FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2051875167> ?p ?o ?g. }

• W2051875167 endingPage "722" @default.
• W2051875167 startingPage "708" @default.
• W2051875167 abstract "•NF-Y uses distinct modes to regulate housekeeping and cell-identity programs•NF-Y co-occupies active enhancers with cell type-specific master TFs•NF-Y promotes master TF binding by facilitating a favorable chromatin conformation•NF-Y is required for the maintenance of ESC identity Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that the ubiquitous CCAAT-binding NF-Y complex is required for the maintenance of embryonic stem cell (ESC) identity and is an essential component of the core pluripotency network. Genome-wide studies in ESCs and neurons reveal that NF-Y regulates not only genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. Mechanistically, NF-Y’s distinct DNA-binding mode promotes master/pioneer TF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a conceptually unique function for histone-fold domain (HFD) protein NF-Y in promoting chromatin accessibility and suggest that other HFD proteins with analogous structural and DNA-binding properties may function in similar ways. Cell type-specific master transcription factors (TFs) play vital roles in defining cell identity and function. However, the roles ubiquitous factors play in the specification of cell identity remain underappreciated. Here we show that the ubiquitous CCAAT-binding NF-Y complex is required for the maintenance of embryonic stem cell (ESC) identity and is an essential component of the core pluripotency network. Genome-wide studies in ESCs and neurons reveal that NF-Y regulates not only genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. Mechanistically, NF-Y’s distinct DNA-binding mode promotes master/pioneer TF binding at enhancers by facilitating a permissive chromatin conformation. Our studies unearth a conceptually unique function for histone-fold domain (HFD) protein NF-Y in promoting chromatin accessibility and suggest that other HFD proteins with analogous structural and DNA-binding properties may function in similar ways. Master transcription factors (TFs) establish and/or maintain cellular identity by orchestrating distinct profiles of gene expression that are faithfully transmitted through cell division. The simplified paradigm for master TFs relies on the premise that their expression is spatiotemporally restricted to one or few cell types or lineages that depend on their activity (Oestreich and Weinmann, 2012Oestreich K.J. Weinmann A.S. Master regulators or lineage-specifying? Changing views on CD4+ T cell transcription factors.Nat. Rev. Immunol. 2012; 12: 799-804Crossref PubMed Scopus (105) Google Scholar). This assumption may have unintentionally led to the underappreciation of the roles “housekeeping” or other essential TFs, expressed ubiquitously or in a multitude of cell types, might play in cell specification. Recent studies, however, have shown that many ubiquitous factors, previously thought to have an exclusive housekeeping function, have additional cell type-specific roles (Chia et al., 2010Chia N.Y. Chan Y.S. Feng B. Lu X. Orlov Y.L. Moreau D. Kumar P. Yang L. Jiang J. Lau M.S. et al.A genome-wide RNAi screen reveals determinants of human embryonic stem cell identity.Nature. 2010; 468: 316-320Crossref PubMed Scopus (364) Google Scholar, Cinghu et al., 2014Cinghu S. Yellaboina S. Freudenberg J.M. Ghosh S. Zheng X. Oldfield A.J. Lackford B.L. Zaykin D.V. Hu G. Jothi R. Integrative framework for identification of key cell identity genes uncovers determinants of ES cell identity and homeostasis.Proc. Natl. Acad. Sci. USA. 2014; 111: E1581-E1590Crossref PubMed Scopus (19) Google Scholar, Golan-Mashiach et al., 2012Golan-Mashiach M. Grunspan M. Emmanuel R. Gibbs-Bar L. Dikstein R. Shapiro E. Identification of CTCF as a master regulator of the clustered protocadherin genes.Nucleic Acids Res. 2012; 40: 3378-3391Crossref PubMed Scopus (50) Google Scholar, Kagey et al., 2010Kagey M.H. Newman J.J. Bilodeau S. Zhan Y. Orlando D.A. van Berkum N.L. Ebmeier C.C. Goossens J. Rahl P.B. Levine S.S. et al.Mediator and cohesin connect gene expression and chromatin architecture.Nature. 2010; 467: 430-435Crossref PubMed Scopus (1383) Google Scholar, Kim et al., 2010aKim J. Woo A.J. Chu J. Snow J.W. Fujiwara Y. Kim C.G. Cantor A.B. Orkin S.H. A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs.Cell. 2010; 143: 313-324Abstract Full Text Full Text PDF PubMed Scopus (518) Google Scholar, Pijnappel et al., 2013Pijnappel W.W. Esch D. Baltissen M.P. Wu G. Mischerikow N. Bergsma A.J. van der Wal E. Han D.W. Bruch Hv. Moritz S. et al.A central role for TFIID in the pluripotent transcription circuitry.Nature. 2013; 495: 516-519Crossref PubMed Scopus (60) Google Scholar). This emerging body of evidence indicates that we are yet to fully appreciate the significance of ubiquitously expressed proteins in many settings, and argues for a comprehensive reassessment of the roles other housekeeping proteins may play in cell specification. NF-Y, also known as the CCAAT-binding factor CBF, is a ubiquitously expressed heterotrimeric TF composed of NF-YA, NF-YB, and NF-YC subunits, all of which are conserved from yeast to human (Maity and de Crombrugghe, 1998Maity S.N. de Crombrugghe B. Role of the CCAAT-binding protein CBF/NF-Y in transcription.Trends Biochem. Sci. 1998; 23: 174-178Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). NF-Y binds to the CCAAT box, which occurs at ∼30% of all eukaryotic promoters (Dolfini et al., 2012aDolfini D. Gatta R. Mantovani R. NF-Y and the transcriptional activation of CCAAT promoters.Crit. Rev. Biochem. Mol. Biol. 2012; 47: 29-49Crossref PubMed Scopus (169) Google Scholar). NF-YB and NF-YC dimerize via their histone-fold domains (HFDs) before associating with NF-YA (Romier et al., 2003Romier C. Cocchiarella F. Mantovani R. Moras D. The NF-YB/NF-YC structure gives insight into DNA binding and transcription regulation by CCAAT factor NF-Y.J. Biol. Chem. 2003; 278: 1336-1345Crossref PubMed Scopus (211) Google Scholar), which harbors both DNA-binding and transactivation domains. The crystal structure of NF-Y bound to DNA shows that while NF-YA makes sequence-specific DNA contacts, NF-YB/NF-YC interacts with DNA via nonspecific HFD-DNA contacts (Nardini et al., 2013Nardini M. Gnesutta N. Donati G. Gatta R. Forni C. Fossati A. Vonrhein C. Moras D. Romier C. Bolognesi M. Mantovani R. Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination.Cell. 2013; 152: 132-143Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The key structural feature of the NF-Y/DNA complex is the minor-groove interaction of NF-YA, which induces an ∼80° bend in the DNA. The structure and DNA-binding mode of NF-YB/NF-YC HFDs are similar to those of the core histones H2A/H2B, TATA-binding protein (TBP)-associated factors (TAFs), the TBP/TATA-binding negative cofactor 2 (NC2α/β), and the CHRAC15/CHRAC17 subunits of the nucleosome remodeling complex CHRAC (Nardini et al., 2013Nardini M. Gnesutta N. Donati G. Gatta R. Forni C. Fossati A. Vonrhein C. Moras D. Romier C. Bolognesi M. Mantovani R. Sequence-specific transcription factor NF-Y displays histone-like DNA binding and H2B-like ubiquitination.Cell. 2013; 152: 132-143Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Yet, unlike H2A/H2B, which lack sequence specificity, NF-YB/NF-YC interaction with NF-YA provides the NF-Y complex with sequence-specific targeting capability as well as nucleosome-like properties of nonspecific DNA binding, a combination that allows for stable DNA binding. NF-Y, largely described as a transcription activator via its promoter-proximal binding, is a key regulator of cell-cycle progression in proliferating cells (Benatti et al., 2011Benatti P. Dolfini D. Viganò A. Ravo M. Weisz A. Imbriano C. Specific inhibition of NF-Y subunits triggers different cell proliferation defects.Nucleic Acids Res. 2011; 39: 5356-5368Crossref PubMed Scopus (58) Google Scholar, Bungartz et al., 2012Bungartz G. Land H. Scadden D.T. Emerson S.G. NF-Y is necessary for hematopoietic stem cell proliferation and survival.Blood. 2012; 119: 1380-1389Crossref PubMed Scopus (52) Google Scholar, Hu and Maity, 2000Hu Q. Maity S.N. Stable expression of a dominant negative mutant of CCAAT binding factor/NF-Y in mouse fibroblast cells resulting in retardation of cell growth and inhibition of transcription of various cellular genes.J. Biol. Chem. 2000; 275: 4435-4444Crossref PubMed Scopus (62) Google Scholar), with its activity often downregulated during cellular differentiation and senescence (Bungartz et al., 2012Bungartz G. Land H. Scadden D.T. Emerson S.G. NF-Y is necessary for hematopoietic stem cell proliferation and survival.Blood. 2012; 119: 1380-1389Crossref PubMed Scopus (52) Google Scholar, Farina et al., 1999Farina A. Manni I. Fontemaggi G. Tiainen M. Cenciarelli C. Bellorini M. Mantovani R. Sacchi A. Piaggio G. Down-regulation of cyclin B1 gene transcription in terminally differentiated skeletal muscle cells is associated with loss of functional CCAAT-binding NF-Y complex.Oncogene. 1999; 18: 2818-2827Crossref PubMed Scopus (96) Google Scholar). In addition to binding core promoters, NF-Y has also been shown to bind enhancer elements away from transcription start sites (TSSs) (Fleming et al., 2013Fleming J.D. Pavesi G. Benatti P. Imbriano C. Mantovani R. Struhl K. NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors.Genome Res. 2013; 23: 1195-1209Crossref PubMed Scopus (99) Google Scholar, Testa et al., 2005Testa A. Donati G. Yan P. Romani F. Huang T.H. Viganò M.A. Mantovani R. Chromatin immunoprecipitation (ChIP) on chip experiments uncover a widespread distribution of NF-Y binding CCAAT sites outside of core promoters.J. Biol. Chem. 2005; 280: 13606-13615Crossref PubMed Scopus (70) Google Scholar), but its function and mechanism of action at these distal regulatory elements remain to be elucidated. Consistent with its role in cell-cycle regulation, NF-Y is required for ESC and hematopoietic stem cell proliferation (Bungartz et al., 2012Bungartz G. Land H. Scadden D.T. Emerson S.G. NF-Y is necessary for hematopoietic stem cell proliferation and survival.Blood. 2012; 119: 1380-1389Crossref PubMed Scopus (52) Google Scholar, Dolfini et al., 2012bDolfini D. Minuzzo M. Pavesi G. Mantovani R. The short isoform of NF-YA belongs to the embryonic stem cell transcription factor circuitry.Stem Cells. 2012; 30: 2450-2459Crossref PubMed Scopus (38) Google Scholar, Grskovic et al., 2007Grskovic M. Chaivorapol C. Gaspar-Maia A. Li H. Ramalho-Santos M. Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.PLoS Genet. 2007; 3: e145Crossref PubMed Scopus (78) Google Scholar). While NF-YA heterozygous mice are normal and fertile, NF-YA null mice die prior to 8.5 dpc (Bhattacharya et al., 2003Bhattacharya A. Deng J.M. Zhang Z. Behringer R. de Crombrugghe B. Maity S.N. The B subunit of the CCAAT box binding transcription factor complex (CBF/NF-Y) is essential for early mouse development and cell proliferation.Cancer Res. 2003; 63: 8167-8172PubMed Google Scholar), suggesting an essential role for NF-Y in early mouse embryonic development. Interestingly, conditional deletion of NF-YA in postmitotic mouse neurons induces progressive neurodegeneration (Yamanaka et al., 2014Yamanaka T. Tosaki A. Kurosawa M. Matsumoto G. Koike M. Uchiyama Y. Maity S.N. Shimogori T. Hattori N. Nukina N. NF-Y inactivation causes atypical neurodegeneration characterized by ubiquitin and p62 accumulation and endoplasmic reticulum disorganization.Nat. Commun. 2014; 5: 3354Crossref PubMed Scopus (29) Google Scholar), which suggests a role for NF-Y that is independent of its role in cell-cycle regulation, as has also been shown in hepatocytes (Luo et al., 2011Luo R. Klumpp S.A. Finegold M.J. Maity S.N. Inactivation of CBF/NF-Y in postnatal liver causes hepatocellular degeneration, lipid deposition, and endoplasmic reticulum stress.Sci. Rep. 2011; 1: 136Crossref PubMed Scopus (22) Google Scholar). Given the relatively high expression of one or more NF-Y subunits in mouse oocytes (Su et al., 2004Su A.I. Wiltshire T. Batalov S. Lapp H. Ching K.A. Block D. Zhang J. Soden R. Hayakawa M. Kreiman G. et al.A gene atlas of the mouse and human protein-encoding transcriptomes.Proc. Natl. Acad. Sci. USA. 2004; 101: 6062-6067Crossref PubMed Scopus (2848) Google Scholar) and the inner cell mass (ICM) of the mouse blastocyst (Yoshikawa et al., 2006Yoshikawa T. Piao Y. Zhong J. Matoba R. Carter M.G. Wang Y. Goldberg I. Ko M.S. High-throughput screen for genes predominantly expressed in the ICM of mouse blastocysts by whole mount in situ hybridization.Gene Expr. Patterns. 2006; 6: 213-224Crossref PubMed Scopus (69) Google Scholar), we set out to determine NF-Y’s role in ESCs, derivatives of the ICM. Although NF-Y has been predominantly studied in the context of its promoter-proximal binding at key cell-cycle genes, given the prevalence of NF-Y targeting sites at greater distances from TSSs (Fleming et al., 2013Fleming J.D. Pavesi G. Benatti P. Imbriano C. Mantovani R. Struhl K. NF-Y coassociates with FOS at promoters, enhancers, repetitive elements, and inactive chromatin regions, and is stereo-positioned with growth-controlling transcription factors.Genome Res. 2013; 23: 1195-1209Crossref PubMed Scopus (99) Google Scholar), we investigated NF-Y’s function and mechanism of action at distal regulatory elements. We demonstrate a requirement for all three NF-Y subunits in the expression of core ESC self-renewal and pluripotency genes, and in the maintenance of ESC identity. Through genome-wide occupancy and transcriptomic analyses in ESCs and neurons, we show that not only does NF-Y regulate genes with housekeeping functions through cell type-invariant promoter-proximal binding, but also genes required for cell identity by binding to cell type-specific enhancers with master TFs. We present evidence that NF-Y’s distinct DNA-binding mode facilitates a permissive chromatin conformation and promotes enhanced binding of master ESC TFs at enhancers. Our studies unearth a function for NF-Y in promoting chromatin accessibility and specification of cell identity. To gain insight into NF-Y-mediated transcriptional regulation in early embryogenesis, we investigated the genome-wide occupancy of all three subunits of the NF-Y complex in mouse ESCs using chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-Seq). ChIP-Seq analyses revealed enrichment for NF-Y occupancy near TSSs of annotated genes (Figure 1A), consistent with NF-Y’s preference for binding and recruiting RNA polymerase II and general TFs to various CCAAT motif-containing promoters (Kabe et al., 2005Kabe Y. Yamada J. Uga H. Yamaguchi Y. Wada T. Handa H. NF-Y is essential for the recruitment of RNA polymerase II and inducible transcription of several CCAAT box-containing genes.Mol. Cell. Biol. 2005; 25: 512-522Crossref PubMed Scopus (71) Google Scholar). Binding of all three subunits to the promoters of known NF-Y targets including Cdc25c, Rnf5, and Zic3 (Grskovic et al., 2007Grskovic M. Chaivorapol C. Gaspar-Maia A. Li H. Ramalho-Santos M. Systematic identification of cis-regulatory sequences active in mouse and human embryonic stem cells.PLoS Genet. 2007; 3: e145Crossref PubMed Scopus (78) Google Scholar, Yamanaka et al., 2014Yamanaka T. Tosaki A. Kurosawa M. Matsumoto G. Koike M. Uchiyama Y. Maity S.N. Shimogori T. Hattori N. Nukina N. NF-Y inactivation causes atypical neurodegeneration characterized by ubiquitin and p62 accumulation and endoplasmic reticulum disorganization.Nat. Commun. 2014; 5: 3354Crossref PubMed Scopus (29) Google Scholar) attested to the sensitivity of the ChIP-Seq data (Figure 1B). ChIP followed by quantitative polymerase chain reaction (qPCR) analysis of certain sites, either previously demonstrated as NF-Y-bound regions (Cdc25c, Rnf5, Zic3) or highly enriched for NF-Y binding (Khsrp), further validated the quality and the reproducibility of the ChIP-Seq data (Figure 1C). Analysis of ChIP-Seq data sets using the SISSRs peak-calling algorithm (Jothi et al., 2008Jothi R. Cuddapah S. Barski A. Cui K. Zhao K. Genome-wide identification of in vivo protein-DNA binding sites from ChIP-Seq data.Nucleic Acids Res. 2008; 36: 5221-5231Crossref PubMed Scopus (445) Google Scholar, Narlikar and Jothi, 2012Narlikar L. Jothi R. ChIP-Seq data analysis: identification of protein-DNA binding sites with SISSRs peak-finder.Methods Mol. Biol. 2012; 802: 305-322Crossref PubMed Scopus (33) Google Scholar) identified a total of 4,642, 2,774, and 2,675 binding sites (peaks) for NF-YA, NF-YB, and NF-YC, respectively, with at least 7-fold or more ChIP over input enrichment (p < 10−3). As expected, de novo sequence motif analysis identified the known RRCCAATVR consensus motif within the binding sites for all three subunits (Figure 1D). Although a vast majority of the binding sites for NF-YA, NF-YB, or NF-YC overlap with those for one or both of the other two subunits (Figure 1E), about one-third of the NF-YA binding sites did not show statistically significant evidence for the cobinding of the other two subunits. To determine whether NF-YA alone binds certain genomic loci, we knocked down NF-YA or NF-YC using siRNAs for 48 hr (see Figures S1A and S1B available online) and analyzed their binding patterns at sites bound by all three NF-Y subunits (Figure 1B) as well as those bound only by NF-YA (Figure S1C). ChIP-qPCR analyses show that upon NF-YC knockdown (KD), NF-YA occupancy on DNA is compromised even at sites that seem to bind only NF-YA (Figure S1D), indicating that NF-YC is essential for NF-YA binding. Low NF-YB and NF-YC occupancy at sites defined as bound only by NF-YA compared to those bound by all three subunits (Figure S1E) suggest that the failure to call peaks for NF-YB/NF-YC at these sites is likely due to weaker ChIP-Seq signals that did not pass the statistical threshold for peak calling, and/or less immunoefficient antibodies for NF-YB/NF-YC compared to that of NF-YA. Together with the fact that nearly all of the NF-YB-only and NF-YC-only sites contain the CCAAT motif, we conclude that NF-Y binding requires all three subunits. Although we cannot completely rule out that a very low and/or transient occupancy of NF-YA alone or NF-YB/NF-YC heterodimer may exist, our data show that such events are highly unlikely if not sparse, consistent with in vitro biochemical studies showing NF-Y binding requiring all three subunits (Sinha et al., 1995Sinha S. Maity S.N. Lu J. de Crombrugghe B. Recombinant rat CBF-C, the third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-A and CBF-B and with yeast HAP2 and HAP3.Proc. Natl. Acad. Sci. USA. 1995; 92: 1624-1628Crossref PubMed Scopus (251) Google Scholar). Therefore, hereafter, we will refer to the union of all NF-YA, NF-YB, and NF-YC binding sites (5,359 in total) as NF-Y binding sites. Relative to annotated RefSeq genes, a vast majority of all NF-Y binding sites are located near genic regions (Figure 1F), suggesting a role for NF-Y in regulation of gene expression. Indeed, the levels of NF-Y binding at gene promoters correlate positively with gene expression (Figure 1G), which is consistent with NF-Y’s established role as a transcription activator (Dolfini et al., 2012aDolfini D. Gatta R. Mantovani R. NF-Y and the transcriptional activation of CCAAT promoters.Crit. Rev. Biochem. Mol. Biol. 2012; 47: 29-49Crossref PubMed Scopus (169) Google Scholar). NF-Y has predominantly been reported to bind in close proximity to TSSs. While we confirm a strong enrichment for NF-Y binding sites around TSSs (within 500 bp of TSSs), with a clear preference for binding to the region immediately upstream of the TSS (Figure 2A), nearly half of all NF-Y binding sites are located at distances greater than 500 bp from the TSSs (Figure 2B). These data suggest a role for NF-Y in ESC transcription regulation by binding to sites distal to TSSs. To investigate the role distal sites may play in the regulation of gene expression, and how they might differ in function compared to that of promoter-proximal NF-Y sites, we examined published ChIP-Seq data sets for histone modifications, DNase I hypersensitivity, Hi-C interaction profiles, 14 different TFs, and other genomic features. As one would expect from their proximity to TSSs, nearly 80% of all promoter-proximal NF-Y sites overlap with CpG islands (Figure S1F), consistent with ∼85% of all promoters overlapping CpG islands. In contrast, only about one-tenth of distal NF-Y sites overlap with CpG islands (Figure S1F), a fraction similar to that observed for enhancers. Furthermore, ChromHMM annotation of chromatin states (Ernst and Kellis, 2012Ernst J. Kellis M. ChromHMM: automating chromatin-state discovery and characterization.Nat. Methods. 2012; 9: 215-216Crossref PubMed Scopus (1325) Google Scholar) shows a majority of distal NF-Y sites with chromatin features reminiscent of enhancers (Figure S1G). Chromatin marks at distal NF-Y sites revealed open chromatin architecture marked by high DNase I activity, accompanied by enhancer marks H3K4me1 and H3K27ac but not the promoter mark H3K4me3 (Figure 2C). Additionally, enrichment of Hi-C interaction density at distal but not proximal NF-Y strongly supports the notion that distal NF-Y sites are within enhancers (Figure 2D). To gain insight into the mechanism underlying NF-Y’s function at enhancers, we studied the frequency of known TF binding motifs within distal and proximal NF-Y sites. We found that distal NF-Y sites are significantly enriched for sequence motifs bound by master ESC TFs known to bind distal enhancers, including Oct4 and Sox2, but not for motifs bound by ubiquitous TFs, such as CTCF (Figure 2E). In contrast, promoter-proximal NF-Y sites are significantly enriched for sequence motifs bound by Klf4, cMyc, and Zfx, all known to preferentially bind to promoters (Figure 2E) (Chen et al., 2008Chen X. Xu H. Yuan P. Fang F. Huss M. Vega V.B. Wong E. Orlov Y.L. Zhang W. Jiang J. et al.Integration of external signaling pathways with the core transcriptional network in embryonic stem cells.Cell. 2008; 133: 1106-1117Abstract Full Text Full Text PDF PubMed Scopus (1940) Google Scholar). Furthermore, analysis of NF-Y binding in conjunction with other TF binding data revealed an extensive colocalization of NF-Y with ESC-specific master TFs Oct4, Sox2, Nanog, and Esrrb at distal NF-Y sites, and with TFs Zfx, cMyc, and Klf4 at promoter-proximal NF-Y sites (Figure 2F). Consistent with NF-Y’s co-occupancy with master ESC TFs at distal NF-Y sites, gene ontology analysis revealed enrichment for genes with roles in early embryonic development among those targeted by distal NF-Y sites (Figure 2G). Interestingly, genes with NF-Y sites at proximal promoters are enriched for housekeeping functions including cell cycle and proliferation. Taken together, these data suggest a potential role for NF-Y in the regulation of the core pluripotency transcription network through distal enhancer binding. Genes targeted by NF-Y include many key pluripotency-associated genes that have been implicated in the control of ESC identity (Figure 3A). These include genes encoding the master ESC TFs Oct4, Nanog, Prdm14, Esrrb, Tcl1, Tbx3, Rex1, Lefty1, and Lefty2. More importantly, in a majority of cases, NF-Y colocalizes with master ESC TFs at distal enhancers previously demonstrated (Kagey et al., 2010Kagey M.H. Newman J.J. Bilodeau S. Zhan Y. Orlando D.A. van Berkum N.L. Ebmeier C.C. Goossens J. Rahl P.B. Levine S.S. et al.Mediator and cohesin connect gene expression and chromatin architecture.Nature. 2010; 467: 430-435Crossref PubMed Scopus (1383) Google Scholar, Zhang et al., 2013Zhang Y. Wong C.H. Birnbaum R.Y. Li G. Favaro R. Ngan C.Y. Lim J. Tai E. Poh H.M. Wong E. et al.Chromatin connectivity maps reveal dynamic promoter-enhancer long-range associations.Nature. 2013; 504: 306-310Crossref PubMed Scopus (314) Google Scholar) to interact with promoters of these ESC identity genes (Figures 3A and S2A). This suggests that NF-Y might be regulating these genes via enhancer-promoter interactions. In addition to its binding at the enhancers of self-renewal and pluripotency genes, NF-Y binds the promoters of genes with known roles in cell cycle (Figure S2B) as well as development (including Gata3, Krt18, Krt8, Srf, Jun, Prom1, and Lef1) (Figures 3B and S2C). To establish a functional role for NF-Y in the regulation of these genes, we used RNAi to knock down subunits of the NF-Y complex individually or in combination (Figures 3C and S2D) and profiled gene expression changes using qPCR following reverse transcription (qRT-PCR). As a positive control, we observed significant changes in the expression of key cell-cycle genes in cells depleted of individual NF-Y subunits (Figure S2E) accompanied by severe proliferation defects and growth arrest (Figures S2F and S2G), corroborating NF-Y’s established role in proliferation (Dolfini et al., 2012aDolfini D. Gatta R. Mantovani R. NF-Y and the transcriptional activation of CCAAT promoters.Crit. Rev. Biochem. Mol. Biol. 2012; 47: 29-49Crossref PubMed Scopus (169) Google Scholar). Depletion of NF-Y subunits decreased the expression of pluripotency-associated factors by 2- to 5-fold, and increased the expression of differentiation genes by up to an order of magnitude (Figure 3D). Together, these findings show that the NF-Y complex is required for the expression of key ESC self-renewal and pluripotency genes, and support the conclusion that NF-Y regulates not only genes with housekeeping functions through its canonical promoter-proximal binding but also genes required for ESC identity by cobinding distal enhancers with cell type-specific master regulators. To investigate whether NF-Y is essential for the maintenance of ESCs in an undifferentiated state, we examined colony morphology and alkaline phosphatase (AP) staining 96 hr after RNAi-mediated silencing of individual or all subunits of the NF-Y complex (Figure S2D). We observed notable self-renewal defects accompanied by loss of characteristic ESC colony morphology and AP staining (ESC marker), all consistent with ESC differentiation (Figure 4A). NF-YA KD ESCs showed profound proliferation defects compared to NF-YB/NF-YC KD ESCs, perhaps due to NF-YA being the limiting subunit of the NF-Y complex with relatively low expression compared to the other two subunits (Figure S2H). Nevertheless, these cellular changes are consistent with NF-Y’s control over both housekeeping and ESC-specific functions through distinct modes of regulation (Figures 3 and S2). At the molecular level, immunostaining experiments after NF-YA silencing revealed a drastic loss of Nanog protein levels, while NF-YC protein levels remain unchanged (Figure 4B). Furthermore, RT-qPCR analysis of ESCs depleted of NF-Y subunits revealed a significant increase in the expression of differentiation and lineage markers (Figure 4C) in addition to a significant decrease in the expression of pluripotency genes including Oct4, Nanog, Prdm14, Esrrb, Tcl1, Tbx3, and nodal antagonists Lefty1 and Lefty2 (Figure 3D), which are among the earliest to be downregulated during ESC differentiation. Based on these data, we conclude that the NF-Y complex is essential to maintain ESC identity, and that the depletion of even one of the three NF-Y subunits leads to the loss of the pluripotent state. To determine the extent to which NF-Y regulates gene expression programs influencing ESC self-renewal and pluripotency, we used microarrays to profile global gene expression in ESCs transfected with a control siRNA or siRNA(s) targeting individual or all of the NF-Y subunits (Figures 3C and S3A). Consistent with gene expression changes observed in the RT-qPCR analysis (Figures 3D and 4C), global gene expression changes upon silencing of all three NF-Y subunits correlated highly with those after individual knockdowns (Figure S3B). Notably, many of the NF-Y targets lie at a “crossroads” of cell-fate determination during embryonic development (Figure S4). Using a stringent criteria (FDR < 0.05; fold change ≥ 2.0), we identified 847 genes that were differentially expressed in cells depleted of all three NF-Y subunits (NF-Y TKD) (Figure 4D), a vast majority of which were also differentially expressed in individual knockdowns (Figure 4E). Notably, while a majority of the downregulated genes harbor NF-Y binding sites either within 5 kb upstream of the TSS or the gene body, only ∼18% of the upregulated genes have NF-Y binding sites (Figure 4D). With much of the upregulation attributable to indirect effects, this is a clear indication that NF-Y plays a predominantly activating role in ESCs. We observed a strong positive correlation between gene expression changes upon NF-Y TKD ESCs and those during the normal course of embryoid body differentiation (Figure 4F). Consistent with this observation, principle component analysis of gene expression profiles during differentiation of ESCs into three different lineages showed ESCs depleted of NF-Y cluster away from undifferentiated ESCs (Figure 4G). Analysis of published gene expression microarray data from ESCs depleted" @default.
• W2051875167 created "2016-06-24" @default.
• W2051875167 creator A5013914345 @default.
• W2051875167 creator A5022233425 @default.
• W2051875167 creator A5027162470 @default.
• W2051875167 creator A5030615993 @default.
• W2051875167 creator A5057038999 @default.
• W2051875167 creator A5069926610 @default.
• W2051875167 creator A5088258550 @default.
• W2051875167 date "2014-09-01" @default.
• W2051875167 modified "2023-10-14" @default.
• W2051875167 title "Histone-Fold Domain Protein NF-Y Promotes Chromatin Accessibility for Cell Type-Specific Master Transcription Factors" @default.
• W2051875167 cites W1979513200 @default.
• W2051875167 cites W1981030303 @default.
• W2051875167 cites W1981088384 @default.
• W2051875167 cites W1981359686 @default.
• W2051875167 cites W1981537690 @default.
• W2051875167 cites W1984018744 @default.
• W2051875167 cites W1987371782 @default.
• W2051875167 cites W1988294855 @default.
• W2051875167 cites W1993252465 @default.
• W2051875167 cites W1993829333 @default.
• W2051875167 cites W1998004003 @default.
• W2051875167 cites W2013340257 @default.
• W2051875167 cites W2014787267 @default.
• W2051875167 cites W2016923780 @default.
• W2051875167 cites W2018363492 @default.
• W2051875167 cites W202181832 @default.
• W2051875167 cites W2022966544 @default.
• W2051875167 cites W2027127452 @default.
• W2051875167 cites W2028200156 @default.
• W2051875167 cites W2030073462 @default.
• W2051875167 cites W2036454804 @default.
• W2051875167 cites W2040326257 @default.
• W2051875167 cites W2040729654 @default.
• W2051875167 cites W2047288220 @default.
• W2051875167 cites W2049779318 @default.
• W2051875167 cites W2050264932 @default.
• W2051875167 cites W2052471217 @default.
• W2051875167 cites W2053441740 @default.
• W2051875167 cites W2054517572 @default.
• W2051875167 cites W2060453586 @default.
• W2051875167 cites W2061475715 @default.
• W2051875167 cites W2063248559 @default.
• W2051875167 cites W2066911215 @default.
• W2051875167 cites W2068674690 @default.
• W2051875167 cites W2077536265 @default.
• W2051875167 cites W2080859509 @default.
• W2051875167 cites W2083839498 @default.
• W2051875167 cites W2104865962 @default.
• W2051875167 cites W2110893450 @default.
• W2051875167 cites W2122097157 @default.
• W2051875167 cites W2134861134 @default.
• W2051875167 cites W2144256548 @default.
• W2051875167 cites W2150822416 @default.
• W2051875167 cites W2155304372 @default.
• W2051875167 cites W2164683872 @default.
• W2051875167 cites W2166811005 @default.
• W2051875167 cites W2175853567 @default.
• W2051875167 cites W2312832763 @default.
• W2051875167 cites W4232123484 @default.
• W2051875167 doi "https://doi.org/10.1016/j.molcel.2014.07.005" @default.
• W2051875167 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4157648" @default.
• W2051875167 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/25132174" @default.
• W2051875167 hasPublicationYear "2014" @default.
• W2051875167 type Work @default.
• W2051875167 sameAs 2051875167 @default.
• W2051875167 citedByCount "142" @default.
• W2051875167 countsByYear W20518751672015 @default.
• W2051875167 countsByYear W20518751672016 @default.
• W2051875167 countsByYear W20518751672017 @default.
• W2051875167 countsByYear W20518751672018 @default.
• W2051875167 countsByYear W20518751672019 @default.
• W2051875167 countsByYear W20518751672020 @default.
• W2051875167 countsByYear W20518751672021 @default.
• W2051875167 countsByYear W20518751672022 @default.
• W2051875167 countsByYear W20518751672023 @default.
• W2051875167 crossrefType "journal-article" @default.
• W2051875167 hasAuthorship W2051875167A5013914345 @default.
• W2051875167 hasAuthorship W2051875167A5022233425 @default.
• W2051875167 hasAuthorship W2051875167A5027162470 @default.
• W2051875167 hasAuthorship W2051875167A5030615993 @default.
• W2051875167 hasAuthorship W2051875167A5057038999 @default.
• W2051875167 hasAuthorship W2051875167A5069926610 @default.
• W2051875167 hasAuthorship W2051875167A5088258550 @default.
• W2051875167 hasBestOaLocation W20518751671 @default.
• W2051875167 hasConcept C10058791 @default.
• W2051875167 hasConcept C104317684 @default.
• W2051875167 hasConcept C138885662 @default.
• W2051875167 hasConcept C179926584 @default.
• W2051875167 hasConcept C41895202 @default.
• W2051875167 hasConcept C54355233 @default.
• W2051875167 hasConcept C552990157 @default.
• W2051875167 hasConcept C64927066 @default.
• W2051875167 hasConcept C83640560 @default.
• W2051875167 hasConcept C84772758 @default.
• W2051875167 hasConcept C86339819 @default.
• W2051875167 hasConcept C86803240 @default.

• next