Our genome-wide evaluation of HDAC1 occupancy in ES and TS cells lends new insight into epigenetic mechanisms that support self-renewal in pluripotent ES and multipotent TS cells. By mapping HDAC1 binding in ES and TS cells and the NuRD component MBD3 in ES cells on a global scale, and evaluating global mRNA expression of wild-type and Hdac1 knockout ES and ES and TS cells cultured in the presence of the HDAC inhibitor, TSA, we found that HDAC1 distributions are present to a larger extent at active genes compared with inactive genes. We also observed upregulation of pluripotency-related genes in Hdac1 knockout ES cells relative to wild-type ES cells, suggesting that Hdac1 may limit the expression level of highly enriched genes in ES cells. Results presented here also show that in ES cells, HDAC1 binds key genes implicit in maintaining ES cell self-renewal such as Oct4, Sox2 and Nanog and in TS cells, HDAC1 binds important trophoblast-lineage genes such as Cdx2, Elf5 and Eomes. Our results also show that MBD3 and HDAC1 co-occupy several pluripotency regulators including Oct4, Nanog and Klf4. Moreover, a subset of HDAC1-occupied genes are overexpressed in both ES and TS cells such as Sox2 and Tbx3. Therefore, by identifying global HDAC1 binding sites in ES and TS cells, our work provides clues about epigenetic phenomena that may contribute to mechanisms of ES and TS cells self-renewal.
ES and TS cells, which are derived from preimplantation embryos, share the capacity to self-renew indefinitely in vitro
in the presence of appropriate external signals including LIF and FGF4, respectively. During preimplantation blastocyst development, LIF produced by trophectodermal (TE) cells promotes ICM cell self-renewal and pluripotency, while FGF4 produced by ICM cells aids in TE cell proliferation and self-renewal. This paracrine signaling mechanism suggests a cooperative relationship between pluripotent ICM and multipotent cells of the TE, which persists upon formation of the blastocyst. Our results implicate a role for HDAC1 in propagating these signals, where HDAC1 was found to bind promoter regions of genes encoding components of the LIF and FGF4 signaling pathways in ES and TS cells. We observed HDAC1 binding to LIFR and FGF4 in ES cells, and HDAC1 binding to FGFR2 in TS cells (Supplementary Tables S1
), demonstrating that HDAC1 occupies extracellular genes including growth factors and cytokines (LIF, FGF4), and receptors (FGFR2), which are critical for maintaining ES and TS cells self-renewal. These results suggest that HDAC1 occupancy of target genes in ES and TS cells may serve to reinforce signaling cascades that participate in the regulation of self-renewal.
The ability of ES cells to differentiate into cells of the three germ layers, and TS cells to differentiate into cells of the trophoblast lineage is determined in part by their distinctive epigenetic programs, where epigenetic modifiers and transcription factors participate in regulating chromatin structure by modifying histones or recruiting histone modifiers. For example, trithorax and Polycomb group proteins (PcGs) regulate regions of histone modifications H3K4me3 and H3K27me3, respectively, which are associated with developmentally repressed genes in ES cells that are primed for activation upon differentiation (31–33
). Additional studies have demonstrated that chromatin remodeling proteins such as BRG1 are important in regulating ES (12
) and TS cells self-renewal (13
), and BRG1 was found to associate with genes involved in ES and TS cells self-renewal. We observed co-binding of HDAC1 and BRG1 at a number of target genes in ES and TS cells (data not shown), suggesting that multiple epigenetic regulators may co-regulate transcription of target genes. These results are in agreement with previous findings, which demonstrated that BRG1 cooperates with HDAC to regulate target gene expression (35
). Moreover, transcription factors including OCT4 and NANOG have been shown to associate with epigenetic regulators including HDAC1 (36
). In this study, we observed co-occupancy of HDAC1 and OCT4, SOX2 and NANOG (37
) at 347 target genes in ES cells, and co-occupancy of HDAC1 and BRG1, EOMES and TCFAP2C (13
) at 295 genes in TS cells, suggesting that co-binding of transcription factors and epigenetic modifiers at target genes may be important in maintaining ES and TS cells self-renewal.
HDACs are generally thought to act as transcriptional repressors, by removing acetyl groups at inactive genes. HDAC1 has been shown to function as a co-repressor in multiprotein complexes, after being recruited to DNA by proteins in complexes such as the NuRD, the SIN3 corepressor, the CoREST, the Nanog- and Oct4-associated deacetylase (NODE) and the SHIP1 complexes (38
). In addition, HDAC1 has been shown to be recruited by additional complexes such as the Polycomb repressive complex 2, which catalyze histone H3 K27 trimethylation at PcG target genes (39
). Because several of these transcriptionally repressive HDAC1-containing complexes, such as NuRD, NODE and PcG, have important roles in normal ES cell function (27
), it is plausible that HDAC1 associates with predominantly inactive genes in ES cells. An alternative interpretation has been suggested from results in T cells where HDACs have been recently shown to bind mainly to active genes but exert regulatory functions on active and inactive genes (11
). These results are unexpected because HDACs are well known for their transcriptionally repressive functions. While these findings may seem to suggest a paradigm shift for understanding HDAC function in somatic cells, the authors conclude that HDACs may be more prevalent at active genes compared with inactive genes to reset the acetylation state, whereas inactive genes may not require continuous binding of HDACs to maintain their inactive state or condensed chromatin structure (11
Because HDACs have a repressive role, it is reasonable to assume that inhibition of HDACs would positively influence the expression state of HDAC target genes. Indeed, we observed upregulation of pluripotency genes in Hdac1 knockout ES cells relative to control ES cells. However, we found that treatment of ES cells with the general HDAC inhibitor TSA resulted in downregulation of pluripotency-related genes and upregulation of lineage-specific genes, which is similar to previous results (26
), demonstrating that HDACs as a whole positively and negatively regulate expression of target genes. It has been also demonstrated that treatment with TSA increases chromatin accessibility by increasing acetylation levels (40
). HDAC inhibitors have been also used to influence chromatin accessibility during reprogramming. Recent work has demonstrated that four transcription factors including OCT4, SOX2, KLF4 and MYC are sufficient to induce reprogramming of somatic cells to a pluripotent state (41
). While the reprogramming process is relatively inefficient, treatment with HDAC inhibitors such as TSA, VPA and SAHA significantly enhances the rate of generating iPS cells (9
). Our results show that HDAC1 binds the four reprogramming factors Oct4, Sox2, Klf4 and c-Myc in ES cells (Supplementary Table S1
). Therefore, treatment of HDAC inhibitors during the reprogramming process may allow Oct4, Sox2, Klf4 and c-Myc to more efficiently activate or repress target genes by regulating chromatin accessibility and relaxing repressive chromatin regions.
HDACs are also thought to regulate cell cycle progression, where HDAC1 null ES cells have reduced proliferation rates and increased expression of the cyclin-dependent kinase inhibitor, p21 (also known as Cdkn1a) (3
). Our results support a role for HDAC1 in regulating proliferation and cell cycle progression, where HDAC1 was found to bind cell cycle regulators such as cyclin-dependent kinase inhibitors (Cdkn1a/b), cyclin dependent kinases (Cdk4, −6), cyclin genes (Ccnd1/2, Ccne1/2, etc.) and Myc (Supplementary Tables S1
), in ES and TS cells. We also observed HDAC1 binding to p53 (also known as Trp53), which induces p21, and Mdm2, another member of the p53 tumor suppressor family. p53 also plays a role in repressing Nanog expression during ES cell differentiation (42
), and p53 is negatively correlated with reprogramming efficiency (43
). By associating with DNA regions encoding cell cycle regulators and tumor suppressors, HDAC1 may regulate the tumorigenic growth properties of ES and TS cells.
In conclusion, results presented here describe a role for HDAC1 in occupying active and inactive genes, including key transcription factors that regulate ES and TS cells self-renewal and differentiation. Consistent with our findings, a recent study also demonstrated that HDACs associated with active and inactive genes, albeit in a different cell context (11
). Through genome-wide identification of HDAC1 binding sites in ES and TS cells, which comprise the first two stem cells populations to form during development, our results describe epigenetic mechanisms of self-renewal and increase our understanding of pluripotency and multipotency.