We identify Xenopus NF‐Y as a key regulator of acetylation responsiveness for the Xenopus hsp70 promoter within chromatin assembled in Xenopus oocyte nuclei. Y‐box sequences are required for the assembly of DNase I‐hypersensitive sites in the hsp70 promoter, and for transcriptional activation both by inhibitors of histone deacetylase and by the p300 acetyltransferase. The viral oncoprotein E1A interferes with both of these activation steps. We clone Xenopus NF‐YA, NF‐YB and NF‐YC and establish that NF‐Y is the predominant Y‐box‐binding protein in Xenopus oocyte nuclei. NF‐Y interacts with p300 in vivo and is itself a target for acetylation by p300. Transcription from the hsp70 promoter in chromatin can be enhanced further by heat shock factor. We suggest two steps in chromatin modification at the Xenopus hsp70 promoter: first the binding of NF‐Y to the Y‐boxes to pre‐set chromatin and second the recruitment of p300 to modulate transcriptional activity.

Gene expression requires binding of transcription factors to their cognate DNA response elements, the latter being often integrated into sequence-specifically positioned nucleosomes. To investigate the constraints imposed on factor-DNA recognition by the nucleosomal organization, we studied the binding of glucocorticoid receptor to a single glucocorticoid response element (GRE) displaying four different rotational frames in three different translational positions in reconstituted nucleosomes. We demonstrate that rotational setting of the GRE per se is important for its accessibility. Furthermore, the effects of rotational positioning of the GRE are different for different translational positions of the GRE in the nucleosome. A GRE placed near the nucleosomal dyad is totally blocked by rotating it 180 degrees so that the major groove of the GRE faces the histone octamer. If, on the other hand, the GRE is placed about 40 bp from the nucleosome dyad, then the 180 degrees rotation of the GRE still allows glucocorticoid receptor binding, albeit with a sixfold lower affinity than the peripherally oriented GRE. This suggests that both the rotational positioning and the translational positioning function as a framework for transcription factor response elements in gene regulation.

The Kruppel-associated box (KRAB)-associated co-repressor KAP1 is an essential nuclear co-repressor for the KRAB zinc finger protein superfamily of transcriptional factors. Ataxia telangiectasia mutated (ATM)-Chk2 and ATM- and Rad3-related (ATR)-Chk1 are two primary kinase signaling cascades activated in response to DNA damage. A growing body of evidence suggests that ATM and ATR phosphorylate KAP1 at Ser-824 in response to DNA damage and regulate KAP1-dependent chromatin condensation, DNA repair, and gene expression. Here, we show that, depending on the type of DNA damage that occurs, KAP1 Ser-473 can be phosphorylated by ATM-Chk2 or ATR-Chk1 kinases. Phosphorylation of KAP1 at Ser-473 attenuated its binding to the heterochromatin protein 1 family proteins and inhibited its transcriptional repression of KRAB-zinc finger protein (KRAB-ZFP) target genes. Moreover, KAP1 Ser-473 phosphorylation induced by DNA damage stimulated KAP1-E2F1 binding. Overexpression of heterochromatin protein 1 significantly inhibited E2F1-KAP1 binding. Elimination of KAP1 Ser-473 phosphorylation increased E2F1-targeted proapoptotic gene expression and E2F1-induced apoptosis in response to DNA damage. Furthermore, loss of phosphorylation of KAP1 Ser-473 led to less BRCA1 focus formation and slower kinetics of loss of γH2AX foci after DNA damage. KAP1 Ser-473 phosphorylation was required for efficient DNA repair and cell survival in response to DNA damage. Our studies reveal novel functions of KAP1 Ser-473 phosphorylation under stress.

Transcriptional co-activator p300, which contains an intrinsic histone acetyltransferase activity, is required for an array of important cellular processes. Tight control of p300 function is critic...

Myogenic regulatory factors (MRF) are the master regulators of skeletal myogenesis. Among the MRFs, Myf5 is the earliest to be expressed and is regulated by a complex set of enhancers. The expressi...