The multi-domain protein Np95 connects DNA methylation and histone modification

DNA methylation and histone modifications play a central role in the epigenetic regulation of gene expression and cell differentiation. Recently, Np95 (also known as UHRF1 or ICBP90) has been found to interact with Dnmt1 and to bind hemimethylated DNA, indicating together with genetic studies a central role in the maintenance of DNA methylation. Using in vitro binding assays we observed a weak preference of Np95 and its SRA (SET- and Ring-associated) domain for hemimethylated CpG sites. However, the binding kinetics of Np95 in living cells was not affected by the complete loss of genomic methylation. Investigating further links with heterochromatin, we could show that Np95 preferentially binds histone H3 N-terminal tails with trimethylated (H3K9me3) but not acetylated lysine 9 via a tandem Tudor domain. This domain contains three highly conserved aromatic amino acids that form an aromatic cage similar to the one binding H3K9me3 in the chromodomain of HP1ss. Mutations targeting the aromatic cage of the Np95 tandem Tudor domain (Y188A and Y191A) abolished specific H3 histone tail binding. These multiple interactions of the multi-domain protein Np95 with hemimethylated DNA and repressive histone marks as well as with DNA and histone methyltransferases integrate the two major epigenetic silencing pathways

Targeted DNA methylation by homology-directed repair in mammalian cells. Transcription reshapes methylation on the repaired gene.

We report that homology-directed repair of a DNA double-strand break within a single copy Green Fluorescent Protein (GFP) gene in HeLa cells alters the methylation pattern at the site of recombination. DNA methyl transferase (DNMT)1, DNMT3a and two proteins that regulate methylation, Np95 and GADD45A, are recruited to the site of repair and are responsible for selective methylation of the promoter-distal segment of the repaired DNA. The initial methylation pattern of the locus is modified in a transcription-dependent fashion during the 15\u201320 days following repair, at which time no further changes in the methylation pattern occur. The variation in DNA modification generates stable clones with wide ranges of GFP expression. Collectively, our data indicate that somatic DNA methylation follows homologous repair and is subjected to remodeling by local transcription in a discrete time window during and after the damage. We propose that DNA methylation of repaired genes represents a DNA damage code and is source of variation of gene expression

Global analysis of estrogen receptor beta binding to breast cancer cell genome reveals an extensive interplay with estrogen receptor alpha for target gene regulation

Background: Estrogen receptors alpha (ERa) and beta (ERb) are transcription factors (TFs) that mediate estrogen signaling and define the hormone-responsive phenotype of breast cancer (BC). The two receptors can be found co-expressed and play specific, often opposite, roles, with ERb being able to modulate the effects of ERa on gene transcription and cell proliferation. ERb is frequently lost in BC, where its presence generally correlates with a better prognosis of the disease. The identification of the genomic targets of ERb in hormone-responsive BC cells is thus a critical step to elucidate the roles of this receptor in estrogen signaling and tumor cell biology. Results: Expression of full-length ERb in hormone-responsive, ERa-positive MCF-7 cells resulted in a marked reduction in cell proliferation in response to estrogen and marked effects on the cell transcriptome. By ChIP-Seq we identified 9702 ERb and 6024 ERa binding sites in estrogen-stimulated cells, comprising sites occupied by either ERb, ERa or both ER subtypes. A search for TF binding matrices revealed that the majority of the binding sites identified comprise one or more Estrogen Response Element and the remaining show binding matrixes for other TFs known to mediate ER interaction with chromatin by tethering, including AP2, E2F and SP1. Of 921 genes differentially regulated by estrogen in ERb+ vs ERb- cells, 424 showed one or more ERb site within 10 kb. These putative primary ERb target genes control cell proliferation, death, differentiation, motility and adhesion, signal transduction and transcription, key cellular processes that might explain the biological and clinical phenotype of tumors expressing this ER subtype. ERb binding in close proximity of several miRNA genes and in the mitochondrial genome, suggests the possible involvement of this receptor in small non-coding RNA biogenesis and mitochondrial genome functions. Conclusions: Results indicate that the vast majority of the genomic targets of ERb can bind also ERa, suggesting that the overall action of ERb on the genome of hormone-responsive BC cells depends mainly on the relative concentration of both ERs in the cell

Correlation of UHRF1 expression in primary prostate cancer patients with adverse prognosis

Background: Cancer of the prostate is a leading cause of cancer death in western countries. There is a great need to understand the clinical course of the disease and to distinguish the indolent tumors from those with aggressive behavior. Epigenetic mechanisms play an essential role in cancer initiation and progression. Our groups have recently provided evidence that over-expression of UHFR1 (ubiquitin-like with PHD and ring finger domain) leads to prostate cancer progression by activating a robust epigenetic switch involving silencing of a network of tumor suppressor genes. The purpose of this study was to evaluate in a clinical setting the prognostic impact of UHFR1 expression. Methods: In a series of 225 eligible patients (median age 63 years, range 44-75) with prostate cancer treated in a single institution with prostatectomy between 1990 and 1999 we evaluated the tumor nuclear expression of UHRF1 by immunohistochemistry (IHC). Clinical and histological data of the series were also reviewed. The UHFR1 expression (evaluated in tissue microarrays by immunohistochemistry) and prognostic factors were analyzed using univariate analyses and multivariate logistic regression analysis to identify association with overall survival (OS). Results: The median FU for the series was 137 months (range 1-229), eighty-one patients died (median FU 85 months). In Ninety-seven patients (43%) the UHRF1 expression was positive. In univariate analyses Gleason Score (<7 vs 7-9), Stage Risk Group (TNM Stage <III vs Stage III and/or N+) and UHFR1 expression (negative vs positive) were significant prognostic factors for OS with p-value <0.0001. In multivariate analyses Gleason Score, Stage Risk Group and UHFR1 expression were independent predictors for OS with respectively HRs of 2.77 (95% CI 1.72-4.46) p<0.0001, HRs of 1.96 (95% CI 1.14-3.37) p=0.014 and Hrs 1.35 (95% CI 1.02-1.78) p=0.030. Conclusions: Our results indicate that expression of UHFR1 protein, independently from historical prognostic factors, is linked with adverse prognosis for overall survival in a homogeneous primary prostate cancer treated group with long-term follow-up