, including a DNA binding domain (DBD) and a dimerization domain (DD) localized on the N- and C-terminus of the protein, respectively. The DBD is typically composed of a helix-wing-helix domain, while the DD consists of three antiparallel β-strands and two α-helices. Collectively, these structures show the presence of two or three functional metal binding sites. The first structural site, referred to as S1, is located at the C-terminus and is coordinated by four cysteine residues. The second structural site, S3, is also found within the DD and plays a role in stabilizing the dimeric form of the regulator. The third metal binding pocket is the regulatory site (S2) and is composed of residues located in a region referred to as the hinge, which links the DBD and DD.Previous structural studies of members of the Fur family of proteins25. Mechanistically, the metal ion binds to Fur (referred to as the holo-form) to induce a structural reorganization of the DBD, leading to DNA binding and gene repression. However, recent studies revealed that Campylobacter jejuni17, Helicobacter pylori28 and Neisseria gonorrhoeae29 Fur proteins both repress and activate gene expression in the absence of regulatory iron (referred to as the apo-form) and also positively regulate gene expression when bound to iron collectively forming four different modes of gene regulation. To better understand the mechanistic basis underlying the extended repertoire of DNA binding by the Fur family of metalloregulators, we undertook structural and biochemical studies of CjFur. In the current study, we show that similar sets of residues are involved in the positive and negative regulation of holo- and apo-regulated genes. Moreover, we demonstrate that metal coordination in the S3 site is important for CjFur DNA binding activity and contributes to processes related to chick ceca colonization by Campylobacter jejuni.Classically, the Fur family of metalloregulators was defined as transcriptional repressors coupling metal ion concentrations to gene downregulation17. Based on our initial observations, we believe that our previously published structure of CjFur is devoid of metal in its regulatory site, S2, and still adopts a conformation conducive for binding DNA  as the DNA binding element , two genes known to be activated and repressed by CjFur, respectively, in the absence of iron 23 structure as the template and SWISS_MODEL software31. Calculation of the holo-CjFur surface electrostatic potential  in which H99 and H137 were substituted with alanine residues . Similarly, CjFurΔS3 binds with less affinity to DNA; however, the impact of its mutation is less deleterious in the holo-conditions compared to the binding reactions performed in the apo-conditions. Overall, these observations suggest that both metal binding sites play a role in properly aligning the CjFur DBD in an orientation conducive for binding DNA.The binding of metals in the regulatory site of Fur proteins is important for the DNA binding activity. Given that the S2 and S3 sites are important for the binding of manganese and zinc, we posited that both sites are important for DNA binding activity. To test this hypothesis, we performed binding reactions with increasing concentrations of wild-type CjFur, CjFuratA Fig. . Similarin vivo, we complemented the CjFur-deleted strain (referred to as Δfur) using CjFur wild-type (Δfur + fur), CjFurS2Δ (Δfur + furΔS2) or CjFurS3Δ (Δfur + furΔS3) genes. Consistent with the in vitro binding assays, stable expression of CjFurΔS2 and CjFurΔS3 in a Δfur background strain failed to maintain the repression of two holo-regulated genes, chuA and cfrA 32, the Δfur background strain mutant exhibited increased sensitivity toward H2O2 relative to the NCTC strain or the Δfur + fur strain. In contrast to the Δfur strain, both the Δfur + furΔS2 and Δfur + furΔS3 strains failed to show H2O2 resistance  value of 0.01 Å, and the angles fall within favored and allowed regions  of the Ramachandran plot  of 0.07. However, upon substitution of R30 with a glutamic acid, an intermediary shifted species migrating at a Rf of 0.31 was observed. While the stoichiometry of binding of apo-CjFur to DNA is still unknown, our data suggest that wild-type CjFur, in apo-conditions, forms oligomers on DNA and that R30 is important for this multimerization. Interestingly, the Protein Interfaces, Surfaces and Assemblies (PISA) analysis revealed that the homologous arginine residue in PaFur, R18, is important for its tetramerization35, suggesting that this residue could also be involved in the multimerization of CjFur on DNA  recognizes DNA through shape readout, while Y56 and R57 (corresponding to Y68 and R69 in CjFur) recognize the substrate through base readout38  and Glu110 (S2 site) resulted in the reduced ability to form higher-order structures42. Collectively, these results suggest that these mutations induce conformational changes that position residues important for the interaction of different CjFur dimers on DNA element in one or multiple orientations non-conducive for protein oligomerization.In addition of blunting metal incorporation, mutation of the S2 and S3 metal binding sites also impairs CjFur ability to form higher molecular weight complexes. As shown in Campylobacter jejuni life cycle, (2) no Fur homologues are found in eukaryotes and 3) the S3 site is essential for the activity of CjFur in terms of contributing to both the metalation of S2 and the correct alignment of the CjFur DNA binding domain, our results collectively highlight a potentially novel approach to block CjFur function in developing antimicrobial agents targeting the S3 metal binding sites.In conclusion, considering that (1) CjFur is essential for gut colonization and E. coli Rosetta cells for 16 hours at 18 °C with 100 μM IPTG. Following overexpression, cells were centrifuged and harvested in 50 mM Tris pH 7.0, 100 mM NaCl and 5 mM β-mercaptoethanol (Bme). CjFur was purified on Strep-TACTIN beads as previously described43. Following elution and TEV cleavage, CjFur was concentrated and further purified by size-exclusion chromatography using a Superdex 75 pre-equilibrated with 20 mM Tris pH 7.0, 250 mM NaCl and 5 mM Bme. The protein was concentrated and stored at −80 °C.To obtain the metal bound form of CjFur, its cDNA was cloned into a homemade vector, enabling the expression of the metalloregulator as an N-terminal TEV-cleavable Strep tag. CjFur was overexpressed in 2 , which was gradually added to the protein, with 5 minutes of incubation on ice between each addition. To remove the excess of free metal ions, a second size exclusion chromatography was performed in 20 mM Tris pH 7.0, 250 mM NaCl and 5 mM Bme. After this second gel filtration, purified holo-CjFur was concentrated and used for electrophoretic mobility shift assays.Purified CjFur was incubated with excess MnClThe metal content of purified CjFur proteins was determined by inductively coupled plasma mass spectrometry (ICP-MS) at the Quantitative Bioelemental Imaging Center, Northwestern University, using a Thermo Fisher X Series II ICP-MS system. Using two serial dilutions (1/20 and 1/100) of purified CjFur and recording 3 spectra per dilution, a total of six MS spectra per protein sample were recorded and used for metal content calculations.katA promoter region were annealed by incubation at 95 °C for 15 minutes and slowly cooled to room temperature. For the gel shift assays, 2 nM Cy5-labeled DNA fragment was incubated with increasing amounts (0–100 nM) of purified recombinant holo-CjFur protein for 45 minutes on ice in binding buffer . Samples were separated on a 6% non-denaturing polyacrylamide gel (19:1) for 1 hour at 100 V and 4 °C. Gels were freshly prepared with 100 mM Bis-Tris borate, pH 7.4, and 100 μM MnCl2 and prerun for 30 minutes at 150 V and 4 °C.To perform EMSAs mirroring the holo-form of the transcription factor, forward (CjkatA60_For_1588) and reverse (CjkatA60_Rev_1589) Table  Cy5-labecj1345c operon (200 bp) was PCR amplified using the Campylobacter jejuni NCTC chromosomal DNA as a template and the cj1345c_For_1692/cj1345c_Rev_1693  for 45 minutes. Samples were then loaded on 5% nondenaturing polyacrylamide gel (19:1) and electrophoresed for 1 hour at 100 V and 4 °C. Gels were freshly prepared with 100 mM Bis-Tris borate, pH 7.4, and prerun for 30 minutes at 150 V and 4 °C.To perform EMSAs in conditions mirroring the apo-form of CjFur, the promoter region upstream of the 93 Table   was added to cell cultures, and the cells were harvested by centrifugation at 4000 RPM for 10 minutes. Cell pellets were resuspended in TE buffer , and the RNA was extracted using the hot phenol-chloroform method45. RNA precipitation was performed by an overnight incubation in absolute ethanol at −80 °C. RNA pellets were subsequently washed four times with 80% cold ethanol and were resuspended in RNase-free water. RNA was then treated with RNase-free DNase I (Invitrogen) to remove contaminating genomic DNA and was further purified using an RNeasy Mini Kit (Qiagen). The absence of contaminating genomic DNA was confirmed by PCR, and the quality of RNA was assessed on agarose gel. Reverse transcription was carried out using the QuantiTect Reverse Transcription Kit (Qiagen). RT-qPCR was performed with the MX3000P platform (Stratagene) using the Syber Green quantification method and ROX normalization. The relative expression levels of katA and cfrA as well as cj1345c and cj0948c, two holo-Fur-regulated and apo-Fur-regulated genes, respectively, were analyzed by RT-qPCR. The relative expression level of each gene was normalized to slyD (endogenous control) and to fur. The primers set used for katA, cfrA, cj1345c and cj0948c are listed in Table Cells were grown under microaerophilic conditions to midlog phase in MEM-α medium supplemented with 10 mM sodium pyruvate with or without 40 μM FeSOCampylobacter jejuni NCTC11168, Δfur deletion mutant and complemented fur, furΔS2 and furΔS3 strains were grown on MH agar plates supplemented with required antibiotics  for three days under microaerophilic conditions. Several colonies from each strain were cultured overnight in MH biphasic flasks. The overnight cell cultures were diluted in MH broth to an optical density at 600 nm of 1.0. For each strain, 4 mL of this diluted bacterial suspension was added to 90 mL of melted MH agar (cooled to approximately 50 °C), and the mixture was poured in equal volumes into three Petri dishes. After solidification, paper discs (6 mm diameter) were placed on the surface of the agar, and 10 μL of 3% hydrogen peroxide (H2O2) was added to each paper disc. MH agar plates were subsequently incubated under microaerophilic conditions for 20 hours, and the diameter of growth inhibition was measured in millimeters for each strain. All experiments were performed in triplicate, and statistical significance was determined using the Student’s t-test. P values < 0.05 were considered significant.34. Briefly, strains corresponding to Campylobacter jejuni NCTC11168 or strains harboring a deletion in the fur gene (Δfur) and complemented with either wild-type fur (Δfur + fur), Δfur + furS2Δ and Δfur + furS3Δ were grown to mid-log phase in biphasic flasks under microaerophilic conditions at 37 °C. Bacterial suspensions were then diluted in fresh MH broth to an optical density at 600 nm of 0.13, which corresponds to approximately 108 CFU/mL.Water and food were withheld for 2 hours prior to inoculation. Three-day-old chicks were inoculated orally with 300 μL of the diluted bacterial suspension. Each bacterial suspension was serially diluted and plated on MH-agar plates to confirm that all the chicks received approximately the same number of viable C. jejuni. The chicks were euthanized seven days post inoculation, and their ceca were collected and weighed. The cecal contents were extracted and homogenized in MH broth. The cecal contents were then serially diluted and plated onto selective Karmali agar (Oxoid) supplemented with chloramphenicol and kanamycin for the complemented strains. The Karmali plates were incubated under microaerophilic conditions for 48 hours at 42 °C, and the resulting colonies were counted and expressed as CFU per gram of ceca. The data were statistically analyzed with a nonparametric Mann-Whitney rank sum test. P values less than 0.05 were considered statistically significant.The chick colonization assay was performed as described previously46. The model was refined using the BUSTER software47, and manual modifications to the model were done in Coot48. The quality of the model was assessed using Molprobity49 and RAMPAGE50. Coordinates were deposited under the accession number 6D57.The crystals of CjFur devoid of metals in the S2 and S3 regulatory sites were obtained as follows. Following gel filtration and concentration, CjFur (7.5 μg/μL) was immediately combined, in a 1:1 ratio, with a mother liquor composed of 0.1 M Bis-Tris pH 5.5, 0.25 M MgFormate, and 25% PEG3350. Crystals were harvested and soaked in the mother liquor supplemented with 7.5% glycerol. A full dataset was collected at the 17-ID beamline at Argonne National Laboratory’s Advanced Photon Source and indexed using HKL-2000 (HKL Research). The structure of CjFurZn was solved by molecular replacement using the CjFur DBD and DD (PDB: 4ETS) as search models and the program Phaser from the PHENIX packageSupplementary Information


Trf2−/− mice are sterile and characterized by arrested spermatogenesis at the transition from late haploid spermatids to early elongating spermatids. Despite this characterization, the molecular function of murine Trf2 remains poorly characterized and no direct evidence exists to show that it acts as a bona fide chromatin-bound transcription factor. We show here that Trf2 forms a stable complex with TFIIA or the testis expressed paralogue ALF chaperoned in the cytoplasm by heat shock proteins. We demonstrate for the first time that Trf2 is recruited to active haploid cell promoters together with Tbp, Taf7l and RNA polymerase II. RNA-seq analysis identifies a set of genes activated in haploid spermatids during the first wave of spermatogenesis whose expression is down-regulated by Trf2 inactivation. We therefore propose that Trf2 is recruited to the preinitiation complex as a testis-specific subunit of TFIIA/ALF that cooperates with Tbp and Taf7l to promote haploid cell gene expression.Mammalian genomes encode two genes related to the TATA-box binding protein (TBP), TBP-related factors 2 and 3 (TRF2 and TRF3). Male  Regulated initiation of transcription by RNA polymerase II (Pol II) involves formation of a preinitiation complex (PIC) over the transcription start site (TSS). In addition to RNA polymerase II (Pol II), the PIC comprises a set of general transcription factors TFIIA, B, D, E, F, and H as well as the mediator complex and a set of DNA repair factors1235891011TBP is a bipartite protein with a variable N-terminal domain, but a highly conserved C-terminal domain shared with the TRFs and forming a saddle-like structure with a concave surface that binds the consensus TATA element DNA and a convex surface that interacts with TAFs as well as the TFIIA and TFIIB components of the PIC131415171920218232425Drosophila, TRF2 binds chromatin at different sites from TBP and ChIP-seq analyses revealed its binding at the DPE motif in a large group of TATA-less promoters272830Trf2−/− mice are morphologically indistinguishable from their wild-type (WT) littermates32Trf2−/− mice are sterile and characterized by an arrest of spermatogenesis at the transition from late haploid spermatids to early elongating spermatids. Chromatin organization of mutant haploid round spermatids is disorganized by fragmentation of the chromocenter, a structure formed by pericentric heterochromatin of all chromosomesThe functions of TRF2 have been studied in different organisms. In Trf2−/− testes suggested that several haploid-expressed genes were down-regulated32bona fide transcription factor and that the effects seen upon its ablation are due to altered gene expression.Despite this characterization of its essential role in spermatogenesis, the molecular function of murine Trf2 remains poorly characterized. Analyses of We show here that Trf2 forms a complex with TFIIA and ALF that is chaperoned in the cytoplasm by several heat shock proteins. We also demonstrate for the first time that Trf2 is recruited to active haploid cell promoters, not as a substitute for Tbp, but together with Tbp, Taf7l and Pol II. RNA-seq analysis identifies a set of testis genes activated around post-natal day 21 when the haploid spermatids are formed in the first wave of spermatogenesis and whose expression is down-regulated by Trf2 inactivation. We therefore propose that Trf2 is recruited to the PIC together with Tbp and Taf7l as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression.Trf2tag/tag mice expressed the Trf2-Tag protein in their testes at levels comparable to WT littermates . More importantly, Trf2tag/tag male mice were fertile indicating that Trf2-tag was functional and supported spermatogenesis. This can be seen in histology sections from testis showing that haploid round spermatids from WT and Trf2tag/tag mice contained an intact chromocenter, whereas those from Trf2−/− mice comprised a fragmented chromocenter  along with TFIIA and ALF . ImportaTrf2−/− testes showed that TFIIA and ALF eluted around the same B5 fraction  cells that do not express Trf2 . Trf2-tag, but not native Trf2 was present in the IP from the Trf2tag/+ extracts, and neither was present in the extracts from the WT testis  and TFIIA (66 kDa) or ALF (82 kDa). The higher molecular mass could be explained if the complex were a dimer formed through dimerization of Trf2 or the TFIIA/ALF components of the complex. To test this, we prepared extracts from heterozygous T testis  indicatiTrf2tag/tag testis . Integration of these data sets showed that Trf2 bound to active H3K4me3-marked promoters along with Tbp, Pol II and Taf7l  to identify active promoters. ChIP was performed on chromatin from nd Taf7l . Around nd Taf7l . Analysind Taf7l . Sites, nd Taf7l .Trf2−/− testis chromatin. While the specific signal for Trf2 was lost in the chromatin from mutant testes, that of Tbp, Pol II and H3K4me3 was virtually unchanged  7 only spermatogonia and somatic cells are present. At PND14, the first pachytene spermatocytes appear. At PND20 the first round spermatids can be detected, and by PND30 spermatids have elongated. Each stage of spermatogenesis is characterised by activation and repression of a set of genes. Laiho Taf7l−/y mice. However, only 15 genes were commonly down-regulated in both lines . All experimental procedures involving mice were performed in accordance with institutional guidelines regarding the care and use of laboratory animals and with National Animal Care Guidelines .A modified pL451vector containing a 6XHisTev3XFlag TAP-TAG followed by a neomycin resistance cassette flanked with two FRT sites was used to construct a targeting vector by sub cloning 5′ and 3′ arms of the The following antibodies were used in this study. For ChIP: Flag- M2 resin (Sigma), Trf2: in house rabbit polyclonal antibody 1727 raised against a peptide corresponding to Trf2 amino acids 115–136, gift from L. Tora, TBP: ab28175, Abcam and ab51841, Abcam, PolII: sc 9001X, Santa Cruz, H3K4Me3: 04-745, Millipore. For immunoblots the following antibodies were used. Trf2; 1727 as described above, TFIIA: rabbit polyclonal J17, gift from H.G. Stunnenberg, ALF: in house mouse monoclonal 2B11 as described20 mouse testes were homogenized by douncing with a loose pestle in 10 mL hypotonic buffer . After 10 min incubation on ice the suspension was centrifuged for 10 min at 4000 rpm and the supernatant kept as cytoplasmic extract. The nuclear pellet was washed once with hypotonic buffer and dounced in 2 mL high salt buffer  with a tight pestle. The suspension was incubated at 4 °C with agitation for 30 minutes and centrifuged at 13000 rpm for 5 min.500 μl of cytoplasmic extracts containing 5–10 mg of protein were injected onto a equilibrated Superose 6 (10/300) column and run at 0.4 ml/min. 500 ul fractions were collected and analysed by western blot.Trf2tag/tag and control Trf2+/+ fractions were resolved by SDS-PAGE and stained using the Silver Quest kit (Invitrogen). Sections of gel from the lowest to highest molecular mass were systematically excised and analysed by mass-spectrometry either at the Taplin Biological Mass Spectrometry Facility  or the IGBMC mass-spectrometry facility.The fractions containing Trf2 and TFIIA/ALF from the gel filtration were pooled and subjected to tandem Flag-His purification. Tagged Trf2 was immunoprecipitated with anti-Flag M2-agarose (Sigma), eluted with Flag peptide (0.5 mg/mL), and further affinity purified with Talon superflow affinity resin (Clontech) and elution with 0.1 M EDTA. Purified complexes from the Chromatin from mouse testes was prepared either by sonication or by digestion with MNase. Mouse testes were decapsulated in PBS and seminiferous tubules were put into a dounce containing 0.4% PFA in 1X PBS (for sonicated chromatin) or 1% PFA in 1X PBS (for MNase digestion) and homogenised with a loose pestle. Cross-linking was stopped with glycine . The cell pellet was collected by centrifugation for 5 min at 4000 rpm, lysed in 5 volumes of lysis buffer  and sonicated for 10 min on a Covaris sonicator. MNase digested chromatin was prepared by resuspending the cell pellet in 7 mL hypotonic buffer  and douncing with tight pestle. The suspension was incubated for 10 min on ice and centrifuged for 5 min at 4000 rpm. Nuclei were washed once with 1X PBS and spin and resuspended in 2 volumes of MNase lysis buffer . The lysate was sonicated for 30 sec as described above, CaCl2 and MgCl2 were added to 5 mM final concentration each and digestion was performed with MNase   for 10 min of incubation. The reaction was stopped by addition of EDTA to 10 mM final concentration. SDS  and protease inhibitors were added and suspension left on ice for 10 min and the centrifuged for 5 min at 13000 rpm. The supernatant was used for MNase digested chromatin.3). For Trf2, the eluate was diluted 10 times with ChIP-dilution buffer  and re-ChIPed with the polyclonal anti-Trf2 antibody 1727. For Tbp the second ChIP was performed using or anti-TBP . ChIP-seq libraries were prepared and sequenced on an Illumina Hi-seq2500 as single-end 50-base reads. After sequencing, peak detection was performed using the MACS softwarehttp://liulab.dfci.harvard.edu/MACS/). Global clustering, meta-analyses and quantitative comparisons were performed using seqMINER and R (http://www.r-project.org/). The Taf7l ChIP-seq data is from GSE50807ChIP experiments were performed on sonicated or MNase digested chromatin as previously described. ChIP-reChIP was performed for Trf2 and Tbp according to the same protocol with minor modifications as follows. After first IP [anti Flag M2 resin (Sigma) or anti-TBP ] chromatin was eluted with a 3-fold smaller volume of elution buffer  and the curated C3 transcription factor target gene set collection . For GSEA of Trf2+/+ versus Trf2−/−, we used the mean of the log2 fold changes of the biological replicates as metric for the H Hallmark gene sets of the BROAD javaGSEA tool with 1,000 permutations and the canonical pathway (cp) subcollection of the C2 curated BROAD molecular signature gene-set collection.Total RNA from mouse testes was isolated using the TriReagent (Molecular research center TR118). Libraries of template molecules suitable for high throughput DNA sequencing were created using “TruSeq™ Stranded Total RNA LT library prep kit with Ribo-Zero Gold” according to manufacturer’s user guide (Illumina Inc.). The libraries were loaded in the flow cell at 7pM concentration and clusters were generated in the Cbot and sequenced on the Illumina Hiseq2500 as single-end 50 base reads. Sequence reads mapped to reference genome mm9/NCBI37 using TophatHow to cite this article: Martianov, I. et al. TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression. Sci. Rep.6, 32069; doi: 10.1038/srep32069 (2016).


RANKL promoter activity. Chromatin immunoprecipitation analysis demonstrated that isoproterenol increased NFAT-binding and ATF4-binding activities on the mouse RANKL promoter, but did not increase CREB1-binding activity. Association of NFATc1 and ATF4 was not observed in a co-immunoprecipitation study. ATF4 knockdown suppressed isoproterenol-induced NFAT binding to the RANKL promoter, whereas NFATc1 knockdown did not suppress isoproterenol-induced ATF4 binding to the RANKL promoter. ATF4 knockdown suppressed isoproterenol-induced expressions of NFATc1 and RANKL. These results suggest that isoproterenol increases RANKL expression in an ATF4/NFATc1-dependent manner.Sympathetic nervous system stimulation-induced β-adrenergic signal transduction is known to induce bone loss and increase of osteoclast activity. Although isoproterenol, a nonspecific β-adrenergic receptor agonist, has been shown to increase receptor activator of NF-κB ligand (RANKL), the details of the regulatory mechanisms remain unclear. In the present study, we investigated the role of the nuclear factor of activated T-cells (NFAT) in isoproterenol-induced RANKL expression in C2C12 and in primary cultured mouse calvarial cells. Isoproterenol increased nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and RANKL expressions at both mRNA and protein levels and increased NFAT reporter activity. NFATc1 knockdown blocked isoproterenol-mediated RANKL expression. Isoproterenol also promoted cAMP response element-binding protein 1 (CREB1) and activating transcription factor 4 (ATF4) phosphorylation. Isoproterenol-mediated transcriptional activation of NFAT was blocked by protein kinase A (PKA) inhibitor H89. Isoproterenol-induced CREB1, ATF4, NFATc1, and RANKL expressions were suppressed by H89. Mutations in cAMP response element-like or NFAT-binding element suppressed isoproterenol-induced  Chronic stress activates a defense mechanism via the release of catecholamines from sympathetic nerves and of glucocorticoids by the adrenal glands. The skeletal system is richly innervated by sympathetic nerve fibers that release norepinephrine as a neurotransmitter . SNS reghenotype . The β-ahenotype ,6,7.RANKL is a critical regulator of osteoclastogenesis. It is primarily produced by stromal cells/osteoblasts in bone tissue. In cooperation with colony-stimulating factor 1, RANKL induces osteoclast differentiation and stimulates its bone-resorbing activity ,9. ActivcAMP response element-binding protein (CREB) is a cellular transcription factor. There are several subtypes of CREBs (CREB1–5) and CREB-like proteins. CREB2 is also known as activating transcription factor 4 (ATF4). Once activated by phosphorylation, CREB protein binds alone or recruits other transcriptional coactivators to bind to cAMP response element (CRE) in the promoter region, thereby regulating various genes’ expression.It has been demonstrated that downstream of cAMP/protein kinase A (PKA) activation, the activated CREB1 mediates PTH1R-induced RANKL expression by binding to multiple distal enhancers of the RANKL gene ,12. We pTo experimentally address that SNS activation stimulates bone resorption by increasing RANKL expression, a pharmacological model has been developed using β-adrenergic receptor agonists. Stimulation of the β-adrenergic receptor in osteoblasts by norepinephrine or isoproterenol, a pharmacological β-adrenergic receptor agonist, enhances osteoclastogenesis via upregulating RANKL expression . In the RANKL promoter.In the present study, we propose another transcriptional regulatory mechanism of β-adrenergic signaling-mediated RANKL expression, in which β-adrenergic signaling-induced NFATc1 upregulates RANKL transcription by directly binding to the mouse First, we examined whether isoproterenol induces RANKL expressions and NFATc1 activation in C2C12 cells. The cells were incubated in the presence of 1 µM isoproterenol for 1, 6, and 24 h. Isoproterenol increased mRNA and protein levels of RANKL expression, which peaked at 24 h. A. In add1-adrenergic receptor blocker) and ICI-118551  on isoproterenol-induced RANKL expression. The induction of RANKL expression by isoproterenol was significantly mitigated by treatment with ACE (1 µM) or ICI (1 µM)  B. SimilaI (1 µM) B. These Since the above data showed that activation of β-adrenergic receptor increases NFATc1 expression levels, we next examined whether isoproterenol enhances transcriptional activity of NFAT using a reporter plasmid containing a NFAT-responsive element . IsoprotWhen stimulated, osteoblast β-adrenergic receptor primarily binds to the G stimulatory (Gs) protein. The Gα subunit activates adenyl cyclase, generating the second messenger cAMP; elevated cAMP levels activate the PKA signaling pathway . We prevRANKL promoter is functional to isoproterenol-induced RANKL expression. Luciferase reporter assays were conducted using the RANKL promoter-reporter construct that contains approximately 2 kb of the DNA sequence located upstream of the transcription start site of the mouse RANKL gene [We next explored whether the NFAT-binding element or CRE-like element in the mouse NKL gene . An apprRANKL promoter. ChIP assay was performed to observe the direct binding of NFATc1 to the RANKL promoter. PCR amplification of the DNA region containing the NFATc1-binding element revealed that isoproterenol enhanced NFATc1 binding to the RANKL promoter  and the NFAT-binding element (−941 to −936 bp) in the murine ic cells . TherefoRANKL promoter by ATF4 knockdown, NFATc1 and RANKL expressions were assessed in the context of ATF4 knockdown. ATF4 knockdown significantly suppressed isoproterenol-induced expressions of NFATc1 and RANKL  cells. Consistent with the results observed in C2C12 cells, isoproterenol induced RANKL expression in both mRNA and protein levels after 24 h of incubation A. Inhibipromoter C. These RANKL promoter. We provided the evidence supporting the functional relevance of the NFAT binding in the RANKL promoter in isoproterenol-induced RANKL expression as follows: (i) isoproterenol enhances expression levels and transcriptional activities of NFATc1 in a PKA-dependent manner; (ii) mutations in the NFAT-binding element suppressed isoproterenol-induced RANKL promoter activity; (iii) NFATc1 knockdown abolished isoproterenol-induced RANKL expression; and (iv) ChIP assays with NFATc1 antibody showed isoproterenol induces NFATc1 binding to the RANKL promoter.In the present study, we demonstrated that, in isoproterenol-stimulated cells, NFATc1 binds to and transactivates the 2-induced activation of NFATc1 is required in the context of TNFα-induced RANKL expression in mouse osteoblasts [Several mechanistic pathways have been demonstrated to elucidate the role NFATc1 in RANKL expression. We previously reported that PGEeoblasts . In the eoblasts . Lee et eoblasts . In addieoblasts .RANKL promoter and that mutations in CRE-like element block isoproterenol induction of RANKL promoter activity. Although CREB1 was also activated by isoproterenol treatment in C2C12 cells, binding of CREB1 to the CRE-like element was not induced by isoproterenol in this study. It is not clear how binding of ATF4, but not that of CREB1, to the CRE-like element is induced in the context of β-adrenergic receptor activation.It is of significance that our results demonstrated that NFAT also plays a crucial role in the context of isoproterenol-induced RANKL expression in mouse osteoblastic cells. In the context of PTHrP-induced or TNFα-induced RANKL expression, CREB1 and NFATc1 binding and their cooperation to transactivate RANKL promoter has been reported ,18, whilRANKL promoter, whereas NFATc1 knockdown did not suppress isoproterenol-induced binding of ATF4 to the RANKL promoter. NFATc1 expression was dramatically decreased by ATF4 knockdown in C2C12 cells. These data imply that ATF4, in the upstream axis, activates NFATc1 gene transcription. These data are similar to the previous report showing that ATF4 acts as an upstream activator of the NFATc1 in osteoclast lineage cells [Our study results revealed the axis of ATF4 and NFATc1 in the isoproterenol- induced RANKL expression. In the present study, ATF4 knockdown suppressed isoproterenol-induced binding of NFATc1 to the ge cells .We previously showed that cAMP/PKA signaling activation increases NFAT transcriptional activity through MEK/ERK activation in the context of PTHrP induction of RANKL expression . In the RANKL promoter in mouse osteoblastic cells. The results of the present study establish a direct and important role for NFAT in regulating isoproterenol-induced RANKL expression. Our data suggest that NFAT inhibition in osteoblasts may contribute to suppress bone resorption by downregulating RANKL expression.Thus, SNS activation stimulates bone resorption, at least through the following pathway. β-adrenergic signal transduction increases RANKL expression in ATF4-NFAT axis-dependent manners, and NFAT directly binds and transactivates the Previous studies have shown that increases in osteoclast number and bone resorption under SNS-activated conditions such as mechanical unloading  and ovarIsoproterenol, acebutolol, ICI-118551, and H89 were purchased from Sigma . Antibodies to RANKL, NFATc1, ATF4, and β-actin were obtained from Santa Cruz Biotechnology , and horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Thermo Fisher Scientific . CREB1 and phospho-CREB1 antibodies were obtained from Cell Signaling Technology , and phospho-ATF4 antibody was purchased from MyBioSource, Inc. .C2C12 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 µg/mL of penicillin/streptomycin).We isolated MC cells from neonatal ICR mice calvaria and cultured in α-minimum essential medium (α-MEM) containing 10% FBS and antibiotics, as previously published . For preRANKL promoter (RANKL-WT) and a function-defective mutant reporter (RANKL-MT-N) containing mutations in the NFAT-binding site  have been described previously [RANKL promoter-reporter (RANKL-MT-C) containing mutations in the CRE-like element  was also used in the present study [Construction of the reporter plasmid containing an NFAT-responsive element has been reported previously . A lucifeviously . Anothernt study ,26.We performed quantitative RT-PCR analysis to observe RANKL, NFATc1, CREB1, and ATF4 mRNA levels. Total RNA was prepared using easy-BLUE RNA extraction reagents . The first-strand cDNA was synthesized with AccuPower RT PreMix  and used for PCR amplification with SYBR premix EX Taq . The primer sequences for real-time PCR were as follows: RANKL-forward (F) 5′-CAG AAG ATG GCA CTC ACT GCA-3′, RANKL-reverse (R) 5′-CAC CAT CGC TTT CTC TGC TCT-3′; NFATc1-F 5′-AAT AAC ATG CGA GCC ATC ATC-3′, NFATc1-R 5′-TCA CCC TGG TGT TCT TCC TC-3′; CREB1-F 5′-AGC TGC CAC TCA GCC GGG TA-3′, CREB1-R 5′-TGG TGC TCG TGG GTG CTG TG-3′; ATF4-F 5′-GCA TGC TCT GTT TCG AAT GGA-3′; ATF4-R 5′-CCA ACG TGG TCA AGA GCT CAT-3′; GAPDH-F 5′-TCA ATG ACA ACT TTG TCA AGC-3′; and GAPDH-R 5′-CCA GGG TTT CTT ACT CCT TGG-3′. The target genes were normalized to the reference housekeeping gene GAPDH.To prepare samples for Western blot analysis, cells were lysed as previously described . Each saTo perform immunoprecipitation, cells were co-transfected with NFATc1 and ATF4 expression plasmids. Cells were lysed with the lysis buffer  and sonicated briefly. Cell lysate was incubated with 10 µg of NFATc1 antibody, ATF4 antibody, or control IgG, followed by incubation with protein G agarose beads. Bound protein samples were subjected to SDS-PAGE and immunoblot analysis.RANKL promoter region, including the NFAT-binding element (amplified region: −1070 to −858 bp) and CRE-like element (amplified region: −1147 to −967 bp), with the following primers: NFAT-binding element forward, 5′-GCA AGC TCC AGG CCA GCC TAG-3′, and reverse, 5′-CCA ATA AGA CGG CTC AGC TG-3′ and CRE-like element forward, 5′-AGG AGG CAG AGA TGG CAG AG-3′, and reverse, 5′-ACA CGC GCG CGC GCA AAT A-3′.The chromatin immunoprecipitation (ChIP) assay was performed according to the protocol previously described . Prepare4 cells/well. After overnight culture, the cells were transiently transfected with the reporter plasmids using LipofectAMINE 2000 . In each transfection, 0.2 µg of reporters  and Renilla luciferase plasmid were used as indicated. The cells were then incubated in the presence of the indicated reagents for 24 h, and luciferase activity was measured using a Dual-Glo luciferase assay kit . Each test condition described was represented by three replicate plates. The relative luciferase activity was calculated by dividing firefly luciferase activity by Renilla luciferase activity to normalize the transfection efficiency.Cells were seeded in a 96-well plate at a density of 1 × 10t-test. For the multiple comparisons, one-way analysis of variance was performed. Post hoc analyses for all statistical tests were performed only when significant main effects were detected with the least squares means error test. Differences or changes were considered significant at p < 0.05. Luciferase reporter assay was designed with n = 6 and other tests were with n = 3. For statistical agreements, the number of their repeated experiments was two or three in this study. Data were analyzed using the SAS program .Statistical significance was determined using Student’s


Pax5 gene, which encodes a master regulator for B cell lineage commitment, in a chicken B cell line, DT40, with the goal of obtaining mechanistic insight into transcriptional regulation through chromosomal interaction. We found that the Pax5 promoter bound to multiple genomic regions using locus-specific chromatin immunoprecipitation (locus-specific ChIP), a method for locus-specific isolation of target genomic regions, in combination with next-generation sequencing (NGS). Comparing chromosomal interactions in wild-type DT40 with those in a macrophage-like counterpart, we found that some of the identified chromosomal interactions were organized in a B cell-specific manner. In addition, deletion of a B cell-specific interacting genomic region in chromosome 11, which was marked by active enhancer histone modifications, resulted in moderate but significant down-regulation of Pax5 transcription. Together, these results suggested that Pax5 transcription in DT40 is regulated by B cell-specific inter-chromosomal interactions. Moreover, these analyses showed that locus-specific ChIP combined with NGS analysis is useful for non-biased identification of functional genomic regions that physically interact with a locus of interest.Chromosomal interactions regulate genome functions, such as transcription, via dynamic chromosomal organization in the nucleus. In this study, we attempted to identify genomic regions that physically bind to the promoter region of the  Elucidation of the molecular mechanisms underlying genome functions, such as transcription, requires identification of molecules that interact with the genomic regions of interest. To this end, several biochemical methods have been developed. For example, proteomics of isolated chromatin (PICh) utilizes oligonucleotide probes to capture target loci for identification of associated proteins.,,,,,,,,In principle, locus-specific ChIP consists of locus-tagging and affinity purification. On the basis of various strategies for locus-tagging, we developed two locus-specific ChIP technologies, insertional ChIP (iChIP)in situ hybridization (FISH)Genome functions are mediated by chromosomal interactions (e.g. interactions between enhancers and promoters). To detect physical chromosomal interactions, several techniques have been utilized to date, including fluorescence Pax5 gene encodes a transcription factor essential for B cell lineage commitment.Pax5 gene inhibits B cell differentiation,,Pax5-deficient B cells can be trans-differentiated into other lymphoid cell types in mice.Pax5 gene, we previously used iChIP-SILAC to identify proteins that interact with the Pax5 promoter region in the chicken B cell line DT40.15 However, the mechanisms underlying regulation of Pax5 transcription by chromosomal interactions remain incompletely understood. Although intron 5 of the mouse Pax5 gene contains enhancers essential for transcription of the gene,Pax5 intron 5 are scarcely conserved between mouse and chicken, it is possible that transcription of Pax5 is controlled in a species-specific manner.The Pax5 promoter region in DT40 cells. Some of the detected chromosomal interactions were independently confirmed by an updated form of enChIP-Seq. In addition, deletion of a B cell-specific interacting genomic region significantly decreased Pax5 transcription in DT40 cells, suggesting that the deleted region is an enhancer and that Pax5 transcription is regulated through chromosomal interactions between this enhancer and the promoter in a B cell-specific manner. Thus, locus-specific ChIP in combination with NGS analysis revealed a mechanism of transcriptional regulation of the chicken Pax5 gene.In this study, we applied iChIP in combination with NGS analysis (iChIP-Seq) to direct identification of genomic regions that interact with the DT40, Non-KI(B), KI(B), and KI(MΦ) were maintained as described previously.7 each) were subjected to the iChIP procedure as described previously.in vitro enChIP procedure as described previously.Pax5 promoter and trans-activating crRNA (tracrRNA) was used as Pax5 gRNA for in vitro enChIP. The gRNA sequences are shown in in vitro enChIP. After purification of DNA, DNA libraries were prepared using TruSeq ChIP Sample Prep Kit (Illumina); in this preparation step, DNA fragments around 0.4 kbp in length were selectively concentrated. The libraries were subjected to DNA sequencing using the HiSeq platform according to the manufacturer’s protocol. NGS and data analysis were performed as described previously.,http://liulab.dfci.harvard.edu/MACS/ ) with default parameters. Images of NGS peaks were generated using Integrative Genomics Veiwer (IGV) ). The accession number of the NGS data is DRA005236.Non-KI(B), KI(B), and KI(MΦ) (2 × 10The Cas9 expression plasmid (Addgene #41815)7) were transfected with a Cas9 expression plasmid (120 µg), sgRNA expression plasmids (120 µg) targeting each end of a target genomic region, and pEGFP-N3  by electroporation using Gene Pulser II (Bio-Rad) at 250 V and 950 µF. One day later, GFP-positive cells were sorted and expanded individually. To confirm targeted locus deletion, genomic DNA was extracted and subjected to genotyping PCR with KOD FX (Toyobo). PCR cycles were as follows: heating at 94 °C for 2 min followed by 30 cycles of 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1 min. Primers used for genotyping PCR are shown in DT40 cells  and each antibody (3.5 µl for H3K4me1 or 2 µg for the others) as described previously.Antibodies against H3K4me1 , H3K27ac , and histone H3  were used. ChIP assays were performed with DT40 cells (2 × 10Pax5 exon 1A in chromosome Z of DT40 cells. (II) 3xFNLDD, which consists of 3xFLAG-tag, a nuclear localization signal (NLS), and LexA DNA-binding and dimerization domains, was expressed in the cells established in Step (I). (III) The resultant cells were crosslinked with formaldehyde and lysed, and chromatin DNA was fragmented by sonication. (IV) The tagged locus (the Pax5 promoter region) was affinity-purified using an anti-FLAG antibody. (V) After reverse crosslinking and DNA purification, genomic regions interacting with the Pax5 promoter region were identified by NGS analysis.The scheme of iChIP-Seq used in this study is as follows : (I) UsiPax5 promoter region;Pax5 promoter region and expressing 3xFNLDD. In our previous study, insertion of LexA BE and expression of 3xFNLDD did not disturb transcription of the endogenous Pax5 gene,Pax5 transcription is retained in both Non-KI(B) and KI(B). In addition, we previously showed that the Pax5 promoter region can be efficiently isolated from KI(B) by iChIP (∼10% of input as DNA yields).Pax5 promoter region by iChIP and subjected the purified DNA samples to NGS analysis using HiSeq. NGS reads corresponding to the Pax5 promoter region were clearly enriched when iChIP was performed with KI(B) but not Non-KI(B)  are used for in vitro locus-tagging rather than in cell locus-tagging   recognizing a 23 bp target site that is 0.1 kb upstream from the TSS of the  exon 1A ; the rechIP-Seq)  and PCR hIP-Seq) . Next, whIP-Seq)  were alsChIP-Seq . Based o of gRNA ; represe of gRNA . Thus, tChIP-Seq  can be c,,Intra-chromosomal interactions would be more frequently observed than inter-chromosomal interactions by 3C-based techniques.Pax5 gene (or chromosome Z on which the Pax5 gene is located) in the nucleus of DT40 cells. On the other hand, some peaks identified by iChIP-Seq were not confirmed by in vitro enChIP-Seq. In this regard, insertion of LexA BE might partially change the chromosomal conformation around the insertion site and organize artificial chromosomal interactions, which would not be confirmed in intact DT40 by in vitro enChIP. Alternatively, in vitro enChIP might fail to confirm some bona fide chromosomal interactions identified by iChIP-Seq. Because the insertion site of LexA BE is 0.2 kb upstream from the target site of the Pax5 gRNA  by iChIP. Visual comparison in IGV revealed that three peaks in chromosome 11 and three peaks in chromosome 21  were observed in a B cell-specific manner, whereas the other eight peaks were constitutively observed both in the B cell and the macrophage-like cell lines , which is DT40 trans-differentiated into a macrophage-like cell by ectopic expression of chicken C/EBPβ (in vitro enChIP with Pax5 gRNA failed to isolate the Pax5 promoter region from DT40(MΦ) (Pax5 transcription is silenced in DT40(MΦ)  , suggestDT40(MΦ) , the PaxPax5 transcription through chromosomal interactions. To examine this possibility, we used CRISPR-mediated genome editing to delete genomic regions that B cell-specifically interacted with the Pax5 promoter region.,Pax5 gene were not changed  suggested that IRC11-1 is involved in transcriptional regulation of the Pax5 gene, acting as an enhancer via inter-chromosomal interaction.The identified genomic regions  might in changed . Next, w changed . Additio of Pax5 . In DT40 in DT40 . The res exon 1A . Thus, tPax5 transcription. Enrichment of the active enhancer marks was also observed in IRC11-3, whereas only H3K4me1 was enriched in IRC11-2 (Pax5.Active enhancers are marked by enrichment of histone H3 lysine 4 mono-methylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27ac). IRC11-2 . TherefoPax5 transcription . Thus, this technology could facilitate elucidation of the molecular mechanisms underlying regulation of genome functions, including transcription.in vitro enChIP-Seq) could be used as one of several methods. In this study, we used in vitro enChIP-Seq just to confirm the results of iChIP-Seq. We believe that in vitro enChIP is potentially useful for identification of chromosomal interactions. However, to make the claim of utility of this technology stronger, more analyses using other gRNAs (e.g. more than two gRNAs targeting one target locus or other transcribed genes as controls) should be necessary. Nevertheless, considering its convenience, in vitro enChIP-Seq may be preferable for future identification of chromosomal interactions.Several methods have been utilized for detection of genome-wide chromosomal interaction. However, observation by only a single method might not accurately reflect physiological chromosomal interactions. In this regard, potential discrepancies have been reported between the results of FISH and those of 3C or its derivatives.https://www.ncbi.nlm.nih.gov/sra/?term=DRA005236 ).The accession number of the NGS data is DRA005236 (Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.Supplementary DataClick here for additional data file.


Intron retention (IR) is the most prevalent form of alternative splicing in plants. IR, like other forms of alternative splicing, has an important role in increasing gene product diversity and regulating transcript functionality. Splicing is known to occur co-transcriptionally and is influenced by the speed of transcription which in turn, is affected by chromatin structure. It follows that chromatin structure may have an important role in the regulation of splicing, and there is preliminary evidence in metazoans to suggest that this is indeed the case; however, nothing is known about the role of chromatin structure in regulating IR in plants. DNase I-seq is a useful experimental tool for genome-wide interrogation of chromatin accessibility, providing information on regions of chromatin with very high likelihood of cleavage by the enzyme DNase I, known as DNase I Hypersensitive Sites (DHSs). While it is well-established that promoter regions are highly accessible and are over-represented with DHSs, not much is known about DHSs in the bodies of genes, and their relationship to splicing in general, and IR in particular.In this study we use publicly available DNase I-seq data in arabidopsis and rice to investigate the relationship between IR and chromatin structure. We find that IR events are highly enriched in DHSs in both species. This implies that chromatin is more open in retained introns, which is consistent with a kinetic model of the process whereby higher speeds of transcription in those regions give less time for the spliceosomal machinery to recognize and splice out those introns co-transcriptionally. The more open chromatin in IR can also be the result of regulation mediated by DNA-binding proteins. To test this, we performed an exhaustive search for footprints left by DNA-binding proteins that are associated with IR. We identified several hundred short sequence elements that exhibit footprints in their DNase I-seq coverage, the telltale sign for binding events of a regulatory protein, protecting its binding site from cleavage by DNase I. A highly significant fraction of those sequence elements are conserved between arabidopsis and rice, a strong indication of their functional importance.In this study we have established an association between IR and chromatin accessibility, and presented a mechanistic hypothesis that explains the observed association from the perspective of the co-transcriptional nature of splicing. Furthermore, we identified conserved sequence elements for DNA-binding proteins that affect splicing.The online version of this article (10.1186/s12864-017-4393-z) contains supplementary material, which is available to authorized users. Recesplicing , 4–6, ansplicing .There is an ongoing effort to understand how alternative splicing is regulated and the factors that contribute to it. Some of these factors include AU-rich and U-rich sequences in plant introns –10, the in-vivo. DHSs have been used to identify several types of regulatory elements such as, promoters, silencers, enhancers, and insulators [The fact that splicing can happen co-transcriptionally suggests that chromatin state is relevant for splicing , 18. Onesulators , 20. It sulators  and leavsulators , 23. Thesulators .When it comes to AS, Mercer et al.  have shoOur goal is to shed light on the regulation of IR from the perspective of chromatin organization. First we test the association between DHSs and IR using DNase I seq data in arabidopsis and rice, and find that DHSs have a highly significant association with IR; we then look for evidence at the DNA level for the footprints of protein binding and find a large collection of hexamers that are conserved across arabidopsis and rice, and likely function as SREs. Finally, we discuss how these observations are consistent with current models that describe the interaction between transcription, splicing, and chromatin organization.p-value of 5.90×10−89 in arabidopsis and a p-value of 8.93×10−25 in rice using the Fisher exact test.Our first goal is to investigate the relationship between IR and chromatin accessibility. For this task we analyzed existing DNase I-seq data in both arabidopsis and rice for which RNA-seq data for the same samples is also available , 27. Firp-value of 1.22×10−56 in arabidopsis, and a p-value of 5.25×10−100 in rice using the Mann–Whitney U test [As a complement to the analysis of DHSs detected using peak calling, we compared IR and IE events on the basis of raw DNase I-seq read depth , demonsy U test .Fig. 3Mp-value of 0.0008 in arabidopsis, and a p-value of 1.07×10−24 in rice, computed using the Fisher exact test).Previous studies have used DNase I-seq data to detect potential transcription factor binding sites in promoter regions by searching for a dip in the DNase I-seq coverage : a regioWe performed a comprehensive analysis across all hexamers to detect those that have a footprint and exhibit an association with IR or IE in the arabidopsis and rice leaf data. Our first observation is that in IR events the majority of the hexamers come from the 3’ exon, while for IE, all the hexamers are intronic . This level of conservation is strong support for the functional importance of these hexamers. We note that for finding conserved hexamers we used a looser threshold for footprint calling, as the requirement of conservation provided an additional level of filtering of potential false positives. Manual inspection of the detected hexamers showed that all of them exhibited valid footprints.Many of the hexamers we identified are conserved in arabidopsis and rice: In the 3’ exon 246 hexamers were common between the two species, while 19 are conserved in the intronic region of IE events. This level of overlap is highly statistically significant  are AT-rich, and some of the rest (3/10) are GC-rich. Both sets of motifs exhibit very different positional preferences: the AT-rich motifs tend to occur at the 3’ end of the exon, while the GC-rich motifs tend to occur in the 5’ end of the exon . We used the xactTest  to quantSplicing occurs co-transcriptionally, and there is increasing evidence indicating that chromatin organization involving epigenetic marks and rate of transcription regulate alternative splicing in mammalian systems . Howevertrans-factors, and also does not account for the much larger number of hexamers with footprints that are associated with IR. For example, in the arabidopsis leaf data we found 118 hexamers with footprints that are enriched in IR, and only 28 in IE.In our data we observe a greater number of DHSs in IR events compared to IE events, and this is most prominent in the 3’ exon. A similar pattern was observed in the raw DNase I-seq data as well. We present two possible hypotheses by which this increase in open chromatin contributes to IR. Splicing is a much slower process than transcription , and we The increased number of footprints that we observed in IR could be the result of one of two factors: 1. Increased PolII pausing and/or, 2. Binding of other chromatin/DNA-interacting proteins. Braunschweig et al. have recently shown that in mammalian systems retained introns are associated with increased PolII pausing . This paChromatin modifications have recently been associated with IR in humans: Braunschweig et al. have shown that the chromatin activation mark H3K27ac is enriched in retained introns . This obThe AT-rich hexamers in IE have a positional preference for the 3’ end of the intron, which suggests they are likely associated with the polypyrimidine tract, which in plants is T-rich , leadingDNA methylation has been shown to regulate alternative splicing, including IR, in plants and animals –40. PartATHB9, which is an HD-Zip class II protein, was shown to have affinity for the sequence GTAAT(G/C)ATTAC; the core AAT(G/C)A segment of this sequence matches multiple conserved hexamers detected in the 3’ exon of retained introns. HD-Zip class IV proteins bind sequences containing a TAAA core, which is consistent with a large number of hexamers both in IR and IE events.In addition to the matches in the Plant Cistrome Database described above, we identified other transcription factors that have DNA binding motifs that match the hexamers discovered by our pipeline. These include Homeodomain-leucine zipper (HD-Zip) proteins, which are a family of transcription factors unique to plants  have DNAAlthough epigenetic changes, including DNA methylation and histone modifications have been shown to be important regulators of AS in animals , 43, 44,In this work we established a clear correlation between IR and chromatin accessbility and DNA methylation in arabidopsis and rice. We found that chromatin is more open in retained introns, which can be explained using a kinetic model of the splicing process. The observed open chromatin in IR is consistent with the reduced methylation levels we observed in these regions. The more open chromatin in IR also suggests that IR is more highly regulated than constitutive splicing, which is supported by the large number of conserved sequence elements that were discovered in footprints associated with IR. A majority of the discovered sequence elements occur in exons immediately downstream of retained introns, indicating its importance in regulating IR events. Further experiments are required in order to establish the biological function of these sequence elements and to experimentally verify the hypothesized connections between intron retention and chromatin organization.GSE53322 was used. For rice, we used data from Wu et al. [GSE26610); The corresponding RNA-seq was published elsewhere [GSE33265). For rice, there were two samples coming from two tissues: leaf and callus. For bisulfite-seq, we used raw data from Zemach et al. [GSE41302) and Chodavarapu et al. [GSE38480), for arabidopsis and rice, respectively.For arabidopsis, read data from Zhang et al.  were dowu et al.   using different alignment tools. All the RNA-seq samples were aligned using Tophat2 [Tophat2 alignments were filtered to obtain only uniquely aligned reads. The arabidiopsis DNase I-seq data was aligned using Bowtie [Bowtie was used with the command-line argument -m 1 to suppress multiple alignments. For the rice DNase I-seq data, we used STAR [Trim Galore! [bismark [In case of data from Zhang et al. , we usedFastQC  and trim-trimmer  when req Tophat2  with defg Bowtie . Bowtie sed STAR  to align Galore! . For ali[bismark . Note thTo extract IR and IE events we used annotated IR events from the gene models as well as evidence from the RNA-seq data found using SpliceGrapher , which iFor the discovery of k-mers that exhibit footprints we chose to focus on hexamers since this provides a good balance of specificity and tractability of exhaustive search. We considered all possible hexamers coming from the three parts of an event: 5’ exon, intron, and 3’ exon. For every hexamer, we generated the DNase I-seq profile. For each occurrence of the hexamer we extracted its DNase I cut at every nucleotide position of the hexamer as well as 100bp upstream and downstream of its location and then took the average over all positions. Note that in going 100bp upstream and downstream, we made sure not to go beyond the boundaries of the event parts: intron or the flanking exons. This was done to avoid introducing any bias coming from the properties of different segments of the event. In case of multiple instances of a hexamer in a sequence, we considered the one which had the lowest DNase I-seq coverage.BG1), the down state (DN), the footprint state (FP), the up state (UP) and finally, the trailing background state (BG2). The HMM was trained on data profiles of hexamers with manually verified footprints and was used to score the rest of the hexamers. Note that all hexamer profiles were standardized to a background score calculated from the training set. To account for tandem motifs, we added additional states to the model to represent secondary footprints upstream or downstream of the primary footprint. The state diagram for the final HMM, which has 13 states, along with complete specification of the model (transition and emission probabilities), and the training and testing protocol, can be found in the Additional file We used the profile of DNase I-seq coverage to call footprints using a continuous HMM. Continuous HMMs are a good modeling tool for sequences of real values such as DNase I-seq coverage, and allow us to detect whether the observed profile contains a feature that can be identified as a protein footprint. Our model was inspired by a similar model  and the CFP is the average standardized coverage at the footprint state and CBG is the average coverage across the background states. A conservative threshold of S=0.30 was used in the analysis of individual hexamers, and the cutoff was lowered to S=0.20 in the cross-species analysis. To cluster the hexamers into motifs, we used complete linkage hierarchical clustering with a distance metric that assigns two k-mers a distance of 0 if they share a 4-mer; their edit distance is used otherwise; clusters were cut at a depth of 4. We used clustalw2 [weblogo [Using the trained HMM we score hexamers as potential footprints using the following expression: lustalw2  to gener[weblogo  to generAll significantly enriched arabidopsis hexamers were searched against each motif from the Plant Cistrome Database  using thp-values were adjusted using the Benjamini-Hochberg method [Whenever testing multiple hypotheses, the resulting g method . All theg method  with popAdditional file 1SupplementaryFile_1.docx. Additional figures and tables are provided in this file. (DOCX 2507 kb)Additional file 2SupplementaryFile_2.xlsx. This file lists footprint-exhibiting hexamers for all four individual samples in separate tabs for IR and IE events. In addition, the footprint-exhibiting hexamers common between the arabidopsis and rice leaf samples are provided in the last two tabs for IR and IE events, respectively. (XLSX 78 kb)Additional file 3SupplementaryFile_3.xlsx. This file provides a list of the footprint-exhibiting hexamers in arabidopsis that match motifs in the Plant Cistrom Database. (XLSX 64 kb)