Jongkyu Choi

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.

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.

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.

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.

Linker Histone H1.2 Cooperates with Cul4A and PAF1 to Drive H4K31 Ubiquitylation-Mediated Transactivation

Increasing evidence suggests that linker histone H1 can influence distinct cellular processes by acting as a gene-specific regulator. However, the mechanistic basis underlying such H1 specificity and whether H1 acts in concert with other chromatin-altering activities remain unclear. Here, we show that one of the H1 subtypes, H1.2, stably interacts with Cul4A E3 ubiquitin ligase and PAF1 elongation complexes and that such interaction potentiates target gene transcription via induction of H4K31 ubiquitylation, H3K4me3, and H3K79me2. H1.2, Cul4A, and PAF1 are functionally cooperative because their individual knockdown results in the loss of the corresponding histone marks and the deficiency of target gene transcription. H1.2 interacts with the serine 2-phosphorylated form of RNAPII, and we argue that it recruits the Cul4A and PAF1 complexes to target genes by bridging the interaction between the Cul4A and PAF1 complexes. These data define an expanded role for H1 in regulating gene transcription and illustrate its dependence on the elongation competence of RNAPII.

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.

Functional interplay between p53 acetylation and H1.2 phosphorylation in p53-regulated transcription

Linker histone H1.2 has been shown to suppress p53-dependent transcription through the modulation of chromatin remodeling; however, little is known about the mechanisms governing the antagonistic effects of H1.2 in DNA damage response. Here, we show that the repressive action of H1.2 on p53 function is negatively regulated via acetylation of p53 C-terminal regulatory domain and phosphorylation of H1.2 C-terminal tail. p53 acetylation by p300 impairs the interaction of p53 with H1.2 and triggers a rapid activation of p53-dependent transcription. Similarly, DNA-PK-mediated phosphorylation of H1.2 at T146 enhances p53 transcriptional activity by impeding H1.2 binding to p53 and thereby attenuating its suppressive effects on p53 transactivation. Consistent with these findings, point mutations mimicking modification states of H1.2 and p53 lead to a significant increase in p53-induced apoptosis. These data suggest that p53 acetylation–H1.2 phosphorylation cascade serves as a unique mechanism for triggering p53-dependent DNA damage response pathways.

Loss of muscleblind-like 1 results in cardiac pathology and persistence of embryonic splice isoforms

Cardiac dysfunction is a prominent cause of mortality in myotonic dystrophy I (DM1), a disease where expanded CUG repeats bind and disable the muscleblind-like family of splice regulators. Deletion of muscleblind-like 1 (Mbnl1ΔE2/ΔE2) in 129 sv mice results in QRS, QTc widening, bundle block and STc narrowing at 2–4 months of age. With time, cardiac function deteriorates further and at 6 months, decreased R wave amplitudes, sinus node dysfunction, cardiac hypertrophy, interstitial fibrosis, multi-focal myocardial fiber death and calcification manifest. Sudden death, where no end point illness is overt, is observed at a median age of 6.5 and 4.8 months in ~67% and ~86% of male and female Mbnl1ΔE2/ΔE2 mice, respectively. Mbnl1 depletion results in the persistence of embryonic splice isoforms in a network of cardiac RNAs, some of which have been previously implicated in DM1, regulating sodium and calcium currents, Scn5a, Junctin, Junctate, Atp2a1, Atp11a, Cacna1s, Ryr2, intra and inter cellular transport, Clta, Stx2, Tjp1, cell survival, Capn3, Sirt2, Csda, sarcomere and cytoskeleton organization and function, Trim55, Mapt, Pdlim3, Pdlim5, Sorbs1, Sorbs2, Fhod1, Spag9 and structural components of the sarcomere, Myom1, Tnnt2, Zasp. Thus this study supports a key role for Mbnl1 loss in the initiation of DM1 cardiac disease.

Vpr-binding protein antagonizes p53-mediated transcription via direct interaction with H3 tail.

ABSTRACT HIV-1 Vpr-binding protein (VprBP) has been implicated in the regulation of both DNA replication and cell cycle progression, but its precise role remains unclear. Here we report that VprBP regulates the p53-induced transcription and apoptotic pathway. VprBP is recruited to p53-responsive promoters and suppresses p53 transactivation in the absence of stress stimuli. To maintain target promoters in an inactive state, VprBP stably binds to nucleosomes by recognizing unacetylated H3 tails. Promoter-localized deacetylation of H3 tails is a prerequisite for VprBP to tether and act as a bona fide inhibitor at p53 target genes. VprBP knockdown leads to activation of p53 target genes and causes an increase in DNA damage-induced apoptosis. Moreover, phosphorylation of VprBP at serine 895 impairs the ability of VprBP to bind H3 tails and to repress p53 transactivation. Our results thus reveal a new role for VprBP in regulation of the p53 signaling pathway, as well as molecular mechanisms of cancer development related to VprBP misregulation.