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Trevor K Archer

Publications and source records attributed to Trevor K Archer.

At least 19 recordsLinked to original sources
PubMed2007

Dynamic histone acetylation/deacetylation with progesterone receptor-mediated transcription.

Histone acetylation is a highly dynamic posttranslational modification that plays an important role in gene expression. Previous work showed that promoter histone deacetylation is accompanied by progesterone receptor (PR)-mediated activation of the mouse mammary tumor virus (MMTV) promoter. We investigated the role of this deacetylation and found that this histone deacetylation is not a singular event. In fact, histone acetylation at the MMTV promoter is highly dynamic, with an initial increase in acetylation followed by an eventual net deacetylation of histone H4. The timing of increase in acetylation of H4 coincides with the time at which PR, RNA polymerase II, and histone acetyltransferases cAMP response element-binding protein (CREB)-binding protein and p300 are recruited to the MMTV promoter. The timing in which histone H4 deacetylation occurs (after PR and RNA polymerase II recruitment) and the limited effect that trichostatin A and small interfering RNA knockdown of histone deacetylase (HDAC)3 have on MMTV transcription suggests that this deacetylation activity is not required for the initiation of PR-mediated transcription. Interestingly, two HDACs, HDAC1 and HDAC3, are already present at the MMTV before transcription activation. HDAC association at the MMTV promoter fluctuates during the hormone treatment. In particular, HDAC3 is temporarily undetected at the MMTV promoter within minutes after hormone treatment when the histone H4 acetylation increases but returns to the promoter near the time when histone acetylation levels start to decline. These results demonstrate the dynamic nature of coactivator/corepressor-promoter association and histone modifications such as acetylation during a transcription activation event.

Acetylation↗
PubMed2006

Chromatin-dependent cooperativity between site-specific transcription factors in vivo.

Accessing binding sites in DNA wrapped around histones in condensed chromatin is an obstacle that transcription factors must overcome to regulate gene expression. Here we demonstrate cooperativity between two transcription factors, the glucocorticoid receptor (GR) and nuclear factor 1 (NF1) to bind the mouse mammary tumor virus promoter organized as regular chromatin in vivo. This cooperativity is not observed when the promoter is introduced transiently into cells. Using RNA interference to deplete NF1 protein levels in the cells, we confirmed that NF1 promotes binding of GR to the promoter. Furthermore, we observed a similar synergism between GR and NF1 binding on the endogenous 11beta-hydroxysteroid dehydrogenase promoter, also regulated by GR and NF1. Our results suggest that the chromatin architecture of the promoters does not permit strong association of GR in the absence of NF1. Therefore we propose that cooperativity among DNA binding factors in binding to their cognate recognition sites in chromatin may be an important feature in the regulation of gene expression.

Animals↗
PubMed2006

Osmotic stress-dependent repression is mediated by histone H3 phosphorylation and chromatin structure.

Histone H3 phosphorylation has been linked to various environmental stress responses and specific chromatin structure. The role of H3 phosphorylation in the osmotic stress response was investigated on the mouse mammary tumor virus (MMTV) promoter in different chromatin configurations. Hormone-dependent transcription from the MMTV promoter is repressed by osmotic stress when the promoter is integrated and has a normal chromatin structure. However, when the MMTV promoter is transiently transfected, the chromatin structure is less organized, and hormone induction is not affected by osmotic stress. On the integrated MMTV promoter, phosphorylation of histone H3 serine 10 and 28 increases in response to osmotic stress, but the transient promoter shows no change. Hormone-dependent glucocorticoid receptor binding is reduced on the repressed promoter, and elevated H3 phosphorylation is temporally correlated with maximal MMTV repression Additionally, the protein kinase C inhibitor rottlerin, but not other kinase inhibitors, blocks both histone H3 phosphorylation and osmotic repression of MMTV transcription. Glucocorticoid receptor binding is inversely correlated with H3 phosphorylation, suggesting that displacement of the glucocorticoid receptor from the promoter is due to H3 phosphorylation and is the mechanism for the osmotic repression of hormone-dependent transcription.

Acetophenones↗
PubMed2006

Transcriptional silencing of the mouse mammary tumor virus promoter through chromatin remodeling is concomitant with histone H1 phosphorylation and histone H3 hyperphosphorylation at M phase.

We examined histone phosphorylation and their effects on glucocorticoid receptor (GR)-mediated activation of the mouse mammary tumor virus promoter (MMTV) in synchronized cells. In vivo protein expression studies suggest that both histones H1 and H3 are highly phosphorylated in mitotic-arrested cells in which GR is unable to remodel chromatin and recruit transcription factor NF1 to the promoter. Postmitotic cells show an open chromatin structure and efficient binding of NF1 to the promoter accompanied by reversing histone H1 and H3 phosphorylation level. In contrast, the acetylation status of histone H3 and H4 did not change in either condition. These results suggest that hyperphosphorylation of histone H1 and H3 leads to inhibition of GR-mediated chromatin remodeling and inactivation of MMTV by preventing the association of transcription factors to the promoter in vivo.

Animals↗
PubMed2005

Modulating molecular chaperone Hsp90 functions through reversible acetylation.

The molecular chaperone protein Hsp90 is a key regulator of approximately 100 'client' proteins crucial for numerous cell signaling processes. Consequently, understanding the molecular underpinnings that regulate Hsp90 activity is an important biological endeavor. Exciting new results now suggest that, at least for nuclear receptor activity, Hsp90 function is directly regulated by histone deacetylase 6 (HDAC6). These observations have consequences for various biological processes and potentially important implications for the development of cancer therapeutics.

Acetylation↗
PubMed2005

Changes in attitude, changes in latitude: nuclear receptors remodeling chromatin to regulate transcription.

Nuclear receptors (NRs) are a large family of ligand-dependent transcription factors that regulate important physiological processes. To activate or repress genes assembled naturally as chromatin, NRs recruit two distinct enzymatic activities, namely histone-modifying enzymes and ATP-dependent chromatin remodeling complexes, to alter local chromatin structure at target gene promoters. In this review, we examine the functional relationship between ATP-dependent chromatin remodeling complexes and NRs in the context of transcriptional regulation. Using the steroid-responsive mouse mammary tumor virus promoter as a model system, we discuss in detail the molecular mechanisms underlying the recruitment of these complexes and subsequent chromatin structure changes catalyzed by this group of enzymes. In addition, we extend the discussion to other NR-regulated promoters including the pS2 promoter. Finally, we summarize specific principles governing this critical relationship, identify unanswered questions and discuss the potential application of these principles in rational drug design.

Animals↗
PubMed2005

Swapping function of two chromatin remodeling complexes.

SWI/SNF- and ISWI-based complexes have distinct yet overlapping chromatin-remodeling activities in vitro and perform different roles in vivo. This leads to the hypothesis that the distinct remodeling functions of these complexes are specifically required for distinct biological tasks. By creating and characterizing chimeric proteins of BRG1 and SNF2h, the motor proteins of human SWI/SNF- and ISWI-based complexes, respectively, we found that a region that includes the ATPase domain specifies the outcome of the remodeling reaction in vitro. A chimeric protein based on BRG1 but containing the SNF2h ATPase domain formed an intact SWI/SNF complex that remodeled like SNF2h. This altered-function complex was active for remodeling and could stimulate expression from some, but not all, SWI/SNF responsive promoters in vivo. Thus, we were able to separate domains of BRG1 responsible for function from those responsible for SWI/SNF complex formation and demonstrate that remodeling functions are not interchangeable in vivo.

Adenosine Triphosphatases↗
PubMed2005

ATP-dependent chromatin remodeling complexes and their role in nuclear receptor-dependent transcription in vivo.

Nuclear receptors (NRs) are ligand-dependent transcription factors that mediate transcription of target genes in chromatin. Modulation of chromatin structure plays an important part in the NR-mediated transcription process. ATP-dependent chromatin remodeling complexes have been shown to be intimately involved in NR-mediated transcription. In this review, we examine the role of chromatin remodeling complexes in facilitating the recruitment of coregulators and basal transcription factors. In addition, the role of subunit specificity within the chromatin remodeling complexes, the complexes' influence on remodeling activity, and complexes' recruitment to the NR-responsive promoters are discussed.

Adenosine Triphosphate↗
PubMed2005

Regulating SWI/SNF subunit levels via protein-protein interactions and proteasomal degradation: BAF155 and BAF170 limit expression of BAF57.

The mammalian SWI/SNF chromatin remodeling complex, whose function is of critical importance in transcriptional regulation, contains approximately 10 protein components. The expression levels of the core SWI/SNF subunits, including BRG1/Brm, BAF155, BAF170, BAF60, hSNF/Ini1, and BAF57, are stoichiometric, with few to no unbound molecules in the cell. Here we report that exogenous expression of the wild type or certain deletion mutants of BAF57, a key subunit that mediates the interaction between the remodeling complex and transcription factors, results in diminished expression of endogenous BAF57. This down-regulation process is mediated by an increase in proteasome-dependent degradation of the BAF57 protein. Furthermore, the protein levels of BAF155/170 dictate the maximum cellular amount of BAF57. We mapped the domains responsible for the interaction between BAF57 and BAF155 and demonstrated that protein-protein interactions between them play an important role in this regulatory process. These findings provide insights into the physiological mechanisms responsible for maintaining the proper stoichiometric levels of the protein components comprising multimeric enzyme complexes.

Base Sequence↗
PubMed2004

Chromatin-dependent E1A activity modulates NF-kappaB RelA-mediated repression of glucocorticoid receptor-dependent transcription.

The role of chromatin-dependent regulatory mechanisms in the repression of glucocorticoid-dependent transcription from the murine mammary tumor virus (MMTV) promoter by p65 and E1A was investigated by using chromatin and transiently transfected reporters. The p65 RelA subunit of NF-kappaB represses MMTV expression on either transient or integrated reporters. In contrast, the viral oncoprotein E1A represses a transient but not an integrated MMTV. E1A repression is attenuated by chromatin, suggesting p65 but not E1A manipulates chromatin appropriately to inhibit the GR. Coexpression of p65 and E1A additively represses the transient MMTV but restores the transcriptional activation of the chromatin MMTV in response to glucocorticoids. This indicates that E1A has a dominant chromatin-dependent activity that attenuates repression by p65. E1A, p65, and GR bind the MMTV promoter, and chromatin remodeling enhances binding on both repressed and activated promoters. In addition, p65 requires Brg for repression of the integrated MMTV. This suggests that neither p65 repression nor E1A attenuation of repression results from an inhibition of remodeling that prevents transcription factor binding. Furthermore, p300/CBP is also required for both repression and attenuation by p65 and E1A. E1A and p65 mutants that do not bind p300/CBP are inactive, indicative of a requirement for p300/CBP-dependent complex formation for both repression and attenuation with chromatin. These data suggest that both the p65-dependent repression and the E1A-mediated attenuation of repression require the Brg1-dependent chromatin remodeling function and p300/CBP-dependent complex formation at a promoter assembled within chromatin.

Cell Line↗
PubMed2004

DNA binding sites for putative methylation boundaries in the unmethylated region of the BRCA1 promoter.

Changes in DNA methylation patterns are frequently observed in human cancers and are associated with a decrease in tumor suppressor gene expression. Hypermethylation of the BRCA1 promoter has been reported in a portion of sporadic breast tumours that correspond to a reduction in BRCA1 transcription and expression. Questions remain concerning the maintenance of methylation free zones in promoter regions of tumor suppressor genes in normal tissues. Sodium bisulfite based analysis of the BRCA1 promoter defines a methylation free zone in normal breast tissue that starts 650 bp upstream of the transcription start site and extends for 1.4 kb through most of the BRCA1 CpG island. We provide data implicating 2 proteins, Sp1 and CTCF, in the maintenance of this methylation-free zone. Both of these proteins have been shown to function as methylation boundaries in other genes. Four Sp1 sites have been identified in the BRCA1 promoter by gel shift assays. In vivo binding of Sp1 has been confirmed at 2 of these sites in the BRCA1 promoter and at 2 CTCF sites that flank the unmethylated region. Our data suggest that these DNA binding sites for Sp1 and CTCF may act as boundary elements that are important in maintaining genomic integrity by delineating the normal methylation free BRCA1 promoter. Inactivation or disruption of these boundaries may facilitate an epigenetic "hit", in this case DNA methylation, leading to BRCA1 downregulation and contributing to tumorigenesis, particularly the genesis of sporadic breast tumours.

BRCA1 Protein↗
PubMed2004

Modifying chromatin to permit steroid hormone receptor-dependent transcription.

Lipophilic hormones, including steroids, exert their physiological effects through binding to high-affinity superfamily of steroid hormone receptor (SR) proteins that function as ligand-dependent DNA binding transcription factors. To date, SR proteins are among a few transcription factors shown to directly interact with higher order chromatin structures to regulate gene expression. To perturb chromatin, SRs employ enzymatic multicomplexes that can either remodel or modify chromatin. Here we examine the current state of knowledge concerning multicomplex chromatin remodeling/modification machines and SR-dependent transcription. We will focus on the role of these protein-protein and chromatin-protein interactions in vivo with the MMTV promoter as a primary model. In addition, we discuss emerging evidence implicating chaperone proteins and proteasome degradation machinery in SR-mediated gene regulation within chromatin.

Adenosine Triphosphate↗
PubMed2004

Understanding global changes in histone H1 phosphorylation using mass spectrometry.

Histone post-translational modifications have been implicated in a variety of biological processes such as gene expression, DNA replication, and chromatin assembly. The modifications include methylation, acetylation, phosphorylation, ubiquitination, glycosylation, and ADP-ribosylation. For several years, we have been investigating the role of histone H1 phosphorylation in transcription using the hormone inducible mouse mammary tumor virus (MMTV) promoter. When mouse cells were exposed to prolonged treatment with dexamethasone, a significant decrease in the level of histone H1 phosphorylation was observed. Traditionally, Western analyses with anti-histone H1 and phospho-specific H1 antibodies were performed to observe changes in phosphorylation levels of the bulk H1 histones. More recently, we have applied electrospray ionization mass spectrometry to the analysis of histone H1 isoforms. Utilizing this approach, we have investigated the phosphorylation state of the specific H1 isoforms before and after prolonged treatment with dexamethasone. Specifically, we could determine that the relative phosphorylation levels of the histone H1.3, H1.4, and H1.5 isoforms decrease after prolonged hormone exposure. Recent advancements in mass spectrometry have proven invaluable toward the analysis of post-translational modifications on proteins. The continued developments in the area of mass spectrometry should provide new insights into not only the function of proteins but also into the basic regulatory mechanisms that control cellular functions.

Animals↗
PubMed2004

Reconstitution of glucocorticoid receptor-dependent transcription in vivo.

We developed a model system to study glucocorticoid receptor (GR)-mediated chromatin remodeling by the BRG1 complex. Introduction of the BRG1 ATPase into the SW-13 cell line initiates the formation of a functional remodeling complex. This complex is able to induce transcriptional activation from a transiently transfected promoter with wild-type and chromatin-remodeling-deficient BRG1 mutants, suggesting that the complex possesses a coactivator function independent from remodeling. Transactivation from a chromatin template requires the BRG1 remodeling function, which induces regions of hypersensitivity and transcription factor loading onto the integrated MMTV promoter. We report that BRG1 remodeling activity is required for GR-mediated transactivation and that this activity cannot be replaced by other ATP-dependent remodeling proteins. Further characterization of the BRG1-associated factors (BAFs) present in these cells (for example, the expression of BAF250 but not BAF180) reveals that the BAF complex rather than the polybromo-associated BAF complex is the necessary and sufficient chromatin-remodeling component with which the receptor functions in vivo. These results in conjunction with previous findings demonstrate that the GR functions with multiple forms of the SWI/SNF complex in vivo.

Adenosine Triphosphatases↗
PubMed2003

Chromatin remodeling by nuclear receptors.

The eukaryotic genome is structurally organized into nucleosomes to form chromatin, which regulates gene expression, in part, by controlling the accessibility of regulatory factors. When packaged as chromatin, many promoters are transcriptionally repressed, thus reducing the access of transcription factors to their binding sites. However, nuclear receptors (NRs) are a group of transcription factors that have the ability to access their binding sites in this repressive chromatin structure. Nuclear receptors are able to bind to their sites and recruit chromatin-remodeling proteins such as ATP-dependent chromatin-remodeling complexes and histone-modifying enzymes, resulting in transcriptional activation. In this review, we present the role of NRs in recruiting these chromatin-modifying enzymes by means of an extensively studied model system, the glucocorticoid receptor-mediated transactivation of the mouse mammary tumor virus (MMTV) promoter. We use these findings as a template to begin to understand the effect of chromatin changes on gene expression during spermatogenesis.

Animals↗
PubMed2003

Estrogen receptor-dependent proteasomal degradation of the glucocorticoid receptor is coupled to an increase in mdm2 protein expression.

Glucocorticoids and estrogens regulate a number of vital physiological processes. We developed a model breast cancer cell line, MCF-7 M, to examine potential mechanisms by which the ligand-bound estrogen receptor (ER) regulates glucocorticoid receptor (GR)-mediated transcription. MCF-7 cells, which endogenously express ERalpha, were stably transfected with mouse mammary tumor virus promoter-luciferase (MMTV-LUC) reporter and GR expression constructs. Our results demonstrate that treatment with estrogen agonists (17beta-estradiol [E2], diethylstilbestrol, genistein), but not antagonists (tamoxifen or raloxifene), for 48 h inhibits GR-mediated MMTV-LUC transcription and chromatin remodeling. Furthermore, estrogen agonists inhibit glucocorticoid induction of p21 mRNA and protein levels, suggesting that the repressive effect applies to other GR-regulated genes and proteins in MCF-7 cells. Importantly, GR transcriptional activity is compromised because treatment with estrogen agonists down regulates GR protein levels. The protein synthesis inhibitor cycloheximide and the proteasome inhibitor MG132 block E2-mediated decrease in GR protein levels, suggesting that estrogen agonists down regulate the GR via the proteasomal degradation pathway. In support of this, we demonstrate that E2-mediated GR degradation is coupled to an increase in p53 and its key regulator protein Mdm2 (murine double minute 2), an E3 ubiquitin ligase shown to target the GR for degradation. Using the chromatin immunoprecipitation assay, we demonstrate an E2-dependent recruitment of ERalpha to the Mdm2 promoter, suggesting a role of ER in the regulation of Mdm2 protein expression and hence the enhanced GR degradation in the presence of estrogen agonists. Our study shows that cross talk between the GR and ER involves multiple signaling pathways, indicative of the mechanistic diversity within steroid receptor-regulated transcription.

Blotting, Western↗
PubMed2003

BAF60a mediates critical interactions between nuclear receptors and the BRG1 chromatin-remodeling complex for transactivation.

Nuclear hormone receptors are ligand-dependent transcriptional regulators that modulate chromatin structure. However, the precise molecular mechanisms by which receptors recruit chromatin-remodeling activity are not fully elucidated. We show that in the absence of its ligand-binding domain, the glucocorticoid receptor (GR) is able to interact with both nuclear receptor coactivators and the BRG1 chromatin-remodeling complex in vivo. Individually, the GR makes direct interactions with BRG1-associated factor 60a (BAF60a) and BAF57, but not with BRG1, BAF155, or BAF170. Further, BAF60a possesses at least two interaction surfaces, one for GR and BRG1 and a second for BAF155 and BAF170. A GR mutant, GR(R488Q), that fails to interact with BAF60a in vitro has reduced chromatin-remodeling activity and reduced transcriptional activity from the promoter assembled as chromatin in vivo. Stable expression of a BAF60a truncation mutant, BAF60a4-140, caused chromatin-specific loss of GR functions in vivo. In the presence of the BAF60a mutant, the GR fails to interact with the BRG1 complex and consequently is also deficient in its ability to activate transcription from chromatin. Thus, in addition to previously identified BAF250, BAF60a may provide another critical and direct link between nuclear receptors and the BRG1 complex that is required for promoter recruitment and subsequent chromatin remodeling.

Binding Sites↗