Kyu Heo

FACT-Mediated Exchange of Histone Variant H2AX Regulated by Phosphorylation of H2AX and ADP-Ribosylation of Spt16

The phosphorylation of histone variant H2AX at DNA double-strand breaks is believed to be critical for recognition and repair of DNA damage. However, little is known about the molecular mechanism regulating the exchange of variant H2AX with conventional H2A in the context of the nucleosome. Here, we isolate the H2AX-associated factors, which include FACT (Spt16/SSRP1), DNA-PK, and PARP1 from a human cell line. Our analyses demonstrate that the H2AX-associated factors efficiently promote both integration and dissociation of H2AX and this exchange reaction is mainly catalyzed by FACT among the purified factors. The phosphorylation of H2AX by DNA-PK facilitates the exchange of nucleosomal H2AX by inducing conformational changes of the nucleosome. In contrast, poly-ADP-ribosylation of Spt16 by PARP1 significantly inhibits FACT activities for H2AX exchange. Thus, these data establish FACT as the major regulator involved in H2AX exchange process that is modulated by H2AX phosphorylation and Spt16 ADP-ribosylation.

CCAR1, a key regulator of Mediator complex recruitment to nuclear receptor transcription complexes

DNA-bound transcription factors recruit many coactivator proteins to remodel chromatin and activate transcription. The Mediator complex is believed to recruit RNA polymerase II to most protein-encoding genes. It is generally assumed that interaction of Mediator subunits with DNA-binding transcription factors is responsible for Mediator recruitment to promoters. However, we report here that Mediator recruitment by nuclear receptors (NR) requires a coactivator protein, CCAR1 (cell-cycle and apoptosis regulator 1). CCAR1 associates with components of the Mediator and p160 coactivator complexes and is recruited to endogenous NR target genes in response to the appropriate hormone. Reduction of endogenous CCAR1 levels inhibited hormone-induced expression of endogenous NR target genes, hormone-induced recruitment of Mediator components and RNA polymerase II to target gene promoters, and estrogen-dependent growth of breast cancer cells. Thus, CCAR1 regulates expression of key proliferation-inducing genes. CCAR1 also functions as a p53 coactivator, suggesting a broader role in transcriptional regulation.

Isolation and Characterization of a Novel H1.2 Complex That Acts as a Repressor of p53-mediated Transcription

Linker histone H1 has been generally viewed as a global repressor of transcription by preventing the access of transcription factors to sites in chromatin. However, recent studies suggest that H1 can interact with other regulatory factors for its action as a negative modulator of specific genes. To investigate these aspects, we established a human cell line expressing H1.2, one of the H1 subtypes, for the purification of H1-interacting proteins. Our results showed that H1.2 can stably associate with sets of cofactors and ribosomal proteins that can significantly repress p53-dependent, p300-mediated chromatin transcription. This repressive action of H1.2 complex involves direct interaction of H1.2 with p53, which in turn blocks p300-mediated acetylation of chromatin. YB1 and PURα, two factors present in the H1.2 complex, together with H1.2 can closely recapitulate the repressive action of the entire H1.2 complex in transcription. Chromatin immunoprecipitation and RNA interference analyses further confirmed that the recruitment of YB1, PURα, and H1.2 to the p53 target gene Bax is required for repression of p53-induced transcription. Therefore, these results reveal a previously unrecognized function of H1 as a transcriptional repressor as well as the underlying mechanism involving specific sets of factors in this repression process.

Requirement of Histone Methyltransferase SMYD3 for Estrogen Receptor-mediated Transcription

SMYD3 is a SET domain-containing protein with histone methyltransferase activity on histone H3–K4. Recent studies showed that SMYD3 is frequently overexpressed in different types of cancer cells, but how SMYD3 regulates the development and progression of these malignancies remains unknown. Here, we report the previously unrecognized role of SMYD3 in estrogen receptor (ER)-mediated transcription via its histone methyltransferase activity. We demonstrate that SMYD3 functions as a coactivator of ERα and potentiates ERα activity in response to ligand. SMYD3 directly interacts with the ligand binding domain of ER and is recruited to the proximal promoter regions of ER target genes upon gene induction. Importantly, our chromatin immunoprecipitation analyses provide compelling evidence that SMYD3 is responsible for the accumulation of di- and trimethylation of H3–K4 at the induced ER target genes. Furthermore, RNA interference-directed down-regulation of SMYD3 reveals that SMYD3 is required for ER-regulated gene transcription in estrogen signaling pathway. Thus, our results identify SMYD3 as a new coactivator for ER-mediated transcription, providing a possible link between SMYD3 overexpression and breast cancer.

Identification of preferential target sites for human DNA methyltransferases

DNA methyltransferases (DNMTs) play an important role in establishing and maintaining DNA methylation. Aberrant expression of DNMTs and their isoforms has been found in many types of cancer, and their contribution to aberrant DNA methylation has been proposed. Here, we generated HEK 293T cells stably transfected with each of 13 different DNMTs (DNMT1, two DNMT3A isoforms, nine DNMT3B isoforms and DNMT3L) and assessed the DNA methylation changes induced by each DNMT. We obtained DNA methylation profiles of DNA repetitive elements and 1505 CpG sites from 808 cancer-related genes. We found that DNMTs have specific and overlapping target sites and their DNA methylation target profiles are a reflection of the DNMT domains. By examining H3K4me3 and H3K27me3 modifications in the 808 gene promoter regions using promoter ChIP-on-chip analysis, we found that specific de novo DNA methylation target sites of DNMT3A1 are associated with H3K4me3 modification that are transcriptionally active, whereas the specific target sites of DNMT3B1 are associated with H3K27me3 modification that are transcriptionally inactive. Our data suggest that different DNMT domains are responsible for targeting DNA methylation to specific regions of the genome, and this targeting might be associated with histone modifications.

Cooperative action of TIP48 and TIP49 in H2A.Z exchange catalyzed by acetylation of nucleosomal H2A

H2A.Z is an evolutionarily conserved H2A variant that plays a key role in the regulation of chromatin transcription. To understand the molecular mechanism of H2A.Z exchange, we purified two distinct H2A.Z-interacting complexes termed the small and big complexes from a human cell line. The big complex contains most components of the SRCAP chromatin remodeling and TIP60 HAT complexes, whereas the small complex possesses only a subset of SRCAP and TIP60 subunits. Our exchange analysis revealed that both small and big complexes enhance the incorporation of H2A.Z-H2B dimer into the nucleosome. In addition, TIP60-mediated acetylation of nucleosomal H2A specifically facilitates the action of the small complex in the H2A.Z exchange reaction. Among factors present in the small complex, we determined that TIP48 and TIP49 play a major role in catalyzing H2A acetylation-induced H2A.Z exchange via their ATPase activities. Overall, our work uncovers the previously-unrecognized role of TIP48 and TIP49 in H2A.Z exchange and a novel epigenetic mechanism controlling this process.

DNA methylation biomarker candidates for early detection of colon cancer.

Promoter CpG island hypermethylation of tumor suppressor genes is a common hallmark of all human cancers. Many researchers have been looking for potential epigenetic therapeutic targets in cancer using gene expression profiling with DNA microarray approaches. Our recent genome-wide platform of CpG island hypermethylation and gene expression in colorectal cancer (CRC) cell lines revealed that FBN2 and TCERG1L gene silencing is associated with DNA hypermethylation of a CpG island in the promoter region. In this study, promoter DNA hypermethylation of FBN2 and TCERG1L in CRC occurs as an early and cancer-specific event in colorectal cancer. Both genes showed high frequency of methylation in colon cancer cell lines (>80% for both of genes), adenomas (77% for FBN2, 90% for TCERG1L, n = 39), and carcinomas (86% for FBN2, 99% for TCERG1L, n = 124). Bisulfite sequencing confirmed cancer-specific methylation of FBN2 and TCERG1L of promoters in colon cancer cell line and cancers but not in normal colon. Methylation of FBN2 and TCERG1L is accompanied by downregulation in cell lines and in primary tumors as described in the Oncomine™ website. Together, our results suggest that gene silencing of FBN2 and TCERG1L is associated with promoter DNA hypermethylation in CRC tumors and may be excellent biomarkers for the early detection of CRC.

Reciprocal roles of DBC1 and SIRT1 in regulating estrogen receptor α activity and co-activator synergy

Estrogen receptor α (ERα) plays critical roles in development and progression of breast cancer. Because ERα activity is strictly dependent upon the interaction with coregulators, coregulators are also believed to contribute to breast tumorigenesis. Cell Cycle and Apoptosis Regulator 1 (CCAR1) is an important co-activator for estrogen-induced gene expression and estrogen-dependent growth of breast cancer cells. Here, we identified Deleted in Breast Cancer 1 (DBC1) as a CCAR1 binding protein. DBC1 was recently shown to function as a negative regulator of the NAD-dependent protein deacetylase SIRT1. DBC1 associates directly with ERα and cooperates synergistically with CCAR1 to enhance ERα function. DBC1 is required for estrogen-induced expression of a subset of ERα target genes as well as breast cancer cell proliferation and for estrogen-induced recruitment of ERα to the target promoters in a gene-specific manner. The mechanism of DBC1 action involves inhibition of SIRT1 interaction with ERα and of SIRT1-mediated deacetylation of ERα. SIRT1 also represses the co-activator synergy between DBC1 and CCAR1 by binding to DBC1 and disrupting its interaction with CCAR1. Our results indicate that DBC1 and SIRT1 play reciprocal roles as major regulators of ERα activity, by regulating DNA binding by ERα and by regulating co-activator synergy.

Gene dysregulation by histone variant H2A.Z in bladder cancer

BackgroundThe incorporation of histone variants into nucleosomes is one of the main strategies that the cell uses to regulate the structure and function of chromatin. Histone H2A.Z is an evolutionarily conserved histone H2A variant that is preferentially localized within nucleosomes at the transcriptional start site (TSS). H2A.Z reorganizes the local chromatin structure and recruits the transcriptional machinery for gene activation. High expression of H2A.Z has been reported in several types of cancers and is causally linked to genomic instability and tumorigenesis. However, it is not entirely clear how H2A.Z overexpression in cancer cells establishes aberrant chromatin states and promotes gene expression.ResultsThrough integration of genome-wide H2A.Z ChIP-seq data with microarray data, we demonstrate that H2A.Z is enriched around the TSS of cell cycle regulatory genes in bladder cancer cells, and this enrichment is correlated with the elevated expression of cancer-promoting genes. RNAi-mediated knockdown of H2A.Z in the cancer cells causes transcriptional suppression of multiple cell cycle regulatory genes with a distinct decrease in cell proliferation. H2A.Z nucleosomes around the TSS have higher levels of H3K4me2/me3, which coincides with the recruitment of two chromatin factors, WDR5 and BPTF. The observed recruitment is functional, as the active states of H2A.Z target genes are largely erased by suppressing the expression of WDR5 or BPTF, effects resembling H2A.Z knockdown.ConclusionsWe conclude that H2A.Z is overexpressed in bladder cancer cells and contributes to cancer-related transcription pathways. We also provide evidence in support of the engagement of H3K4me2/me3 and WDR5/BPTF in H2A.Z-induced cancer pathogenesis. Further studies are warranted to understand how H2A.Z overexpression contributes to the recruitment of the full repertoire of transcription machinery to target genes in bladder cancer cells.

Isolation and Characterization of Proteins Associated with Histone H3 Tails in Vivo

The histone H3 amino-terminal tails play an important role in regulating chromatin transcription. Although the mechanisms by which the H3 tail modulates transcription are not well understood, recent discoveries of specific interactions of regulatory factors with H3 tails suggest that H3 tails are a key player in the precise regulation of transcription activity. To investigate the recruitment-based action of H3 tails in chromatin transcription, we purified H3 tail-associated proteins from HeLa cells that stably express epitope-tagged H3 tails. This approach resulted in the identification of multiple histone methyltransferase activities and transcription regulatory factors that are specifically associated with expressed H3 tail domains. Point mutations of Lys-9 and Lys-27 to block cellular modifications of the tail domains completely abolished the association of specific factors, including HP1 and several repressors. Importantly, our transcription analysis revealed that the purified factors can significantly stimulate p300-mediated transcription from chromatin templates. These results implicate that the H3 tail, when accessible in relaxed chromatin, acts as a transcriptional regulator by mediating recruitment of specific sets of cofactors.