scholarly journals Transcriptional gene silencing in mammalian cells by miRNA mimics that target gene promoters

2011 ◽  
Vol 39 (13) ◽  
pp. 5682-5691 ◽  
Author(s):  
Scott T. Younger ◽  
David R. Corey
Keyword(s):  
Gene Silencing ◽  
Mammalian Cells ◽  
Target Gene ◽  
Transcriptional Gene Silencing ◽  
Gene Promoters

scholarly journals MicroRNA-directed transcriptional gene silencing in mammalian cells

2008 ◽  
Vol 105 (42) ◽  
pp. 16230-16235 ◽  
Author(s):  
D. H. Kim ◽  
P. Saetrom ◽  
O. Snove ◽  
J. J. Rossi
Keyword(s):  
Gene Silencing ◽  
Mammalian Cells ◽  
Transcriptional Gene Silencing

scholarly journals siRNA Induced Transcriptional Gene Silencing in Mammalian Cells

Cell Cycle ◽  
2005 ◽  
Vol 4 (3) ◽  
pp. 442-448 ◽  
Author(s):  
Hiroaki Kawasaki ◽  
Kazumari Taira ◽  
Kevin V. Morris
Keyword(s):  
Gene Silencing ◽  
Mammalian Cells ◽  
Transcriptional Gene Silencing

A Common Regulatory Signature Associated with Barrier Insulators in Human Primary Erythroid Cells

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3868-3868
Author(s):  
Laurie A. Steiner ◽  
Yelena Maksimova ◽  
Vincent Schulz ◽  
Patrick G. Gallagher
Keyword(s):  
Gene Silencing ◽  
Mammalian Cells ◽  
Gene Promoters ◽  
Erythroid Cells ◽  
Chromatin Domains ◽  
Genome Wide ◽  
A Genome ◽  
Enhancer Blocking ◽  
In Erythroid Cells ◽  
Proximal Promoters

Abstract Abstract 3868 Insulators are DNA sequences and associated binding proteins that establish and/or maintain boundaries between regions of active and silenced chromatin domains. In higher organisms, there are 2 types of insulators, enhancer-blocking insulators, which establish chromatin domains to separate enhancers and promoters, and barrier insulators, which create a barrier to protect against heterochromatin-mediated gene silencing. Despite their role as critical regulators of tissue-specific gene expression, barrier insulators are poorly understood in mammalian cells, with much of our knowledge from studies of the barrier insulator in the chicken β-globin locus, cHS4. The DNA region of cHS4 that functions as a barrier binds upstream stimulatory factor (USF) proteins that recruit histone methyltransferase (HMT) activity, and histone acetyltransferase (HAT) activity, supporting a model that recruitment of enzymes and other proteins associated with activating histone modifications block the mechanism(s) that lead to spreading of gene-silencing. Our goal is to identify a regulatory signature associated with barrier insulators in erythroid cells. We utilized chromatin immunoprecipitation coupled with ultrahigh throughput Solexa sequencing (ChIP-seq) to generate genome-wide maps of regulatory and chromatin modifying proteins in erythroid cells. To generate cells for ChIP, human CD34+ cells were cultured in serum free media with erythropoietin to yield a population of CD71+/GPA+ erythroid cells (R3/R4 population). First, a genome-wide map of USF1 and USF2 occupancy in primary erythroid cells was created. A total of 19213 USF1 and 20115 USF2 sites of occupancy were identified. USF1 and USF2 frequently heterodimerize; co-localization was present at 15882 sites (83% of USF1 and 79% of USF2 sites). USF heterodimers were commonly located near proximal promoters (within 1KB of TSS, 48% of sites) and enhancers (>1kb from RefSeq gene, 30% of sites). To analyze co-localizing barrier-associated arginine methyltransferases from erythroid cells, ChIP-seq was performed with PRMT1 and PRMT4/CARM1. A total of 7062 PRMT1 sites and 15900 PRMT4 sites were identified. PRMT1 and PRMT4 were commonly found at sites of USF occupancy, with 6120 sites demonstrating occupancy of all four factors, consistent with the hypothesis that the USF proteins frequently recruit HMT's in mammalian cells. Sites of PRMT/USF co-occupancy were more likely to be at proximal promoters (68%) than sites of USF occupancy alone. Genome-wide occupancy of four acetyltransferases commonly found in erythroid cells, CBP, p300, PCAF, and SRC1, was also studied using ChIP-seq. 6804, 46932, 25688, and 25833 sites of occupancy were found for CBP, p300, PCAF, and SRC1 respectively. Co-localization with the p300, PCAF, and SRC1 with the USF/PRMT binding sites was common, occurring in 3825 sites. These sites were most commonly located near proximal promoters (71%) and enhancers (17%). In contrast, CBP co-localized with the USF/PRMT/p300/PCAF/SRC regions in only 10 locations and sites of CBP occupancy were more commonly found at enhancers (64%) and introns (29%) than at promoters (0.4%). Detection of barrier insulators near gene promoters is not surprising. Recent studies have revealed many similarities between barriers and promoters, including binding of specific transcription factors, and have led to the suggestion that barrier insulators have evolved as specialized derivatives of gene promoters, each with specific, yet discrete function. The regulatory protein CTCF mediates enhancer-blocking insulator activity. ChIP-seq was utilized to create a genome-wide map of CTCF binding in erythroid cells. 38503 sites of CTCF occupancy were identified. These sites were located at enhancers (41%), introns, (28%) and proximal promoters (18%). 4459 CTCF sites (12%) co-localized with regions of USF/PRMT/p300/PCAF/SRC binding. These sites most commonly occurred at promoters (65%) and enhancers (19%). The role of CTCF in barrier insulator function is controversial; our data are consistent with recent data demonstrating its mark at chromatin boundaries. The signature composed of USF/PRMT/p300/PCAF/SRC/CTCF was found in the well characterized functional erythroid barrier located in the ankyrin-1 gene proximal promoter region. These data indicate that a common regulatory signature is likely associated with barrier elements in erythroid cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The DREAM complex through its subunit Lin37 cooperates with Rb to initiate quiescence

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Christina FS Mages ◽  
Axel Wintsche ◽  
Stephan H Bernhart ◽  
Gerd A Müller
Keyword(s):  
Cell Cycle ◽  
Mammalian Cells ◽  
Target Gene ◽  
Gene Promoters ◽  
Essential Factor ◽  
Rb Protein ◽  
Essential Component ◽  
Related Proteins

The retinoblastoma Rb protein is an important factor controlling the cell cycle. Yet, mammalian cells carrying Rb deletions are still able to arrest under growth-limiting conditions. The Rb-related proteins p107 and p130, which are components of the DREAM complex, had been suggested to be responsible for a continued ability to arrest by inhibiting E2f activity and by recruiting chromatin-modifying enzymes. Here, we show that p130 and p107 are not sufficient for DREAM-dependent repression. We identify the MuvB protein Lin37 as an essential factor for DREAM function. Cells not expressing Lin37 proliferate normally, but DREAM completely loses its ability to repress genes in G0/G1 while all remaining subunits, including p130/p107, still bind to target gene promoters. Furthermore, cells lacking both Rb and Lin37 are incapable of exiting the cell cycle. Thus, Lin37 is an essential component of DREAM that cooperates with Rb to induce quiescence.

scholarly journals A Truncated Form of the Human CAF-1 p150 Subunit Impairs the Maintenance of Transcriptional Gene Silencing in Mammalian Cells

2001 ◽  
Vol 21 (6) ◽  
pp. 1953-1961 ◽  
Author(s):  
Thierry Tchénio ◽  
Jean-François Casella ◽  
Thierry Heidmann
Keyword(s):  
Gene Silencing ◽  
Reporter Gene ◽  
Mammalian Cells ◽  
Cell Model ◽  
Methylation Status ◽  
Dominant Negative ◽  
Transcriptional Gene Silencing ◽  
Dominant Negative Mutant ◽  
Control Of Gene Expression ◽  
Gene Copies

ABSTRACT Chromatin assembly factor 1 (CAF-1) is a protein complex formed of three subunits, p150, p60, and p48, conserved from the yeast Saccharomyces cerevisiae to humans, which can promote nucleosome assembly onto newly replicated DNA. In S. cerevisiae, deletion of the genes encoding any of the three CAF-1 subunits (cacΔ mutants), although nonlethal, results in a silencing defect of genes packaged into heterochromatin. Here we report on a mammalian cell model that we devised to monitor gene silencing and its reversal in a quantitative manner. This model relies on the use of a cell line stably transfected with a reporter gene in a silenced state. Reversal of reporter gene silencing was achieved upon treatment of the cells with 5-azacytidine, which resulted in the demethylation of the reporter gene copies. We show that expression of a cDNA for the human p150 CAF-1 subunit harboring 5′ truncations, but not that of a cDNA encoding the full-length p150 CAF-1 subunit, increases by more than 500-fold the frequency at which transcriptional silencing of the reporter gene copies is reversed in these cells. Reversal of gene silencing is dependent upon expression of a truncated protein, possibly acting as a dominant negative mutant of the wild-type CAF-1, is associated with alterations in chromatin structure as measured by an endonuclease sensitivity assay and is not associated with detectable changes in the methylation status of the silenced genes. These results suggest that the role of CAF-1 in the epigenetic control of gene expression has been conserved between yeast and mammals, despite the lack of DNA methylation in yeast chromatin.

VIGS technology: an attractive tool for functional genomics studies in legumes

2013 ◽  
Vol 40 (12) ◽  
pp. 1234 ◽  
Author(s):  
Stéphanie Pflieger ◽  
Manon M. S. Richard ◽  
Sophie Blanchet ◽  
Chouaib Meziadi ◽  
Valérie Geffroy
Keyword(s):  
Gene Silencing ◽  
Functional Genomics ◽  
Target Gene ◽  
Reverse Genetics ◽  
Plant Viruses ◽  
Transcriptional Gene Silencing ◽  
Full Potential ◽  
Legume Species ◽  
Virus Induced Gene Silencing ◽  
Apple Latent Spherical Virus

Legume species are among the most important crops worldwide. In recent years, six legume genomes have been completely sequenced, and there is now an urgent need for reverse-genetics tools to validate genes affecting yield and product quality. As most legumes are recalcitrant to stable genetic transformation, virus-induced gene silencing (VIGS) appears to be a powerful alternative technology for determining the function of unknown genes. VIGS technology is based on the property of plant viruses to trigger a defence mechanism related to post-transcriptional gene silencing (PTGS). Infection by a recombinant virus carrying a fragment of a plant target gene will induce homology-dependent silencing of the endogenous target gene. Several VIGS systems have been developed for legume species since 2004, including those based on Bean pod mottle virus, Pea early browning virus, and Apple latent spherical virus, and used in reverse-genetics studies of a wide variety of plant biological processes. In this work, we give an overview of the VIGS systems available for legumes, and present their successful applications in functional genomics studies. We also discuss the limitations of these VIGS systems and the future challenges to be faced in order to use VIGS to its full potential in legume species.

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