During the last year we have made significant progress on this project both with respect to the understanding of the mechanisms by which steroid receptors function in vivo and the importance of chromatin to the regulation of transcription in cancer cells. The results from our work can be highlighted by exploring our progress in three specific areas indicated below. We developed a model breast cancer cell lines to examine potential mechanisms by which the ligand bound estrogen receptor (ERa) regulates glucocorticoid receptor (GRa)-mediated transcription. MCF-7 cells, which endogenously express ERa, were stably transfected with MMTV-luciferase reporter and GR expression constructs. Treatment with estrogen agonists [17b-estradiol (E2), diethylstilbestrol, genistein], but not antagonists [tamoxifen or raloxifene], inhibits GR-mediated MMTV 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/proteins in MCF-7 cells. GR transcriptional activity is compromised because treatments with estrogen agonists down regulate 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 both p53 and it?s key regulator protein Mdm2, an E3 ubiquitin ligase shown to target the GR for degradation. Using ChIP assay we demonstrate an E2-dependent recruitment of ERa 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. The molecular mechanisms by which nuclear hormone receptors recruit chromatin-remodeling activity are not fully elucidated. We showed that in the absence of its ligand-binding domain the glucocorticoid receptor (GR) is able to interact with both nuclear receptor co-activators and the BRG1 chromatin-remodeling complex in vivo. Individually, the GR makes direct interactions with 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. To dissect the precise role of nuclear factor one (NF1) in chromatin remodeling and transcriptional activation, we used linker-scanning mutants of transcription factor binding sites on the MMTV promoter. We compared the NF1 mutant promoter in the context of non-chromatin templates (transient transfection) and templates organized as chromatin (stable transfection) to understand the effect of chromatin on factor binding and transcription. We showed that on a non-chromatin template, mutation in the NF1 binding site reduces both basal and hormone dependent transcription. This suggests that NF1 is required for transcription in the absence of organized chromatin. We also found that binding of NF1 on a non-chromatin template is independent of mutation in hormone response elements (HRE) or the octamer transcription factor (OTF) binding site. In contrast, the binding of OTF proteins to a non-chromatin template was found to be dependent on the binding of NF1, which may imply that NF1 has a stabilizing effect on OTF binding. On a chromatin template, mutation in the NF1 binding site does not affect the positioning of nucleosomes on the promoter. We also show that in the absence of NF1 binding, GR mediated chromatin remodeling of nucleosome B is reduced and hormone dependent activation of transcription is abolished. Further, we demonstrate that NF1 is required for the association of BRG1 complex on the promoter. These results suggest that NF1 may participate in chromatin remodeling activities in addition to directly enhancing transcription and that in the absence of its binding site GR is unable to effectively recruit the remodeling complex to the promoter.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES071006-05
Application #
6838568
Study Section
(LRDT)
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2003
Total Cost
Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
United States
Zip Code
Singh, Ajeet Pratap; Foley, Julie; Tandon, Arpit et al. (2017) A role for BRG1 in the regulation of genes required for development of the lymphatic system. Oncotarget 8:54925-54938
Singh, Ajeet P; Foley, Julie F; Rubino, Mark et al. (2016) Brg1 Enables Rapid Growth of the Early Embryo by Suppressing Genes That Regulate Apoptosis and Cell Growth Arrest. Mol Cell Biol 36:1990-2010
Lavender, Christopher A; Cannady, Kimberly R; Hoffman, Jackson A et al. (2016) Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters. PLoS Genet 12:e1006224
Takaku, Motoki; Grimm, Sara A; Shimbo, Takashi et al. (2016) GATA3-dependent cellular reprogramming requires activation-domain dependent recruitment of a chromatin remodeler. Genome Biol 17:36
Yang, Jun; Bennett, Brian D; Luo, Shujun et al. (2015) LIN28A Modulates Splicing and Gene Expression Programs in Breast Cancer Cells. Mol Cell Biol 35:3225-43
Wade, Staton L; Langer, Lee F; Ward, James M et al. (2015) MiRNA-Mediated Regulation of the SWI/SNF Chromatin Remodeling Complex Controls Pluripotency and Endodermal Differentiation in Human ESCs. Stem Cells 33:2925-35
Zhang, Xiaoli; Li, Bing; Li, Wenguo et al. (2014) Transcriptional repression by the BRG1-SWI/SNF complex affects the pluripotency of human embryonic stem cells. Stem Cell Reports 3:460-74
Singh, Ajeet Pratap; Archer, Trevor K (2014) Analysis of the SWI/SNF chromatin-remodeling complex during early heart development and BAF250a repression cardiac gene transcription during P19 cell differentiation. Nucleic Acids Res 42:2958-75
Rana, Ritu; Coulter, Sherry; Kinyamu, Harriet et al. (2013) RBCK1, an E3 ubiquitin ligase, interacts with and ubiquinates the human pregnane X receptor. Drug Metab Dispos 41:398-405
Singh, Ajeet Pratap; Cummings, Connie A; Mishina, Yuji et al. (2013) SOX8 regulates permeability of the blood-testes barrier that affects adult male fertility in the mouse. Biol Reprod 88:133

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