Reading and Function of a Histone Code Involved in Targeting Corepressor Complexes for Repression

ABSTRACT A central question in histone code theory is how various codes are recognized and utilized in vivo. Here we show that TBL1 and TBLR1, two WD-40 repeat proteins in the corepressor SMRT/N-CoR complexes, are functionally redundant and essential for transcriptional repression by unliganded thyroid hormone receptors (TR) but not essential for transcriptional activation by liganded TR. TBL1 and TBLR1 bind preferentially to hypoacetylated histones H2B and H4 in vitro and have a critical role in targeting the corepressor complexes to chromatin in vivo. We show that targeting SMRT/N-CoR complexes to the deiodinase 1 gene (D1) requires at least two interactions, one between unliganded TR and SMRT/N-CoR and the other between TBL1/TBLR1 and hypoacetylated histones. Neither interaction alone is sufficient for the stable association of the corepressor complexes with the D1 promoter. Our data support a feed-forward working model in which deacetylation exerted by initial unstable recruitment of SMRT/N-CoR complexes via their interaction with unliganded TR generates a histone code that serves to stabilize their own recruitment. Similarly, we find that targeting of the Sin3 complex to pericentric heterochromatin may also follow this model. Our studies provide an in vivo example that a histone code is not read independently but is recognized in the context of other interactions.

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Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.4.2091-2103.1993 ◽

1993 ◽

Vol 13 (4) ◽

pp. 2091-2103

Author(s):

S Türkel ◽

P J Farabaugh

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcriptional Activation ◽

Binding Sites ◽

Transcriptional Repression ◽

Physiological Control ◽

Reading Frame ◽

Negative Region ◽

Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

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Abstract 356: Krueppel-Like Factor 15 Regulates Wnt/β-Catenin Transcription and Controls Cardiac Progenitor Cell Fate in the Postnatal Heart

Circulation Research ◽

10.1161/res.111.suppl_1.a356 ◽

2012 ◽

Vol 111 (suppl_1) ◽

Author(s):

Claudia Noack ◽

Maria P Zafiriou ◽

Anke Renger ◽

Hans J Schaeffer ◽

Martin W Bergmann ◽

...

Keyword(s):

Cell Fate ◽

Transcriptional Activation ◽

Progenitor Cell ◽

Cellular Localization ◽

Target Genes ◽

Critical Role ◽

Isolated Cells ◽

Cardiac Progenitor Cell

Wnt/β-catenin signaling controls adult heart remodeling partly by regulating cardiac progenitor cell (CPC) differentiation. We now identified and characterized a novel cardiac interaction of the transcription factor Krueppel-like factor 15 (KLF15) with the Wnt/β-catenin signaling on adult CPCs. In vitro mutation, reporter gene assays and co-localization studies revealed that KLF15 requires two distinct domains for nuclear localization and for repression of β-catenin-mediated transcription. KLF15 had no effect on β-catenin stability or cellular localization, but interacted with its co-factor TCF4, which is required for activation of β-catenin target gene expression. Moreover, increased TCF4 ubiquitination was induced by KLF15. In line with this finding we found KLF15 to interact with the Nemo-like kinase, which was shown to phosphorylate and target TCF4 for degradation. In vivo analyses of adult Klf15 functional knock-out (KO) vs. wild-type (WT) mice showed a cardiac β-catenin-mediated transcriptional activation and reduced TCF4 degradation along with cardiac dysfunction assessed by echocardiography (n=10). FACS analysis of the CPC enriched-population of KO vs. WT mice revealed a significant reduction of cardiogenic-committed precursors identified as Sca1+/αMHC+ (0.8±0.2% vs. 1.8±0.1%) and Tbx5+ (3.5±0.3% vs. 5.2±0.5%). In contrast, endothelial Sca1+/CD31+ cells were significantly higher in KO mice (11.3±0.4% vs. 8.6±0.4%; n≥9). In addition, Sca1+ isolated cells of Klf15 KO showed increased RNA expression of endothelial markers von Willebrand Factor, CD105, and Flk1 along with upregulation of β-catenin target genes. CPCs co-cultured on adult fibroblasts resulted in increased endothelial Flk1 cells and reduction of αMHC and Hand1 cardiogenic cells in KO vs. WT CPCs (n=9). Treating these co-cultures with Quercetin, an inhibitor of nuclear β-catenin, resulted in partial rescue of the observed phenotype. This study uncovers a critical role of KLF15 for the maintenance of cardiac tissue homeostasis. Via inhibition of β-catenin transcription, KLF15 controls cardiomyogenic cell fate similar to embryonic cardiogenesis. This knowledge may provide a tool for activation of endogenous CPCs in the postnatal heart.

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HIF-1–dependent repression of equilibrative nucleoside transporter (ENT) in hypoxia

Journal of Experimental Medicine ◽

10.1084/jem.20050177 ◽

2005 ◽

Vol 202 (11) ◽

pp. 1493-1505 ◽

Cited By ~ 227

Author(s):

Holger K. Eltzschig ◽

Parween Abdulla ◽

Edgar Hoffman ◽

Kathryn E. Hamilton ◽

Dionne Daniels ◽

...

Keyword(s):

Transcriptional Repression ◽

Molecular Mechanisms ◽

Hypoxia Inducible Factor ◽

Nucleoside Transporter ◽

In Vivo Models ◽

Equilibrative Nucleoside Transporter ◽

Hypoxia Inducible Factor 1 ◽

And Function

Extracellular adenosine (Ado) has been implicated as central signaling molecule during conditions of limited oxygen availability (hypoxia), regulating physiologic outcomes as diverse as vascular leak, leukocyte activation, and accumulation. Presently, the molecular mechanisms that elevate extracellular Ado during hypoxia are unclear. In the present study, we pursued the hypothesis that diminished uptake of Ado effectively enhances extracellular Ado signaling. Initial studies indicated that the half-life of Ado was increased by as much as fivefold after exposure of endothelia to hypoxia. Examination of expressional levels of the equilibrative nucleoside transporter (ENT)1 and ENT2 revealed a transcriptionally dependent decrease in mRNA, protein, and function in endothelia and epithelia. Examination of the ENT1 promoter identified a hypoxia inducible factor 1 (HIF-1)–dependent repression of ENT1 during hypoxia. Using in vitro and in vivo models of Ado signaling, we revealed that decreased Ado uptake promotes vascular barrier and dampens neutrophil tissue accumulation during hypoxia. Moreover, epithelial Hif1α mutant animals displayed increased epithelial ENT1 expression. Together, these results identify transcriptional repression of ENT as an innate mechanism to elevate extracellular Ado during hypoxia.

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The microtubule modulator RanBP10 plays a critical role in regulation of platelet discoid shape and degranulation

Blood ◽

10.1182/blood-2009-04-216804 ◽

2009 ◽

Vol 114 (27) ◽

pp. 5532-5540 ◽

Cited By ~ 24

Author(s):

Stefan Kunert ◽

Imke Meyer ◽

Silke Fleischhauer ◽

Martin Wannack ◽

Janine Fiedler ◽

...

Keyword(s):

Fetal Liver ◽

Critical Role ◽

Mutant Mice ◽

Axis Ratio ◽

Prolonged Bleeding Time ◽

Normal Platelet ◽

And Function ◽

Granule Release

Abstract Terminally mature megakaryocytes undergo dramatic cellular reorganization to produce hundreds of virtually identical platelets. A hallmark feature of this process is the generation of an elaborate system of branched protrusions called proplatelets. We recently identified RanBP10 as a tubulin-binding protein that is concentrated along polymerized microtubules in mature megakaryocytes. RanBP10 depletion in vitro caused the disturbance of polymerized filaments. Here we study the function of RanBP10 in vivo by generating deficient mice using a gene-trap approach. Mutant mice show normal platelet counts, and fetal liver-derived megakaryocytes reveal only slightly reduced proplatelet formation. However, ultrastructural analysis unveiled a significantly increased geometric axis ratio for resting platelets, and many platelets exhibited disorders in microtubule filament numbers and localization. Mutant mice showed a markedly prolonged bleeding time. Granule release, a process that depends on internal contraction of the microtubule marginal coil, also was reduced. Flow cytometry analysis revealed reduced expression of CD62P and CD63 after PAR4-peptide stimulation. These data suggest that RanBP10 plays an essential role in hemostasis and in maintaining microtubule dynamics with respect to both platelet shape and function.

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Adenovirus Protein VII Condenses DNA, Represses Transcription, and Associates with Transcriptional Activator E1A

Journal of Virology ◽

10.1128/jvi.78.12.6459-6468.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6459-6468 ◽

Cited By ~ 36

Author(s):

Jeffrey S. Johnson ◽

Yvonne N. Osheim ◽

Yuming Xue ◽

Margaux R. Emanuel ◽

Peter W. Lewis ◽

...

Keyword(s):

Transcriptional Activation ◽

Transcriptional Repression ◽

Function Analysis ◽

Specific Protein ◽

In Vitro Translation ◽

Major Protein ◽

Lampbrush Chromosomes ◽

Binding Studies

ABSTRACT Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.

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Distinct Tissue-Specific Roles for Thyroid Hormone Receptors β and α1 in Regulation of Type 1 Deiodinase Expression

Molecular Endocrinology ◽

10.1210/mend.15.3.0605 ◽

2001 ◽

Vol 15 (3) ◽

pp. 467-475 ◽

Cited By ~ 4

Author(s):

Lori L. Amma ◽

Angel Campos-Barros ◽

Zhendong Wang ◽

Björn Vennström ◽

Douglas Forrest

Keyword(s):

Thyroid Hormone ◽

Hormone Receptors ◽

Critical Role ◽

Thyroid Hormone Receptors ◽

Minor Role ◽

Tissue Specific ◽

Mice Liver

Abstract Type 1 deiodinase (D1) metabolizes different forms of thyroid hormones to control levels of T3, the active ligand for thyroid hormone receptors (TR). The D1 gene is itself T3-inducible and here, the regulation of D1 expression by TRα1 and TRβ, which act as T3-dependent transcription factors, was investigated in receptor-deficient mice. Liver and kidney D1 mRNA and activity levels were reduced in TRβ−/− but not TRα1−/− mice. Liver D1 remained weakly T3 inducible in TRβ–/– mice whereas induction was abolished in double mutant TRα1–/–TRβ–/– mice. This indicates that TRβ is primarily responsible for regulating D1 expression whereas TRα1 has only a minor role. In kidney, despite the expression of both TRα1 and TRβ, regulation relied solely on TRβ, thus revealing a marked tissue restriction in TR isotype utilization. Although TRβ and TRα1 mediate similar functions in vitro, these results demonstrate differential roles in regulating D1 expression in vivo and suggest that tissue-specific factors and structural distinctions between TR isotypes contribute to functional specificity. Remarkably, there was an obligatory requirement for a TR, whether TRβ or TRα1, for any detectable D1 expression in liver. This suggests a novel paradigm of gene regulation in which the TR sets both basal expression and the spectrum of induced states. Physiologically, these findings suggest a critical role for TRβ in regulating the thyroid hormone status through D1-mediated metabolism.

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A proline-rich sequence unique to MEK1 and MEK2 is required for raf binding and regulates MEK function.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5214 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5214-5225 ◽

Cited By ~ 137

Author(s):

A D Catling ◽

H J Schaeffer ◽

C W Reuter ◽

G R Reddy ◽

M J Weber

Keyword(s):

Protein Interactions ◽

Critical Role ◽

Phosphorylation Site ◽

Specific Protein ◽

Protein Protein Interactions ◽

Serum Stimulation ◽

And Function

Mammalian MEK1 and MEK2 contain a proline-rich (PR) sequence that is absent both from the yeast homologs Ste7 and Byr1 and from a recently cloned activator of the JNK/stress-activated protein kinases, SEK1/MKK4. Since this PR sequence occurs in MEKs that are regulated by Raf family enzymes but is missing from MEKs and SEKs activated independently of Raf, we sought to investigate the role of this sequence in MEK1 and MEK2 regulation and function. Deletion of the PR sequence from MEK1 blocked the ability of MEK1 to associate with members of the Raf family and markedly attenuated activation of the protein in vivo following growth factor stimulation. In addition, this sequence was necessary for efficient activation of MEK1 in vitro by B-Raf but dispensable for activation by a novel MEK1 activator which we have previously detected in fractionated fibroblast extracts. Furthermore, we found that a phosphorylation site within the PR sequence of MEK1 was required for sustained MEK1 activity in response to serum stimulation of quiescent fibroblasts. Consistent with this observation, we observed that MEK2, which lacks a phosphorylation site at the corresponding position, was activated only transiently following serum stimulation. Finally, we found that deletion of the PR sequence from a constitutively activated MEK1 mutant rendered the protein nontransforming in Rat1 fibroblasts. These observations indicate a critical role for the PR sequence in directing specific protein-protein interactions important for the activation, inactivation, and downstream functioning of the MEKs.

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Residue R113 Is Essential for PhoP Dimerization and Function: a Residue Buried in the Asymmetric PhoP Dimer Interface Determined in the PhoPN Three-Dimensional Crystal Structure

Journal of Bacteriology ◽

10.1128/jb.185.1.262-273.2003 ◽

2003 ◽

Vol 185 (1) ◽

pp. 262-273 ◽

Cited By ~ 27

Author(s):

Yinghua Chen ◽

Catherine Birck ◽

Jean-Pierre Samama ◽

F. Marion Hulett

Keyword(s):

Crystal Structure ◽

Dna Binding ◽

Transcriptional Activation ◽

Salt Bridge ◽

Pho Regulon ◽

Dimer Interface ◽

And Function ◽

Negative Phenotype

ABSTRACT Bacillus subtilis PhoP is a member of the OmpR/PhoB family of response regulators that is directly required for transcriptional activation or repression of Pho regulon genes in conditions under which Pi is growth limiting. Characterization of the PhoP protein has established that phosphorylation of the protein is not essential for PhoP dimerization or DNA binding but is essential for transcriptional regulation of Pho regulon genes. DNA footprinting studies of PhoP-regulated promoters showed that there was cooperative binding between PhoP dimers at PhoP-activated promoters and/or extensive PhoP oligomerization 3′ of PhoP-binding consensus repeats in PhoP-repressed promoters. The crystal structure of PhoPN described in the accompanying paper revealed that the dimer interface between two PhoP monomers involves nonidentical surfaces such that each monomer in a dimer retains a second surface that is available for further oligomerization. A salt bridge between R113 on one monomer and D60 on another monomer was judged to be of major importance in the protein-protein interaction. We describe the consequences of mutation of the PhoP R113 codon to a glutamate or alanine codon and mutation of the PhoP D60 codon to a lysine codon. In vivo expression of either PhoPR113E, PhoPR113A, or PhoPD60K resulted in a Pho-negative phenotype. In vitro analysis showed that PhoPR113E was phosphorylated by PhoR (the cognate histidine kinase) but was unable to dimerize. Monomeric PhoPR113E∼P was deficient in DNA binding, contributing to the PhoPR113E in vivo Pho-negative phenotype. While previous studies emphasized that phosphorylation was essential for PhoP function, data reported here indicate that phosphorylation is not sufficient as PhoP dimerization or oligomerization is also essential. Our data support the physiological relevance of the residues of the asymmetric dimer interface in PhoP dimerization and function.

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Gut microbiota regulation of P-glycoprotein in the intestinal epithelium in maintenance of homeostasis

Microbiome ◽

10.1186/s40168-021-01137-3 ◽

2021 ◽

Vol 9 (1) ◽

Author(s):

Sage E. Foley ◽

Christine Tuohy ◽

Merran Dunford ◽

Michael J. Grey ◽

Heidi De Luca ◽

...

Keyword(s):

Microbial Community ◽

Intestinal Epithelium ◽

Critical Role ◽

Secondary Bile Acid ◽

Core Microbiome ◽

P Glycoprotein ◽

Mucosal Barrier Function ◽

And Function

Abstract Background P-glycoprotein (P-gp) plays a critical role in protection of the intestinal epithelia by mediating efflux of drugs/xenobiotics from the intestinal mucosa into the gut lumen. Recent studies bring to light that P-gp also confers a critical link in communication between intestinal mucosal barrier function and the innate immune system. Yet, despite knowledge for over 10 years that P-gp plays a central role in gastrointestinal homeostasis, the precise molecular mechanism that controls its functional expression and regulation remains unclear. Here, we assessed how the intestinal microbiome drives P-gp expression and function. Results We have identified a “functional core” microbiome of the intestinal gut community, specifically genera within the Clostridia and Bacilli classes, that is necessary and sufficient for P-gp induction in the intestinal epithelium in mouse models. Metagenomic analysis of this core microbial community revealed that short-chain fatty acid and secondary bile acid production positively associate with P-gp expression. We have further shown these two classes of microbiota-derived metabolites synergistically upregulate P-gp expression and function in vitro and in vivo. Moreover, in patients suffering from ulcerative colitis (UC), we find diminished P-gp expression coupled to the reduction of epithelial-derived anti-inflammatory endocannabinoids and luminal content (e.g., microbes or their metabolites) with a reduced capability to induce P-gp expression. Conclusion Overall, by means of both in vitro and in vivo studies as well as human subject sample analysis, we identify a mechanistic link between cooperative functional outputs of the complex microbial community and modulation of P-gp, an epithelial component, that functions to suppress overactive inflammation to maintain intestinal homeostasis. Hence, our data support a new cross-talk paradigm in microbiome regulation of mucosal inflammation.

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Simple biochemical features underlie transcriptional activation domain diversity and dynamic, fuzzy binding to Mediator

eLife ◽

10.7554/elife.68068 ◽

2021 ◽

Vol 10 ◽

Author(s):

Adrian L Sanborn ◽

Benjamin T Yeh ◽

Jordan T Feigerle ◽

Cynthia V Hao ◽

Raphael J L Townshend ◽

...

Keyword(s):

Transcriptional Activation ◽

Structural Constraints ◽

Sequence Motifs ◽

Activation Domains ◽

Activator Proteins ◽

Transcriptional Activation Domain ◽

Shape Complementarity ◽

And Function

Gene activator proteins comprise distinct DNA-binding and transcriptional activation domains (ADs). Because few ADs have been described, we tested domains tiling all yeast transcription factors for activation in vivo and identified 150 ADs. By mRNA display, we showed that 73% of ADs bound the Med15 subunit of Mediator, and that binding strength was correlated with activation. AD-Mediator interaction in vitro was unaffected by a large excess of free activator protein, pointing to a dynamic mechanism of interaction. Structural modeling showed that ADs interact with Med15 without shape complementarity ('fuzzy' binding). ADs shared no sequence motifs, but mutagenesis revealed biochemical and structural constraints. Finally, a neural network trained on AD sequences accurately predicted ADs in human proteins and in other yeast proteins, including chromosomal proteins and chromatin remodeling complexes. These findings solve the longstanding enigma of AD structure and function and provide a rationale for their role in biology.

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