RPH1 and GIS1 are damage-responsive repressors of PHR1.
The Saccharomyces cerevisiae DNA repair gene PHR1 encodes a photolyase that catalyzes the light-dependent repair of pyrimidine dimers. PHR1 expression is induced at the level of transcription by a variety of DNA-damaging agents. The primary regulator of the PHR1 damage response is a 39-bp sequence called URS(PHR1) which is the binding site for a protein(s) that constitutes the damage-responsive repressor PRP. In this communication, we report the identification of two proteins, Rph1p and Gis1p, that regulate PHR1 expression through URS(PHR1). Both proteins contain two putative zinc fingers that are identical throughout the DNA binding region, and deletion of both RPH1 and GIS1 is required to fully derepress PHR1 in the absence of damage. Derepression of PHR1 increases the rate and extent of photoreactivation in vivo, demonstrating that the damage response of PHR1 enhances cellular repair capacity. In vitro footprinting and binding competition studies indicate that the sequence AG(4) (C(4)T) within URS(PHR1) is the binding site for Rph1p and Gis1p and suggests that at least one additional DNA binding component is present in the PRP complex. (+info)
An ARID family protein binds to the African swine fever virus encoded ubiquitin conjugating enzyme, UBCv1.
The NH(2)-terminal end of a protein, named SMCp, which contains an ARID (A/T rich interaction domain) DNA binding domain and is similar to the mammalian SMCY/SMCX proteins and retinoblastoma binding protein 2, was shown to bind the African swine fever virus encoded ubiquitin conjugating enzyme (UBCv1) using the yeast two hybrid system and in in vitro binding assays. Antisera raised against the SMCp protein were used to show that the protein is present in the cell nucleus. Immunofluorescence showed that although UBCv1 is present in the nucleus in most cells, in some cells it is in the cytoplasm, suggesting that it shuttles between the nucleus and cytoplasm. The interaction and co-localisation of UBCv1 with SMCp suggest that SMCp may be a substrate in vivo for the enzyme. (+info)
Saccharomyces cerevisiae Ras/cAMP pathway controls post-diauxic shift element-dependent transcription through the zinc finger protein Gis1.
The Saccharomyces cerevisiae protein kinase Rim15 was identified previously as a component of the Ras/cAMP pathway acting immediately downstream of cAMP-dependent protein kinase (cAPK) to control a broad range of adaptations in response to nutrient limitation. Here, we show that the zinc finger protein Gis1 acts as a dosage-dependent suppressor of the rim15Delta defect in nutrient limitation-induced transcriptional derepression of SSA3. Loss of Gis1 results in a defect in transcriptional derepression upon nutrient limitation of various genes that are negatively regulated by the Ras/cAMP pathway (e.g. SSA3, HSP12 and HSP26). Tests of epistasis as well as transcriptional analyses of Gis1-dependent expression indicate that Gis1 acts in this pathway downstream of Rim15 to mediate transcription from the previously identified post-diauxic shift (PDS) element. Accordingly, deletion of GIS1 partially suppresses, and overexpression of GIS1 exacerbates the growth defect of mutant cells that are compromised for cAPK activity. Moreover, PDS element-driven expression, which is negatively regulated by the Ras/cAMP pathway and which is induced upon nutrient limitation, is almost entirely dependent on the presence of Gis1. (+info)
Phosphorylation of Rph1, a damage-responsive repressor of PHR1 in Saccharomyces cerevisiae, is dependent upon Rad53 kinase.
Rph1, a Cys2-His2 zinc finger protein, binds to an upstream repressing sequence of the photolyase gene PHR1, and represses its transcription in response to DNA damage in Saccharomyces cerevisiae. In this report, we have demonstrated that the phosphorylation of Rph1 protein was increased in response to DNA damage. The DNA damage-induced phosphorylation of Rph1 was missing in most damage checkpoint mutants including rad9, rad17, mec1 and rad53. These results indicate that Rph1 phosphorylation is under the control of the Mec1-Rad53 damage checkpoint pathway. Rph1 phosphorylation required the kinase activity of Rad53 since it was significantly decreased in rad53 checkpoint mutant. Furthermore, loss of other kinases including Dun1, Tel1 and Chk1, which function downstream of Mec1, did not affect the Rph1 phosphorylation. This contrasts with the derepression of Crt1-regulated genes, which requires both Rad53 and Dun1 protein kinases. These results imply that post-translational modification of Rph1 repressor is regulated by a potentially novel damage checkpoint pathway that is distinct from the RAD53-DUN1-CRT1 cascade implicated in the DNA damage-dependent transcription of ribonucleotide reductase genes. (+info)
Regulation of the yeast DPP1-encoded diacylglycerol pyrophosphate phosphatase by transcription factor Gis1p.
The Saccharomyces cerevisiae DPP1-encoded diacylglycerol pyrophosphate phosphatase catalyzes the dephosphorylation of diacylglycerol pyrophosphate to form phosphatidate and Pi. The enzyme also dephosphorylates phosphatidate to form diacylglycerol and Pi. Because diacylglycerol pyrophosphate, phosphatidate, and diacylglycerol have roles as lipid signal molecules in higher eukaryotic cells, it is important to understand how diacylglycerol pyrophosphate phosphatase is regulated. Analysis of DPP1 expression using PDPP1-lacZ reporter genes with a series of deletions from the 5' end of the promoter indicated sequences responsible for enzyme expression. Three binding sites (URSPDS) for transcription factor Gis1p were identified in the DPP1 promoter (consensus sequence of 5'-T(A/T)AGGGAT-3'). A gis1 Delta mutant exhibited elevated levels of DPP1 expression and diacylglycerol pyrophosphate phosphatase activity. Direct interaction between Gis1p and DPP1 promoter elements was demonstrated by electrophoretic mobility shift assays. Mutations in the three URSPDS elements within the DPP1 promoter abolished Gis1p-DNA interactions in vitro and abolished the regulation of DPP1 in vivo. These data indicated that Gis1p was a repressor of DPP1 expression. Phospholipid composition analysis of the gis1 Delta mutant showed that Gis1p played a role in regulating the cellular level of diacylglycerol pyrophosphate, as well as the levels of the major phospholipids phosphatidylethanolamine and phosphatidylcholine. (+info)
Gender-specific gene expression in post-mortem human brain: localization to sex chromosomes.
Gender differences in brain development and in the prevalence of neuropsychiatric disorders such as depression have been reported. Gender differences in human brain might be related to patterns of gene expression. Microarray technology is one useful method for investigation of gene expression in brain. We investigated gene expression, cell types, and regional expression patterns of differentially expressed sex chromosome genes in brain. We profiled gene expression in male and female dorsolateral prefrontal cortex, anterior cingulate cortex, and cerebellum using the Affymetrix oligonucleotide microarray platform. Differentially expressed genes between males and females on the Y chromosome (DBY, SMCY, UTY, RPS4Y, and USP9Y) and X chromosome (XIST) were confirmed using real-time PCR measurements. In situ hybridization confirmed the differential expression of gender-specific genes and neuronal expression of XIST, RPS4Y, SMCY, and UTY in three brain regions examined. The XIST gene, which silences gene expression on regions of the X chromosome, is expressed in a subset of neurons. Since a subset of neurons express gender-specific genes, neural subpopulations may exhibit a subtle sexual dimorphism at the level of differences in gene regulation and function. The distinctive pattern of neuronal expression of XIST, RPS4Y, SMCY, and UTY and other sex chromosome genes in neuronal subpopulations may possibly contribute to gender differences in prevalence noted for some neuropsychiatric disorders. Studies of the protein expression of these sex-chromosome-linked genes in brain tissue are required to address the functional consequences of the observed gene expression differences. (+info)
Histone demethylation mediated by the nuclear amine oxidase homolog LSD1.
Posttranslational modifications of histone N-terminal tails impact chromatin structure and gene transcription. While the extent of histone acetylation is determined by both acetyltransferases and deacetylases, it has been unclear whether histone methylation is also regulated by enzymes with opposing activities. Here, we provide evidence that LSD1 (KIAA0601), a nuclear homolog of amine oxidases, functions as a histone demethylase and transcriptional corepressor. LSD1 specifically demethylates histone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs via an oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1 causes an increase in H3 lysine 4 methylation and concomitant derepression of target genes, suggesting that LSD1 represses transcription via histone demethylation. The results thus identify a histone demethylase conserved from S. pombe to human and reveal dynamic regulation of histone methylation by both histone methylases and demethylases. (+info)
Vascular endothelial cells have impaired capacity to present immunodominant, antigenic peptides: a mechanism of cell type-specific immune escape.
Vascular endothelial cells (EC) are an exposed target tissue in the course of CTL-mediated alloimmune diseases such as graft-vs-host disease (GVHD) or solid organ transplant rejection. The outcome of an interaction between CTL and target cells is determined by the amount of Ag presented and the costimulatory signals delivered by the target cells. We compared human EC with leukocytes and epithelial cells as targets for peptide-specific, MHC class I-restricted CTL clones. EC were poor targets for immunodominant CTL. Both endogenously processed antigenic proteins and exogenously added antigenic peptides are presented at 50- to 5000-fold lower levels on EC compared with any other target cell analyzed. This quantitative difference fully explained the poor CTL-mediated killing of EC. There was no evidence that lack of costimulation would contribute significantly to this cell type-specific difference in CTL activation. An HLA-A2-specific CTL clone that killed a broad selection of HLA A2-positive target cells equally well, killed EC less efficiently. Our data suggest that EC present a different Ag repertoire compared with other cell types. By this mechanism, these cells may escape an attack by effector CTL, which have been educated by professional APCs and are specific for immunodominant antigenic peptides. (+info)