scholarly journals Postintegrative Gene Silencing within the Sleeping Beauty Transposition System

2007 ◽  
Vol 27 (24) ◽  
pp. 8824-8833 ◽  
Author(s):  
Brian S. Garrison ◽  
Stephen R. Yant ◽  
Jacob Giehm Mikkelsen ◽  
Mark A. Kay
Keyword(s):  
Gene Silencing ◽  
Mammalian Cells ◽  
Transgene Expression ◽  
Trichostatin A ◽  
Sleeping Beauty ◽  
Histone Deacetylation ◽  
Phenotypic Analysis ◽  
Clonal Cell Lines ◽  
Mammalian Genomes

ABSTRACT The Sleeping Beauty (SB) transposon represents an important vehicle for in vivo gene delivery because it can efficiently and stably integrate into mammalian genomes. In this report, we examined transposon expression in human cells using a novel nonselective fluorescence-activated cell sorter-based method and discovered that SB integrates ∼20 times more frequently than previously reported within systems that were dependent on transgene expression and likely subject to postintegrative gene silencing. Over time, phenotypic analysis of clonal integrants demonstrated that SB undergoes additional postintegrative gene silencing, which varied based on the promoter used for transgene expression. Molecular and biochemical studies suggested that transposon silencing was influenced by DNA methylation and histone deacetylation because both 5-aza-2′-deoxycytidine and trichostatin A partially rescued transgene silencing in clonal cell lines. Collectively, these data reveal the existence of a multicomponent postintegrative gene silencing network that efficiently targets invading transposon sequences for transcriptional silencing in mammalian cells.

scholarly journals Position Effects Are Influenced by the Orientation of a Transgene with Respect to Flanking Chromatin

2001 ◽  
Vol 21 (1) ◽  
pp. 298-309 ◽  
Author(s):  
Yong-Qing Feng ◽  
Matthew C. Lorincz ◽  
Steve Fiering ◽  
John M. Greally ◽  
Eric E. Bouhassira
Keyword(s):  
Mammalian Cells ◽  
Transgene Expression ◽  
Methylation Status ◽  
Regulatory Elements ◽  
Cell Populations ◽  
Methylation Analysis ◽  
Position Effects ◽  
Methylation State ◽  
Cassette Exchange

ABSTRACT We have inserted two expression cassettes at tagged reference chromosomal sites by using recombinase-mediated cassette exchange in mammalian cells. The three sites of integration displayed either stable or silencing position effects that were dominant over the different enhancers present in the cassettes. These position effects were strongly dependent on the orientation of the construct within the locus, with one orientation being permissive for expression and the other being nonpermissive. Orientation-specific silencing, which was observed at two of the three site tested, was associated with hypermethylation but not with changes in chromatin structure, as judged by DNase I hypersensitivity assays. Using CRE recombinase, we were able to switch in vivo the orientation of the transgenes from the permissive to the nonpermissive orientation and vice versa. Switching from the permissive to the nonpermissive orientation led to silencing, but switching from the nonpermissive to the permissive orientation did not lead to reactivation of the transgene. Instead, transgene expression occurred dynamically by transcriptional oscillations, with 10 to 20% of the cells expressing at any given time. This result suggested that the cassette had been imprinted (epigenetically tagged) while it was in the nonpermissive orientation. Methylation analysis revealed that the methylation state of the inverted cassettes resembled that of silenced cassettes except that the enhancer had selectively lost some of its methylation. Sorting of the expressing and nonexpressing cell populations provided evidence that the transcriptional oscillations of the epigenetically tagged cassette are associated with changes in the methylation status of regulatory elements in the transgene. This suggests that transgene methylation is more dynamic than was previously assumed.

scholarly journals Induction of H3K9me3 and DNA methylation by tethered heterochromatin factors in Neurospora crassa

2017 ◽  
Vol 114 (45) ◽  
pp. E9598-E9607 ◽  
Author(s):  
Jordan D. Gessaman ◽  
Eric U. Selker
Keyword(s):  
Dna Methylation ◽  
Gene Silencing ◽  
Neurospora Crassa ◽  
Dna Methyltransferase ◽  
Catalytic Domain ◽  
De Novo ◽  
Histone Deacetylation ◽  
Heterochromatin Formation ◽  
Histone Deacetylase 1

Functionally different chromatin domains display distinct chemical marks. Constitutive heterochromatin is commonly associated with trimethylation of lysine 9 on histone H3 (H3K9me3), hypoacetylated histones, and DNA methylation, but the contributions of and interplay among these features are not fully understood. To dissect the establishment of heterochromatin, we investigated the relationships among these features using an in vivo tethering system in Neurospora crassa. Artificial recruitment of the H3K9 methyltransferase DIM-5 (defective in methylation-5) induced H3K9me3 and DNA methylation at a normally active, euchromatic locus but did not bypass the requirement of DIM-7, previously implicated in the localization of DIM-5, indicating additional DIM-7 functionality. Tethered heterochromatin protein 1 (HP1) induced H3K9me3, DNA methylation, and gene silencing. The induced heterochromatin required histone deacetylase 1 (HDA-1), with an intact catalytic domain, but HDA-1 was not essential for de novo heterochromatin formation at native heterochromatic regions. Silencing did not require H3K9me3 or DNA methylation. However, DNA methylation contributed to establishment of H3K9me3 induced by tethered HP1. Our analyses also revealed evidence of regulatory mechanisms, dependent on HDA-1 and DIM-5, to control the localization and catalytic activity of the DNA methyltransferase DIM-2. Our study clarifies the interrelationships among canonical aspects of heterochromatin and supports a central role of HDA-1–mediated histone deacetylation in heterochromatin spreading and gene silencing.

scholarly journals Dynamic Analysis of Proviral Induction and De Novo Methylation: Implications for a Histone Deacetylase-Independent, Methylation Density-Dependent Mechanism of Transcriptional Repression

2000 ◽  
Vol 20 (3) ◽  
pp. 842-850 ◽  
Author(s):  
Matthew C. Lorincz ◽  
Dirk Schübeler ◽  
Scott C. Goeke ◽  
Mark Walters ◽  
Mark Groudine ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone Acetylation ◽  
Histone Deacetylase ◽  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
De Novo ◽  
Trichostatin A ◽  
Histone Deacetylation ◽  
Over Time

ABSTRACT Methylation of cytosines in the CpG dinucleotide is generally associated with transcriptional repression in mammalian cells, and recent findings implicate histone deacetylation in methylation-mediated repression. Analyses of histone acetylation in in vitro-methylated transfected plasmids support this model; however, little is known about the relationships among de novo DNA methylation, transcriptional repression, and histone acetylation state. To examine these relationships in vivo, we have developed a novel approach that permits the isolation and expansion of cells harboring expressing or silent retroviruses. MEL cells were infected with a Moloney murine leukemia virus encoding the green fluorescent protein (GFP), and single-copy, silent proviral clones were treated weekly with the histone deacetylase inhibitor trichostatin A or the DNA methylation inhibitor 5-azacytidine. Expression was monitored concurrently by flow cytometry, allowing for repeated phenotypic analysis over time, and proviral methylation was determined by Southern blotting and bisulfite methylation mapping. Shortly after infection, proviral expression was inducible and the reporter gene and proviral enhancer showed a low density of methylation. Over time, the efficacy of drug induction diminished, coincident with the accumulation of methyl-CpGs across the provirus. Bisulfite analysis of cells in which 5-azacytidine treatment induced GFP expression revealed measurable but incomplete demethylation of the provirus. Repression could be overcome in late-passage clones only by pretreatment with 5-azacytidine followed by trichostatin A, suggesting that partial demethylation reestablishes the trichostatin-inducible state. These experiments reveal the presence of a silencing mechanism which acts on densely methylated DNA and appears to function independently of histone deacetylase activity.

scholarly journals p53-Dependent Transcriptional Repression of c-myc Is Required for G1 Cell Cycle Arrest

2005 ◽  
Vol 25 (17) ◽  
pp. 7423-7431 ◽  
Author(s):  
Jenny S. L. Ho ◽  
Weili Ma ◽  
Daniel Y. L. Mao ◽  
Samuel Benchimol
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Trichostatin A ◽  
Histone Deacetylation ◽  
Dependent Manner ◽  
Cycle Arrest ◽  
Mouse Tissues

ABSTRACT The ability of p53 to promote apoptosis and cell cycle arrest is believed to be important for its tumor suppression function. Besides activating the expression of cell cycle arrest and proapoptotic genes, p53 also represses a number of genes. Previous studies have shown an association between p53 activation and down-regulation of c-myc expression. However, the mechanism and physiological significance of p53-mediated c-myc repression remain unclear. Here, we show that c-myc is repressed in a p53-dependent manner in various mouse and human cell lines and mouse tissues. Furthermore, c-myc repression is not dependent on the expression of p21WAF1. Abrogating the repression of c-myc by ectopic c-myc expression interferes with the ability of p53 to induce G1 cell cycle arrest and differentiation but enhances the ability of p53 to promote apoptosis. We propose that p53-dependent cell cycle arrest is dependent not only on the transactivation of cell cycle arrest genes but also on the transrepression of c-myc. Chromatin immunoprecipitation assays indicate that p53 is bound to the c-myc promoter in vivo. We report that trichostatin A, an inhibitor of histone deacetylases, abrogates the ability of p53 to repress c-myc transcription. We also show that p53-mediated transcriptional repression of c-myc is accompanied by a decrease in the level of acetylated histone H4 at the c-myc promoter and by recruitment of the corepressor mSin3a. These data suggest that p53 represses c-myc transcription through a mechanism that involves histone deacetylation.

scholarly journals A conserved BAH module within mammalian BAHD1 connects H3K27me3 to Polycomb gene silencing

2021 ◽  
Author(s):  
Huitao Fan ◽  
Yiran Guo ◽  
Yi-Hsuan Tsai ◽  
Aaron J. Storey ◽  
Arum Kim ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Histone Acetylation ◽  
Mammalian Cells ◽  
Target Genes ◽  
Direct Interaction ◽  
Histone Deacetylation ◽  
List Type ◽  
Bah Domain ◽  
Organismal Development ◽  
Point Mutagenesis

ABSTRACTTrimethylation of histone H3 lysine 27 (H3K27me3) is important for gene silencing and imprinting, (epi)genome organization and organismal development. In a prevalent model, the functional readout of H3K27me3 in mammalian cells is achieved through the H3K27me3-recognizing chromodomain harbored within the chromobox (CBX) component of canonical Polycomb repressive complex 1 (cPRC1), which induces chromatin compaction and gene repression. Here, we report that binding of H3K27me3 by a Bromo Adjacent Homology (BAH) domain harbored within BAH domain-containing protein 1 (BAHD1) is required for overall BAHD1 targeting to chromatin and for optimal repression of the H3K27me3-demarcated genes in mammalian cells. Disruption of direct interaction between BAHD1BAH and H3K27me3 by point mutagenesis leads to chromatin remodeling, notably, increased histone acetylation, at its Polycomb gene targets. Mice carrying an H3K27me3-interaction-defective mutation of Bahd1BAH causes marked embryonic lethality, showing a requirement of this pathway for normal development. Altogether, this work demonstrates an H3K27me3-initiated signaling cascade that operates through a conserved BAH “reader” module within BAHD1 in mammals.Key PointsBAHD1BAH is a functionally validated mammalian “reader” of H3K27me3, mediating BAHD1 targeting for gene silencing.BAHD1BAH connects H3K27me3 together with histone deacetylation, an integral step of gene silencing.BAHD1BAH-mediated functional readout of H3K27me3 is essential for organismal development.Graphic abstractA mammalian H3K27me3-transduction pathway operates through an H3K27me3-specific ‘reader’ module (BAH) of BAHD1, which assembles a complex with corepressors (HDACs and others) for suppressing histone acetylation and repressing expression at Polycomb target genes.

scholarly journals The Growth Suppressor PML Represses Transcription by Functionally and Physically Interacting with Histone Deacetylases

2001 ◽  
Vol 21 (7) ◽  
pp. 2259-2268 ◽  
Author(s):  
Wen-Shu Wu ◽  
Sadeq Vallian ◽  
Edward Seto ◽  
Wen-Ming Yang ◽  
Diane Edmondson ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Cell Cycle Progression ◽  
Trichostatin A ◽  
Specific Inhibitor ◽  
Promyelocytic Leukemia ◽  
Histone Deacetylation ◽  
Growth Suppressor

ABSTRACT The growth suppressor promyelocytic leukemia protein (PML) is disrupted by the chromosomal translocation t(15;17) in acute promyelocytic leukemia (APL). PML plays a key role in multiple pathways of apoptosis and regulates cell cycle progression. The present study demonstrates that PML represses transcription by functionally and physically interacting with histone deacetylase (HDAC). Transcriptional repression mediated by PML can be inhibited by trichostatin A, a specific inhibitor of HDAC. PML coimmunoprecipitates a significant level of HDAC activity in several cell lines. PML is associated with HDAC in vivo and directly interacts with HDAC in vitro. The fusion protein PML-RARα encoded by the t(15;17) breakpoint interacts with HDAC poorly. PML interacts with all three isoforms of HDAC through specific domains, and its expression deacetylates histone H3 in vivo. Together, the results of our study show that PML modulates histone deacetylation and that loss of this function in APL alters chromatin remodeling and gene expression. This event may contribute to the development of leukemia.

scholarly journals Development of a Novel Helper-Dependent Adenovirus-Epstein-Barr Virus Hybrid System for the Stable Transformation of Mammalian Cells

2004 ◽  
Vol 78 (12) ◽  
pp. 6556-6566 ◽  
Author(s):  
Oliver Dorigo ◽  
Jose S. Gil ◽  
Sean D. Gallaher ◽  
Brenton T. Tan ◽  
Maria G. Castro ◽  
...  
Keyword(s):  
Hybrid System ◽  
Mammalian Cells ◽  
Transgene Expression ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Genetic Defects ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.

Effect of serum triiodothyronine on regulation of cardiac gene expression: role of histone acetylation

2005 ◽  
Vol 289 (4) ◽  
pp. H1506-H1511 ◽  
Author(s):  
Sara Danzi ◽  
Peter Dubon ◽  
Irwin Klein
Keyword(s):  
Thyroid Hormone ◽  
Thyroid Hormone Receptor ◽  
Trichostatin A ◽  
Histone Deacetylation ◽  
Constant Infusion ◽  
Histone H4 ◽  
Time Treatment ◽  
Cardiac Gene ◽  
Relationship Of

Thyroid hormone regulates the transcription of several important cardiac genes. Although the thyroid gland produces predominantly thyroxine (T4), it is triiodothyronine (T3) that is transported across the sarcolemma and binds to nuclear thyroid hormone receptor proteins; yet various studies suggest that serum T3 levels do not accurately reflect cellular T3 action. To address this question, we studied the dose-response relationship of T3 administered by constant infusion in hypothyroid animals with the simultaneous in vivo transcription rate of the cardiac-specific α-myosin heavy chain (MHC) gene, measured by quantitating α-MHC heteronuclear (hn)RNA content. Constant infusion of 4 μg T3·kg body wt−1·day−1 for 3 days normalized serum T3 and restored transcription to euthyroid levels; in contrast, daily injections of the same dose increased α-MHC transcription by only 55% of that obtained by infusion. Although infusion of T3 at 1.25 μg T3·kg body wt−1·day−1 was not sufficient to restore serum T3 to normal, it was capable of restoring transcription to normal at 3 days, but when administered for 12 days, transcription of α-MHC was found to be 50% of euthyroid levels, demonstrating a decreased sensitivity to T3 over time. Treatment with trichostatin A (TSA) to inhibit histone deacetylation increased levels of total nuclear acetylated histone H4 by almost 50% but was without effect on the real-time PCR measures of α-MHC hnRNA. TSA administered together with T3 (10 μg T3/kg body wt) significantly increased transcription of α-MHC after 30 h, thus demonstrating a potential role for histones as cofactors in the T3 regulation of cardiac α-MHC transcription.

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