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Scholarly Works (5 results)

  • Thesis
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UC Riverside Electronic Theses and Dissertations (2012)

ABSTRACT OF THE DISSERTATION

Mechanisms of Core Promoter Sequence-dependent RNA Polymerase II Transcription

by

Muyu Xu

Doctor of Philosophy, Graduate Program in Biochemistry and Molecular Biology

University of California, Riverside, December 2012

Dr. Ernest Martinez, Chairperson

The TATA-box, Initiator (INR), Downstream Promoter Element (DPE), Motif Ten Element (MTE), TFIIB Recognition Element (BRE) and the other core promoter elements contribute to the diverse architecture of core promoters and are paramount for transcriptional activation. Diverse core promoters can communicate with enhancer-bound activators to contribute in the second level gene expression regulation. However, the mechanisms of transcription initiation catalyzed by different core promoters remain unknown and/or controversial. Because TFIID and TFIIB bind most of the core promoter elements, many scientists believe that different core promoters are regulated by the same set of general transcription factors. In contrast, other scientists including us insist that additional core promoter sequence-specific transcription factors besides the general transcription machinery are required to regulate transcription from different core promoters. Several lines of evidence support this hypothesis: a TAFs and Initiator dependent Cofactor 1 (TIC1) fraction requires for the TATA/INR synergy; a TIC2 fraction supports TATA-less core promoter directed transcription; CK2, PC4 and Mediator facilitate the Sp1-activated INR/DPE transcription in mammalian system and NC2 mediates the INR/DPE transcription in Drosophila system. Here, we further purify the TIC1 fraction and identified HMGA1 and Mediator as the effective components that support TATA/INR synergy in vitro. In addition, we also verify the TATA/INR specific role of HMGA1 in mammalian cells. Furthermore, we demonstrate HMGA1 interacts with TFIID and Mediator, and the acidic COOH-tail of HMGA1 is required but not sufficient for HMGA1 to interact with TFIID and Mediator. Accordingly, HMGA1 COOH-tail is also required to support the maximal transcriptional synergy between the TATA-box and the INR. Finally, analysis of activated transcription by Gal4-fusion activators and the beta-Actin gene (ACTB) promoter upstream activating sequences further demonstrates that preferential communication between activators and core promoters contributes to the gene expression regulation. Amazingly, a strict TATA-specificificity by the ACTB upstream activating sequences is revealed for the first time.

    Cover page: Mechanisms of Core Promoter Sequence-dependent RNA Polymerase II Transcription
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2016)
    Gene-specific activation by enhancers involves their communication with the basal RNA polymerase II transcription machinery at the core promoter. Core promoters are diverse and may contain a variety of sequence elements such as the TATA box, the Initiator (INR), and the downstream promoter element (DPE) recognized, respectively, by the TATA-binding protein (TBP) and TBP-associated factors of the TFIID complex. Core promoter elements contribute to the gene selectivity of enhancers, and INR/DPE-specific enhancers and activators have been identified. Here, we identify a TATA box-selective activating sequence upstream of the human β-actin (ACTB) gene that mediates serum response factor (SRF)-induced transcription from TATA-dependent but not INR-dependent promoters and requires the TATA-binding/bending activity of TBP, which is otherwise dispensable for transcription from a TATA-less promoter. The SRF-dependent ACTB sequence is stereospecific on TATA promoters but activates in an orientation-independent manner a composite TATA/INR-containing promoter. More generally, we show that SRF-regulated genes of the actin/cytoskeleton/contractile family tend to have a TATA box. These results suggest distinct TATA-dependent and INR-dependent mechanisms of TFIID-mediated transcription in mammalian cells that are compatible with only certain stereospecific combinations of activators, and that a TBP-TATA binding mechanism is important for SRF activation of the actin/cytoskeleton-related gene family.
      Cover page: Core promoter-specific gene regulation: TATA box selectivity and Initiator-dependent bi-directionality of serum response factor-activated transcription
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2016)
      Epstein-Barr virus (EBV) nuclear antigen 2 (EBNA2) plays an important role in driving immortalization of EBV-infected B cells through regulating the expression of many viral and cellular genes. We report a structural study of the tumor suppressor BS69/ZMYND11 C-terminal region, comprised of tandem coiled-coil-MYND domains (BS69CC-MYND), in complex with an EBNA2 peptide containing a PXLXP motif. The coiled-coil domain of BS69 self-associates to bring two separate MYND domains in close proximity, thereby enhancing the BS69 MYND-EBNA2 interaction. ITC analysis of BS69CC-MYND with a C-terminal fragment of EBNA2 further suggests that the BS69CC-MYND homodimer synergistically binds to the two EBNA2 PXLXP motifs that are respectively located in the conserved regions CR7 and CR8. Furthermore, we showed that EBNA2 interacts with BS69 and down-regulates its expression at both mRNA and protein levels in EBV-infected B cells. Ectopic BS69CC-MYND is recruited to viral target promoters through interactions with EBNA2, inhibits EBNA2-mediated transcription activation, and impairs proliferation of lymphoblastoid cell lines (LCLs). Substitution of critical residues in the MYND domain impairs the BS69-EBNA2 interaction and abolishes the BS69 inhibition of the EBNA2-mediated transactivation and LCL proliferation. This study identifies the BS69 C-terminal domains as an inhibitor of EBNA2, which may have important implications in development of novel therapeutic strategies against EBV infection.
        Cover page: BS69/ZMYND11 C-Terminal Domains Bind and Inhibit EBNA2
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2018)
        HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.
          Creative Commons 'BY-ND' version 4.0 license
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2018)
          HIV-1 Tat is a key regulator of viral transcription, however little is known about the mechanisms that control its turnover in T cells. Here we use a novel proteomics technique, called DiffPOP, to identify the molecular target of JIB-04, a small molecule compound that potently and selectively blocks HIV-1 Tat expression, transactivation, and virus replication in T cell lines. Mass-spectrometry analysis of whole-cell extracts from 2D10 Jurkat T cells revealed that JIB-04 targets Serine Hydroxymethyltransferase 2 (SHMT2), a regulator of glycine biosynthesis and an adaptor for the BRCC36 K63Ub-specific deubiquitinase in the BRISC complex. Importantly, knockdown of SHMT1,2 or BRCC36, or exposure of cells to JIB-04, strongly increased Tat K63Ub-dependent destruction via autophagy. Moreover, point mutation of multiple lysines in Tat, or knockdown of BRCC36 or SHMT1,2, was sufficient to prevent destruction of Tat by JIB-04. We conclude that HIV-1 Tat levels are regulated through K63Ub-selective autophagy mediated through SHMT1,2 and the BRCC36 deubiquitinase.
            Cover page: SHMT2 and the BRCC36/BRISC deubiquitinase regulate HIV-1 Tat K63-ubiquitylation and destruction by autophagy
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