Mbd1 is recruited to both methylated and nonmethylated CpGs via distinct DNA binding domains

MBD1 is a vertebrate methyl-CpG binding domain protein (MBD) that can bring about repression of methylated promoter DNA sequences. Like other MBD proteins, MBD1 localizes to nuclear foci that in mice are rich in methyl-CpG. In methyl-CpG-deficient mouse cells, however, Mbd1 remains localized to heterochromatic foci whereas other MBD proteins become dispersed in the nucleus. We find that Mbd1a, a major mouse isoform, contains a CXXC domain (CXXC-3) that binds specifically to nonmethylated CpG, suggesting an explanation for methylation-independent localization. Transfection studies demonstrate that the CXXC-3 domain indeed targets nonmethylated CpG sites in vivo. Repression of nonmethylated reporter genes depends on the CXXC-3 domain, whereas repression of methylated reporters requires the MBD. Our findings indicate that MBD1 can interpret the CpG dinucleotide as a repressive signal in vivo regardless of its methylation status

Telomerase Maintains Telomere Structure in Normal Human Cells

AbstractIn normal human cells, telomeres shorten with successive rounds of cell division, and immortalization correlates with stabilization of telomere length. These observations suggest that human cancer cells achieve immortalization in large part through the illegitimate activation of telomerase expression. Here, we demonstrate that the rate-limiting telomerase catalytic subunit hTERT is expressed in cycling primary presenescent human fibroblasts, previously believed to lack hTERT expression and telomerase activity. Disruption of telomerase activity in normal human cells slows cell proliferation, restricts cell lifespan, and alters the maintenance of the 3′ single-stranded telomeric overhang without changing the rate of overall telomere shortening. Together, these observations support the view that telomerase and telomere structure are dynamically regulated in normal human cells and that telomere length alone is unlikely to trigger entry into replicative senescence

An embryonic stem cell–like gene expression signature in poorly differentiated aggressive human tumors

Cancer cells possess traits reminiscent of those ascribed to normal stem cells. It is unclear, however, whether these phenotypic similarities reflect the activity of common molecular pathways. Here, we analyze the enrichment patterns of gene sets associated with embryonic stem (ES) cell identity in the expression profiles of various human tumor types. We find that histologically poorly differentiated tumors show preferential overexpression of genes normally enriched in ES cells, combined with preferential repression of Polycomb-regulated genes. Moreover, activation targets of Nanog, Oct4, Sox2 and c-Myc are more frequently overexpressed in poorly differentiated tumors than in well-differentiated tumors. In breast cancers, this ES-like signature is associated with high-grade estrogen receptor (ER)-negative tumors, often of the basal-like subtype, and with poor clinical outcome. The ES signature is also present in poorly differentiated glioblastomas and bladder carcinomas. We identify a subset of ES cell-associated transcription regulators that are highly expressed in poorly differentiated tumors. Our results reveal a previously unknown link between genes associated with ES cell identity and the histopathological traits of tumors and support the possibility that these genes contribute to stem cell–like phenotypes shown by many tumors

Stimulation of epidermal hyperplasia and tumorigenesis by resident p16INK4a-expressing cells

p16INK4a (CDKN2A) is a central tumor-suppressor and activator of senescence. We recently found that prolonged expression of p16INK4a in epidermal cells induces hyperplasia and dysplasia through Wnt-mediated stimulation of neighboring keratinocytes. The study suggests a pro-tumorigenic function of p16INK4a in early epidermal lesions, which could potentially be targeted by senolytic therapy

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Cells entering a state of senescence undergo a permanent cell cycle arrest, accompanied by a set of functional and morphological changes. Senescence of cells occurs following an extended period of proliferation in culture or in response to various physiologic stresses, yet little is known about the role this phenomenon plays in vivo. The study of senescence has focused largely on its hypothesized role as a barrier to extended cell division, governed by a division-counting mechanism in the form of telomere length. Here, we discuss the biological functions of cellular senescence and suggest that it should be viewed in terms of its role as a general cellular stress response program, rather than strictly as a barrier to unlimited cycles of cell growth and division. We also discuss the relative roles played by telomere shortening and telomere uncapping in the induction of senescence

CTCF Elements Direct Allele-Specific Undermethylation at the Imprinted H19 Locus

AbstractThe H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation [1], and replication timing [2]. This ICR becomes de novo methylated during late spermatogenesis in the male [3] but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development [4] and in all tissues of the mouse [5]. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation [6]. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR [7, 8] have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated [9, 10], this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo

Vav1 Fine Tunes p53 Control of Apoptosis versus Proliferation in Breast Cancer

<div><p>Vav1 functions as a signal transducer protein in the hematopoietic system, where it is exclusively expressed. Vav1 was recently implicated in several human cancers, including lung, pancreatic and neuroblasoma. In this study, we analyzed the expression and function of Vav1 in human breast tumors and breast cancer cell lines. Immunohistochemical analysis of primary human breast carcinomas indicated that Vav1 is expressed in 62% of 65 tumors tested and is correlated positively with estrogen receptor expression. Based on published gene profiling of 50 breast cancer cell lines, several Vav1-expressing cell lines were identified. RT-PCR confirmed Vav1 mRNA expression in several of these cell lines, yet no detectable levels of Vav1 protein were observed due to cbl-c proteasomal degradation. We used two of these lines, MCF-7 (Vav1 mRNA negative) and AU565 (Vav1 mRNA positive), to explore the effect of Vav1 expression on breast cell phenotype and function. Vav1 expression had opposite effects on function in these two lines: it reduced proliferation and enhanced cell death in MCF-7 cells but enhanced proliferation in AU565 cells. Consistent with these findings, transcriptome analysis revealed an increase in expression of proliferation-related genes in Vav1-expressing AU565 cells compared to controls, and an increase in apoptosis-related genes in Vav1-expressing MCF-7 cells compared with controls. TUNEL and γ-H2AX foci assays confirmed that expression of Vav1 increased apoptosis in MCF-7 cells but not AU565 cells and shRNA experiments revealed that p53 is required for this pro-apoptotic effect of Vav1 in these cells. These results highlight for the first time the potential role of Vav1 as an oncogenic stress activator in cancer and the p53 dependence of its pro-apoptotic effect in breast cells.</p> </div

Vav1 as a signal transducer protein in breast cancer cells.

<p>(A) MCF-7Vector, MCF-7Vav1, AU565Vector and AU565Vav1 were stimulated with EGF or CSF1, respectively, for various times as indicated. Cell lysates were immunoprecipitated with anti-Vav1 antibody and then immunoblotted with either anti-Vav1 antibody or anti- pTyr antibody (top 2 immunoblots). In addition, total cell lysates were separated on SDS-PAGE and immunoblotted with anti-Vav1, anti-pERK or anti-ERK antibodies (lower 3 immunoblots). (B) Immunofluorescence of 145 MCF-7Vector, 176 MCF-7Vav1, 355 AU565Vector and 174 AU565Vav1 with anti-Vav1 antibody. Actin filaments were detected by phalloidin and nuclei were stained with Hoechst. The difference in morphology between MCF-7Vav1, AU565Vav1 and their corresponding control cells were highly significant (two-tailored pValue; 0.0002 and 0.0024 respectively). Representative photographs taken with a Zeiss LSM 710 confocal microscope and analyzed by the ZEN 2010 program are shown. (C) MCF-7Vector, MCF-7Vav1, AU565Vector and AU565Vav1 were transiently transfected with Flag-Rac. 48 hours later, cell lysates were incubated with GST–PAK bacterial fusion proteins immobilized on glutathione sepharose beads. Bound proteins (+) and unbound proteins (−) were separated on SDS–PAGE and immunoblotted with anti-Flag mAbs. Numbers indicate mean (+/− S.E.) relative binding from three different experiments. Unpaired Student's <i>t</i>-test was used. (*) indicates p<0.05 value.</p