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BBA - Gene Regulatory Mechanisms (v.1789, #6-8)
Sin3: Master scaffold and transcriptional corepressor by Adrienne Grzenda; Gwen Lomberk; Jin-San Zhang; Raul Urrutia (pp. 443-450).
Sin3 was isolated over two decades ago as a negative regulator of transcription in budding yeast. Subsequent research has established the protein as a master transcriptional scaffold and corepressor capable of transcriptional silencing via associated histone deacetylases (HDACs). The core Sin3–HDAC complex interacts with a wide variety of repressors and corepressors, providing flexibility and expanded specificity in modulating chromatin structure and transcription. As a result, the Sin3/HDAC complex is involved in an array of biological and cellular processes, including cell cycle progression, genomic stability, embryonic development, and homeostasis. Abnormal recruitment of this complex or alteration of its enzymatic activity has been implicated in neoplastic transformation.
Keywords: Sin3; Transcription; Deacetylation; Repression; Histone; Chromatin
SUMO association with repressor complexes, emerging routes for transcriptional control by Mario Garcia-Dominguez ⁎; Jose C. Reyes ⁎ (pp. 451-459).
Covalent attachment of the small ubiquitin-like modifier (SUMO) to proteins constitutes a posttranslational modification intensely studied during the last decade. From the many proteins that serve as SUMO substrates, multiple functions have been assigned to this 100-amino acid polypeptide. Among them, a salient role in transcriptional regulation, and essentially in repression, prevails. Association of histone deacetylases (HDACs) with SUMO closely ties sumoylation with transcriptional repression. However, repressive effects linked to SUMO modification are not exclusively attributable to HDAC recruitment. Recently, several reports have revealed the importance of SUMO in the function of a variety of repressor complexes. In this respect, sumoylation is usually coupled to the establishment of heterochromatic states in the DNA. In this review, we summarize these recent reports and previous results concerning SUMO-mediated transcriptional repression. The analysis of the available data uncovers the importance not only of the covalent attachment of SUMO to proteins, but also of the presence in many proteins of SUMO interacting motifs (SIMs) that mediate non-covalent association with SUMO. In light of these findings we raise key questions and discuss why SUMO adopts a prominent role in establishing transcriptional repression in context of the activity, localization and architecture of chromatin-associated repressor complexes.
Keywords: SUMO; SIM; Transcription repression; Chromatin remodelling; Heterochromatin; HDAC
Identification of key DNA elements involved in promoter recognition by Mxr1p, a master regulator of methanol utilization pathway in Pichia pastoris by Balla Venkata Kranthi; Ritesh Kumar; Nallani Vijay Kumar; Desirazu N. Rao; Pundi N. Rangarajan ⁎ (pp. 460-468).
Mxr1p (methanol expression regulator 1) functions as a key regulator of methanol metabolism in the methylotrophic yeast Pichia pastoris. In this study, a recombinant Mxr1p protein containing the N-terminal zinc finger DNA binding domain was overexpressed and purified from E. coli cells and its ability to bind to promoter sequences of AOXI encoding alcohol oxidase was examined. In the AOX1 promoter, Mxr1p binds at six different regions. Deletions encompassing these regions result in a significant decrease in AOXI promoter activity in vivo. Based on the analysis of AOXI promoter sequences, a consensus sequence for Mxr1p binding consisting of a core 5′ CYCC 3′ motif was identified. When the core CYCC sequence is mutated to CYCA, CYCT or CYCM (M = 5-methylcytosine), Mxr1p binding is abolished. Though Mxr1p is the homologue of Saccharomyces cerevisiae Adr1p transcription factor, it does not bind to Adr1p binding site of S. cerevisiae alcohol dehydrogenase promoter ( ADH2UAS1). However, two point mutations convert ADH2UAS1 into an Mxr1p binding site. The identification of key DNA elements involved in promoter recognition by Mxr1p is an important step in understanding its function as a master regulator of the methanol utilization pathway in P. pastoris.
Keywords: Abbreviations; Mxr1p; methanol expression regulator 1; AOXI; alcohol oxidase I; P; AOX; promoter of; AOXI; DHAS; dihydroxyacetone synthase; PEX8; peroxin 8; MXRE; MXR response element; EMSA; electrophoretic mobility shift assay; Adr1p; alcohol dehydrogenase II synthesis regulator; HP0051; Helicobacter pylori; C; 5; cytosine methyltransferaseAlcohol oxidase; Pichia pastoris; Zinc finger protein; Methanol; Transcription; DNase I foot printing
Compensatory mutations in the L30e kink-turn RNA–protein complex by James J. Schweppe; Chaitanya Jain; Susan A. White ⁎ (pp. 469-476).
The S. cerevisiae ribosomal protein L30e is an autoregulatory protein that binds to its own pre-mRNA and mature mRNA to inhibit splicing and translation, respectively. The L30e RNA-binding element is a stem-asymmetric loop–stem that forms a kink-turn. A bacterial genetic system was designed to test the ability of protein variants to repress the expression of reporter mRNAs containing the L30e RNA-binding element. Initial screens revealed that changes in several RNA nucleotides had a measurable effect on repression of the reporter by the wild type protein. RNA mutants that reduce repression were screened against libraries of randomly mutagenized L30e proteins. These screens identified a glycine to serine mutation of L30e, which specifically restores activity to an RNA variant containing a U that replaces a helix-capping G. Similarly, an asparagine to alanine mutation was found to suppress a substitution at a position where the L30e RNA nucleotide extends out into the protein pocket. In addition, a compensatory RNA mutation within a defective RNA variant was found. The identification of these suppressors provides new insights into the architecture of a functional binding element and its recognition by an important RNA-binding protein.
Keywords: Abbreviations; BLAST; Basic Local Search Alignment Tool; BSA; Bovine Serum Albumin; DTT; Dithiothreitol; IPTG; Isopropyl β-; d; -thiogalactopyranoside; K; d; Dissociation constant; LB; Luria–Bertani; LBE; L30e RNA-binding element; MBP; Maltose-Binding Protein; NC; Non-canonical; OD; Optical density; ONPG; o-nitrophenyl-β-galactoside; RR; Repression Ratio; S/D; Shine–Dalgarno; TE; Tris–Ethylenediaminetetraacetate; X-gal; 5-bromo-4-chloro-3-indolyl-β-; d; -galactopyranosideRNA–protein interaction; Kink-turn RNA; Ribosomal protein; Two-plasmid screen; Suppressor mutation; L7Ae superfamily
Characterization of the region of the aryl hydrocarbon receptor required for ligand dependency of transactivation using chimeric receptor between Drosophila and Mus musculus by Kyoko Kudo; Takeshi Takeuchi; Yusuke Murakami; Masayuki Ebina; Hideaki Kikuchi ⁎ (pp. 477-486).
The aryl hydrocarbon receptor (AhR) is a ligand-activated transcriptional factor. Although 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) is high affinity and toxic to many vertebrate animals, invertebrate AhRs including Drosophila melanogaster AhR (spineless) have no ability to bind exogenous chemicals as ligands. To analyze the ligand-binding domain (LBD) of AhR, we used chimeras between mouse and Drosophila AhR. The chimeric AhR revealed that the LBD determines constitutive transactivation in Drosophila AhR or ligand-dependent activation in mouse AhR. The LBD was further divided into three blocks that corresponded to amino acids 230–300, 301–361, and 361–420 of the mouse sequence. Six chimeric proteins clarified that amino acids 291–350 of the Drosophila LBD, i.e. the middle region, were required to keep the protein in the active form in the absence of ligand binding, whereas in the mouse AhR, this region was required to maintain the protein in the inactive form in the absence of ligand. Furthermore, Arg346 in the middle region of the mouse LBD, was identified as amino acids that were critical for AhR activation by site-directed mutagenesis.
Keywords: Aryl hydrocarbon receptor; TCDD; Mouse; Drosophila; Hepa-1c1c7; S2
Interactome for auxiliary splicing factor U2AF65 suggests diverse roles by Justin R. Prigge; Sonya V. Iverson; Ashley M. Siders; Edward E. Schmidt (pp. 487-492).
U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is an essential component of the splicing machinery that is composed of two protein subunits, the 35 kDa U2AF35 (U2AF1) and the 65 kDa U2AF65 (U2AF2). U2AF interacts with various splicing factors within this machinery. Here we expand the list of mammalian splicing factors that are known to interact with U2AF65 as well as the list of nuclear proteins not known to participate in splicing that interact with U2AF65. Using a yeast two-hybrid system, we found fourteen U2AF65-interacting proteins. The validity of the screen was confirmed by identification of five known U2AF65-interacting proteins, including its heterodimeric partner, U2AF35. In addition to binding these known partners, we found previously unrecognized U2AF65 interactions with four splicing-related proteins (DDX39, SFRS3, SFRS18, SNRPA), two zinc finger proteins (ZFP809 and ZC3H11A), a U2AF65 homolog (RBM39), and two other regulatory proteins (DAXX and SERBP1). We report which regions of U2AF65 each of these proteins interacts with and we discuss their potential roles in regulation of pre-mRNA splicing, 3′-end mRNA processing, and U2AF65 sub-nuclear localization. These findings suggest expanded roles for U2AF65 in both splicing and non-splicing functions.
Keywords: U2AF; Splicing; Interactome