Cheng Ming Chiang

The general transcription machinery and general cofactors.

ABSTRACT In eukaryotes, the core promoter serves as a platform for the assembly of transcription preinitiation complex (PIC) that includes TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase II (pol II), which function collectively to specify the transcription start site. PIC formation usually begins with TFIID binding to the TATA box, initiator, and/or downstream promoter element (DPE) found in most core promoters, followed by the entry of other general transcription factors (GTFs) and pol II through either a sequential assembly or a preassembled pol II holoenzyme pathway. Formation of this promoter-bound complex is sufficient for a basal level of transcription. However, for activator-dependent (or regulated) transcription, general cofactors are often required to transmit regulatory signals between gene-specific activators and the general transcription machinery. Three classes of general cofactors, including TBP-associated factors (TAFs), Mediator, and upstream stimulatory activity (USA)-derived positive cofactors (PC1/PARP-1, PC2, PC3/DNA topoisomerase I, and PC4) and negative cofactor 1 (NC1/HMGB1), normally function independently or in combination to fine-tune the promoter activity in a gene-specific or cell-type-specific manner. In addition, other cofactors, such as TAF1, BTAF1, and negative cofactor 2 (NC2), can also modulate TBP or TFIID binding to the core promoter. In general, these cofactors are capable of repressing basal transcription when activators are absent and stimulating transcription in the presence of activators. Here we review the roles of these cofactors and GTFs, as well as TBP-related factors (TRFs), TAF-containing complexes (TFTC, SAGA, SLIK/SALSA, STAGA, and PRC1) and TAF variants, in pol II-mediated transcription, with emphasis on the events occurring after the chromatin has been remodeled but prior to the formation of the first phosphodiester bond.

The Double Bromodomain-containing Chromatin Adaptor Brd4 and Transcriptional Regulation

Brd4 is a double bromodomain-containing protein that binds preferentially to acetylated chromatin. It belongs to the BET (bromodomains and extraterminal) family that includes mammalian Brd2, Brd3, Brd4, Brdt, Drosophila Fsh, yeast Bdf1, Bdf2, and corresponding homologues in other species. Brd4 is essential for cellular growth and has been implicated in cell cycle control, DNA replication, and gene rearrangement found in t(15;19)-associated carcinomas. Recently, Brd4 has been found in several transcription complexes, including the general cofactor Mediator and the P-TEFb elongation factor, and is capable of stimulating HIV-1 transcription in a Tat-independent manner. In addition, Brd4 is used as a cellular adaptor by some animal and human papillomaviruses (HPV) for anchoring viral genomes to mitotic chromosomes. This tethering, mediated by Brd4 interaction with virus-encoded E2 protein, facilitates viral genome segregation during mitosis. Interestingly, Brd4 is also identified in a transcriptional silencing complex assembled by HPV E2 and turns out to be the long sought cellular corepressor that inhibits the expression of HPV-encoded E6 and E7 oncoproteins that antagonize p53 and pRB tumor suppressor activity, respectively. The dual role of Brd4 in gene activation and repression illustrates how a dynamic chromatin-binding adaptor is able to recruit distinct transcriptional regulators to modulate promoter activity through cell cycle progression.

SIRT1 Deacetylates and Inhibits SREBP-1C Activity in Regulation of Hepatic Lipid Metabolism

The SIRT1 deacetylase inhibits fat synthesis and stimulates fat oxidation in response to fasting, but the underlying mechanisms remain unclear. Here we report that SREBP-1c, a key lipogenic activator, is an in vivo target of SIRT1. SIRT1 interaction with SREBP-1c was increased during fasting and decreased upon feeding, and consistently, SREBP-1c acetylation levels were decreased during fasting in mouse liver. Acetylated SREBP-1c levels were also increased in HepG2 cells treated with insulin and glucose to mimic feeding conditions, and down-regulation of p300 by siRNA decreased the acetylation. Depletion of hepatic SIRT1 by adenoviral siRNA increased acetylation of SREBP-1c with increased lipogenic gene expression. Tandem mass spectrometry and mutagenesis studies revealed that SREBP-1c is acetylated by p300 at Lys-289 and Lys-309. Mechanistic studies using acetylation-defective mutants showed that SIRT1 deacetylates and inhibits SREBP-1c transactivation by decreasing its stability and its occupancy at the lipogenic genes. Remarkably, SREBP-1c acetylation levels were elevated in diet-induced obese mice, and hepatic overexpression of SIRT1 or treatment with resveratrol, a SIRT1 activator, daily for 1 week decreased acetylated SREBP-1c levels with beneficial functional outcomes. These results demonstrate an intriguing connection between elevated SREBP-1c acetylation and increased lipogenic gene expression, suggesting that abnormally elevated SREBP-1c acetylation increases SREBP-1c lipogenic activity in obese mice. Reducing acetylation of SREBP-1c by targeting SIRT1 may be useful for treating metabolic disorders, including fatty liver, obesity, and type II diabetes.

FXR acetylation is normally dynamically regulated by p300 and SIRT1 but constitutively elevated in metabolic disease states.

The nuclear bile acid receptor FXR is critical for regulation of lipid and glucose metabolism. Here, we report that FXR is a target of SIRT1, a deacetylase that mediates nutritional and hormonal modulation of hepatic metabolism. Lysine 217 of FXR is the major acetylation site targeted by p300 and SIRT1. Acetylation of FXR increases its stability but inhibits heterodimerization with RXRalpha, DNA binding, and transactivation activity. Downregulation of hepatic SIRT1 increased FXR acetylation with deleterious metabolic outcomes. Surprisingly, in mouse models of metabolic disease, FXR interaction with SIRT1 and p300 was dramatically altered, FXR acetylation levels were elevated, and overexpression of SIRT1 or resveratrol treatment reduced acetylated FXR levels. Our data demonstrate that FXR acetylation is normally dynamically regulated by p300 and SIRT1 but is constitutively elevated in metabolic disease states. Small molecules that inhibit FXR acetylation by targeting SIRT1 or p300 may be promising therapeutic agents for metabolic disorders.

Phospho Switch Triggers Brd4 Chromatin Binding and Activator Recruitment for Gene-Specific Targeting

Bromodomain-containing protein 4 (Brd4) is an epigenetic reader and transcriptional regulator recently identified as a cancer therapeutic target for acute myeloid leukemia, multiple myeloma, and Burkitts lymphoma. Although chromatin targeting is a crucial function of Brd4, there is little understanding of how bromodomains that bind acetylated histones are regulated, nor how the gene-specific activity of Brd4 is determined. Via interaction screen and domain mapping, we identified p53 as a functional partner of Brd4. Interestingly, Brd4 association with p53 is modulated by casein kinase II (CK2)-mediated phosphorylation of a conserved acidic region in Brd4 that selectively contacts either a juxtaposed bromodomain or an adjacent basic region to dictate the ability of Brd4 binding to chromatin and also the recruitment of p53 to regulated promoters. The unmasking of bromodomains and activator recruitment, concurrently triggered by the CK2 phospho switch, provide an intriguing mechanism for gene-specific targeting by a universal epigenetic reader.

CTCF Interacts with and Recruits the Largest Subunit of RNA Polymerase II to CTCF Target Sites Genome-Wide

ABSTRACT CTCF is a transcription factor with highly versatile functions ranging from gene activation and repression to the regulation of insulator function and imprinting. Although many of these functions rely on CTCF-DNA interactions, it is an emerging realization that CTCF-dependent molecular processes involve CTCF interactions with other proteins. In this study, we report the association of a subpopulation of CTCF with the RNA polymerase II (Pol II) protein complex. We identified the largest subunit of Pol II (LS Pol II) as a protein significantly colocalizing with CTCF in the nucleus and specifically interacting with CTCF in vivo and in vitro. The role of CTCF as a link between DNA and LS Pol II has been reinforced by the observation that the association of LS Pol II with CTCF target sites in vivo depends on intact CTCF binding sequences. “Serial” chromatin immunoprecipitation (ChIP) analysis revealed that both CTCF and LS Pol II were present at the β-globin insulator in proliferating HD3 cells but not in differentiated globin synthesizing HD3 cells. Further, a single wild-type CTCF target site (N-Myc-CTCF), but not the mutant site deficient for CTCF binding, was sufficient to activate the transcription from the promoterless reporter gene in stably transfected cells. Finally, a ChIP-on-ChIP hybridization assay using microarrays of a library of CTCF target sites revealed that many intergenic CTCF target sequences interacted with both CTCF and LS Pol II. We discuss the possible implications of our observations with respect to plausible mechanisms of transcriptional regulation via a CTCF-mediated direct link of LS Pol II to the DNA.

Recruitment of TFIIH to the HIV LTR is a rate-limiting step in the emergence of HIV from latency

Latently infected cells rapidly initiate HIV transcription after exposure to signals that induce NF‐κB. To investigate the role of TFIIH during HIV reactivation in vivo, we developed a population of Jurkat cells containing integrated, but transcriptionally silent, HIV proviruses. Surprisingly, the HIV promoter in unactivated Jurkat T cells is partially occupied and carries Mediator containing the CDK8 repressive module, TFIID and RNAP II that is hypophosphorylated and confined to the promoter region. Significantly, the promoter is devoid of TFIIH. Upon stimulation of the cells by TNF‐α, NF‐κB and TFIIH are rapidly recruited to the promoter together with additional Mediator and RNAP II, but CDK8 is lost. Detailed time courses show that the levels of TFIIH at the promoter fluctuate in parallel with NF‐κB recruitment to the promoter. Similarly, recombinant p65 activates HIV transcription in vitro and stimulates phosphorylation of the RNAP II CTD by the CDK7 kinase module of TFIIH. We conclude that the recruitment and activation of TFIIH represents a rate‐limiting step for the emergence of HIV from latency.

Human papillomavirus (HPV) origin-binding protein associates with mitotic spindles to enable viral DNA partitioning

Human papillomaviruses (HPVs) establish long-term infections in patients. The mechanism for extrachromosomal HPV DNA persistence in cycling cells is unknown. We show that HPV origin-containing plasmids partition as minichromosomes, attributable to an association of the viral origin recognition protein E2 with mitotic spindles. α-, β-, and γ-tubulins were pulled down with a tagged E2. The N-terminal transacting and C-terminal protein dimerization/DNA binding domains independently associated with the spindles. We suggest that this E2 property enables these viruses to establish persistence. Its implication for HPV oncogenesis is presented.

Crosstalk between sumoylation and acetylation regulates p53‐dependent chromatin transcription and DNA binding

Covalent modification by small ubiquitin‐related modifiers (SUMO) regulates p53 transcription activity through an undefined mechanism. Using reconstituted sumoylation components, we purified SUMO‐1‐conjugated p53 (Su‐p53) to near homogeneity. Su‐p53 exists in solution as a tetramer and interacts with p300 histone acetyltransferase as efficiently as the unmodified protein. Nevertheless, it fails to activate p53‐dependent chromatin transcription because of its inability to bind DNA. With sequential modification assays, we found that sumoylation of p53 at K386 blocks subsequent acetylation by p300, whereas p300‐acetylated p53 remains permissive for ensuing sumoylation at K386 and alleviates sumoylation‐inhibited DNA binding. While preventing the free form of p53 from accessing its cognate sites, sumoylation fails to disengage prebound p53 from DNA. The sumoylation‐deficient K386R protein, when expressed in p53‐null cells, exhibits higher transcription activity and binds better to the endogenous p21 gene compared with the wild‐type protein. These studies unravel a molecular mechanism underlying sumoylation‐regulated p53 function and further uncover a new role of acetylation in antagonizing the inhibitory effect of sumoylation on p53 binding to DNA.

The SUMO E3-ligase PIAS1 regulates the tumor suppressor PML and its oncogenic counterpart PML-RARA

The ubiquitin-like SUMO proteins covalently modify protein substrates and regulate their functional properties. In a broad spectrum of cancers, the tumor suppressor PML undergoes ubiquitin-mediated degradation primed by CK2 phosphorylation. Here, we report that the SUMO E3-ligase inhibitor PIAS1 regulates oncogenic signaling through its ability to sumoylate PML and the PML-RARA oncoprotein of acute promyelocytic leukemia (APL). PIAS1-mediated SUMOylation of PML promoted CK2 interaction and ubiquitin/proteasome-mediated degradation of PML, attenuating its tumor suppressor functions. In addition, PIAS1-mediated SUMOylation of PML-RARA was essential for induction of its degradation by arsenic trioxide, an effective APL treatment. Moreover, PIAS1 suppression abrogated the ability of arsenic trioxide to trigger apoptosis in APL cells. Lastly, PIAS1 was also essential for PML degradation in non-small cell lung carcinoma (NSCLC) cells, and PML and PIAS1 were inversely correlated in NSCLC cell lines and primary specimens. Together, our findings reveal novel roles for PIAS1 and the SUMOylation machinery in regulating oncogenic networks and the response to leukemia therapy.