ABC953 Sigma-AldrichAnti-AATF Antibody/Rb-binding protein Che-1

To combat threats posed by DNA damage, cells have evolved mechanisms, collectively termed DNA damage response (DDR). These mechanisms detect DNA lesions, signal their presence, and promote their repair. Centrosomes integrate G2/M checkpoint control and repair signals in response to genotoxic stress, acting as an efficient control mechanism when G2/M checkpoint function fails and mitosis begins in the presence of damaged DNA. Che-1 is an RNA polymerase II-binding protein involved in the regulation of gene transcription, induction of cell proliferation, and DDR. Here we provide evidence that in addition to its nuclear localization, Che-1 localizes at interphase centrosomes, where it accumulates following DNA damage or spindle poisons. We show that Che-1 depletion generates supernumerary centrosomes, multinucleated cells, and multipolar spindle formation. Notably, Che-1 depletion abolishes the ability of Chk1 to bind pericentrin and to localize at centrosomes, which, in its turn, deregulates the activation of centrosomal cyclin B-Cdk1 and advances entry into mitosis. Our results reinforce the notion that Che-1 plays an important role in DDR and that its contribution seems to be relevant for the spindle assembly checkpoint.

We have recently identified apoptosis-antagonizing transcription factor (AATF), tumor-susceptibility gene 101 (TSG101) and zipper-interacting protein kinase (ZIPK) as novel coactivators of the androgen receptor (AR). The mechanisms of coactivation remained obscure, however. Here we investigated the interplay and interdependence between these coactivators and the AR using the endogenous prostate specific antigen (PSA) gene as model for AR-target genes. Chromatin immunoprecipitation in combination with siRNA-mediated knockdown revealed that recruitment of AATF and ZIPK to the PSA enhancer was dependent on AR, whereas recruitment of TSG101 was dependent on AATF. Association of AR and its coactivators with the PSA enhancer or promoter occurred in cycles. Dissociation of AR-transcription complexes was due to degradation because inhibition of the proteasome system by MG132 caused accumulation of AR at enhancer/promoter elements. Moreover, inhibition of degradation strongly reduced transcription, indicating that continued and efficient transcription is based on initiation, degradation and reinitiation cycles. Interestingly, knockdown of ZIPK by siRNA had a similar effect as MG132, leading to reduced transcription but enhanced accumulation of AR at androgen-response elements. In addition, knockdown of ZIPK, as well as overexpression of a dominant-negative ZIPK mutant, diminished polyubiquitination of AR. Furthermore, ZIPK cooperated with the E3 ligase Mdm2 in AR-dependent transactivation, assembled into a single complex on chromatin and phosphorylated Mdm2 in vitro. These results suggest that ZIPK has a crucial role in regulation of ubiquitination and degradation of the AR, and hence promoter clearance and efficient transcription.

hRPB11 is a core subunit of RNA polymerase II (pol II) specifically down-regulated on doxorubicin (dox) treatment. Levels of this protein profoundly affect cell differentiation, cell proliferation, and tumorigenicity in vivo. Here we describe Che-1, a novel human protein that interacts with hRPB11. Che-1 possesses a domain of high homology with Escherichia coli RNA polymerase final sigma-factor 70 and SV40 large T antigen. In addition, we report that Che-1 interacts with the retinoblastoma susceptibility gene (Rb) by two distinct domains. Functionally, we demonstrate that Che-1 represses the growth suppression function of Rb, counteracting the inhibitory action of Rb on the trans-activation function of E2F1. These results identify a novel protein that binds Rb and the core of pol II, and suggest that Che-1 may be part of transcription regulatory complex.