A Barcode Screen for Epigenetic Regulators Reveals a Role for the NuB4/HAT-B Histone Acetyltransferase Complex in Histone Turnover

Dynamic modification of histone proteins plays a key role in regulating gene expression. However, histones themselves can also be dynamic, which potentially affects the stability of histone modifications. To determine the molecular mechanisms of histone turnover, we developed a parallel screening method for epigenetic regulators by analyzing chromatin states on DNA barcodes. Histone turnover was quantified by employing a genetic pulse-chase technique called RITE, which was combined with chromatin immunoprecipitation and high-throughput sequencing. In this screen, the NuB4/HAT-B complex, containing the conserved type B histone acetyltransferase Hat1, was found to promote histone turnover. Unexpectedly, the three members of this complex could be functionally separated from each other as well as from the known interacting factor and histone chaperone Asf1. Thus, systematic and direct interrogation of chromatin structure on DNA barcodes can lead to the discovery of genes and pathways involved in chromatin modification and dynamics

Regulation of Androgen Receptor-Mediated Transcription by RPB5 Binding Protein URI/RMP ▿

Androgen receptor (AR)-mediated transcription is modulated by interaction with coregulatory proteins. We demonstrate that the unconventional prefoldin RPB5 interactor (URI) is a new regulator of AR transcription and is critical for antagonist (bicalutamide) action. URI is phosphorylated upon androgen treatment, suggesting communication between the URI and AR signaling pathways. Whereas depletion of URI enhances AR-mediated gene transcription, overexpression of URI suppresses AR transcriptional activation and anchorage-independent prostate cancer cell growth. Repression of AR-mediated transcription is achieved, in part, by URI binding and regulation of androgen receptor trapped clone 27 (Art-27), a previously characterized AR corepressor. Consistent with this idea, genome-wide expression profiling in prostate cancer cells upon depletion of URI or Art-27 reveals substantially overlapping patterns of gene expression. Further, depletion of URI increases the expression of the AR target gene NKX-3.1, decreases the recruitment of Art-27, and increases AR occupancy at the NKX-3.1 promoter. While Art-27 can bind AR directly, URI is bound to chromatin prior to hormone-dependent recruitment of AR, suggesting a role for URI in modulating AR recruitment to target genes

Phosphorylation by Cdk2 is required for Myc to repress Ras-induced senescence in cotransformation

The MYC and RAS oncogenes are frequently activated in cancer and, together, are sufficient to transform rodent cells. The basis for this cooperativity remains unclear. We found that although Ras interfered with Myc-induced apoptosis, Myc repressed Ras-induced senescence, together abrogating two main barriers of tumorigenesis. Inhibition of cellular senescence required phosphorylation of Myc at Ser-62 by cyclin E/cyclin-dependent kinase (Cdk) 2. Cdk2 interacted with Myc at promoters, where it affected Myc-dependent regulation of genes, including Bmi-1, p16, p21, and hTERT, which encode proteins known to control senescence. Repression of senescence by Myc was abrogated by the Cdk inhibitor p27Kip1, which is induced by antiproliferative signals like IFN-γ or by pharmacological inhibitors of Cdk2 but not by inhibitors of other Cdks. In contrast, a phospho-mimicking Myc-S62D mutant was resistant to these manipulations. Inhibition of cyclin E/Cdk2 reversed the senescence-associated gene expression pattern imposed by Myc/cyclin E/Cdk2. This indicates a role of Cdk2 as a transcriptional cofactor and activator of the antisenescence function of Myc and provides mechanistic insight into the Myc-p27Kip1 antagonism. Finally, our findings highlight that pharmacological inhibition of Cdk2 activity is a potential therapeutical principle for cancer therapy, in particular for tumors with activated Myc or Ras

Role of the Unconventional Prefoldin Proteins URI and UXT in Transcription Regulation

The Unconventional prefoldin RPB5 interacting protein (URI), also known as RPB5-Mediating Protein (RMP) has been shown to play several regulatory roles in different cellular compartments including the mitochondria, as a phosphatase binding protein; in the cytoplasm, as a chaperone-like protein; and in the nucleus, as a transcriptional regulator through binding to RPB5 and RNA polymerase II (polII). This chapter focuses on the role URI plays in transcriptional regulation in the prostate cell. In prostate cells, URI is tightly bound to another prefoldin-like protein called UXT, a known androgen receptor (AR) cofactor. Part of a multiprotein complex, URI and UXT act as transcriptional repressors, and URI regulates KAP1 through PP2A phosphatase activity. The discovery of the interaction of URI and UXT with KAP1, AR, and PP2A, as well as the numerous interactions between URI and components of the R2TP/prefoldin-like complex, RPB5, and nuclear proteins involved in DNA damage response, chromatin remodeling and gene transcription, reveal a pleiotropic effect of the URI/UXT complex on nuclear processes. The mechanisms by which URI/UXT affect transcription, chromatin structure and regulation, and genome stability, remain to be elucidated but will be of fundamental importance considering the many processes affected by alterations of URI/UXT and other prefoldins and prefoldin-like proteins