The basic helix–loop–helix transcription factor (bHLH TF) family is involved in tissue development, cell differentiation, and disease. These factors have transcriptionally positive, negative, and inactive functions by combining dimeric interactions among family members. The best known bHLH TFs are the E-protein homodimers and heterodimers with the tissue-specific TFs or ID proteins. These cooperative and dynamic interactions result in a complex transcriptional network that helps define the cell’s fate. Here, the reported dimeric interactions of 67 vertebrate bHLH TFs with other family members are summarized in tables, including specifications of the experimental techniques that defined the dimers. The compilation of these extensive data underscores homodimers of tissue-specific bHLH TFs as a central part of the bHLH regulatory network, with relevant positive and negative transcriptional regulatory roles. Furthermore, some sequence-specific TFs can also form transcriptionally inactive heterodimers with each other. The function, classification, and developmental role for all vertebrate bHLH TFs in four major classes are detailed. Transcription factors (TFs) are proteins that are directly involved in the activation or repression of RNA synthesis from a DNA template , most ofTFs can be broadly classified as either basal  or sequence-specific TFs . The genThe sequence-specific DNA-binding TFs have also been classified based on their well-defined DNA-binding protein domains . These TTFs usually cooperate and synergize with other TFs through extensive protein–protein interactions within their TF family and different families . This coThe latest, 2019, bHLH TF family comprehensive review  summarizThe bHLH family is one of the most prominent among transcription factors ,5 and isDrosophila and mammals were initially classified into three groups: Class A, B, and ID [Back in the 1990s, the bHLH TFs from , and ID . Class A, and ID .Drosophila’s bHLH TFs hairy and enhancer of split and generally functioned as repressors . Later, Class VII was added to accommodate the PAS-domain proteins [With the burgeoning number of bHLH TFs being identified, several other classification schemes have emerged to accommodate our growing understanding. The Murre Lab initially classified the bHLH TFs into six classes I–VI,  based upproteins .Early on, the bHLH TFs were also classified as groups A to D  based onC. elegans and mouse were included in the phylogenetic analyses. These additions resulted in further classification of the bHLH TFs into orthology families [Ledent and collaborators  expandedfamilies , where tfamilies .Afterward, the complete amino acid sequences of the bHLH TFs of seven different species  were used to carry out phylogenetic analyses that identified six new clades . Clades The bHLH TFs function cooperatively as homodimers (E47/E47), heterodimers (MYOD1/E47), trimers (TAL1/E47/LIM), or multimeric structures ,177,178.Dimeric interactions within the bHLH TF family are summarized in In many cases, phosphorylation of the bHLH TF regulates its dimerization. NEUROG2 homodimers are efficient transactivators. However, when NEUROG2 is phosphorylated, it heterodimerizes with E47, reducing its transactivator capacity . For OLIThe Murre classification scheme  was selePredictions of the functionality of bHLH TFs could be made based on the classification above and the other phylogenetic classifications; however, this could be misleading as each bHLH dimer’s function depends on the protein–protein interactions established. Thus, the relevance of this review originates from the need to summarize experimentally corroborated dimeric interactions among this TF family.From Diverse experimental approaches, including in vivo and in vitro assays, have defined the dimeric interactions of bHLH transcription factors. The most common in vitro assay for testing dimeric bHLH interactions in the presence of DNA is the electrophoretic mobility shift assay (EMSA). This approach has been used since the discovery of the bHLH TFs and has demonstrated most E-protein homodimeric and heterodimeric interactions with Class II bHLH TFs. These interactions include all DNA-binding myogenic and neurogenic bHLH heterodimers. A major drawback of EMSA is that it cannot detect DNA-independent interactions or dimers that bind non-consensus or untested DNA sequences. Thus, unless a broader repertoire of DNA sequences was tested, such as in the CASTing assay , the posSome bHLH TFs dimers were discovered by alternate in vitro approaches, including GST-pulldown (GST), methylation interference footprinting (MIF), co-immunoprecipitation . For example, the interaction demonstrated by coIP between ATOH8 and NEUROD1 could not be reproduced using in vitro translated proteins . SimilarIn vivo assays preserve the native surrounding in which the interaction takes place. However, these assays also have drawbacks, such as the expression under non-physiological conditions  and the influence of the cell context. Sometimes, an interaction can be observed in one cellular context but not in another. Reasons for these inconsistencies could be a requirement for additional interacting factors or an altered bHLH network composition due to a TF overexpression. TAL1 is an example of a TF capable of activating or repressing transcription in a context-dependent manner through differential interactions with HDACs and HATs ,123,124 In the transcriptional reporter assays, the most common approach to define the function of the dimeric bHLH TFs, the main drawback is the presence of a specific endogenous pool of bHLH TFs in the cell. This TF pool may contain TFs able to influence the function of the TF tested, a condition that must be considered by the scientist when analyzing homodimeric TFs.In compiling this review, information was gathered about the dimeric interactions of bHLH TFs from individual publications since their discovery at the end of the 1980s. This exhaustive literature search was complemented by a manual search in global protein–protein interaction databases to guarantee a thorough summary of the bHLH dimer diversity. These web databases summarize experimentally corroborated and predicted vertebrate protein–protein interactions, with none of them being devoted to TFs or specifically to bHLH TFs. For this work, IntAct , String Class I TFs can also heterodimerize among each other, still functioning as transactivators . UnfortuBesides heterodimerizing with E-proteins, some bHLH TFs also form heterodimers with other Class II, V, and VI TFs . This diDll3 [From analyzing Dll3  and TAL1Dll3 ,245. TheDll3 ,245. TheDll3 ,82, HANDDll3 ,89,90, aDll3  can sequDll3 ,150, homDll3 ,150. In contrast, 100% of Class II TFs interactions with ID proteins and 95% of the interactions with Class VI TFs, negatively affect transcription . The ClaThe comprehensive 2019 review by Murre  summarizDrosophila were described as transcriptionally inactive [Homodimerizing tissue-specific bHLH proteins in inactive ,276. Theinactive ,66. Expeinactive , CD [231inactive , and LC-inactive . The MYOinactive . It was inactive . These Dinactive . Unfortuinactive . Thus, fNo systematic reviews about bHLH TF homodimerization exist. Homodimers of the myogenic factors  and six other factors  are transcriptionally inactive or act as repressors. Within this group, only ASCL3 does not bind duplex DNA as a homodimer. In contrast, five TFs bind DNA to remain transcriptionally inactive or repress gene expression . Thus, some Class II homodimers affect gene expression negatively through diverse mechanisms that can be dependent or independent of DNA binding, including the possible sequestration of components of active dimers.BHLHA15 homodimers can transactivate or repress transcription through direct DNA binding in a context-dependent manner ,116,254.Six Class II homodimers can function as transactivators through direct DNA binding: NEUROD6, NEUROG2, BHLHA15, ASCL1, NHLH1, and NHLH2. Thus, Class II homodimers can be both transcriptionally inactive and active. On the other hand, homodimerization of 27 (of 48) class II TFs is uncertain, either because it has never been tested or because the experimental evidence is inconclusive. The untested factors include FIGLA, NEUROD1, NEUROD2, ATOH8, ASCL4, ASCL5, TCF23, TCF24, BHLHE23, OLIG1, and OLIG3 gray in . SimilarFinally, 9 of 11 Class VI factors function as homodimers repressing transcription . HomodimAlthough bHLH TFs have been studied for over 30 years, there remain extensive gaps in our knowledge either because some dimeric interactions have never been tested or due to the inherent limitations of the techniques used. Furthermore, due to the bHLH TFs’ ability to interact with multiple partners, dissecting the function of each dimer pair requires carefully designed experiments. I anticipate that the field will be accelerated by increasingly powerful technologies such as cryo-electron microscopy/tomography and genome-wide interactomes in different cell types and conditions. Another layer of complexity is added by the fact that alternate dimerizing partners are usually co-expressed in vivo, establishing a dynamic pool of TFs, whose balance defines the outcome of the assays. This indicates the need for more live-cell approaches to allow the visualization of interaction dynamics and computational approaches using available dimer structures to predict bHLH interactions ,280 and


NANOG 3′UTR, four possible target sites of MIR630 were identified. To examine the functional interaction between MIR630 and NANOG mRNA, the predicted MIR630 target sites in the NANOG 3′UTR were deleted and the activity of the reporters were compared. After targeted mutation of the predicted MIR630 target sites, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. It is worth noting that mutation of a single putative binding site in the 3′UTR of NANOG did not completely abolish MIR630-mediated suppression, suggesting that MIR630 in the NANOG 3′UTR may have multiple binding sites and act together to maximally repress NANOG expression. Interestingly, MIR630 mimics significantly downregulated NANOG gene transcription. Exogenous expression of OCT4, SOX2, and NANOG lacking the 3′UTR almost completely rescued the reduced transcriptional activity of MIR630. MIR630 mediated the expression of differentiation markers in NT2/D1 cells, suggesting that MIR630 leads to the differentiation of NT2/D1 cell. Our findings show that MIR630 represses NANOG through transcriptional and post-transcriptional regulation, suggesting a direct link between core pluripotency factors and MIR630.The pluripotent transcription factor NANOG is essential for maintaining embryonic stem cells and driving tumorigenesis. We previously showed that PKC activity is involved in the regulation of NANOG expression. To explore the possible involvement of microRNAs in regulating the expression of key pluripotency factors, we performed a genome-wide analysis of microRNA expression in the embryonal carcinoma cell line NT2/D1 in the presence of the PKC activator, PMA. We found that MIR630 was significantly upregulated in PMA-treated cells. Experimentally, we showed that transfection of MIR630 mimic into embryonal carcinoma cell lines directly targeted the 3′UTR of OCT4, SOX2, and NANOG and markedly suppressed their expression. RNAhybrid and RNA22 algorithms were used to predict miRNA target sites in the  NANOG has been shown to be regulated by the OCT4/SOX2 complexes through a highly conserved adjacent pair of OCT4 and SOX2 binding elements in the NANOG promoter [A number of transcription factors have been shown to play critical roles in the self-renewal and maintenance of pluripotency in embryonic stem cell (ESCs), among them OCT4, NANOG, and SOX2  have beepromoter . Expresspromoter . Cancer promoter  but alsopromoter , seminompromoter , oral capromoter , ovary cpromoter , renal cpromoter , and malpromoter . Overexppromoter ,12. Nevepromoter . We showMicroRNAs (miRNAs) are a large family of non-coding RNAs, typically 19–25 nucleotides in length. They regulate gene expression at the post-transcriptional level and are one of the crucial regulators necessary to maintain ESC self-renewal. Genetic deletion of the major miRNA processing enzymes DGCR8  or DicerNANOG and repressed its expression. This regulation was found to be at the transcriptional and post-transcriptional levels.In the current study, we investigated the possible involvement of miRNAs in the regulation of NANOG expression by PKC. We first identified 20 miRNAs that were significantly upregulated in PKC activator-treated NT2/D1 cells. Computational predictions indicated that miRNA 630 (MIR630) targeted multiple core pluripotency factors, including NANOG, OCT4. and SOX2. Further experimental work showed that MIR630 directly targeted the 3′UTR of Recent studies have shown that miRNAs play a critical role in the regulatory circuit that controls pluripotency and self-renewal of ESCs ,18. DuriWe performed a genome-wide analysis of miRNA expression in NT2/D1 cells in the presence of PKC activator, phorbol 12-myristate 13-acetate (PMA), in an attempt to link this change to altered NANOG expression. MiRNA microarray was used to identify the miRNA species that were up- or down-regulated after PMA treatment .Among the miRNAs upregulated by PMA treatment, the expression of MIR630 was upregulated 28-fold, while MIR335-3p and MIR335-5p were upregulated by 17.1- and 21.9-fold, respectively, compared to untreated cells. The upregulation of MIR335-5p during mouse ESC differentiation is thought to play an important role in antagonizing OCT4-promoted mouse ESC self-renewal . In addiNANOG/POU5F1(OCT4)/SOX2. One possible target site was identified in POU5F1 and SOX2, while four target sites of MIR630 were identified in NANOG , and SOX2 reporters ranging from 27% to 80% (NANOG and POU5F1(OCT4) to regulate their expression.To investigate whether NANOG, OCT4, and SOX2 are indeed targeted by MIR630, we constructed a luciferase reporter containing the 3′UTRs of these genes A. The lu% to 80% B. The reA negative control mimic or miRNA mimic was transfected into human embryonal carcinoma cell lines, NCCIT and NT2/D1. Protein expression was examined by Western blotting. In NT2/D1 cells, the expression of NANOG, OCT4, and SOX2 proteins was inhibited by 69%, 70%, and 32%, respectively, after transfection with MIR630 mimic compared to control cells C. SimilaNANOG mRNA, the predicted MIR630 target sites in the NANOG 3′UTR region were deleted and the activity of the reporter was compared in negative control mimic or cells transfected with MIR630 mimic. As expected, MIR630 repressed 70% of the activity of the reporter construct containing the full-length wild-type NANOG 3′UTR . In contrast, no statistically significant attenuated repressive effect was observed in the reporter that was deleted in 570–605.To examine the functional interaction between MIR630 and OG 3′UTR . CorrespNANOG 3′UTR . Among the putative binding sites, we showed that MIR630 forms the most stable base pairing with 570–605 in the OG 3′UTR B, which NANOG 3′UTR, all of which may be involved in MIR630-mediated downregulation of NANOG in a coordinated manner.Notably, mutation of a single putative binding site did not completely abolish the MIR630-mediated inhibition of reporter activity. This suggests that MIR630 may have multiple binding sites in the NANOG promoter. We cotransfected pGL3-NANOG-Luc [NANOG promoter region from –993 to +231 relative to the NANOG transcriptional start site. Cotransfection of the pGL3-NANOG-Luc construct with the MIR630 mimic resulted in a significant 40% reduction in luciferase activity compared to cells transfected with negative control mimic  in the 5′-flanking region of the NANOG promoter [NANOG promoter activity is driven by a reduced cellular PKC activity, an effect that depends on the presence of the composite Sox-Octamer element [ element . In addi element .NANOG transcription repressed by MIR630, we carried out “rescue” experiments in NT2/D1 cells. We assumed that NANOG, OCT4, and SOX2 are involved in MIR630-repressed NANOG transcription. Then, re-expression of NANOG, OCT4, and SOX2 could rescue the inhibitory effect of MIR630 on NANOG transcriptional activity. For this purpose, we used constructs containing pCMV-OCT4, pCMV-SOX2, and pCMV-NANOG expressing full-length cDNA but without the 3′UTR. Thus, this construct expressed ectopic OCT4, SOX2, and NANOG and should not be affected by the MIR630 putative binding sites. Cotransfection of MIR630 mimic and constructs overexpressing OCT4 and SOX2 in NT2/D1 cells partially restored the reduction in luciferase reporter activity induced by MIR630 transfection , and SOX2 cDNAs. As shown in To determine whether NANOG, OCT4, and SOX2 proteins are involved in sfection . OverexpTo further determine whether MIR630 plays a role in NT2/D1 cell differentiation, cells were transfected with negative control mimic or MIR630 mimic. After 3 days, total cellular proteins were extracted and the expression of some selected differentiation markers was analyzed by Western blot. NeuroD1, the earliest marker of neuronal differentiation, was found to be upregulated in MIR630-transfected cells . MoreoveNANOG has been shown to be a central regulator in maintaining the self-renewal properties and pluripotency of human ESCs . In addiNANOG, POU5F1 (OCT4), and SOX2 for putative target sites complementary to MIR630. We identified putative target sites of MIR630 in the 3′UTR of NANOG, POU5F1 (OCT4), and SOX2 mRNA. In this regard, we showed that PKC activators induce the expression of MIR630, while MIR630 mimic represses the luciferase activity of NANOG, POU5F1, and SOX2 3′UTRs reporters. We further showed that ectopic MIR630 mimic directly targets NANOG, POU5F1, and SOX2 3′UTRs to regulate their expression. Furthermore, after targeted mutation of the predicted MIR630 target sites in the 3′UTR of NANOG, the MIR630 mimic inhibited NANOG significantly less than the wild-type reporters. Our results suggest that MIR630 negatively regulates the expression of NANOG, OCT4, and SOX2 in a post-transcriptional manner and that their upstream regulator may be PKC.Accumulating evidence strongly suggests that alterations in miRNA expression are associated with cancer initiation and progression. In addition to regulating the expression of tumor suppressors and oncogenes, recent studies have indicated that miRNAs play a role in the maintenance of pluripotency and cellular reprogramming in ESC. On the other hand, NANOG, OCT4, and SOX2 have all been recognized to play a pivotal role in pluripotency maintenance. Therefore, it is important to investigate whether there is cross-talk between these two regulatory systems. In the present study, we scanned the genomic loci of NANOG promoter vector abolished the upregulation of promoter activity by PKC inhibitors. Consistently, knockdown of OCT4 and SOX2 diminished the PKC inhibitor-induced enhancement of NANOG expression, suggesting that the OCT4/SOX2 complexes are involved in the downregulation of NANOG expression by PKC. Furthermore, NANOG was reported to autoregulate its expression by binding to its proximal downstream promoter [NANOG transcription by MIR630. The results showed that MIR630 mimic significantly reduced the NANOG transcription compared to negative control mimic-transfected cells. Intriguingly, ectopic expression of POU5F1(OCT4), SOX2, and NANOG lacking the 3′UTR almost completely restored the reduced transcription of MIR630. These results strongly suggest that NANOG and OCT4–SOX2 complexes are involved in MIR630-repressed NANOG gene transcription. Taken together, this suggests that MIR630 represses NANOG expression at the transcriptional and post-transcriptional levels  were obtained from the Bioresource Collection and Research Center in Taiwan. Cells were incubated in Dulbecco’s Modified Eagle’s Medium (DMEM)  supplemented with 10% Fetal bovine serum, 1 mM pyruvate, and 2 mM L-glutamine at 37 °C in a humidified 5% C02/95% air incubator. The undifferentiated cells were split at 1:10 by trypsinization.NT2/D1 cells were treated with 50 nM PMA for 24 h. Total RNA was purified with Trizol reagent (invitrogen). RNA was quantified using a NanoDrop1000 spectrophotometer  at OD260nm. RNA integrity was characterized using an RNA 6000 Pico Chip Kit  on an Agilent 2100 Bioanalyzer. RNA samples with 28S:18S rRNA ratios between 1.4 and 1.8 were used. Briefly, each 100 ng of total RNA was dephosphorylated and labeled with Cy3-pCp using a miRNA Complete Labeling and hybridization Kit (Agilent Technologies). Samples were hybridized using a human miRNA Microarray Kit R16 (Agilent Technologies), which screens for the expression of 1205 human miRNAs and 142 human viral miRNAs. After hybridization, slides were washed with Gene Expression Wash Buffer 1 for 5 min at room temperature, followed by Gene Expression Wash Buffer 2 for 5 min at 37 °C. The slides were scanned on an Agilent G2505C microarray scanner (Agilent Technologies) with default settings. The scanned images were analyzed by GeneSpring 7.3.1 by Welgene Biotech Co., Ltd. (Agilent Technologies). The total gene signal was normalized to the 75th percentile . The norTotal RNA was extracted using TRIzol reagent (Invitrogen) according to the protocol recommended by the supplier and reverse-transcribed by Superscript III First-Strand Synthesis System (Invitrogen). RT-qPCR was used to quantify the level of MIR630. Aliquots (0.1 μL) of the cDNA samples were performed in iQ SYBR Green Supermix  using a Roche LightCycler 480 System. The program consisted of 95 °C for 3 min and 40 cycles of 95 °C for 15 s and 60 °C for 1 min. Data were averaged and relative quantification was determined by the ΔΔCt method. Results were normalized to β2-microglobulin mRNA. Primers used for MIR630 precursor were sense, 5′-CTGAGGTTAAATAACTCCCTC-3′ and antisense, 5′-GTATAGAGCAACCTCTAACAG-3′. The primers used for β2-microglobulin were sense, 5′-GCCGTGTGAACCATGTGACTTT-3′ and antisense, 5′-CCAAATGCGGCATCTTCAAA -3′.NANOG, POU5F1 (OCT4), or SOX2 mRNA 3′UTR. To analyze possible microRNA target sites in the 3′ untranslated region (3′UTR) of the NANOG, POU5F1 (OCT4), and SOX2 genes, the 3′UTR-luciferase reporter vectors were constructed and the effect of miRNA on its activity was evaluated in NT2/D1 cell lines. The 3′UTR fragments of NANOG, POU5F1 (OCT4), and SOX2 genes were amplified by PCR from genomic DNA of NT2/D1 cells using the following primer pairs containing a XbaI site and a FseI site for the subsequent cloning reactions.To contract the luciferase reporters expressing firefly-luciferase in a 3′UTR-dependent manner, pGL3-GAPDH-Luc containing the constitutive GAPDH promoter (16) was modified to replace vector-specific 3′UTR luciferase mRNA with NANOG 3′UTR, Forward (XbaI): 5′-cccTCTAGAgaacatgcaacctgaagacgtg-3′ andReverse (FseI): 5′-cccGGCCGGCCctgtatatttactcatcgaaac-3′;POU5F1 3′UTR, Forward (XbaI): 5′-cccTCTAGAgctctcccat gcattcaaactg-3′ andReverse (FseI): cccGGCCGGCCctgtgtcccaggcttctttat;SOX2 3′UTR, Forward (XbaI): 5′-cccTCTAGAcacactgcccctctcacacatg-3′ andReverse (FseI): 5′-cccGGCCGGCCgtgtccatatttcaaaaatttat-3′.PCR reactions were carried out at 94 °C for 5 min, followed by 35 cycles at 95 °C for 30 s, 50–55 °C for 45 s, and 72 °C for 1 min. PCR products were digested by XbaI and FseI. Desired DNA fragments were PCR purified and inserted into luciferase reporter vector pGL3-GAPDH-Luc via the XbaI and FseI site to generate pGL3-GAPDH-NANOG 3′UTR-Luc, pGL3-GAPDH-POU5F1-3′UTR-Luc, and pGL3-GAPDH-SOX2-3′UTR-Luc. The inserts were positioned in sense orientation relative to the luciferase coding sequence between the XbaI and FseI sites. Proper insertion was verified by direct DNA sequencing.NANOG, POU5F1 (OCT4), and SOX2 genes, the effect of miRNA mimic on their activity was evaluated in NT2/D1 cell lines. The mirVana miRNA mimics (MIR630 or negative control) were obtained from Ambion . Transient transfection of the 3′UTR-luciferase reporter vector was performed using Lipofectamine 2000 (Invitrogen) according to the protocol recommended by the supplier. The cells were seeded in 12-well tissue culture plates at 2 × 105 per well 24 h before transfection. On the day of transfection, cells were exposed to DNA–lipofectamine 2000 mixtures containing 0.5 µg of luciferase reporter plasmids, 0.5 µg of pSV-β-galactosidase control vector , and 30 nM miRNA mimics. After incubation for 24 h, cells were rinsed with PBS and lysed in 120 µL 1 × reporter lysis buffer (Promega). The lysates were used directly for the Luciferase activity assay (Promega), performed according to the manufacturer’s protocols. The β-Galactosidase enzyme assay (Promega) was performed with the same lysates to standardize transfection efficiencies.To analyze possible microRNA target sites in the 3′ untranslated region (3′UTR) of NANOG were deleted. Site-directed mutagenesis of the 3′UTR luciferase reporter vectors was performed using PCR methods. The PCR reaction mixture contained 1 μg pGL3-GAPDH-Luc containing the 3′UTR of either NANOG, POU5F1, or SOX2, 12.5 µL 2X Phusion High-Fidelity DNA Polymerase , and 0.1 μg of each primer. The synthetic oligonucleotide primers used were: 5′-TGCCTATCCAGTCAATCTCATGGGTTGGAGCCTAATCAGCGAGGT-3′ (sense) and 5′-ACCTCGCTGATTAGGCTCCAACCCATGAGATTGACTGGATAGGCA-3′ (antisense) for NANOG 3′ UTR (124–158), 5′-GAGACGGAGTCTTGCTCTGTCGGTGGCGCGGTCTTGGCTCACTG-3′ (sense) and 5′-CAGTGAGCCAAGACCGCGCCACCGACAGAGCAAGACTCCGTCTC-3′ (antisense) for NANOG 3′UTR (254–277), 5′-CTGCTAAGGACAACATTGATAGAATAAGTAGATCTAATACTAGTTTGG-3′(sense) and 5′-CCAAACTAGTATTAGATCTACTTATTCTATCAATGTTGTCCTTAGCAG-3′(antisense) for NANOG 3′UTR (570–605), and 5′-AGTTGATTTTACCCTGATTTCTCGATGAGTAAATATACAGGG-3′ (sense) and 5′-CCCTGTATATTTACTCATCGAGAAATCAGGGTAAAATCAACT-3′ (antisense) for NANOG 3′UTR (930–952). PCR was performed at 94 °C for 30 s, 55°C for 60 s, and 68 °C for 8 min for 10 cycles. The methylated parental DNA templates were then digested with 20 U DpnI (NEB) at 37 °C for 6 h. The resulting nicked DNA fragments containing the mutation were transformed into competent E. coli DH5α cells. The presence of the mutation was verified by direct DNA sequencing.To construct a 3′UTR-luciferase reporter vector lacking the predicted MIR630 binding site, the predicted MIR630 binding sites on Cells were washed with twice pre-cold PBS and lysed in RIPA Lysis Buffer . Protein concentrations were quantified by the Bio-Rad protein assay kit . Protein lysates were resolved on a 10% SDS-polyacrylamide gel and electroblotted onto Immobilon-P PVDF membranes (Millipore). The membranes were blocked in TTBS containing 5% nonfat milk and incubated with diluted primary antibodies overnight at 4 °C. The membranes were then reacted with species-specific HRP-conjugated secondary antibodies (1:5000–10000 in TTBS), and the immunoreactive protein bands were detected by Amersham ECL™ Prime Western Blotting Detection Reagent . The primary antibodies used included Brachyury monoclonal antibody , GAPDH monoclonal antibody , GFAP monoclonal antibody , NANOG polyclonal antibody , NeuroD1 polyclonal antibody , OCT4 monoclonal antibody , Pax6 monoclonal antibody , and SOX2 polyclonal antibody .p values < 0.05 were considered statistically significant. All analyses were performed using SigmaStat 3.5 .Data were presented as the mean ± standard deviation of at least three independent experiments, each performed in triplicate. To compare the data between two groups, a two-tailed Student’s t-test was performed. Data from different groups were compared using a one-way ANOVA and a post hoc test with the Holm–Sidak method. The


TNF, IL17A, IL17B, IL17C, IL17F, IL17RA, IL12A, IL12B and IL23A). SNPClinic predictions were run with DNAseI-HUP chromatin accessibility data in eight psoriasis/epithelia-relevant cell lines from ENCODE including keratinocytes (NHEK), TH1 and TH17 lymphocytes. Results were ranked quantitatively by transcriptional relevance according to our novel Functional Impact Factor (FIF) parameter. We found six rSNPs in five genes  and each was confirmed as true rSNP in at least one public eQTL database including GTEx portal and ENCODE (Phase 3). Predicted regulatory SNPs in cathelicidin, IL17C and IL17RA genes may explain hyperproliferation of keratinocytes. Predicted rSNPs in psoriasin, IL17C and cathelicidin may contribute to activation and polarization of lymphocytes. Predicted rSNPs in TNF gene are concordant with the epithelium-mesenchymal transition. In spite that these results must be validated in vitro and in vivo with a functional genomics approach, we propose FOXP2, RUNX2, NR2F1, ELF1 and HESX1 transcription factors (those with the highest FIF on each gene) as novel drug targets for psoriasis. Furthermore, four out of six rSNPs uncovered by SNPClinic v.1.0 software, could also be validated in the clinic as companion diagnostics/pharmacogenetics assays for psoriasis prescribed drugs that block TNF-α (e.g. Etanercept), IL-17 (e.g. Secukinumab) and IL-17 receptor .Psoriasis is an autoimmune disease associated with interleukins, their receptors, key transcription factors and more recently, antimicrobial peptides (AMPs). Cathelicidin LL-37 is an AMP proposed to play a fundamental role in psoriasis etiology. With our proprietary software SNPClinic v.1.0, we analyzed 203 common SNPs (MAF frequency > 1%) in proximal promoters of 22 genes associated with psoriasis. These include nine genes which protein products are classic drug targets for psoriasis ( •S100A7), IL17C, IL17RA and TNF genes.We found six putative regulatory SNPs in cathelicidin (LL-37), psoriasin  may contribute to T-cell polarization.Regulatory SNP rs12049559 in psoriasin ( This will allow us to propose those TFs as novel therapeutic targets and those rSNPs as potential companion diagnostics.High-throughput techniques have facilitated to annotate and analyze human genetic variations such as single nucleotide polymorphisms (SNPs) . Severalpression . GWAS anpression . Furtherreg lymphocytes are dysfunctional in psoriasis, with decreased suppressive capacity, suggesting that psoriasis may result from the inability to suppress autoinflammation [Psoriasis is a chronic inflammatory disease caused by an exacerbated immune system response ,7 that aammation . Also, rammation , as wellWe applied our software SNPClinic to 22 psoriasis-related genes. In order to have a more specific search of the TFBSs that could be affected by the rSNPs in psoriasis disease, we focused our analysis in cell lines and tissues implicated in skin inflammation from the Encyclopedia of DNA Elements (ENCODE) Project.SNPClinic could be used to efficiently identify rSNPs in promoter regions and the transcription factor binding sites (TFBSs) disturbed by the SNP. This information could be useful to discover transcription factors as novel drug targets and rSNPs that could be co-developed as companion diagnostics.22.1in silico human proximal  pseudo-promoters comprising all common SNPs (MAF >1%) in the averaged world population (code=ALL) according to the 1000 Genomes Project for each of the 22 psoriasis genes, including transcription factors, antimicrobial peptides/proteins and interleukins and their receptors (H0), helper T lymphocytes 1 (TH1), helper T lymphocytes 2 (TH2) and helper T lymphocytes 17 (TH17). Transcription factor Position Frequency Matrices (PFMs) were obtained from JASPAR database [We generated eceptors . Then, tdatabase . Thus wedatabase ) to Pyth2.2M) and RBS for the Minor Allele (RBSm). SNPClinic scans base-per-base the proximal promoter for each gene for each of the two DNA strands calculating the local DNA affinity to each of the 396 human transcription factors from JASPAR Database [M, RBSm, affinity impact (%), homotypic redundance (HR), homotypic redundance weight factor (HWF) [We followed the method of Flores Saiffe et al., . BrieflyDatabase . The outor (HWF)  and our 2.3The hypothesis for genomic interpretation of data obtained in 3In this work we applied the software SNPClinic v.1.0, useful in the identification of altered transcription factor binding sites (TFBSs) by the presence of a certain regulatory SNP (rSNP). Our group has demonstrated the utility of SNPClinic in the prediction of rSNPs to dissect comorbidities ,in the pp-value filtering and/or ranking. The obtained results suggest that statistical ranking methods alone are not sufficient to predict correctly, because they should integrate as much biological context as possible to obtain relevant biological results, such as the chromatin accessibility, cell line/tissue specificity and the DNA sequence itself.The selection and ranking of the TF-SNP association is mainly based on the calculated percentage of the binding affinity impact taking in account also the homotypic redundancy which is useful in the determination of the possible impact that a polymorphism could have in the activation or repression of a gene, unlike other less sensitive methods which are mainly based on CAMP, IL17C, IL17RA,S100A7 and TNF , DEFB1 and DEFB4 (defensins), S100A7A,S100A7 (psoriasin) as well as S100A8 and S100A9  in this study. From these, we found rSNP in CAMP andS100A7 genes. In the promoter region of CAMP gene in TH1 cell line, TFBS were altered for transcription factors HMBOX1, TFCP2 and ARNT:HIF1A when SNP rs9844566 is present. The same SNP is regulatory in NHEK altering TFBS for forkhead box P2 (FOXP2), SP8 and ZBTB7C, among others. Of these TFs just the HIF1A and FOXP2 were previously suggested by our group and others [CAMP gene expression. SNP rs9844566 also was suggested as regulatory in A549, HL60 and CD14 cell lines [In vitro evidence suggest that FOXP2 is involved in pentose phosphate metabolism by binding to ribose 5-phosphate isomerase A (RPIA) [It has been found three families of innate antimicrobial peptides and proteins: Cathelicidin (LL-37), S100 proteins and defensins that have been identified as potentially important players in psoriasis. These AMPs can be found in higher concentrations in psoriatic skin lesions, mainly in keratinocytes, phagocytes, T-cells, NK-cells, monocytes and mast cells .We have d others ,19,20 asll lines . FOXP2 ill lines . FOXP2 rA (RPIA) ,50. It iA (RPIA) .CAMP  is uncontrolled [CAMP gene, has binding sites for hypoxia-inducible factor 1 (HIF-1α) in the promoter region [In psoriatic lesions, gene expression of ntrolled . LL-37 introlled . CAMP ger region ,20. HIF-r region . Besidesr region .IL17 gene family plays an important role against certain pathogens through the stimulation of the release of antimicrobial peptides, pro-inflammatory cytokines and chemokines [. IL17C isoform increases the production of itself and of hBD-2 (DEFB4 gene), S100A7/A8/A9 and IL-19 among other proteins enabling a pro-inflammatory positive feedback loop [IL-17 is a family of cytokines consisting of six members, with both amino acid sequence homology and highly conserved cysteine residues . One of emokines . IL17C iack loop .H1, but more recently has been proved to be a clearly defined TH17 profile [H17 cells are differentiated from Naive CD4+ T cells that were stimulated by cytokines such as TGF-β, IL-6, IL-23 and have the ability to express IL-17, IL-21, IL-22, TNF- α, IL-6 and GM-CSF [IL17C gene, we found one putative rSNP in melanocytes and keratinocytes, with alteration in both cell lines of RUNX2 binding site, which is part of a set of “hub” transcription factors, whose motifs are enriched in many super-enhancers involved in epidermal differentiation [It was previously thought that the inflammatory profile of psoriasis was T profile . TH17 ced GM-CSF . In IL17ntiation . These rIL17RA), is a single-pass transmembrane receptor expressed in all tissues examined to date. When IL17RA is in contact with its ligand, leads to an activation of the NF-κB pathway, inducing secretion of other proinflammatory cytokines [IL17RA, eight SNPs were evaluated, but we found only rs4819958 as putative regulatory in the TH1 and monocyte cell line, with altered TFBS for paired box gene 1 (PAX1) and NR2F1. The ortholog gene in mice, Pax1, together with Pax9, are involved in organogenesis of thymus [TFAP2C and TFAP2B genes [The interleukin 17 receptor A gene . It is known that Msx2 play a role in promoting EMT, inducing changes in cell morphology and disruption of cell-cell contacts in transfected cells [IFNG gene, we found no accessible chromatin in the promoters of the analyzed cell lines.We included also inflammatory cytokines coding genes  lesions . In TNF ed cells , c-JUN aed cells . The seted cells . On the ed cells . TNFα aned cells . These Ted cells . RegardiThe remaining genes with no predicted rSNP by SNPClinic, may not have transcriptional relevance in psoriasis but instead may be mainly regulated by metal ions , dimerization and abundance , homotypic redundance (STAT1 and NFKB1 transcription factors) or an altered alternative layer of gene regulation as Pathogen-Associated Molecular Pattern-dependent expression .https://www.ebi.ac.uk/training-beta/online/courses/functional-genomics-i-introduction-and-design/what-is-functional-genomics/) to develop a more rational model to link both genotype plus other personalized data in order to predict in a most trustable way the prognosis of clinic phenotypes in psoriasis. These in silico results are interesting and have strong in vitro support since SNPclinic uses ENCODE and JASPAR databases as inputs (which are supported by an extensive collection of in vitro experiments). In spite of its limitations, SNPClinic results are a more rational starting point that can propose novel in vitro and in vivo experiments as well as research and development projects which results will allow to validate novel drugs targets and companion diagnostics. Tools like SNPClinic software v.1.0 open new avenues to a comprehensive personalized medicine for psoriasis patients.Even when the SNPClinic v.1.0 software is a powerful tool that aid for a better understanding of the molecular mechanism between rSNPs and gene expression, the relationship of rSNPs/gene expression with pathogenicity is far more complex and is a topic that our group is very interested to elucidate and to predict more accurately. We pretend to apply this “pathogenicity score” to e.g. polygenic risk scores  and pharmacogenetics . Prediction of pathogenicity based on both genetic and non-genetic variants must include many other features as non-coding RNAs, transcription factor grammar, protein synthesis and degradation rates, proteomics (proteins present in a sample at a given time and condition), 3D interactions, metabolome and other relevant data as well. The above mentioned massive data must be wisely weighted and integrated with a functional genomics approach, which main aim is to understand the dynamic expression of gene products in a specific context  and their companion diagnostics (rSNPs). Furthermore, the putative four rSNPs involved in TNF, IL17C and IL17RA regulation that we found, could be validated in the clinic as companion diagnostics/pharmacogenetics assays for prescribed drugs for current psoriasis therapy as brodalumab, secukinumab, ixekizumab, infliximab, certolizumab, etanercept, golimumab and adalimumab.Results obtained with traditional available software is very limited in the prediction of rSNPs with a cell line/tissue- and disease-specific approach. We found six rSNPs in five genes that may explain hallmark processes in psoriasis as hyperproliferation of keratinocytesAndrea Virginia Ruiz Ramírez: Investigation, interpretation of data, Visualization, Writing – original draft preparation; Adolfo Flores Saiffe Farías: Software, Investigation; Rocío del Carmen Chávez Álvarez: Software, Investigation; Ernesto Prado Montes de Oca: Conceptualization, design, Funding acquisition, Resources, interpretation of data, writing-review, editing & revision.CAMP gene (cathelicidin LL-37) transcription.EPM has a granted patent for a small molecule that inhibits


HTT) gene. The typical motor symptoms have been associated with basal ganglia pathology. However, psychiatric and cognitive symptoms often precede the motor component and may be due to changes in the limbic system. Recent work has indicated pathology in the hypothalamus in HD but other parts of the limbic system have not been extensively studied. Emerging evidence suggests that changes in HD also include white matter pathology. Here we investigated if the main white matter tract of the limbic system, the fornix, is affected in HD. We demonstrate that the fornix is 34% smaller already in prodromal HD and 41% smaller in manifest HD compared to controls using volumetric analyses of MRI of the IMAGE-HD study. In post-mortem fornix tissue from HD cases, we confirm the smaller fornix volume in HD which is accompanied by signs of myelin breakdown and reduced levels of the transcription factor myelin regulating factor but detect no loss of oligodendrocytes. Further analyses using RNA-sequencing demonstrate downregulation of oligodendrocyte identity markers in the fornix of HD cases. Analysis of differentially expressed genes based on transcription-factor/target-gene interactions also revealed enrichment for binding sites of SUZ12 and EZH2, components of the Polycomb Repressive Complex 2, as well as RE1 Regulation Transcription Factor. Taken together, our data show that there is early white matter pathology of the fornix in the limbic system in HD likely due to a combination of reduction in oligodendrocyte genes and myelin break down.Huntington disease (HD) is a fatal neurodegenerative disorder caused by an expanded CAG repeat in the huntingtin (The online version contains supplementary material available at 10.1007/s00401-021-02362-8. HTT) gene. There are no disease-modifying treatments available. Clinical diagnosis is based on the manifestation of typical motor signs in combination with a genetic test. Motor symptoms are associated with neuronal loss in the striatum of the basal ganglia and the cerebral cortex. Psychiatric and cognitive symptoms often precede the motor component of HD by many years but less is known about the underlying biological mechanisms. The hypothalamus and the limbic system have been suggested to play a role for non-motor symptoms and signs of HD  and myelin . The volume of the column of the fornix within the hypothalamic region was determined on blind-coded serial myelin stained sections using the Computer Assisted Toolbox Software (New CAST) module in VIS software  and applying the optical fractionator using the Cavalieri method . The bor2O. Finally, sections were stained in 0.5% lead citrate for 2 min at room temperature. EM images were obtained using a FEI Tecnai at 120 kV. Axon and myelin fiber diameters were measured using ImageJ (NIH). 125 axons were measured per case. The integrity of myelin rings of the fornix was evaluated on 46 randomly selected EM images from 4 HD and 4 controls. For each image the total number of complete intact myelin rings as well as misshapen myelin rings were determined. Myelin rings in which myelin sheets contained swellings, disruptions, folds and irregular shapes were classified as misshapen.For electron microscopy (EM), a 2-mm punch using a disposable biopsy punch (Miltex) of the fornix was collected from 50 μm-thick free-floating formalin sections of 4 HD and 4 controls . The frozen hypothalamic tissue was fixed in frozen state in a coronal position using OCT Cryomount (Histolab) and serially sectioned from a rostral to caudal direction at − 15 °C on a Cryostat (Microm HM 560). The tissue was cut at a thickness of 200 μm and collected on pre-cooled glass slides and stored at − 20 °C overnight before being micro dissected. Every 1 mm, a 20-μm section was collected and stained for Nissl substance  for orientation purposes during the dissection. The CV-stained sections were assessed under a light microscope and then scanned and printed on A4 paper. The anatomical position of the fornix in the scanned images was determined using the atlas of Mai et al.  as referTissue collected from the fornix was used for analyses using the MBP-QD9 antibody, which has been shown to detect fragmented myelin , 35, 58 Total RNA was isolated from tissue samples using RNeasy Lipid Tissue Kit (Qiagen) with an on-column DNase digestion  according to supplier’s recommendations. RNA quantity was measured on a NanoDrop 2000 spectrophotometer (Thermo Scientific). RNA integrity number (RIN), an indicator for appropriate preservation of RNA integrity, was used to assess the RNA quality of the human postmortem tissue , 16, 51 For RNA sequencing, subsequent library preparation using TruSeq Stranded mRNA LT Sample Prep Kit and paired-end 150-bp sequencing and 25 million reads/per sample were performed by Macrogen (Singapore). We performed RNA sequencing quantification directly from the reads with Salmon v0.9.1  using GRp < 0.05. After verifying normal distributions using Kolmogorov–Smirnov tests, one-way ANOVA followed by a Tukey’s post hoc test or Kruskal–Wallis followed by Dunn’s post hoc test were performed. For correlation analyses, Spearmans’ ρ tests were performed.Data are shown as mean ± SD, unless otherwise specified. GraphPad Prism7 and PASW19 statistical package (SPSS) was used for statistical analyses. Significance was considered for t test p < 0.001)   in the pre-HD group, 70 ± 18 mm3 in the symp-HD group and 118 ± 26 mm3 in the control group  but no significant effect of sex, age or ICV on the fornix volume estimates . Tukey’s post hoc analyses revealed significant differences between both HD groups and the control group . As previous research has shown WM changes in the corpus callosum in HD [ρ, p = n.s.), indicating an early independent volumetric change in this structure.We began by investigating whether the fornix would be affected in prodromal and manifest HD in MRI from the IMAGE-HD cohort, and confirm atrophy of the corpus callosum , 45. We ρ for the fornix volumes and the clinical, neurocognitive and neuropsychiatric data collected from all the participants. We found small but statistically significant correlations between fornix volume and age , UHDRS , SDMT , STROOP , HADS-A , BDI II  as well as with symptom duration for the symp-HD group   as well as neuropsychiatric parameters . Correlation analyses were performed using Spearman’s Taken together, the volume of the fornix is smaller in HD at an early stage before the clinical onset of motor symptoms compared to controls and the fornix volumes show discrete but statistically significant correlations to measures of depression as well as cognition.t test, n.s.). We detected an average 27% smaller volume of the fornix in HD cases . In order to further investigate underlying mechanisms of the smaller fornix volume in HD, we wanted to examine whether there is axonal damage within this region. We examined protein levels of neurofilaments, which are neuron-specific cytoskeletal components present in axons. We found no significant differences in the protein levels of neurofilament light or heavy chain between HD and control cases . Taken together, these data indicate myelin breakdown in the fornix in HD.We then assessed the fornix at an ultrastructural level using EM in tissue from 4 HD cases compared to four controls. We detected a 27% increase in the percentage of misshapen myelin rings in HD cases compared to controls Fig. a–c. Therses Fig. d, e, whiNext, we determined the protein levels of myelin regulating factor, MRF, a major transcription factor for oligodendrocyte genes, found to be reduced in mouse models of HD . We founTo gain insight into the cellular and biological processes impacted in the fornix region in HD, we performed an unbiased, global transcriptome analysis using RNAseq on fornix samples from five HD cases and four control cases Table . PrincipTo identify transcription factors and/or epigenetic mediators that may account for the differential gene expression patterns observed, we used EnrichR  to perfoWM changes have emerged as important aspects of pathology in HD. Previous research has mainly focused on WM pathology in the corpus callosum and tracts related to the movement disorder. Psychiatric and cognitive symptoms are common in HD and are often present before motor disturbances. The biological origins of psychiatric symptoms in HD are not known but changes in the limbic system have been suggested to play a role (for review see ). In theThe limbic system, including the fornix, is heavily involved in the regulation of emotion and cognition , 55. ThiOligodendrocytes and the myelin they generate are critical for axonal function. These cells are mainly generated during the postnatal period and differentiate from proliferative and migratory oligodendrocyte precursors (OPC). Myelination not only occurs in early life but is a plastic process in the adult brain important for learning, memoryand as response to injury . DysfuncAlterations in white matter and myelin may be due to direct effects of mutant HTT in oligodendrocytes in HD. This would also explain why there may be effects on myelin without apparent axonal damage. Reduced expression of a number of oligodendrocyte genes in the fornix of HD cases compared to controls as revealed by RNA sequencing is a key finding of this study. Analysis of differentially expressed genes based on transcription-factor/target-gene interactions revealed enrichment for binding sites of SUZ12 and EZH2, components of PRC2, as well as REST. These results are consistent with our recent findings in the BACHD mouse model of HD in which these genes were implicated in mutant HTT-mediated defects in oligodendroglia . AbnormaWe have shown that a number of molecular effects of PRC2 are increased in callosal tissues of BACHD mice and that inactivation of mutant HTT in oligodendroglia reverses these changes and rescues the demyelination and WM abnormalities in HD mice . Our finWe also found reduced protein levels of MRF, a key transcriptional regulator that activates and maintains the expression of myelin genes in mature oligodendrocytes , 23. MRFWM pathology has been demonstrated in several rodent models of HD including the YAC128 mouse and the BACHD mouse and rat models , 53, 54.In conclusion, the present data show that the fornix in the limbic system is affected already at an early stage in the disease process in HD. The smaller fornix volume correlated with measures of psychiatric and cognitive changes, suggesting a role for demyelination of connections in the limbic system in these non-motor aspects of HD. Our data demonstrate in human post-mortem tissue that there is a significant downregulation of oligodendrocyte genes as well as signs of myelin breakdown in the fornix. Hence, demyelination of WM in HD is a significant part of the disease pathology and may, therefore, provide a promising target for therapeutic interventions.Figure 1, online resource. Correlation analysis for fornix volume with cognitive and neuropsychiatric parameters. Overview of correlation analyses for fornix volume and selected clinical cognitive and neuropsychiatric data for all the participants in the IMAGE-HD cohort. No significant correlations were seen for ICV (a) and hypothalamic volume (b). Significant correlations were seen for age (c), UHDRS (d), SDMT (g), SCOPI (h), STROOP (i), HADS-A (j) and Beck Depression Inventory score Version II (j). Correlation analyses were made by Spearman’s rho tests. Abbreviations, HADS-A: Hospital Anxiety and Depression scale - anxiety sub score; SCOPI: Schedule of Compulsions Obsessions and Pathological Impulses; SDMT: Symbol Digit Modalities Test; STROOP, speeded word reading task (number of correct words); UHDRS-M: Unified Huntington’s Disease Rating Scale - motor subscale score  (TIFF 2363 kb)Supplementary file2 (DOCX 24 kb)Supplementary file3 (XLSX 1136 kb)Supplementary file4 (XLSX 26 kb)Below is the link to the electronic supplementary material.


Development of cortical areas begins in cortical stem cells through the action of morphogens controlling the graded expression of transcription factors (TFs). Here, we have systematically identified the TFs and gene regulatory elements (REs) that together control regional pattering of the cortical progenitor zone; these data have led us to propose a cortical regionalization TF network. To identify REs active in this network, we performed TF chromatin immunoprecipitation followed by sequencing (ChIP-seq) and chromatin-looping conformation experiments as well as assays for epigenomic marks and DNA accessibility in purified ventricular zone (VZ) progenitor cells in wild-type and patterning mutant mice. This integrated approach has laid the foundations to identify a TF network and cortical VZ REs involved in cortical regional patterning. Emx2, Nr2f1, and Pax6), which enabled us to define a cortical regionalization TF network (CRTFN). To identify genomic programming underlying this network, we performed TF ChIP-seq and chromatin-looping conformation to identify enhancer–gene interactions. To map enhancers involved in regional patterning of cortical progenitors, we performed assays for epigenomic marks and DNA accessibility in VZ cells purified from wild-type and patterning mutant mice. This integrated approach has identified a CRTFN and VZ enhancers involved in cortical regional patterning in the mouse.We uncovered a transcription factor (TF) network that regulates cortical regional patterning in radial glial stem cells. Screening the expression of hundreds of TFs in the developing mouse cortex identified 38 TFs that are expressed in gradients in the ventricular zone (VZ). We tested whether their cortical expression was altered in mutant mice with known patterning defects ( Cortical development is an intricate choreography that integrates finely tuned molecular states such as gradients of transcription factors (TFs) and the chromatin landscape with a variety of cellular processes . Together, these developmental processes result in the establishment of an adult cerebral cortex and associated pallial structures, which are organized into discrete areas and laminae distinguished by specific molecular signatures . The ear27Emx2  and/or dorsoventral (DV) axes. We refer to these 38 TFs as the cortical regionalization TF network (CRTFN) E. TowardEmx2, Nr2f1, and Pax6 mutants, we identified enhancers likely to participate in cortical patterning. Together, this study establishes the foundations of the transcriptional circuitry operating in radial glial stem cells and underlying cortical regionalization.To identify the genomic REs tying together the CRTFN, we performed TF ChIP-seq for EMX2, LHX2, NR2F1, PAX6, and PBX1. This analysis, coupled to histone ChIP-seq and assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), identified ∼2,000 pREs in the CRTFN. Combinatorial TFs binding was tightly associated with the activity of cortical enhancers. Furthermore, coordinated binding of EMX2, LHX2, NR2F1, PAX6, and PBX1 provided evidence for a combinatorial signature regulating enhancer activity along the rostroventral–caudodorsal axes. Finally, by assessing epigenetic changes of the 2,000 CRTFN pREs in To systematically identify TFs that could participate in cortical regional patterning through their activity in neural progenitors, we used the Allen Brain Developing Mouse Atlas to study the RNA expression of 698 TFs based on in situ hybridization (ISH) analyses on sagittal sections of E11.5 mouse embryos.Dataset S1). Herein, we focus on progenitor zone expression, as we wished to concentrate on mechanisms that operate in progenitors and control pallial regional patterning. We assessed the level of expression based on both the intensity and the density of expression . Id3; n = 134), with many showing either RC  . We chose to study the 31 TFs based on the following three criteria: 1) TFs previously not well-known to be expressed in the E11.5 pallium; 2) TFs whose neurodevelopmental functions were poorly known; and 3) TFs with clear E11.5 expression gradients.Next, we investigated how CRTFN TFs are regulated by TFs with known functions in cortical patterning. We studied the expression of 31 TFs in Emx2, Nr2f1, and Pax6 mutations on the expression of the 31 TFs are organized based on changes of their RC, CR, or regional expression. We annotated the expression changes based on the independent assessment of three experts using a five-level qualitative expression scale: increased expression ; no change; decreased expression   showed differential RNA expression in at least one of the three mutants.The phenotypic descriptions of the effects of the  − or −) B. The ex−/−Emx2 (9/13) and in −/−Nr2f1 (8/13) than in −/−Pax6 (5/13). Likewise, genes with RC gradients showed reduced expression in −/−Pax6 (8/12) and increased expression in −/−Emx2 (9/12) . However, unlike in −/−Emx2, loss of Nr2f1 led to a decrease in expression of RC TFs (9/12).During cortical patterning, EMX2 and NR2F1 promote caudal identity, whereas PAX6 promotes rostral identity . ConsideNr2f1 and Emx2 are expressed in CR gradients, they have opposite DV gradients. Emx2 has a DV gradient, whereas Nr2f1, like Pax6, has a ventrodorsal (VD) gradient . Thus, perhaps the TF expression differences between the Emx2 and Nr2f1 mutants might in part relate to their differences in DV patterning functions. Indeed, in the Nr2f1 knockout, several down-regulated RC TFs (including Npas3) were also down-regulated in the Pax6 mutant  suggest that TFs with rostral-ventral gradients (such as Npas3) are similarly regulated by Pax6 and Nr2f1. Loss-of-function analyses show that Pax6 has prominent roles in promoting LVP properties . We first assessed whether they coregulated Pax6 expression in the LVP at E11.5 but detected no change in PAX6 immunostaining .We next examined whether some of the rostroventral TFs that were coregulated by −/− mice . We founpression D and F. Dataset S2). Our goal was to determine whether they directly regulate these genes and to identify pREs for the CRTFN. We performed ChIP-seq using PAX6-, EMX2-, and NR2F1-specific antibodies on E12.5 cortical tissue . A description of the ChIP-seq for PAX6, PBX1, and NR2F1 was previously published . Representative ChIP-seq peaks are depicted around the Pax6 locus in Pax6 locus, and called peaks are annotated beneath the genomic tracks. Overlapping TF ChIP-seq peaks were merged into discrete pREs (highlighted in pink). We assessed the reproducibility of the ChIP-seq experiments by pairwise Pearson genome-wide read count correlation of biological replicates run individually, as depicted in SI Appendix, Fig. S2D. For each TF, ChIP binding sites were located predominantly at distal rather than proximal genomic loci . We defined the size of each genomic locus by defining its boundaries by 1) the distal point of contact for promoter–enhancer looping obtained by a chromatin conformation assay (see Promoter–Enhancer Binding around 35 TFs of the CRTFN) 2) ±100 kb from the limits of the gene body.Next, we investigated whether PAX6, EMX2, and NR2F1 proteins bind to regulatory regions of the CRTFN genes  supporting direct TF binding. Beyond the primary motifs associated with TFs profiled here, many additional motifs were enriched within pREs, including motifs for TFs that are crucial for cortical development , as well as for basal ganglia development  . Notably, when NR2F1 was bound at loci in conjunction with other TFs, the nuclear NR2F1 motif was rare, indicating that NR2F1 is likely to bind indirectly to such pREs .We used Homer  to perfo30SI Appendix, Fig. S2E). These pREs were predominantly found at distal loci . They were highly enriched around TF genes that are expressed in the embryonic pallium . Of particular note, pREs with combinatorial binding were enriched around TFs that we identified as being a part of the CRTFN (dark blue bubbles in Dataset S2). Based on the proximity ligation assisted chromatin-immunoprecipitation (PLAC-seq)-defined interactome (see Promoter–Enhancer Binding around 35 TFs of the CRTFN), 33/38 CRTFN genomic loci had at least one pRE that was co-occupied by at least three to five TFs , Subpallial (n = 56), and Nontelencephalic (n = 75). We also identified Inactive enhancers . We found that TF binding (one to five TFs) was present on 91% of Pallial, 73% of Subpallial, 51% of Nontelencephalic, and 31% of Inactive enhancers . This indicates that nearly all genomic loci from this unbiased set of validated pallial enhancers exhibit binding of at least one of the CRTFN TFs.To assess the activity of pREs defined by ChIP binding, we explored whether they are located in known enhancers that are active at E11.5 [VISTA enhancers—.lbl.gov ]. These SI Appendix, Fig. S5C and Dataset S3). Thus, binding by four to five TFs appears to be a potent indicator of enhancer activity. Furthermore, we estimate that there are 115 such loci associated with the CRTFN. Thus, CRTFN pREs bound by multiple TFs are likely to have pallial activity.Strikingly, 35% of the VISTA enhancers with Pallial activity were bound by four or five TFs. In comparison, only 23% of Subpallial, 1% of Nontelencephalic, and 6% of Inactive VISTA enhancers showed this pattern . Next, we correlated the ChIP-seq peak heights, normalized for coverage depth differences across TFs, and found association between ChIP-seq signal strength and cortical regional activity  or small peaks (<25% percentile for peak enrichment) in ∼44% of cases (n = 6). Peaks that were over enhancers with DP activity  or in mixed regions  displayed more heterogeneity in peak height but were often between both extremes observed in LVP and MP VISTA enhancers. TF binding and peak height enrichment for each individual VISTA enhancers that have regional activity is shown in Enhancers with LVP activity had the most robust peaks . To model our findings, we examined the correlation of TF binding, enhancer spatial activity gradients, and regionally defined enhancer activity. We found that peak presence was correlated with RC and LVP enhancer activity . In contrast, the presence of EMX2, LHX2, NR2F1, and PBX1 was anticorrelated with MP and DV enhancers . This implies that the binding of these five TFs promotes activity of enhancers with RC and/or LVP activity but not enhancers with MP activity. These results provide a gene regulatory logic where TF–RE interactions generate spatial gradients across the developing pallium.Some VISTA enhancers had spatial activity gradients along the pallial RC and DV axes. For instance, hs798 has both CR and DV gradients, whereas hs636 has RC and VD gradients . From these VZ cells, we made nuclei and performed a chromatin accessibility assay (ATAC-seq) and histone ChIP-seq with antibodies to histone modifications associated with active pRE state (H3K27ac) and repressed pRE state (H3K27me3)  , 36. In A3K27me3 SI Appendix, Fig. S6.In general, there was a high degree of overlap of TF binding, H3K27ac, and ATAC peaks; H3K27me3 peaks were less likely to be centered on TF, ATAC, and H3K27ac peaks. The proportion of overlap with TF binding (one to five TFs) was 40% for ATAC peaks, 26% for H3K27ac, and 10% for H3K27me3 C. IntereSI Appendix, Fig. S4C). Certain loci were enriched for H3K27me3 , whereas others were highly enriched for H3K27ac  . In general, genes with MP expression had pREs that were significantly more repressed than pREs in other genes . Thus, the analysis of histone modifications in the CRTFN on purified VZ cells has provided evidence for which REs are active and repressed during cortical VZ patterning.Overall, active marks and repressive marks were evenly represented across the 2,083 pREs identified in the CRTFN (27% for each histone modification). At individual genomic loci though, there was a range of acetylated and repressed pREs . PLAC-seq loops terminated at regions of chromatin accessibility, TF binding, and H3K27Ac, as well as at VISTA enhancers with cortical activity  (Dataset S5). In addition, we complemented the PLAC-seq results with previously computationally derived enhancer–gene associations based on correlation between the putative enhancer activity and the expression level of the genes residing in the same topologically associating domain (Dataset S5) (Enhancer–promoter loops show points of contact between TF gene promoters and multiple pREs (see  hs1050) B. The PLaset S5) , 40. ThuDataset S3. As with TF binding, Pallial VISTA enhancers were more likely to have ATAC and H3K27ac peaks than Subpallial, Nontelencephalic, or Inactive enhancers . In contrast, H3K27me3 was not enriched on cortical VISTA enhancers compared to other enhancers (active in other tissues or inactive).We tested whether chromatin state was predictive of cortical progenitor activity for pREs by examining open chromatin and histone marks on VISTA enhancers in Dataset S6). Of those, 5/15 had activity in the cortex, and an additional 8/15 had activity in surrounding regions such as the pretectum or subpallium (Dataset S6). Two of these enhancers are shown in Etv5) as well as the CDP and MP (mm1789 near Npas3)  E. Thus, −/−Emx2, −/−Nr2f1, and −/−Pax6, an approach we previously applied in Nkx2-1 and Dlx1/2 mutants (Dataset S7).Having identified 2,083 CRTFN pREs in cortical progenitor cells, we next sought evidence for which of these may participate in regulating regional patterning. To this end, we looked for changes in pRE chromatin state in  mutants , 41. We Dataset S7). The annotated changes in epigenetic state of differential pREs for each mutant are presented in Pax6−/− showed a 69% loss of H3K27ac marks (81/118) and no increases and a balanced loss and gain of H3K27me3 . Consistent with histone changes, loss of chromatin accessibility (25%) occurred more frequently than an increase in chromatin accessibility (2%). Emx2−/− showed a 44% loss and a 17% gain of H3K27ac and a 42% loss and 47% gain of H3K27me3. Chromatin accessibility was more often lost (25%) than gained (7%). Finally, Nr2f1−/− showed a 45% loss of H3K27ac and an 11% loss of H3K27me3 and no gains in either mark. Chromatin accessibility only showed a change in 3% of peaks  in enhancers that lost H3K27Ac (P = 1 × 105) and enhancers that gained H3K27me3 (P = 1 × 102) in −/−Emx2. Conversely, the LHX2 binding motif was not detected in enhancers that became more active in −/−Emx2. Taken together, this suggests that enhancers that have LHX2 binding are more likely to be turned off than those that do not have LHX2 binding in −/−Emx2.We found that enhancers that lose H3K27ac in ed Homer  to perfo−/−Emx2, 14 TFs had reduced RNA expression, and 86% of these genomic loci exhibited an overall loss of active and/or gain of repressive marks on pREs (Dataset S7). In −/−Pax6, 15 TF genes had a reduction in RNA expression, and 87% of these genomic loci showed an overall loss of active and/or gain of repressive marks. Finally in −/−Nr2f1, 19 TF genes had a reduction in RNA expression, and 13 (68%) of these genomic loci showed an overall loss of active marks at pREs. This indicates that for a given TF gene, reduction of RNA expression in a mutant is correlated with a reduction of the H3K27ac/H3K27me3 ratio in the pREs of that locus.We next checked whether these epigenomic changes were consistent with changes in RNA expression within the CRTFN F. In gen−/−Emx2 met this expectation; 9/11 TF genes that had increased RNA expression also had pREs that lost H3K27me3 and/or gained H3K27ac. On the other hand in −/−Pax6, increased RNA expression was not associated with an increased H3K27ac/H3K27me3 ratio.In contrast, for genes showing an increase in RNA expression, we expect a loss of H3K27me3 and/or a gain of H3K27ac (increased H3K27ac/H3K27me3 ratio). −/−Emx2, −/−Nr2f1, and −/−Pax6 are presented in −/−Pax6). The number in the center of the rings represents the number of pREs investigated. Overall, we suggest that loss of H3K27ac is associated with reduced RNA expression and with TF protein binding to pREs in the −/−Emx2, Nr2f1−/−, and −/−Pax6.The summary of changes in epigenomic profiles for pREs based on changes in ISH RNA expression in Dmrt3 and Sp8) in SI Appendix, Fig. S6 for all 38 TF loci in the CRTFN). In the genomic loci of these 2 TFs, we highlighted pREs that showed epigenomic changes in yellow. Dmrt3 RNA expression is down-regulated in all mutants. At the Dmrt3 locus, hs112 showed a decreased H3K27ac/H3K27me3 ratio and a loss of chromatin accessibility in both −/−Emx2 and −/−Pax6. Sp8 is down-regulated in Pax6−/− but increased in Emx2−/−. pRE epigenetic changes were in synchrony with this. For instance, the H3K27ac/H3K27me3 ratio on hs844 was reduced in −/−Pax6 and increased in −/−Emx2. Other pREs around the Sp8 locus showed epigenetic changes in only one or two mutants and were unchanged in the other knockouts.Next, we show two gene loci . Understanding how individual pREs regulate transcription at a single gene locus will require investigating these pREs individually and in combination across normal and abnormal development.We identified 20 to 120 pREs per locus, with 10% showing epigenetic changes in the TF mutants  on the expression of TF genes and the epigenomic state of pREs. Within the CRTFN, 30/31 of the TF genes were sensitive to genetic ablation of at least one of these three TFs. pREs were identified using TF ChIP-seq for EMX2, LHX2, NR2F1, PAX6, and PBX1, and epigenomic assays for chromatin accessibility, active pREs (H3K27ac), and repressive pREs (H3K27me3). pREs were further assessed by PLAC-seq–defined promoter–enhancer looping or computationally derived enhancer–gene associations. We found that pREs with combinatorial binding of different TFs were enriched at enhancers displaying cortical activity (VISTA enhancers) and that their binding was correlated with activity in the LVP and RC gradients and anticorrelated with activity in the MP and DV gradients. We also showed that five CRTFN pREs had cortical activity in transient transgenic assays. Approximately10% pREs were sensitive to loss of EMX2, NR2F1 or PAX6; these showed a change in the H3K27ac/H3K27me3 ratio. Taken together, our integrated approach has identified a TF network involved in regional patterning of the cortex.We have identified the key elements (TFs and target genomic REs) in a transcriptional network that regulates early steps in cortical regional patterning in mice. This CRTFN is composed of TFs and their pREs. We tested the validity of this network by characterizing the impact of genetic ablation of three key members (developingmouse.brain-map.org). TFs at E11.5 were expressed either in gradients (∼70) or homogeneously across cortical regions (∼150). We did not find TFs with sharp intrapallial boundaries, except for six whose expression was restricted to the MP. This suggests that cortical areas do not originate from progenitor domains with spatially restricted TF expression but rather from a process that integrates information from TF gradients to create sharp boundaries.We carried out an unbiased screen to discover TFs that have informative patterns in the E11.5 pallium using the Developing Mouse Brain Atlas , nor is it entirely due to a bias in cell numbers since LVP and MP are both small structures in comparison to the DP, which had a level of TF binding intermediate to that of the LVP and MP. We propose that there is a distinct set of MP TFs, such as those found in our screen, Id3 and Lmx1a, that promote the activity of MP enhancers  degree of TF binding  on VISTA enhancers with regional activity and 2) an unbiased computational approach . We founnhancers  45). Th. ThEmx2,SI Appendix, Fig. S5). We suggest that pREs of MP genes are repressed in the DP and LVP, and we predict that a focused analysis on the VZ of the MP would find a H3K27Ac/H3K27me3 > 1.Toward establishing an epigenetic metric for a gene’s activity, we calculated the H3K27Ac/H3K27me3 ratio within the interactomes of each CRTFN TF. We found that pREs within the genetic locus of TFs with expression in the MP had a lower H3K27Ac/H3K27me3 ratio than that of non-MP TF gene loci . This indicates that pREs with combinatorial TF binding are accessible in the VZ and are likely to be active. We estimated that CRTFN gene loci have between 20 and 100 pREs, thus underscoring the complexity of regulatory landscapes (Dataset S2).To gain insights into the function of the pREs, we assessed their epigenomic state. We restricted our study to VZ cells to bypass issues relating to cellular heterogeneity. We found that VZ ATAC and H3K27ac peaks were proportionally enriched at pREs that have increased cobinding by TFs . This suggests that these pREs are responsive to ablation of these specific TFs and likely important for regional patterning of the cortical neuroepithelium.Enhancers are spatial and temporal integrators of combinatorial TF binding and chromatin state . While ths112 in Dmrt3 showed loss of H3K27ac in the three mutants, gain of H3K27me3 in −/− and Emx2−/−Pax6, and loss of chromatin accessibility in −/−Pax6 . For instance,  Pax6−/− . Other p mutants G. Thus,  mutants C, it wasDmrt3 locus in −/−Pax6 and −/−Emx2. Perhaps, some of these pREs can compensate for one another making their regulatory programs more robust to mutations, such as in the Arx loci  was regulated by the modulation of epigenetic mechanisms in a time and area-specific manner by Lmo4 (Here, we define TFs within the CRTFN that exhibit regional gradients in the developing mouse cortical neuroepithelium. We demonstrate via genomic interaction mapping and genetic knockout that expression patterns of these TFs are regulated, at least in part, via the interaction of specific TFs with target REs. Considering the lack of TFs that are restricted to specific cortical VZ regions, pREs must encode regional activity via integrating the effects of combinatorial TF binding. Based on this, we hypothesize that the VZ enhancers identified here may serve as a protomap of cortical regions by integrating TF signaling. We previously postulated that enhancers serve as protein-binding modules that translate gradients of TFs in cortical progenitors into region-specific expression in cortical neurons , 53. Oural plate , 55. Liklization . Moreove by Lmo4 . Here, wSI Appendix.All procedures and animal care were approved and performed in accordance with NIH and the University of California San Francisco Laboratory Animal Research Center guidelines. Cortical expression analysis was performed using the Allen Brain Institute Developing Mouse Brain database. Histology, transgenic assays, TF ChIP-seq, epigenomic, and PLAC-seq assays were performed and analyzed according to published protocols. More detail regarding specific experimental procedures and analyses can be found in Supplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary FileSupplementary File


Arabidopsis as well as in crops.ELONGATED HYPOCOTYL5 (HY5), a bZIP-type transcription factor, acts as a master regulator that regulates various physiological and biological processes in plants such as photomorphogenesis, root growth, flavonoid biosynthesis and accumulation, nutrient acquisition, and response to abiotic stresses. HY5 is evolutionally conserved in function among various plant species. HY5 acts as a master regulator of light-mediated transcriptional regulatory hub that directly or indirectly controls the transcription of approximately one-third of genes at the whole genome level. The transcription, protein abundance, and activity of HY5 are tightly modulated by a variety of factors through distinct regulatory mechanisms. This review primarily summarizes recent advances on HY5-mediated molecular and physiological processes and regulatory mechanisms on HY5 in the model plant  Plants utilize light as the predominant energy source for photosynthesis. Besides, light signal acts as an essential external factor that mediates a variety of physiological and developmental processes in plants . Plants Arabidopsis  and external signals   and controls the expression of numerous target genes in response to light signals, which in turn serves to modulate distinct light-regulated physiological and developmental processes in plants.HY5 is originally identified as a positive regulator of photomorphogenesis, root gravitropic response, and lateral root development in the model plant bidopsis . HY5 gene genome . Extensie genome . The orte genome . HY5 froe genome . These iratures) . Sweet wratures) . The bioArabidopsis. More importantly, its abundance is correlated with seedling photomorphogenesis. A variety of factors converge on HY5, which indirectly or directly controls the expression of over 3,000 genes, to ensure normal seedling photomorphogenic development in response to dynamically changing light signals.Light tightly controls the seedling development including inhibition of hypocotyl growth, promotion of cotyledon expansion, and accumulation of chlorophyll which is totally termed photomorphogenesis. HY5 acts as an essential and indispensable regulator of this developmental process in In the darkness, CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1)-SUPPRESSOR OF PHYTOCHROME A-105 (COP1-SPA) E3 ligase complex directly targets HY5 for polyubiquitination and degradation, and thus, the abundance of HY5 remains at an extremely low level . COP1 SUHY5 to activate its transcription, while HY5 positively regulates the expression of BBX11, BBX21, and itself, thus forming a transcriptional feedback loop in controlling downstream target gene expression  work in concert to control the expression of numerous downstream target genes as well as multiple molecular and biological events . BBXs fupression . These fal level , suggestHY5 to promote photomorphogenesis  and SHI-RELATED SEQUENCE 5 (SRS5) positively control the transcription of ogenesis . INOSITOelopment . COLD REelopment . HY5 asselopment . Similarcription . MYC2, Melopment . These rG-box cis-element present in TANDEM ZINC-FINGER/PLUS3 (TZP) promoter to activate its expression in the far-red light. In turn, TZP competes with COP1 for binding of HY5, thus leading to the accumulation of HY5 that promotes phyA signaling , which is a negative regulator of anthocyanin biosynthesis (CONSTANS-LIKE 11 (MdCOL11), MdMYBDL1, MdMYB10, and itself but represses MdWRKY41 transcript level, which in turn promote anthocyanin biosynthesis (PyMYB10 and WD40 PROTEIN GENE (PyWD40), leading to the anthocyanin biosynthesis  and AaWRKY9 in a light-dependent manner to promote artemisinin biosynthesis (TERPENE SYNTHASE 03 (TPS03), a terpene biosynthetic gene, to facilitate the terpenoid biosynthesis in Arabidopsis , NITRATE TRANSPORTER 2.1 (NRT2.1), AMMONIUM TRANSPORTER 1, 2 , NITRATE REDUCTASE 2 (NIA2), and NITRITE REDUCTASE 1  and SUCROSE TRANSPORTER 12 (SWEET12) by directly associating with their promoters  to activate their transcription (FER transcription factor, leading to the increase of Fe uptake (In apple, MdHY5 promotes nitrate assimilation by positively regulating the expression of MdNRT1.1 . In tomacription . SIphyB e uptake . Similare uptake . Altogete uptake .Arabidopsis. Low temperatures trigger the COP1 translocate from the nucleus to the cytoplasm, thus leading to the inactivation of COP1 and accumulation of HY5 (BBX7 and BBX8. In turn, BBX7 and BBX8 regulate the transcription of a set of cold-responsive genes to promote freezing tolerance in plants (SIMYB15 transcription. On the other hand, both SIHY5 and SIMYB15 upregulate the transcript levels of SICBF1, SICBF2, and SICBF3. Thus, these molecular events increase the cold tolerance in tomatoes (As sessile organisms, plants have to cope with the fluctuating temperatures in adapting to daily and seasonal changing cycles. Low temperature is one of the most impactful environmental cues that affect plant growth and development. HY5 controls the expression of approximately 10% of all cold-induced genes to promote cold acclimation in n of HY5 . PREFOLDn of HY5 . Low temn plants . In tomatomatoes . MdHY5 atomatoes . Thus, HAt elevated high temperatures, HY5 abundance is dramatically reduced due to the inhibition of COP1 activity, resulting in thermomorphogenic development . In contArabidopsis, rice, and soybean, leading to drastically deficient in many facets of development and growth. Increasing HY5 abundance or activity may be a helpful strategy to improve specific agronomic traits in crops. On the one hand, genetic engineering techniques could be applied to generate specific plants expressing appropriately increased HY5 abundance. On the other hand, manipulation of positive or negative regulators of HY5 could be used to enhance the HY5 action. In view of the complexity of HY5 signaling, further studies are required to clarify the detailed HY5 signaling network in diverse plant species.Numerous studies have established that HY5 plays pleiotropic roles in regulating various physiological and developmental processes and responses to diverse internal and external signals in plants. A group of components converges on HY5 to modulate its abundance, activity, and transcription in maintaining its appropriate biological action. HY5 acts as a signaling hub that controls the expression of a large number of genes in response to dynamic changing developmental, hormonal, and environmental signals. This mechanistic regulation may ensure the plants adapt to the intracellular and surrounding fluctuating cues throughout their entire life cycles. Increasing studies have shown that HY5 functions are evolutionally conserved among various plant species. The HY5 orthologs in crops control multiple agronomic traits such as stem growth, root growth, nutrient uptake, and fruit ripening. Fulfilling a comprehensive understanding of HY5 functions and signaling will provide novel knowledge and strategies for the improvement of specific agronomic traits in crops. According to current fundamental knowledge on HY5 function and HY5-mediated signaling network, HY5 most likely have positive roles in the control of various physiological and developmental processes. It is therefore loss of HY5 function in different plant species such as All authors listed have made a substantial, direct, and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Chromatin undergoes drastic structural organization and epigenetic reprogramming during embryonic development. We present here a consistent view of the chromatin structural change, epigenetic reprogramming, and the corresponding sequence-dependence in both mouse and human embryo development. The two types of domains, identified earlier as forests (CGI-rich domains) and prairies (CGI-poor domains) based on the uneven distribution of CGI in the genome, become spatially segregated during embryonic development, with the exception of zygotic genome activation (ZGA) and implantation, at which point significant domain mixing occurs. Structural segregation largely coincides with DNA methylation and gene expression changes. Genes located in mixed prairie domains show proliferation and ectoderm differentiation-related function in ZGA and implantation, respectively. The chromatin of the ectoderm shows the weakest and the endoderm the strongest domain segregation in germ layers. This chromatin structure difference between different germ layers generally enlarges upon further differentiation. The systematic chromatin structure establishment and its sequence-based segregation strongly suggest the DNA sequence as a possible driving force for the establishment of chromatin 3D structures that profoundly affect the expression profile. Other possible factors correlated with or influencing chromatin structures, including transcription, the germ layers, and the cell cycle, are discussed for an understanding of concerted chromatin structure and epigenetic changes in development. In mammals, chromatin undergoes drastic organizational and epigenetic reprogramming after fertilization ,2. TheseThe progression of the mammalian embryo from fertilization to germ-layer formation, concurrent with transcriptional changes and cell fate transitions , involveThanks to recent developments in low-input chromatin analysis technologies, the chromatin structural, epigenetic and transcriptional properties have been roundly explored in the early embryonic development process . AssuminIn our previous study, we analyzed the DNA sequence dependence in the formation of 3D chromatin structures for different cell types . Based oIn the present study, we conducted an analysis of global chromosome structural changes during early embryonic development. Two specific stages, ZGA and implantation, during which gene activation and lineage specification occur, respectively, were both found to involve the mixing of the two types of genomic domains, with the latter showing a more significant change than the former. The segregation level positively correlates with the proportion of prairie domains residing in compartment B, the larger value of which suggests a more segregated chromatin structure. We also found that, compared to prairie regions constantly residing in compartment B, genes in switchable prairie regions tend to be tissue-specific. The DNA methylation distribution in early embryonic development also correlates with the trend of domain segregation in this process, which indicates that the chromatins of the endoderm and mesoderm are more segregated than that of the ectoderm. Moreover, the domain segregation levels of the earliest fate-committed germ layers correlate with those of the differentiated cells. We further investigated in detail the chromatin structure changes at different scales during ZGA and found a correlation between LAD mixing and domain mixing in ZGA, which was related to the functions of cell proliferation. The detailed functions of genes residing in more mixed domains during implantation were then analyzed, all of which are ectoderm-related. Finally, we present a consistent view of the structural change of chromatin from birth to senescence and discuss possible factors influencing global chromatin structure, such as transcription, germ layers, and cell division.Based on the Hi-C contact matrix, the inter-domain contact ratio between the same domain types was calculated as: The inter-domain contact between different types was calculated similarly as: The overall segregation ratio lization .Detailed information can be found in our previous work . BrieflyFor each forest/prairie domain In the above equations, The segregation ratio at each distance We quantified the methylation difference between adjacent forests and prairies by: Overall relative segregation ratios (To identify forest or prairie domains that are significantly more (or less) segregated in one tissue when compared to another, we first identified the threshold values to distinguish more (or less) segregated domains at certain stages. We calculated the logarithm ratio of We calculated the logarithm ratio of To quantify the degree of compartmentalization, we defined a compartment index (C-index) https://david.ncifcrf.gov, accessed on 16 March 2021), which yielded similar results. The results were demonstrated via GO terms with p-values. The functional annotation clustering was analyzed using DAVID, and the results were shown via GO terms with enrichment scores.We analyzed the functional enrichment of genes located in various selected regions using ClusterProfiler and DAVID , we calculated its CpG and SINE density. Bins where the CpG (SINE) density was larger than the median value of CpG (SINE) density were regarded as high-CpG (SINE) bins, and the remaining bins were low-CpG (SINE) bins. For the two groups of bins, such as hChS and hClS, the segregation extent was calculated as the average contact probability between hChS and hChS, divided by hChS and hClS, and the average contact probability between hClS and hClS, divided by hChS and hClS, respectively.Previous studies on chromatin structures during early embryonic development were mainly about changes to structural elements, such as compartments and TADs ,5. Here In our previous study , based oi-C data ,6,20 of ous work  to show As seen from e stages . The two alleles . For the alleles A. IntereThe overall chromatin segregation follows a similar trend in human embryonic development B. The ORi-C data B. Again,Besides the overall structure parameter Furthermore, to investigate chromatin segregation at different genomic distances, we calculated the segregation ratio as a function of genomic distance elopment , and an observed . Finally, the top 10% of contact probabilities under the genomic distance ncreases F, showinncreases G. These ncreases .By means of the Hi-C measurement, chromatin can be partitioned into two spatial compartments A,B 27]..27]. It The corresponding four types of DNA sequences are named as Af, Bf, Ap, and Bp, respectively, with the first letter denoting the compartments and the second, the forest (f) or prairie (p) domain. One can see from ze of Bp . A recenSince the genome compartmentalization is weak at the early stages of development , for theIn comparison with stable Bp regions, the prairie domains that switch the compartment type are particularly interesting, as they may be regarded as mediators of nuclear architecture establishment during development. The analysis of compartment transformation during mouse preimplantation development shows that 7.7% of prairies belong to compartment A (stable Ap), 26.3% of prairies always reside in compartment B (stable Bp), and the remaining 66.0% switch compartment type at least once (switchable p) B. Genes Next, to further investigate the differences between switchable prairie and stable prairie regions, we compared the chromatin features of these two kinds of regions between two adjacent stages. The cells we analyzed here include PN5, early 2-cell, late 2-cell, 8-cell and ICM, due to the availability of mouse Hi-C data. For every two adjacent stages , we identified stable Bp regions and switchable p regions . Although these two kinds of prairie regions are both located in compartment B in the cell of the earlier stage, the PC1 value of switchable p regions was significantly more positive than stable Bp regions ; accordiIn the earlier study, we showed that differences in the methylation levels between forests and prairies correlate well with chromatin spatial packing . In the Following such an observation, the extent of domain segregation of three germ layers can be inferred from the DNA methylation difference between the forest and prairie domains. As can be seen from t-test) and endoderm-derived t-test) tissues, in the same as the segregation degree of the three embryonic germ layers. The data on the prairie domains of human tissues show the same trend, as do those on both the forest and prairie domains of mouse tissues , the F–P ratio for the late 2-cell is larger than that for early 2-cell, while the P–P ratio is smaller for the former , as is consistent with our previous finding on the vital role of forest–prairie spatial interactions in prairie gene activation [As for ironment C. Next, Profiler  and DAVIProfiler ,40  forest–prairie spatial interactions increase, which event was thought to be associated with gene activation since our previous work has revealed that prairie genes tend to move to a more forest environment for activation . BesidesAn analysis of Hi-C data in differentiated and senescent cells  showed aIn this study, we also investigated the association between transcription and chromatin structure. The increase in the F–P ratio at small genomic distances (<500 kb) tends to be associated with ZGA. The effect of transcription on domain mixing during ZGA can also be tested using available mouse α-amanitin-treated Hi-C data. To investigate how transcription inhibition affects the chromatin architecture, we compared the F–P and P–P ratios within the top 10% contact probabilities under one certain genomic distance between the PN3, normal late 2-cell, and α-amanitin-treated-20 h cell (α-amanitin-treated late 2-cell). The critical genomic distance of α-amanitin-treated-20 h cell is significantly smaller than that of the normal late 2-cell, and the F–P and P–P ratios of α-amanitin-treated-20 h cell are between PN3 and normal late 2-cell , indicatSequence-based chromatin-domain segregation suggests that factors affecting domain interactions could lead to functional chromatin structure changes. The cell cycle is an important factor that could influence chromatin domain segregation. During embryonic development, the establishment of a TAD structure was reported as requiring DNA replication but not zygotic transcription ,5, implyDuring mouse embryonic development (from zygote to blastocyst), the nucleocytoplasmic ratio gradually increases, accompanied by a decrease in nuclear size . The chaSince precise gene expression profiles are essential for normal embryo development, factors influencing gene regulation were suggested as impacting the development process . For insIn this study, we delineated chromatin structural and epigenetic reprogramming in early embryonic development, based on the sequence-based domain segregation model, and correlated the overall chromatin structural changes with functional implementation during ZGA and implantation. The two types of DNA domains gradually separate from each other during embryonic development, but they show a tendency to mix when transcription and implantation happen. Increased interactions between the two types of domains indicate the start of transcription or set the stage for further cell differentiation, leading to lineage commitment. Genes of changed segregation states show lineage-relevant functions and important roles in the corresponding processes; thus, more detailed analyses on gene expression and the regulation of transcription factors are needed for understanding the related molecular mechanisms and, more importantly, to predict biological functions at the molecular level from the perspective of domain segregation and mixing-related chromatin structure formation.