Critical Role of Transcription Cofactor PC4 in Mammals

摘要

PC4 is a small protein with unique DNA-binding properties that affects transcription and has presumptive roles in DNA repair and genome stability. It was originally isolated from a cofactor fraction termed the ‘‘upstream stimulatory activity’’ (USA) of HeLa cell nuclear extracts. The cofactor has been shown to broadly enhance RNA polymerase II-driven gene transcription in the presence of activators (e.g., hormone receptors, viral activators, cell-specific and ubiquitous activators). Although such data imply that PC4 is a very important factor in vivo, human tumor cell lines with PC4 knockdowns are without obvious phenotypes. To further study the in vivo role of PC4, we constructed constitutive and conditional knockout mouse models as well as knockout embryonic stem cells. Mammalian PC4 is here shown to be an essential factor during early embryogenesis. PC4-/- embryos develop normally until E5.5, but then degenerated around E7.5. PC4 knockout ES cell lines were generated from PC4-/- blastocysts (E3.5), which develop normally from 2-cell stage embryos. All PC4 knockout ES cell lines displayed a severe proliferation deficit phenotype, which could be partially rescued by re-expression of human PC4. The reduced proliferation was not due to an increase in cell apoptosis. Occasionally, PC4 knockout ES cells undergo tetraploidy apparently as a survival mechanism to circumvent the loss of PC4. Knocking down PC4 in mouse embryonic fibroblasts also resulted in reduced proliferation rates. These data indicate that PC4 is important for cell proliferation in embryos. Moreover, in vitro embryoid body formation and in vivo teratoma formation assays provided preliminary evidence for an important role of PC4 in differentiation. Differentiated ES cells displayed alterations in germ-layer specific gene expression, that are in agreement with morphological abnormalities observed in histological analyses of PC4-/- embryos at E6.5 and E7.5. Thus, depletion of PC4 results in reduced proliferation and impaired differentiation, the consequence of which appears to be of gastrulation arrest in early embryos. In an attempt to understand the underlying mechanisms of this phenotype, differential gene expression in ES knockout and wild-type cells was studied. Microarray and qRT-PCR analyses revealed more than 2 fold alterations in expression of many genes in knockout ES cells as compared with wild-type cells. These include enhanced expression of p21, Rb1, and Ddit4l, and lower expression of Sfmbt2, Tdrd12, and Dppa3, suggesting a specific direct or indirect physiological role of the cofactor. Nevertheless, the previously proposed role of PC4 in p53 expression and function was not confirmed using the knockout model. Taken together, this work represents the first description of the physiological functions of PC4 during mammalian embryogenesis.