Jiemin Wong

Regulation of SRC-3 (pCIP/ACTR/AIB-1/RAC-3/TRAM-1) Coactivator Activity by IκB Kinase

ABSTRACT In the past few years, many nuclear receptor coactivators have been identified and shown to be an integral part of receptor action. The most frequently studied of these coactivators are members of the steroid receptor coactivator (SRC) family, SRC-1, TIF2/GRIP1/SRC-2, and pCIP/ACTR/AIB-1/RAC-3/TRAM-1/SRC-3. In this report, we describe the biochemical purification of SRC-1 and SRC-3 protein complexes and the subsequent identification of their associated proteins by mass spectrometry. Surprisingly, we found association of SRC-3, but not SRC-1, with the IκB kinase (IKK). IKK is known to be responsible for the degradation of IκB and the subsequent activation of NF-κB. Since NF-κB plays a key role in host immunity and inflammatory responses, we therefore investigated the significance of the SRC-3-IKK complex. We demonstrated that SRC-3 was able to enhance NF-κB-mediated gene expression in concert with IKK. In addition, we showed that SRC-3 was phosphorylated by the IKK complex in vitro. Furthermore, elevated SRC-3 phosphorylation in vivo and translocation of SRC-3 from cytoplasm to nucleus in response to tumor necrosis factor alpha occurred in cells, suggesting control of subcellular localization of SRC-3 by phosphorylation. Finally, the hypothesis that SRC-3 is involved in NF-κB-mediated gene expression is further supported by the reduced expression of interferon regulatory factor 1, a well-known NF-κB target gene, in the spleens of SRC-3 null mutant mice. Taken together, our results not only reveal the IKK-mediated phosphorylation of SRC-3 to be a regulated event that plays an important role but also substantiate the role of SRC-3 in multiple signaling pathways.

Progesterone and Glucocorticoid Receptors Recruit Distinct Coactivator Complexes and Promote Distinct Patterns of Local Chromatin Modification

ABSTRACT It is well established that steroid receptor function requires interaction with coactivators. However, the mechanisms through which steroid receptors elicit precise assembly of coactivator complexes and the way the steroid activation signal is transduced remain elusive. Using a T47D cell line stably integrated with a mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter, we demonstrate that specific steroid receptors exhibit preferential recruitment of SRC-1 family coactivators, which determines the subsequent recruitment of specific downstream coregulator molecules. Upon ligand treatment, progesterone receptor (PR) interacted preferentially with SRC-1, which recruited CBP and significantly enhanced acetylation at K5 of histone H4. In contrast, activated glucocorticoid receptor (GR) preferentially associated with SRC-2 (TIF-2/GRIP-1), which subsequently recruited pCAF and led to specific modification of histone H3, suggesting that specific coactivators recruit distinct histone acetyltransferases to modulate the transcription of steroid-responsive genes. Loss-of-function experiments further support the predicted roles of SRC-1 and SRC-2 in, respectively, PR- and GR-mediated transcription on the MMTV promoter. This study indicates that differential recruitment of coactivators by nuclear receptors determines the assembly of coactivator complexes on target promoters to mediate specific transcription signals.

UHRF1 targets DNMT1 for DNA methylation through cooperative binding of hemi-methylated DNA and methylated H3K9

Epigenetic inheritance of DNA methylation in mammals requires a multifunctional protein UHRF1, which is believed to recruit DNMT1 to DNA replication forks through a unique hemi-methylated CpG-binding activity. Here we demonstrate that the UHRF1 mutants deficient in binding either hemi-methylated CpG or H3K9me2/3, but not both, are able to associate with pericentric heterochromatin, recruit Dnmt1 and partially rescue DNA methylation defects in mouse Uhrf1 null ES cells. Furthermore, we present evidence that the flip out of the methylated cytosine induced by UHRF1 binding is unlikely essential for subsequent DNA methylation by DNMT1. Together, our study demonstrates that UHRF1 can target DNMT1 for DNA maintenance methylation through binding either H3K9me2/3 or hemi-methylated CpG, and that the presence of both binding activities ensures high fidelity DNA maintenance methylation. In addition, our study indicates that UHRF1 mediates cross-talk between H3K9 methylation and DNA methylation at the level of DNA methylation maintenance.

p300 Requires Its Histone Acetyltransferase Activity and SRC-1 Interaction Domain To Facilitate Thyroid Hormone Receptor Activation in Chromatin

ABSTRACT We have characterized the mechanism by which coactivator p300 facilitates transcriptional activation mediated by the heterodimer of thyroid hormone (T3) receptor and 9-cis retinoid acid receptor (TR-RXR) in the context of chromatin. We demonstrate that, while p300 can enhance the transcriptional activation mediated by both liganded TR-RXR and GAL4-VP16, its histone acetyltransferase activity (HAT) is required for its ability to facilitate liganded TR-RXR- but not GAL4-VP16-mediated transcriptional activation. To understand how p300 is recruited by liganded TR-RXR, we have analyzed the interactions between TR-RXR and p300 as well as SRC-1 family coactivators. We show that, in contrast to a strong hormone-dependent interaction between TR-RXR and SRC-1 family coactivators, p300 displays minimal, if any, T3-dependent interaction with TR-RXR. However, p300 can be recruited by liganded TR-RXR through its interaction with SRC-1 family coactivators. Consistent with the protein-protein interaction profile described above, we demonstrate that the SRC-1 interaction domain of p300 is important for its ability to facilitate transcriptional activation mediated by TR-RXR, whereas its nuclear receptor interaction domain is dispensable. Collectively, these results reveal the functional significance of the HAT activity of p300 and define an indirect mode for the action of p300 in TR-RXR activation.

The X-linked mental retardation gene PHF8 is a histone demethylase involved in neuronal differentiation

Recent studies have identified mutations in PHF8, an X-linked gene encoding a JmjC domain-containing protein, as a causal factor for X-linked mental retardation (XLMR) and cleft lip/cleft palate. However, the underlying mechanism is unknown. Here we show that PHF8 is a histone demethylase and coactivator for retinoic acid receptor (RAR). Although activities for both H3K4me3/2/1 and H3K9me2/1 demethylation were detected in cellular-based assays, recombinant PHF8 exhibited only H3K9me2/1 demethylase activity in vitro, suggesting that PHF8 is an H3K9me2/1 demethylase whose specificity may be modulated in vivo. Importantly, a mutant PHF8 (phenylalanine at position 279 to serine) identified in the XLMR patients is defective in enzymatic activity, indicating that the loss of histone demethylase activity is causally linked with the onset of disease. In addition, we show that PHF8 binds specifically to H3K4me3/2 peptides via an N-terminal PHD finger domain. Consistent with a role for PHF8 in neuronal differentiation, knockdown of PHF8 in mouse embryonic carcinoma P19 cells impairs RA-induced neuronal differentiation, whereas overexpression of the wild-type but not the F279S mutant PHF8 drives P19 cells toward neuronal differentiation. Furthermore, we show that PHF8 interacts with RARα and functions as a coactivator for RARα. Taken together, our results suggest that histone methylation modulated by PHF8 plays a critical role in neuronal differentiation.

Differential Regulation of the Ten-Eleven Translocation (TET) Family of Dioxygenases by O-Linked β-N-Acetylglucosamine Transferase (OGT)

Background: TET proteins have been shown to target OGT to chromatin, but whether OGT regulates the TET proteins is not clear. Results: OGT regulates the subcellular localization and enzymatic activity of TET3 but not TET1 and TET2. Conclusion: The TET family of proteins is differentially regulated by OGT. Significance: We reveal a potential mechanism by which glucose metabolism regulates TET3 activity. The ten-eleven translocation (TET) family of dioxygenases (TET1/2/3) converts 5-methylcytosine to 5-hydroxymethylcytosine and provides a vital mechanism for DNA demethylation. However, how TET proteins are regulated is largely unknown. Here we report that the O-linked β-GlcNAc (O-GlcNAc) transferase (OGT) is not only a major TET3-interacting protein but also regulates TET3 subcellular localization and enzymatic activity. OGT catalyzes the O-GlcNAcylation of TET3, promotes TET3 nuclear export, and, consequently, inhibits the formation of 5-hydroxymethylcytosine catalyzed by TET3. Although TET1 and TET2 also interact with and can be O-GlcNAcylated by OGT, neither their subcellular localization nor their enzymatic activity are affected by OGT. Furthermore, we show that the nuclear localization and O-GlcNAcylation of TET3 are regulated by glucose metabolism. Our study reveals the differential regulation of TET family proteins by OGT and a novel link between glucose metabolism and DNA epigenetic modification.

Interaction of nucleolin with ribosomal RNA genes and its role in RNA polymerase I transcription

Nucleolin is a multi-functional nucleolar protein that is required for ribosomal RNA gene (rRNA) transcription in vivo, but the mechanism by which nucleolin modulates RNA polymerase I (RNAPI) transcription is not well understood. Nucleolin depletion results in an increase in the heterochromatin mark H3K9me2 and a decrease in H4K12Ac and H3K4me3 euchromatin histone marks in rRNA genes. ChIP-seq experiments identified an enrichment of nucleolin in the ribosomal DNA (rDNA) coding and promoter region. Nucleolin is preferentially associated with unmethylated rRNA genes and its depletion leads to the accumulation of RNAPI at the beginning of the transcription unit and a decrease in UBF along the coding and promoter regions. Nucleolin is able to affect the binding of transcription termination factor-1 on the promoter-proximal terminator T0, thus inhibiting the recruitment of TIP5 and HDAC1 and the establishment of a repressive heterochromatin state. These results reveal the importance of nucleolin for the maintenance of the euchromatin state and transcription elongation of rDNA.

S phase-dependent interaction with DNMT1 dictates the role of UHRF1 but not UHRF2 in DNA methylation maintenance

Recent studies demonstrate that UHRF1 is required for DNA methylation maintenance by targeting DNMT1 to DNA replication foci, presumably through its unique hemi-methylated DNA-binding activity and interaction with DNMT1. UHRF2, another member of the UHRF family proteins, is highly similar to UHRF1 in both sequence and structure, raising questions about its role in DNA methylation. In this study, we demonstrate that, like UHRF1, UHRF2 also binds preferentially to methylated histone H3 lysine 9 (H3K9) through its conserved tudor domain and hemi-methylated DNA through the SET and Ring associated domain. Like UHRF1, UHRF2 is enriched in pericentric heterochromatin. The heterochromatin localization depends to large extent on its methylated H3K9-binding activity and to less extent on its methylated DNA-binding activity. Coimmunoprecipitation experiments demonstrate that both UHRF1 and UHRF2 interact with DNMT1, DNMT3a, DNMT3b and G9a. Despite all these conserved functions, we find that UHRF2 is not able to rescue the DNA methylation defect in Uhrf1 null mouse embryonic stem cells. This can be attributed to the inability for UHRF2 to recruit DNMT1 to replication foci during S phase of the cell cycle. Indeed, we find that while UHRF1 interacts with DNMT1 in an S phase-dependent manner in cells, UHRF2 does not. Thus, our study demonstrates that UHRF2 and UHRF1 are not functionally redundant in DNA methylation maintenance and reveals the cell-cycle-dependent interaction between UHRF1 and DNMT1 as a key regulatory mechanism targeting DNMT1 for DNA methylation.

Deleted in Breast Cancer 1, a Novel Androgen Receptor (AR) Coactivator That Promotes AR DNA-binding Activity

Androgen receptor (AR) plays a critical role in development and maintenance of male reproductive functions and the etiology of prostate cancer. As a ligand-regulated transcription factor, identification and characterization of AR coregulators are essential for understanding the molecular mechanisms underlying its diverse biological functions. Here we reported the identification of a novel AR coactivator, deleted in breast cancer 1 (DBC1), through a biochemical approach. DBC1 interacts with AR in a ligand-stimulated manner and facilitates AR transcriptional activation in transfected cells as well as in Xenopus oocytes. In in vitro gel shift experiments, recombinant DBC1 drastically enhanced AR DNA-binding activity. Expression of DBC1 also enhanced the binding of AR to chromatinized template in vivo, whereas knockdown of DBC1 impaired the binding of AR to endogenous prostate-specific antigen (PSA) gene in the prostate cancer cell line LNCaP. Thus, our data identify DBC1 as a novel AR coactivator.

Reprogramming of somatic cells via TAT-mediated protein transduction of recombinant factors

Generation of induced pluripotent stem cells (iPSCs) holds great promise to regenerative medicine. However, before this technology can be applied for clinical purpose, the issues of iPSC efficiency and safety need to be addressed. In this study, we have compared a simple TAT- and 11 arginine (R)-protein transduction domain (PTD) for somatic cell reprogramming and explored the optimal conditions for the PTD to transduce reprogramming factors (RFs). We show that all recombinant TAT- and 11R-fused RFs are transcriptionally active as they activate their corresponding reporter genes in reporter assays. The TAT-RFs are in general transcriptionally more active than the corresponding 11R-RFs, but less active than the corresponding retroviral transduced RFs. Furthermore, each of TAT-RFs can substitute for their corresponding retrovirus in reprogramming. Finally, using five TAT-RFs together with an HDAC inhibitor, we can generate iPSC-like colonies from human fibroblast cells with high efficiency approximately 2 weeks after the first protein transduction. These colonies exhibit unique features of pluripotent stem cells including the morphology and the expression of pluripotency-associated markers. This characterization of recombinant RFs in reprogramming should facilitate the generation of clinically useful and genetic material-free human iPSCs.