scholarly journals Analysis of In-Vivo LacR-Mediated Gene Repression Based on the Mechanics of DNA Looping

PLoS ONE ◽  
2006 ◽  
Vol 1 (1) ◽  
pp. e136 ◽  
Author(s):  
Yongli Zhang ◽  
Abbye E. McEwen ◽  
Donald M. Crothers ◽  
Stephen D. Levene
Keyword(s):  
Dna Looping ◽  
Gene Repression

scholarly journals TAF7L modulates brown adipose tissue formation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Haiying Zhou ◽  
Bo Wan ◽  
Ivan Grubisic ◽  
Tommy Kaplan ◽  
Robert Tjian
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Core Promoter ◽  
Uncoupling Protein ◽  
Dna Looping ◽  
Chromatin Interactions ◽  
Brown Adipose ◽  
Important Regulator

Brown adipose tissue (BAT) plays an essential role in metabolic homeostasis by dissipating energy via thermogenesis through uncoupling protein 1 (UCP1). Previously, we reported that the TATA-binding protein associated factor 7L (TAF7L) is an important regulator of white adipose tissue (WAT) differentiation. In this study, we show that TAF7L also serves as a molecular switch between brown fat and muscle lineages in vivo and in vitro. In adipose tissue, TAF7L-containing TFIID complexes associate with PPARγ to mediate DNA looping between distal enhancers and core promoter elements. Our findings suggest that the presence of the tissue-specific TAF7L subunit in TFIID functions to promote long-range chromatin interactions during BAT lineage specification.

scholarly journals Gene repression by minimal lac loops in vivo

2010 ◽  
Vol 38 (22) ◽  
pp. 8072-8082 ◽  
Author(s):  
Laura M. Bond ◽  
Justin P. Peters ◽  
Nicole A. Becker ◽  
Jason D. Kahn ◽  
L. James Maher
Keyword(s):  
Gene Repression

FOG-1 Directly Binds to a Chromatin Remodeling and Deacetylase-Containing Complex To Mediate Transcriptional Repression by GATA-1.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 355-355
Author(s):  
Wei Hong ◽  
Minako Nakazawa ◽  
Ying-Yu Chen ◽  
Rajashree Kori ◽  
Carrie Rakowski ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Histone Deacetylation ◽  
Gene Repression ◽  
Single Point Mutation ◽  
Mutant Form ◽  
Erythroid Cells ◽  
Binding Studies ◽  
Nucleosome Remodeling ◽  
N Terminus

Abstract Terminal erythroid maturation requires coordinated activation of erythroid marker genes and repression of genes associated with the undifferentiated state. These gene expression patterns are mediated by the concerted action of the erythroid transcription factor GATA-1 and its cofactor FOG-1 that can activate or repress transcription depending on promoter context. We and others showed previously that one mechanism by which FOG-1 functions is to facilitate GATA-1 association with certain DNA target sites in vivo. Using gene complementation studies of GATA-1-ablated erythroid cells, we show that at several GATA-1-repressed target genes (c-kit, c-myc and GATA-2) FOG-1 is dispensable for GATA-1 occupancy in vivo but essential for gene repression and histone deacetylation. To examine how FOG-1 functions as co-repressor we performed affinity chromatography, conventional protein purification and in vitro binding studies to identify proteins that bind FOG-1. We discovered that FOG-1 directly associates with the nucleosome remodeling and histone deacetylase complex NURD. This interaction is mediated by a small conserved domain at the N-terminus of FOG-1 and the MTA-1 subunit of NURD. Association of FOG-1 with NURD occurs in vivo and depends on an intact N-terminus of FOG-1. A series of point mutations across the N-terminus of FOG-1 revealed a tight correlation between NURD binding and transcriptional repression. In particular, a single point mutation at the N-terminus of FOG-1 that abrogated NURD binding also blocked gene repression by FOG-1. Finally, the ability of GATA-1 to repress transcription was impaired in erythroid cells expressing a mutant form of FOG-1 that is defective for NURD binding. Together, these studies show that FOG-1 and very likely other FOG proteins are bona fide co-repressors that link GATA proteins to histone deacetylation and nucleosome remodeling via a novel protein interaction module.

scholarly journals Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and   repressors

2013 ◽  
Vol 111 (1) ◽  
pp. 349-354 ◽  
Author(s):  
D. G. Priest ◽  
L. Cui ◽  
S. Kumar ◽  
D. D. Dunlap ◽  
I. B. Dodd ◽  
...  
Keyword(s):  
Long Range ◽  
Dna Looping

scholarly journals Reconcilingin vitroandin vivoactivities of engineered, LacI-based repressor proteins: Contributions of DNA looping and operator sequence variation

2018 ◽  
Author(s):  
Sudheer Tungtur ◽  
Kristen M. Schwingen ◽  
Joshua J. Riepe ◽  
Chamitha J. Weeramange ◽  
Liskin Swint-Kruse
Keyword(s):  
Amino Acid ◽  
Dna Looping ◽  
Published Data ◽  
Binding Affinities ◽  
Ligand Specificity ◽  
Primary Operator ◽  
Repressor Proteins ◽  
Operator Sequence

AbstractOne way to create new components for synthetic transcription circuits is to re-purpose naturally occurring transcription factor proteins and their cognate DNA operators. For the proteins, re-engineering can be accomplished via domain recombination (to create chimeric regulators) and/or amino acid substitutions. The resulting activities of new protein regulators are often assessedin vitrousing a representative operator. However, when functioningin vivo, transcription factors can interact with multiple operators. We comparedin vivoandin vitroresults for two LacI-based transcription repressor proteins, their mutational variants, and four operator sequences. The two sets of repressor variants differed in their overallin vivorepression, even though theirin vitrobinding affinities for the primary operator spanned the same range. Here, we show that the offset can be explained by different abilities to simultaneously bind and “loop” two DNA operators. Furtherin vitrostudies of the looping-competent repressors were carried out to measure binding to a secondary operator sequence. Surprisingly, binding to this operator was largely insensitive to amino acid changes in the repressor protein.In vitroexperiments with additional operators and analyses of published data indicates that amino acid changes in these repressor proteins leads to complicated changes in ligand specificity. These results raise new considerations for engineering components of synthetic transcription circuits and – more broadly – illustrate difficulties encountered when trying to extrapolate information about specificity determinant positions among protein homologs.

scholarly journals Topoisomerases I and II facilitate condensin DC translocation to organize and repress X chromosomes in C. elegans

2021 ◽  
Author(s):  
Ana Karina Morao ◽  
Jun Kim ◽  
Daniel Obaji ◽  
Siyu Sun ◽  
Sevinc Ercan
Keyword(s):  
Long Range ◽  
X Chromosome ◽  
Molecular Motors ◽  
Topoisomerase Ii ◽  
Dna Looping ◽  
Loop Formation ◽  
X Chromosomes ◽  
C Elegans ◽  
And Control

Condensin complexes are evolutionarily conserved molecular motors that translocate along DNA and form loops. While condensin-mediated DNA looping is thought to direct the chain-passing activity of topoisomerase II to separate sister chromatids, it is not known if topological constraints in turn regulate loop formation in vivo. Here we applied auxin inducible degradation of topoisomerases I and II to determine how DNA topology affects the translocation of an X chromosome specific condensin that represses transcription for dosage compensation in C. elegans (condensin DC). We found that both topoisomerases colocalize with condensin DC and control its movement at different genomic scales. TOP-2 depletion hindered condensin DC translocation over long distances, resulting in accumulation around its X-specific recruitment sites and shorter Hi-C interactions. In contrast, TOP-1 depletion did not affect long-range spreading but resulted in accumulation of condensin DC within expressed gene bodies. Both TOP-1 and TOP-2 depletions resulted in X chromosome transcriptional upregulation indicating that condensin DC translocation at both scales is required for its function in gene repression. Together the distinct effects of TOP-1 and TOP-2 on condensin DC distribution revealed two distinct modes of condensin DC association with chromatin: long-range translocation that requires decatenation/unknotting of DNA and short-range translocation across genes that requires resolution of transcription-induced supercoiling.

Physical and Functional Association of PLZF with Histone Methyl Transferase and Histone Demethylase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 731-731
Author(s):  
Itsaso Hormaeche ◽  
Kim Rice ◽  
Joti Marango ◽  
Fabien Guidez ◽  
Arthur Zelent ◽  
...  
Keyword(s):  
Histone Methylation ◽  
Transcriptional Repression ◽  
Target Genes ◽  
Histone H3 ◽  
Promyelocytic Leukemia ◽  
Gene Repression ◽  
Histone Demethylases ◽  
Methyl Transferase ◽  
Histone Methyl Transferase

Abstract The promyelocytic leukemia zinc finger protein (PLZF) is a transcription factor fused to RARα in the t(11;17) translocation associated with retinoic acid resistant acute promyelocytic leukemia (APL). As a result of this chromosomal abnormality, two oncogenic proteins are produced, PLZF-RARα and RARα-PLZF. Wild type PLZF is expressed in CD34+ progenitor cells and declines during differentiation. PLZF is a tumor suppressor that causes cell cycle arrest, downregulating genes such as cyclinA2 and c-myc. We previously showed that transcriptional repression by PLZF is mediated by the recruitment of histone deacetylases to target genes, this being critical for its ability to control growth and affect RAR target genes. We now show that PLZF alters the methylation state of histones in its target genes. A biotinylated form of PLZF co-purified in cells along with a histone methyl transferase (HMT) activity for native histones. Using mutant histone H3 tail peptides, we showed that this activity methylated histone H3 on lysine 9 (H3K9me). Tagged forms of PLZF as well as endogenous PLZF co-precipitated in vivo with G9a histone methyl transferase, an enzyme that can mono and dimethylate H3K9 in euchromatin subject to gene repression. The interaction of PLZF with G9a required the presence of the N-terminal BTB/POZ domain as well as a second, more C-terminal, repression domain of PLZF. Given the newly found role of active histone demethylation in gene control we also tested the interaction of PLZF with LSD1, an enzyme associated with gene repression that demethylates H3K4. As in the case of G9a, the interaction of PLZF with LSD1 required both repression domains, suggesting, that these proteins may be part of a multi-protein complex containing multiple contact points with PLZF. Expression of G9a or LSD1 augmented transcriptional repression mediated by PLZF on reporter genes, indicating a functional interaction between histone methylation modifiers and PLZF. To determine the ability of PLZF to affect chromatin methylation in vivo, a Gal4-PLZF fusion protein was expressed in cells containing a chromatin-embedded Gal4-tk-Luciferase reporter gene. In the presence of PLZF, a chromatin immunoprecipitation experiment showed an increase in H3K9 methylation of the target gene while H3K4 methylation decreased, consistent with the ability of PLZF to interact with LSD1 and G9a. Lastly we compared the ability of the histone modifying proteins to interact with the APL fusion proteins PLZF-RARα, PML-RARα and NPM-RARα. Co-precipitation experiments showed a robust interaction between PLZF-RARα and G9a and LSD1 while the PML-RARα and NPM-RARα fusions bound these proteins significantly less avidly. Collectively all these data indicate that specific histone methylation is an important mode of action of PLZF in gene repression. The retinoic acid resistance of t(11;17)-APL may be related to its ability to interact with HMTs and histone demethylases. Hence therapeutic targeting of HMTs and histone demethylases might be considered as a novel mode of therapy in APL and other hematological malignancies.

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