Biomedical subjects

Kevin W Trotter

Publications and source records attributed to Kevin W Trotter.

5 recordsLinked to original sources

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↗

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

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

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↗