Recruitment of chromatin remodelling factors during gene activation via the glucocorticoid receptor N-terminal domain.

We have shown that yeast mutants with defects in the Ada adaptor proteins are defective in hormone-dependent gene activation by ectopically expressed human glucocorticoid receptor (GR). Others have shown that the Ada2 protein is required for physical interactions between some activation domains and TBP (TATA-binding protein), whereas the Gcn5 (Ada4) protein has a histone acetyltransferase (HAT) activity. Although all HAT enzymes are able to acetylate histone substrates, some also acetylate non-histone proteins. Taken together, these observations suggest that the Ada proteins have the ability to effect different steps in the process of gene activation. It has recently been shown that the Ada proteins are present in two distinct protein complexes, the Ada complex and a larger SAGA complex. Our recent work has focused on determining (1) which of the Ada-containing complexes mediates gene activation by GR, (2) whether the HAT activity encoded by GCN5 is required for GR-dependent gene activation, (3) whether the Ada proteins contribute to GR-mediated activation at the level of chromatin remodelling and (4) how the role of these HAT complexes is integrated with other chromatin remodelling activities during GR-mediated gene activation. Our results suggest a model in which GR recruits the SAGA complex and that this contributes to chromatin remodelling via a mechanism involving the acetylation of histones. Furthermore, recruitment of the SWI/SNF remodelling complex also has a role in GR-mediated activation that is independent of the role of SAGA. These complexes are similar to analogous mammalian complexes and therefore these results are likely to be relevant to the human system.

Comparison of nucleosome remodeling by the yeast transcription factor Pho4 and the glucocorticoid receptor.

Chromatin reorganization of the PHO5 and murine mammary tumor virus (MMTV) promoters is triggered by binding of either Pho4 or the glucocorticoid receptor (GR), respectively. In order to compare the ability of Pho4 and GR to remodel chromatin and activate transcription, hybrid promoter constructs were created by insertion of the MMTV B nucleosome sequence into the PHO5 promoter and then transformed into a yeast strain expressing GR. Activation of either Pho4 (by phosphate depletion) or GR (by hormone addition) resulted in only slight induction of hybrid promoter activity. However, simultaneous activation of both Pho4 and GR resulted in synergistic activation to levels exceeding that of the wild type PHO5 promoter. Under these conditions, Pho4 completely disrupted the nucleosome containing its binding site. In contrast, GR had little effect on the stability of the MMTV B nucleosome. A minimal transactivation domain of the GR fused to the Pho4 DNA-binding domain is capable of efficiently disrupting the nucleosome with a Pho4-binding site, whereas the complementary hybrid protein (Pho4 activation domain, GR DNA-binding domain) does not labilize the B nucleosome. Therefore, we conclude that significant activation by Pho4 requires nucleosome disruption, whereas equivalent transcriptional activation by GR is not accompanied by overt perturbation of nucleosome structure. Our results show that the DNA-binding domains of the two factors play critical roles in determining how chromatin structure is modified during promoter activation.

myc boxes, which are conserved in myc family proteins, are signals for protein degradation via the proteasome.

Cellular levels of the rapidly degraded c-myc protein play an important role in determining the proliferation status of cells. Increased levels of c-myc are frequently associated with rapidly proliferating tumor cells. We show here that myc boxes I and II, found in the N termini of all members of the myc protein family, function to direct the degradation of the c-myc protein. Both myc boxes I and II contain sufficient information to independently direct the degradation of otherwise stably expressed proteins to which they are fused. At least part of the myc box-directed degradation occurs via the proteasome. The mechanism of myc box-directed degradation appears to be conserved between yeast and mammalian cells. Our results suggest that the myc boxes may play an important role in regulating the level and activity of the c-myc protein.