Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

CTCF-binding element regulates ESC differentiation via orchestrating long-range chromatin interaction between enhancers and HoxA

2021; Elsevier BV; Volume: 296; Linguagem: Inglês

10.1016/j.jbc.2021.100413

ISSN

1083-351X

Autores

Guangsong Su, Wenbin Wang, Jun Chen, Man Liu, Jian Zheng, Danielle R. Reed, Jinfang Bi, Zhongfang Zhao, Jiandang Shi, Lei Zhang, Wange Lu,

Tópico(s)

CRISPR and Genetic Engineering

Resumo

Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer–enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure. Proper expression of Homeobox A cluster genes (HoxA) is essential for embryonic stem cell (ESC) differentiation and individual development. However, mechanisms controlling precise spatiotemporal expression of HoxA during early ESC differentiation remain poorly understood. Herein, we identified a functional CTCF-binding element (CBE+47) closest to the 3'-end of HoxA within the same topologically associated domain (TAD) in ESC. CRISPR-Cas9-mediated deletion of CBE+47 significantly upregulated HoxA expression and enhanced early ESC differentiation induced by retinoic acid (RA) relative to wild-type cells. Mechanistic analysis by chromosome conformation capture assay (Capture-C) revealed that CBE+47 deletion decreased interactions between adjacent enhancers, enabling formation of a relatively loose enhancer–enhancer interaction complex (EEIC), which overall increased interactions between that EEIC and central regions of HoxA chromatin. These findings indicate that CBE+47 organizes chromatin interactions between its adjacent enhancers and HoxA. Furthermore, deletion of those adjacent enhancers synergistically inhibited HoxA activation, suggesting that these enhancers serve as an EEIC required for RA-induced HoxA activation. Collectively, these results provide new insight into RA-induced HoxA expression during early ESC differentiation, also highlight precise regulatory roles of the CTCF-binding element in orchestrating high-order chromatin structure. Embryonic stem cell (ESC), which are derived from the inner cell mass (ICM) of blastocysts, exhibit unlimited self-renewal and differentiation into the three embryonic germ layers (1Martin G.R. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 7634-7638Crossref PubMed Scopus (4146) Google Scholar, 2Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Abstract Full Text Full Text PDF PubMed Scopus (17708) Google Scholar, 3Takahashi K. Tanabe K. Ohnuki M. Narita M. Ichisaka T. Tomoda K. Yamanaka S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors.Cell. 2007; 131: 861-872Abstract Full Text Full Text PDF PubMed Scopus (14043) Google Scholar). Therefore, ESC are of great utility to the fields of regenerative medicine and tissue engineering (4Sato N. Meijer L. Skaltsounis L. Greengard P. Brivanlou A.H. Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor.Nat. Med. 2004; 10: 55-63Crossref PubMed Scopus (1699) Google Scholar, 5Metcalfe A.D. Ferguson M.W. Tissue engineering of replacement skin: The crossroads of biomaterials, wound healing, embryonic development, stem cells and regeneration.J. R. Soc. Interface. 2007; 4: 413-437Crossref PubMed Scopus (547) Google Scholar). ESC pluripotency requires expression of master control transcription factors such as Oct4/Nanog/Sox2 (6Loh Y.H. Wu Q. Chew J.L. Vega V.B. Zhang W. Chen X. Bourque G. George J. Leong B. Liu J. Wong K.Y. Sung K.W. Lee C.W. Zhao X.D. Chiu K.P. et al.The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells.Nat. Genet. 2006; 38: 431-440Crossref PubMed Scopus (1880) Google Scholar, 7Boyer L.A. Lee T.I. Cole M.F. Johnstone S.E. Levine S.S. Zucker J.P. Guenther M.G. Kumar R.M. Murray H.L. Jenner R.G. Gifford D.K. Melton D.A. Jaenisch R. Young R.A. Core transcriptional regulatory circuitry in human embryonic stem cells.Cell. 2005; 122: 947-956Abstract Full Text Full Text PDF PubMed Scopus (3379) Google Scholar), while expression of HoxA/Gata4/Pax6 genes is essential for ESC differentiation (8Martinez-Ceballos E. Chambon P. Gudas L.J. Differences in gene expression between wild type and Hoxa1 knockout embryonic stem cells after retinoic acid treatment or leukemia inhibitory factor (LIF) removal.J. Biol. Chem. 2005; 280: 16484-16498Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 9Fujikura J. Yamato E. Yonemura S. Hosoda K. Masui S. Nakao K. Miyazaki Ji J. Niwa H. Differentiation of embryonic stem cells is induced by GATA factors.Genes Dev. 2002; 16: 784-789Crossref PubMed Scopus (390) Google Scholar, 10Gajovic S. St-Onge L. Yokota Y. Gruss P. Retinoic acid mediates Pax6 expression during in vitro differentiation of embryonic stem cells.Differ. Res. Biol. Divers. 1997; 62: 187-192Crossref Google Scholar). Recently, studies indicate that high-order chromatin structures play a critical role in regulating gene expression and maintaining ESC status (11Levasseur D.N. Wang J. Dorschner M.O. Stamatoyannopoulos J.A. Orkin S.H. Oct4 dependence of chromatin structure within the extended Nanog locus in ES cells.Genes Dev. 2008; 22: 575-580Crossref PubMed Scopus (89) Google Scholar, 12Wei Z. Gao F. Kim S. Yang H. Lyu J. An W. Wang K. Lu W. Klf4 organizes long-range chromosomal interactions with the Oct4 locus in reprogramming and pluripotency.Cell Stem Cell. 2013; 13: 36-47Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Numerous studies report that topologically associated domains (TADs) are the basic unit of nuclear chromatin (13Pope B.D. Ryba T. Dileep V. Yue F. Wu W. Denas O. Vera D.L. Wang Y. Hansen R.S. Canfield T.K. Thurman R.E. Cheng Y. Gulsoy G. Dennis J.H. Snyder M.P. et al.Topologically associating domains are stable units of replication-timing regulation.Nature. 2014; 515: 402-405Crossref PubMed Scopus (489) Google Scholar, 14Luppino J.M. Park D.S. Nguyen S.C. Lan Y. Xu Z. Yunker R. Joyce E.F. Cohesin promotes stochastic domain intermingling to ensure proper regulation of boundary-proximal genes.Nat. Genet. 2020; 52: 840-848Crossref PubMed Scopus (24) Google Scholar). Functional DNA elements, such as enhancers, within the same TAD are more likely to promote target gene expression via direct long-range chromatin interactions (15Lupianez D.G. Kraft K. Heinrich V. Krawitz P. Brancati F. Klopocki E. Horn D. Kayserili H. Opitz J.M. Laxova R. Santos-Simarro F. Gilbert-Dussardier B. Wittler L. Borschiwer M. Haas S.A. et al.Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions.Cell. 2015; 161: 1012-1025Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, 16Ji X. Dadon D.B. Powell B.E. Fan Z.P. Borges-Rivera D. Shachar S. Weintraub A.S. Hnisz D. Pegoraro G. Lee T.I. Misteli T. Jaenisch R. Young R.A. 3D chromosome regulatory landscape of human pluripotent cells.Cell Stem Cell. 2016; 18: 262-275Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). Long-range chromatin interactions require proteins to mediate those interactions, among them CTCF. Using auxin-induced degradation of CTCF protein, Nora et al. (17Nora E.P. Goloborodko A. Valton A.L. Gibcus J.H. Uebersohn A. Abdennur N. Dekker J. Mirny L.A. Bruneau B.G. Targeted degradation of CTCF decouples local insulation of chromosome domains from genomic compartmentalization.Cell. 2017; 169: 930-944.e922Abstract Full Text Full Text PDF PubMed Scopus (672) Google Scholar) found that CTCF loss promoted disappearance of TADs, revealing CTCT as essential for maintaining TAD formation. CTCF forms a chromatin loop through directly binding CTCF-binding elements (CBEs) enriched at TAD boundaries and relies on binding polarity to maintain TAD formation (18Guo Y. Xu Q. Canzio D. Shou J. Li J. Gorkin D.U. Jung I. Wu H. Zhai Y. Tang Y. Lu Y. Wu Y. Jia Z. Li W. Zhang M.Q. et al.CRISPR inversion of CTCF sites alters genome topology and enhancer/promoter function.Cell. 2015; 162: 900-910Abstract Full Text Full Text PDF PubMed Scopus (528) Google Scholar, 19de Wit E. Vos E.S. Holwerda S.J. Valdes-Quezada C. Verstegen M.J. Teunissen H. Splinter E. Wijchers P.J. Krijger P.H. de Laat W. CTCF binding polarity determines chromatin looping.Mol. Cell. 2015; 60: 676-684Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar, 20Nanni L. Ceri S. Logie C. Spatial patterns of CTCF sites define the anatomy of TADs and their boundaries.Genome Biol. 2020; 21: 197Crossref PubMed Scopus (11) Google Scholar). Although many report that CBEs at TAD boundaries play key regulatory roles in biological development (21Zheng H. Xie W. The role of 3D genome organization in development and cell differentiation.Nat. Rev. Mol. Cell Biol. 2019; 20: 535-550Crossref PubMed Scopus (161) Google Scholar), analysis of CBE activity inside a TAD is lacking. Retinoic acid (RA) rapidly induces ESC differentiation (22Tay Y. Zhang J. Thomson A.M. Lim B. Rigoutsos I. MicroRNAs to Nanog, Oct4 and Sox2 coding regions modulate embryonic stem cell differentiation.Nature. 2008; 455: 1124-1128Crossref PubMed Scopus (1107) Google Scholar, 23Bain G. Kitchens D. Yao M. Huettner J.E. Gottlieb D.I. Embryonic stem cells express neuronal properties in vitro.Dev. Biol. 1995; 168: 342-357Crossref PubMed Scopus (988) Google Scholar) and activates HoxA expression (24Kashyap V. Gudas L.J. Brenet F. Funk P. Viale A. Scandura J.M. Epigenomic reorganization of the clustered Hox genes in embryonic stem cells induced by retinoic acid.J. Biol. Chem. 2011; 286: 3250-3260Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 25De Kumar B. Parrish M.E. Slaughter B.D. Unruh J.R. Gogol M. Seidel C. Paulson A. Li H. Gaudenz K. Peak A. McDowell W. Fleharty B. Ahn Y. Lin C. Smith E. et al.Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells.Genome Res. 2015; 25: 1229-1243Crossref PubMed Scopus (45) Google Scholar). Recent studies show that CBE activity functions in tumorigenesis (26Luo H. Wang F. Zha J. Li H. Yan B. Du Q. Yang F. Sobh A. Vulpe C. Drusbosky L. Cogle C. Chepelev I. Xu B. Nimer S.D. Licht J. et al.CTCF boundary remodels chromatin domain and drives aberrant HOX gene transcription in acute myeloid leukemia.Blood. 2018; 132: 837-848Crossref PubMed Scopus (35) Google Scholar, 27Li Y. Liao Z. Luo H. Benyoucef A. Kang Y. Lai Q. Dovat S. Miller B. Chepelev I. Li Y. Zhao K. Brand M. Huang S. Alteration of CTCF-associated chromatin neighborhood inhibits TAL1-driven oncogenic transcription program and leukemogenesis.Nucleic Acids Res. 2020; 48: 3119-3133Crossref PubMed Scopus (8) Google Scholar), immunogenesis (28Jain S. Ba Z. Zhang Y. Dai H.Q. Alt F.W. CTCF-binding elements mediate accessibility of RAG substrates during chromatin scanning.Cell. 2018; 174: 102-116.e114Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 29Ba Z. Lou J. Ye A.Y. Dai H.-Q. Dring E.W. Lin S.G. Jain S. Kyritsis N. Kieffer-Kwon K.-R. Casellas R. Alt F.W. CTCF orchestrates long-range cohesin-driven V(D)J recombinational scanning.Nature. 2020; 586: 305-310Crossref PubMed Scopus (27) Google Scholar), and stem cell development (21Zheng H. Xie W. The role of 3D genome organization in development and cell differentiation.Nat. Rev. Mol. Cell Biol. 2019; 20: 535-550Crossref PubMed Scopus (161) Google Scholar, 30Arzate-Mejia R.G. Recillas-Targa F. Corces V.G. Developing in 3D: The role of CTCF in cell differentiation.Development. 2018; 145dev137729Crossref PubMed Scopus (60) Google Scholar) by controlling target gene expression through organizing high-order chromatin structures. We and others have found that multiple enhancers are required for RA-induced HoxA expression and early ESC differentiation via long-range chromatin interactions with HoxA genes (31Su G. Guo D. Chen J. Liu M. Zheng J. Wang W. Zhao X. Yin Q. Zhang L. Zhao Z. Shi J. Lu W. A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.Nucleic Acids Res. 2019; 47: 6737-6752Crossref PubMed Scopus (10) Google Scholar, 32Yin Y. Yan P. Lu J. Song G. Zhu Y. Li Z. Zhao Y. Shen B. Huang X. Zhu H. Orkin S.H. Shen X. Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.Cell Stem Cell. 2015; 16: 504-516Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 33Cao K. Collings C.K. Marshall S.A. Morgan M.A. Rendleman E.J. Wang L. Sze C.C. Sun T. Bartom E.T. Shilatifard A. SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.Genes Dev. 2017; 31: 787-801Crossref PubMed Scopus (29) Google Scholar, 34Liu G.Y. Zhao G.N. Chen X.F. Hao D.L. Zhao X. Lv X. Liu D.P. The long noncoding RNA Gm15055 represses Hoxa gene expression by recruiting PRC2 to the gene cluster.Nucleic Acids Res. 2016; 44: 2613-2627Crossref PubMed Scopus (32) Google Scholar). Previous studies have also reported many regulatory patterns for HoxA activation during ESC differentiation; for example, several CBEs (also known as CBS or CTCF-binding sites) within the HoxA locus can organize HoxA chromatin structures and play significant regulatory roles in ESC differentiation and leukemogenesis (26Luo H. Wang F. Zha J. Li H. Yan B. Du Q. Yang F. Sobh A. Vulpe C. Drusbosky L. Cogle C. Chepelev I. Xu B. Nimer S.D. Licht J. et al.CTCF boundary remodels chromatin domain and drives aberrant HOX gene transcription in acute myeloid leukemia.Blood. 2018; 132: 837-848Crossref PubMed Scopus (35) Google Scholar, 35Ghasemi R. Struthers H. Wilson E.R. Spencer D.H. Contribution of CTCF binding to transcriptional activity at the HOXA locus in NPM1-mutant AML cells.Leukemia. 2021; 35: 404-416Crossref PubMed Scopus (8) Google Scholar, 36Narendra V. Rocha P.P. An D. Raviram R. Skok J.A. Mazzoni E.O. Reinberg D. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.Science. 2015; 347: 1017-1021Crossref PubMed Scopus (330) Google Scholar). However, it is unknown whether CBEs function to regulate HoxA expression by orchestrating enhancer chromatin structures during RA-induced early ESC differentiation. Here, we identified a novel functional CBE+47 required for RA to activate HoxA expression and promote early ESC differentiation. Relevant to mechanism, we report that CBE+47 regulates HoxA expression by organizing precise chromatin interactions between its adjacent enhancers and HoxA. Our study increases understanding of mechanisms underlying RA-induced HoxA activation and early ESC differentiation and highlights the unique and precise regulatory roles of CBE in high-order chromatin structure. TADs are basic structural units in high-order chromatin, and DNA functional elements such as enhancers or insulators in the same TAD likely regulate expression of neighboring genes (15Lupianez D.G. Kraft K. Heinrich V. Krawitz P. Brancati F. Klopocki E. Horn D. Kayserili H. Opitz J.M. Laxova R. Santos-Simarro F. Gilbert-Dussardier B. Wittler L. Borschiwer M. Haas S.A. et al.Disruptions of topological chromatin domains cause pathogenic rewiring of gene-enhancer interactions.Cell. 2015; 161: 1012-1025Abstract Full Text Full Text PDF PubMed Scopus (1023) Google Scholar, 16Ji X. Dadon D.B. Powell B.E. Fan Z.P. Borges-Rivera D. Shachar S. Weintraub A.S. Hnisz D. Pegoraro G. Lee T.I. Misteli T. Jaenisch R. Young R.A. 3D chromosome regulatory landscape of human pluripotent cells.Cell Stem Cell. 2016; 18: 262-275Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). To identify functional elements that regulate HoxA expression, we first analyzed Hi-C data in ESC. We found that the HoxA 5'-end was located at a TAD boundary region, while the 3'-end was within the TAD (Fig. 1A). Others had shown that CBEs located at the HoxA locus regulate HoxA expression and ESC differentiation (36Narendra V. Rocha P.P. An D. Raviram R. Skok J.A. Mazzoni E.O. Reinberg D. CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation.Science. 2015; 347: 1017-1021Crossref PubMed Scopus (330) Google Scholar, 37Rousseau M. Crutchley J.L. Miura H. Suderman M. Blanchette M. Dostie J. Hox in motion: Tracking HoxA cluster conformation during differentiation.Nucleic Acids Res. 2014; 42: 1524-1540Crossref PubMed Scopus (34) Google Scholar). Interestingly, we identified many significant CBEs in the same TAD at the HoxA 3'-end (Fig. 1B), but their function was not previously characterized. Here, we focused on a significant CBE closest to the HoxA 3'-end, ∼47 kb from HoxA and located in the second intron of Halr1; we designate that element CBE+47 (Fig. 1B and Fig. S1). We observed significant binding of MED1, MED12, and YY1 upstream of CBE+47 (Fig. 1B) as well as enrichment for the epigenetic modification-related markers H3K27ac and H3K4me1/2/3 (Fig. 1C). We also observed significant chromatin accessibility (based on DNase and ATAC-seq analysis) and transcriptional activity marked by PolⅡenrichment in these regions (Fig. 1D). These data indicate that CBE+47 is located between HoxA and potential regulatory elements. Others had shown that CBEs regulate target gene expression by organizing chromatin interactions between a functional element and a target gene (38Hark A.T. Schoenherr C.J. Katz D.J. Ingram R.S. Levorse J.M. Tilghman S.M. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus.Nature. 2000; 405: 486-489Crossref PubMed Scopus (1189) Google Scholar, 39Kurukuti S. Tiwari V.K. Tavoosidana G. Pugacheva E. Murrell A. Zhao Z. Lobanenkov V. Reik W. Ohlsson R. CTCF binding at the H19 imprinting control region mediates maternally inherited higher-order chromatin conformation to restrict enhancer access to Igf2.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 10684-10689Crossref PubMed Scopus (382) Google Scholar). Therefore, we postulated that CBE+47 may play a regulatory role. To assess CBE+47 function, we employed CRISPR-Cas9 knockout methodology (31Su G. Guo D. Chen J. Liu M. Zheng J. Wang W. Zhao X. Yin Q. Zhang L. Zhao Z. Shi J. Lu W. A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.Nucleic Acids Res. 2019; 47: 6737-6752Crossref PubMed Scopus (10) Google Scholar). Specifically, we designed two sgRNAs, upstream and downstream of CBE+47 (Fig. 2A) and after Cas9 cleavage and DNA recombination, obtained two homozygous CBE+47 knockout lines (CBE+47-KO). PCR of genomic DNA using specific primers and Sanger sequencing confirmed CBE+47 deletion (Fig. 2B). AP staining of CBE+47 knockout lines revealed no significant differences in cell morphology relative to WT cells in self-renewing culture conditions (Fig. 2C). Moreover, expression of Halr1 and Sakp2 mRNAs was comparable in WT and KO lines, based on qRT-PCR analysis (Fig. 2D). However, most of the 3'-end genes of HoxA, including Hoxa2-a6, were significantly upregulated, while the TAD boundary gene Hoxa9 was significantly downregulated in CBE+47-KO relative to WT cells (Fig. 2E). These data show that CBE+47 deletion significantly changes HoxA expression and indicate that even in an undifferentiated ESC state, CBE+47 is required to maintain proper HoxA expression. Given the critical function of HoxA in ESC differentiation (8Martinez-Ceballos E. Chambon P. Gudas L.J. Differences in gene expression between wild type and Hoxa1 knockout embryonic stem cells after retinoic acid treatment or leukemia inhibitory factor (LIF) removal.J. Biol. Chem. 2005; 280: 16484-16498Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 32Yin Y. Yan P. Lu J. Song G. Zhu Y. Li Z. Zhao Y. Shen B. Huang X. Zhu H. Orkin S.H. Shen X. Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.Cell Stem Cell. 2015; 16: 504-516Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 40De Kumar B. Parker H.J. Parrish M.E. Lange J.J. Slaughter B.D. Unruh J.R. Paulson A. Krumlauf R. Dynamic regulation of Nanog and stem cell-signaling pathways by Hoxa1 during early neuro-ectodermal differentiation of ES cells.Proc. Natl. Acad. Sci. U. S. A. 2017; 114: 5838-5845Crossref PubMed Scopus (34) Google Scholar) and the observation that CBE+47 deletion significantly increased expression of a subset of HoxA genes, we asked whether ESC pluripotency was perturbed by CBE+47 deletion by evaluating expression of pluripotency- and differentiation-related genes by qRT-PCR (Fig. 2, F–K). We observed no significant differences in expression of pluripotency-regulated genes in CBE+47-KO versus WT cells, except for slight upregulation of Sox2. However, mesoderm and trophectoderm genes were significantly upregulated in CBE+47-KO cells. Expression of other germ layer genes such as Otx2 (epiblast), Sox17 (endoderm), and Sox11 (ectoderm) also significantly increased in CBE+47-KO cells. These results indicate that CBE+47 deletion does not regulate ESC pluripotency but may alter its differentiation capacity. During early RA-induced differentiation of ESC, HoxA genes become significantly activated (25De Kumar B. Parrish M.E. Slaughter B.D. Unruh J.R. Gogol M. Seidel C. Paulson A. Li H. Gaudenz K. Peak A. McDowell W. Fleharty B. Ahn Y. Lin C. Smith E. et al.Analysis of dynamic changes in retinoid-induced transcription and epigenetic profiles of murine Hox clusters in ES cells.Genome Res. 2015; 25: 1229-1243Crossref PubMed Scopus (45) Google Scholar, 32Yin Y. Yan P. Lu J. Song G. Zhu Y. Li Z. Zhao Y. Shen B. Huang X. Zhu H. Orkin S.H. Shen X. Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.Cell Stem Cell. 2015; 16: 504-516Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) (Fig. S2). Therefore, we asked whether CBE+47 regulates those activities. To do so, we treated CBE+47-KO and WT ESC with RA for 12, 24, and 48 h (Fig. S3A) and then compared HoxA expression levels by qRT-PCR. HoxA 3'-end genes (Hoxa2-a6) showed significantly higher expression in CBE+47-KO cells, as did the TAD boundary genes Hoxa7 and Hoxa9. However, expression of HoxA 5'-end genes (Hoxa10, Hoxa11, and Hoxa13) was comparable in both genotypes (Fig. 3, A–C). Also, antisense long noncoding RNAs (such as Hoxaas3) at the HoxA locus were significantly upregulated in CBE+47-KO relative to WT cells (Fig. S3, B–D). These data suggest overall that CBE+47 functions to restrict RA-induced overexpression of HoxA. To assess whether CBE+47 knockout alters ESC differentiation, we used qRT-PCR to analyze potential changes in pluripotency- or differentiation-related master control genes in WT and CBE+47-KO ESC under RA induction conditions. Expression of pluripotency genes (Nanog and Klf4) did not change significantly at 12 h after ESC differentiation but significantly decreased in CBE+47-KO relative to WT cells by 24 and 48 h. The epiblast master control gene Otx2, endoderm master control gene Gata4, and ectoderm master control gene Pax6 showed significantly higher expression levels in CBE+47-KO versus WT cells, while the mesoderm master gene T and the trophectoderm master gene Hand1 were significantly downregulated (Fig. 3, D and E). These findings suggest overall that CBE+47 is essential to maintain RA-induced HoxA expression and proper early ESC differentiation. To further assess effects of CBE+47 loss on early (24 h) RA-induced ESC differentiation, we performed transcriptome analysis using RNA-seq after RA-induced differentiation of WT and CBE+47-KO cells. Relative to WT cells, statistical analysis of CBE+47-KO cells revealed 869 genes upregulated and 360 downregulated (fold-change ≥ 2, p < 0.05) (Fig. 4A). Among them, the most significantly upregulated genes were at the 3'-end of HoxA (Fig. 4B). Because these genes located in the same TAD with CBE+47, we hypothesized that CBE+47 deletion significantly promotes gene expression restricted to that TAD. These findings reveal that CBE+47 serves as a cis-regulatory element playing a local regulatory role (Fig. S4). Moreover, relative to WT cells, CBE+47 KO cells expressed significantly higher levels of HoxB/C/D cluster genes (Fig. S5A). Genes relevant to RA signaling, such as Crabp2, Cyp26a1, and Stra6, were also significantly overexpressed in CBE+47 KO cells, as were the differentiation-related genes Cbx4 and Foxa1 (Fig. S5B). By contrast, pluripotency genes such as Myc, Lin28a, and Etv4 were significantly suppressed in CBE+47 KO relative to WT cells (Fig. S5C). These data suggest that in normal cells CBE+47 functions to enable proper differentiation. To assess mechanisms underlying these activities, we performed bioinformatic analysis of up- and downregulated genes in CBE+47 KO cells (Fig. S6). GO analysis indicated that biological processes (BP) relevant to upregulated genes include gland development and positive regulation of nervous system development (Fig. 4C). GO-BPs of downregulated genes mainly involved embryonic placenta development, positive regulation of vasoconstriction, and epithelial cell differentiation (Fig. 4D). KEGG pathway enrichment analysis revealed upregulated genes to be enriched in pathways including cGMP-PKG signaling, neuroactive ligand–receptor interaction, axon guidance, stem cell pluripotency, Hippo signaling, cAMP signaling, Wnt and Hedgehog signaling (Fig. S7A). Enrichment results relevant to downregulated genes revealed pathways related to proteoglycans in cancer, transcriptional dysregulation in cancer, arachidonic acid metabolism, HTLV-I infection, Hippo signaling, and thyroid hormone signaling (Fig. S7A). These findings, which were further validated by Gene Set Enrichment Analysis (GSEA) (Fig. 4E and Fig. S7B), suggest that CBE+47 deletion promotes abnormal gene expression and perturbs proper RA-induced early ESC differentiation. Previous studies report that CTCF regulates target gene expression by controlling chromatin interaction between enhancers and target genes and that this activity occurs within a TAD (41Zhao H. Li Z. Zhu Y. Bian S. Zhang Y. Qin L. Naik A.K. He J. Zhang Z. Krangel M.S. Hao B. A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra–Tcrd locus.Nucleic Acids Res. 2020; 48: 9621-9636Crossref PubMed Scopus (5) Google Scholar). Studies by us and others have identified three enhancers (E1, E2, and E3) at the HoxA 3'-end (Fig. 5A) required for HoxA expression following RA-induced ESC differentiation (31Su G. Guo D. Chen J. Liu M. Zheng J. Wang W. Zhao X. Yin Q. Zhang L. Zhao Z. Shi J. Lu W. A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.Nucleic Acids Res. 2019; 47: 6737-6752Crossref PubMed Scopus (10) Google Scholar, 32Yin Y. Yan P. Lu J. Song G. Zhu Y. Li Z. Zhao Y. Shen B. Huang X. Zhu H. Orkin S.H. Shen X. Opposing roles for the lncRNA haunt and its genomic locus in regulating HOXA gene activation during embryonic stem cell differentiation.Cell Stem Cell. 2015; 16: 504-516Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 33Cao K. Collings C.K. Marshall S.A. Morgan M.A. Rendleman E.J. Wang L. Sze C.C. Sun T. Bartom E.T. Shilatifard A. SET1A/COMPASS and shadow enhancers in the regulation of homeotic gene expression.Genes Dev. 2017; 31: 787-801Crossref PubMed Scopus (29) Google Scholar, 34Liu G.Y. Zhao G.N. Chen X.F. Hao D.L. Zhao X. Lv X. Liu D.P. The long noncoding RNA Gm15055 represses Hoxa gene expression by recruiting PRC2 to the gene cluster.Nucleic Acids Res. 2016; 44: 2613-2627Crossref PubMed Scopus (32) Google Scholar). Among them, E1 and E3 reportedly mediate significant long-range interactions with HoxA chromatin. Remarkably, CBE+47 is located between those three enhancers and HoxA (Fig. 5A). Therefore, we speculate that the CBE+47 may be involved in organizing the interactions between the three enhancers and HoxA chromatin. Next we used Capture-C methodology, which is widely used to define chromatin interactions between enhancers and target genes (31Su G. Guo D. Chen J. Liu M. Zheng J. Wang W. Zhao X. Yin Q. Zhang L. Zhao Z. Shi J. Lu W. A distal enhancer maintaining Hoxa1 expression orchestrates retinoic acid-induced early ESCs differentiation.Nucleic Acids Res. 2019; 47: 6737-6752Crossref PubMed Scopus (10) Google Scholar, 42Hughes J.R. Roberts N. McGowan S. Hay D. Giannoulatou E. Lynch M. De Gobbi M. Taylor S. Gibbons R. Higgs D.R. Analysis of hundreds of cis-regulatory landscapes at high resolution in a single, high-throughput experiment.Nat. Genet. 2014; 46: 205-212Crossref PubMed Scopus (289) Google Scholar,

Referência(s)