Knockdown of MBP-1 in Human Foreskin Fibroblasts Induces p53-p21 Dependent Senescence

MBP-1 acts as a general transcriptional repressor. Overexpression of MBP-1 induces cell death in a number of cancer cells and regresses tumor growth. However, the function of endogenous MBP-1 in normal cell growth regulation remains unknown. To unravel the role of endogenous MBP-1, we knocked down MBP-1 expression in primary human foreskin fibroblasts (HFF) by RNA interference. Knockdown of MBP-1 in HFF (HFF-MBPsi-4) resulted in an induction of premature senescence, displayed flattened cell morphology, and increased senescence-associated beta-galactosidase activity. FACS analysis of HFF-MBPsi-4 revealed accumulation of a high number of cells in the G1-phase. A significant upregulation of cyclin D1 and reduction of cyclin A was detected in HFF-MBPsi-4 as compared to control HFF. Senescent fibroblasts exhibited enhanced expression of phosphorylated and acetylated p53, and cyclin-dependent kinase inhibitor, p21. Further analysis suggested that promyolocytic leukemia protein (PML) bodies are dramatically increased in HFF-MBPsi-4. Together, these results demonstrated that knockdown of endogenous MBP-1 is involved in cellular senescence of HFF through p53-p21 pathway

YY1 Regulates Melanocyte Development and Function by Cooperating with MITF

Studies of coat color mutants have greatly contributed to the discovery of genes that regulate melanocyte development and function. Here, we generated Yy1 conditional knockout mice in the melanocyte-lineage and observed profound melanocyte deficiency and premature gray hair, similar to the loss of melanocytes in human piebaldism and Waardenburg syndrome. Although YY1 is a ubiquitous transcription factor, YY1 interacts with M-MITF, the Waardenburg Syndrome IIA gene and a master transcriptional regulator of melanocytes. YY1 cooperates with M-MITF in regulating the expression of piebaldism gene KIT and multiple additional pigmentation genes. Moreover, ChIP–seq identified genome-wide YY1 targets in the melanocyte lineage. These studies mechanistically link genes implicated in human conditions of melanocyte deficiency and reveal how a ubiquitous factor (YY1) gains lineage-specific functions by co-regulating gene expression with a lineage-restricted factor (M-MITF)—a general mechanism which may confer tissue-specific gene expression in multiple lineages

PI 3 Kinase Related Kinases-Independent Proteolysis of BRCA1 Regulates Rad51 Recruitment during Genotoxic Stress in Human Cells

The function of BRCA1 in response to ionizing radiation, which directly generates DNA double strand breaks, has been extensively characterized. However previous investigations have produced conflicting data on mutagens that initially induce other classes of DNA adducts. Because of the fundamental and clinical importance of understanding BRCA1 function, we sought to rigorously evaluate the role of this tumor suppressor in response to diverse forms of genotoxic stress.We investigated BRCA1 stability and localization in various human cells treated with model mutagens that trigger different DNA damage signaling pathways. We established that, unlike ionizing radiation, either UVC or methylmethanesulfonate (MMS) (generating bulky DNA adducts or alkylated bases respectively) induces a transient downregulation of BRCA1 protein which is neither prevented nor enhanced by inhibition of PIKKs. Moreover, we found that the proteasome mediates early degradation of BRCA1, BARD1, BACH1, and Rad52 implying that critical components of the homologous recombination machinery need to be functionally abrogated as part of the early response to UV or MMS. Significantly, we found that inhibition of BRCA1/BARD1 downregulation is accompanied by the unscheduled recruitment of both proteins to chromatin along with Rad51. Consistently, treatment of cells with MMS engendered complete disassembly of Rad51 from pre-formed ionizing radiation-induced foci. Following the initial phase of BRCA1/BARD1 downregulation, we found that the recovery of these proteins in foci coincides with the formation of RPA and Rad51 foci. This indicates that homologous recombination is reactivated at later stage of the cellular response to MMS, most likely to repair DSBs generated by replication blocks.Taken together our results demonstrate that (i) the stabilities of BRCA1/BARD1 complexes are regulated in a mutagen-specific manner, and (ii) indicate the existence of mechanisms that may be required to prevent the simultaneous recruitment of conflicting signaling pathways to sites of DNA damage

The Transcription Factor YY1 Is a Substrate for Polo-Like Kinase 1 at the G2/M Transition of the Cell Cycle

Yin-Yang 1 (YY1) is an essential multifunctional zinc-finger protein. It has been shown over the past two decades to be a critical regulator of a vast array of biological processes, including development, cell proliferation and differentiation, DNA repair, and apoptosis. YY1 exerts its functions primarily as a transcription factor that can activate or repress gene expression, dependent on its spatial and temporal context. YY1 regulates a large number of genes involved in cell cycle transitions, many of which are oncogenes and tumor-suppressor genes. YY1 itself has been classified as an oncogene and was found to be upregulated in many cancer types. Unfortunately, our knowledge of what regulates YY1 is very minimal. Although YY1 has been shown to be a phosphoprotein, no kinase has ever been identified for the phosphorylation of YY1. Polo-like kinase 1 (Plk1) has emerged in the past few years as a major cell cycle regulator, particularly for cell division. Plk1 has been shown to play important roles in the G/M transition into mitosis and for the proper execution of cytokinesis, processes that YY1 has been shown to regulate also. Here, we present evidence that Plk1 directly phosphorylates YY1 in vitro and in vivo at threonine 39 in the activation domain. We show that this phosphorylation is cell cycle regulated and peaks at G2/M. This is the first report identifying a kinase for which YY1 is a substrate

Immunological determination and size characterization of poly(ADP-ribose) synthesized in vitro and in vivo

Poly(ADP-ribose) polymerase is a DNA break detecting enzyme playing a role in the surveillance of genome integrity. Poly(ADP-ribose) is synthesized rapidly and transiently from beta-NAD in response to DNA damaging agents. In order to study the physiological significance of poly(ADP-ribose) metabolism, we have developed immunological methods which enable us to study endogenous poly(ADP-ribose) without interfering with cell metabolism and integrity. For this purpose, we produced a highly specific polyclonal anti-poly(ADP-ribose) antibody which immunoreacts with polymers and oligomers. In addition to the immunodot blot method recently described by us (Affar et al., Anal. Biochem. 259 (1998) 280-283), other applications were investigated in cells: (i) detection of poly(ADP-ribose) by ELISA; (ii) characterization of poly(ADP-ribose) size using high resolution gel electrophoresis of polymers, followed by its transfer onto a positively charged membrane and detection with anti-poly(ADP-ribose) antibody; (iii) immunocytochemistry and flow cytometry analyses allowing poly(ADP-ribose) study at the level of individual cells