Promoter methylation regulates SAMHD1 gene expression in human CD4+ T cells.
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ABSTRACT:
SUBMITTER: de Silva S
PROVIDER: S-EPMC3610999 | biostudies-literature | 2013 Mar
REPOSITORIES: biostudies-literature
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Project description:BackgroundClooxygenase-2 (COX-2) expression is overexpressed in human prostate cancer, and aberrant methylation of the COX-2 promoter has also been elucidated. However, how the methylation of CpG islands at COX-2 regulates its expression in prostate cancer is still unclear. We will determine the methylated 5' CpG island of the COX-2 gene and its role in the expression of COX-2 in prostate androgen-dependent and androgen-independent cancer cells, LNCaP and DU145.MethodsWe used western blotting and quantitative reverse transcription polymerase chain reaction (qRT-PCR) to confirm the COX-2 expression in prostate cancer cell lines, including LNCaP (androgen-dependent) and DU145 (androgen-independent) cells. To investigate whether the COX-2 expression was regulated by the methylation status of the 5' CpG island, we treated LNCaP and DU145 cells with the DNA methylation inhibitor, 5-aza-2'-deoxycytidine, and determined COX-2 expression in the treated/untreated cells by western blotting and qRT-PCR. Subsequently, bisulfite sequencing was performed to study the methylation sites in the treated/untreated cells. The effects of 5-aza-2'-deoxycytidine to cell proliferation, cell migration and cell cycle process in DU145 and LNCaP cells were determined using Cell Counting Kit-8 (CCK-8) assay, transwell assay and flow cytometry, respectively.ResultsThe results revealed that the expression of COX-2 in androgen-dependent LNCaP cells was 5.44-fold (in protein level) and 2.46-fold (in mRNA level) higher than that in androgen-independent DU145 cells. After 5-aza-2'-deoxycytidine treatment, COX-2 expression in DU145 cells was elevated significantly, but no change was found in LNCaP cells. The A and C regions of the COX-2 CpG island exhibited reduced methylation along with that an increased expression of COX-2 was noted in DU145 cell after 5-aza-2'-deoxycytidine treatment. Also, the treatment with 5-aza-2'-deoxycytidine inhibited cell proliferation, cell migration and influenced the cell cycle progression in both DU145 and LNCaP cells.ConclusionsOur results reveal that androgen receptor (AR)-dependent/independent prostate cancer cell lines exhibit different regulation of methylation in COX-2 that regulate its expression. Additionally, 5-aza-2'-deoxycytidine treatment of DU145 and LNCaP cells inhibits their ability of tumor progression.
| S-EPMC9239499 | biostudies-literature
Project description:The epigenetic determinants driving the responses of CD4 T cells to antigen are currently an area of active research. Much has been done to characterize helper T-cell subsets and their associated genome-wide epigenetic patterns. In contrast, little is known about the dynamics of histone modifications during CD4 T-cell activation and the differential kinetics of these epigenetic marks between naive and memory T cells. In this study, we have detailed the dynamics of genome-wide promoter H3K4me2 and H3K4me3 over a time course during activation of human naive and memory CD4 T cells. Our results demonstrate that changes to H3K4 methylation occur relatively late after activation (5 days) and reinforce activation-induced upregulation of gene expression, affecting multiple pathways important to T-cell activation, differentiation and function. The dynamics and mapped pathways of H3K4 methylation are distinctly different in memory cells, which have substantially more promoters marked by H3K4me3 alone, reinforcing their more differentiated state. Our study provides the first data examining genome-wide histone modification dynamics during CD4 T-cell activation, providing insight into the cross talk between H3K4 methylation and gene expression, and underscoring the impact of these marks upon key pathways integral to CD4 T-cell activation and function.
| S-EPMC4956548 | biostudies-literature
Project description:The interplay between multiple transcription factors precisely regulates eukaryotic transcription. Here, we report that the protein methyltransferases, MLL2 and PRMT1, interact directly and act collectively to regulate gene expression. PRMT1 binds to the N-terminal region of MLL2, considered an intrinsically disordered region, and methylates multiple arginine residues within its RGG/RG motifs. Notably, overexpression of PRMT1 decreased poly-ubiquitylation of MLL2, whereas mutations on methylation sites in MLL2 increased MLL2 poly-ubiquitylation, suggesting that PRMT1-mediated methylation stabilizes MLL2. MLL2 and PRMT1 cooperatively stimulated the expression of a chromosomal reporter gene in a PRMT1-mediated, MLL2-methylation–dependent manner. RNA-seq analysis found that MLL2 and PRMT1 jointly regulate the expression of genes involved in cell membrane and extracellular matrix functions, and depletion of either resulted in impaired cell migration and invasion. Our study provides evidence that PRMT1-mediated MLL2 methylation regulates MLL2 protein stability and the expression of their target genes.
2024-10-21 | PXD055018 | JPOST Repository
Project description:Blockade of IL-2R with humanized anti-CD25 Abs, such as daclizumab, inhibits Th2 responses in human T cells. Recent murine studies have shown that IL-2 also plays a significant role in regulating Th2 cell differentiation by activated STAT5. To explore the role of activated STAT5 in the Th2 differentiation of primary human T cells, we studied the mechanisms underlying IL-2 regulation of C-MAF expression. Chromatin immunoprecipitation studies revealed that IL-2 induced STAT5 binding to specific sites in the C-MAF promoter. These sites corresponded to regions enriched for markers of chromatin architectural features in both resting CD4 and differentiated Th2 cells. Unlike IL-6, IL-2 induced C-MAF expression in CD4 T cells with or without prior TCR stimulation. TCR-induced C-MAF expression was significantly inhibited by treatment with daclizumab or a JAK3 inhibitor, R333. Furthermore, IL-2 and IL-6 synergistically induced C-MAF expression in TCR-activated T cells, suggesting functional cooperation between these cytokines. Finally, both TCR-induced early IL4 mRNA expression and IL-4 cytokine expression in differentiated Th2 cells were significantly inhibited by IL-2R blockade. Thus, our findings demonstrate the importance of IL-2 in Th2 differentiation in human T cells and support the notion that IL-2R-directed therapies may have utility in the treatment of allergic disorders.
| S-EPMC3432901 | biostudies-literature
Project description:Activation-induced Fas ligand (FasL) mRNA expression in CD4+ T cells is mainly controlled at transcriptional initiation. To elucidate the epigenetic mechanisms regulating physiologic and pathologic FasL transcription, TCR stimulation-responsive promoter histone modifications in normal and alcohol-exposed primary human CD4+ T cells were examined. TCR stimulation of normal and alcohol-exposed cells led to discernible changes in promoter histone H3 lysine trimethylation, as documented by an increase in the levels of transcriptionally permissive histone 3 lysine 4 trimethylation and a concomitant decrease in the repressive histone 3 lysine 9 trimethylation. Moreover, acetylation of histone 3 lysine 9 (H3K9), a critical feature of the active promoter state that is opposed by histone 3 lysine 9 trimethylation, was significantly increased and was essentially mediated by the p300-histone acetyltransferase. Notably, the degree of these coordinated histone modifications and subsequent recruitment of transcription factors and RNA polymerase II were significantly enhanced in alcohol-exposed CD4+ T cells and were commensurate with the pathologic increase in the levels of FasL mRNA. The clinical relevance of these findings is further supported by CD4+ T cells obtained from individuals with a history of heavy alcohol consumption, which demonstrate significantly greater p300-dependent H3K9 acetylation and FasL expression. Overall, these data show that, in human CD4+ T cells, TCR stimulation induces a distinct promoter histone profile involving a coordinated cross-talk between histone 3 lysine 4 and H3K9 methylation and acetylation that dictates the transcriptional activation of FasL under physiologic, as well as pathologic, conditions of alcohol exposure.
| S-EPMC5096587 | biostudies-literature
Project description:Regulation of expression of HTLV-1 gene products from integrated proviruses plays an important role in HTLV-1-associated disease pathogenesis. Previous studies have shown that T cell receptor (TCR)- and phorbol ester (PMA) stimulation of chronically infected CD4 T cells increases the expression of integrated HTLV-1 proviruses in latently infected cells, however the mechanism remains unknown. Analysis of HTLV-1 RNA and protein species following PMA treatment of the latently HTLV-1-infected, FS and SP T cell lines demonstrated rapid induction of tax/rex mRNA. This rapid increase in tax/rex mRNA was associated with markedly enhanced tax/rex mRNA stability while the stability of unspliced or singly spliced HTLV-1 RNAs did not increase. Tax/rex mRNA in the HTLV-1 constitutively expressing cell lines exhibited high basal stability even without PMA treatment. Our data support a model whereby T cell activation leads to increased HTLV-1 gene expression at least in part through increased tax/rex mRNA stability.
| S-EPMC5522752 | biostudies-literature
Project description:Unlike activated CD4(+) T cells, resting CD4(+) T cells are highly resistant to productive HIV-1 infection. Early after HIV-1 entry, a major block limits reverse transcription of incoming viral genomes. Here we show that the deoxynucleoside triphosphate triphosphohydrolase SAMHD1 prevents reverse transcription of HIV-1 RNA in resting CD4(+) T cells. SAMHD1 is abundantly expressed in resting CD4(+) T cells circulating in peripheral blood and residing in lymphoid organs. The early restriction to infection in unstimulated CD4(+) T cells is overcome by HIV-1 or HIV-2 virions into which viral Vpx is artificially or naturally packaged, respectively, or by addition of exogenous deoxynucleosides. Vpx-mediated proteasomal degradation of SAMHD1 and elevation of intracellular deoxynucleotide pools precede successful infection by Vpx-carrying HIV. Resting CD4(+) T cells from healthy donors following SAMHD1 silencing or from a patient with Aicardi-Goutières syndrome homozygous for a nonsense mutation in SAMHD1 were permissive for HIV-1 infection. Thus, SAMHD1 imposes an effective restriction to HIV-1 infection in the large pool of noncycling CD4(+) T cells in vivo. Bypassing SAMHD1 was insufficient for the release of viral progeny, implicating other barriers at later stages of HIV replication. Together, these findings may unveil new ways to interfere with the immune evasion and T cell immunopathology of pandemic HIV-1.
| S-EPMC3828732 | biostudies-literature
Project description:BackgroundFrontotemporal lobar degeneration (FTLD) is a heterogeneous group of neurodegenerative diseases associated with personality changes and progressive dementia. Loss-of-function mutations in the growth factor progranulin (GRN) cause autosomal dominant FTLD, but so far the pathomechanism of sporadic FTLD is unclear.ResultsWe analyzed whether DNA methylation in the GRN core promoter restricts GRN expression and, thus, might promote FTLD in the absence of GRN mutations. GRN expression in human lymphoblast cell lines is negatively correlated with methylation at several CpG units within the GRN promoter. Chronic treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) strongly induces GRN mRNA and protein levels. In a reporter assay, CpG methylation blocks transcriptional activity of the GRN core promoter. In brains of FTLD patients several CpG units in the GRN promoter are significantly hypermethylated compared to age-matched healthy controls, Alzheimer and Parkinson patients. These CpG motifs are critical for GRN promoter activity in reporter assays. Furthermore, DNA methyltransferase 3a (DNMT3a) is upregulated in FTLD patients and overexpression of DNMT3a reduces GRN promoter activity and expression.ConclusionThese data suggest that altered DNA methylation is a novel pathomechanism for FTLD that is potentially amenable to targeted pharmacotherapy.
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Project description:Spermatogenesis is a highly regulated process that involves both mitotic and meiotic divisions, as well as cellular differentiation to yield mature spermatozoa from undifferentiated germinal stem cells. Although Gpat2 was originally annotated as encoding a glycerol-3-phosphate acyltransferase by sequence homology to Gpat1, GPAT2 is highly expressed in testis but not in lipogenic tissues and is not up-regulated during adipocyte differentiation. New data show that GPAT2 is required for the synthesis of piRNAs (piwi-interacting RNAs), a group of small RNAs that protect the germ cell genome from retrotransposable elements. In order to understand the relationship between GPAT2 and its role in the testis, we focused on Gpat2 expression during the first wave of mouse spermatogenesis. Gpat2 expression was analysed by qPCR (quantitative real-time PCR), in situ hybridization, immunohistochemistry and Western blotting. Gpat2 mRNA content and protein expression were maximal at 15 dpp (days post-partum) and were restricted to pachytene spermatocytes. To achieve this transient expression, both epigenetic mechanisms and trans-acting factors are involved. In vitro assays showed that Gpat2 expression correlates with DNA demethylation and histone acetylation and that it is up-regulated by retinoic acid. Epigenetic regulation by DNA methylation was confirmed in vivo in germ cells by bisulfite sequencing of the Gpat2 promoter. Consistent with the initiation of meiosis at 11 dpp, methylation decreased dramatically. Thus, Gpat2 is expressed at a specific stage of spermatogenesis, consistent with piRNA synthesis and meiosis I prophase, and its on-off expression pattern responds predominantly to epigenetic modifications.
| S-EPMC4613502 | biostudies-literature
Project description:DNA methylation, and consequent down-regulation, of tumour suppressor genes occurs in response to epigenetic stimuli during cancer development. Similarly, human oncoviruses, including human papillomavirus (HPV), up-regulate and augment DNA methyltransferase (DNMT) and histone deacetylase (HDAC) activities, thereby decreasing tumour suppressor genes (TSGs) expression. Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1), an epigenetic regulator of DNA methylation, is overexpressed in HPV-induced cervical cancers. Here, we investigated the role of UHRF1 in cervical cancer by knocking down its expression in HeLa cells using lentiviral-encoded short hairpin (sh)RNA and performing cDNA microarrays. We detected significantly elevated expression of thioredoxin-interacting protein (TXNIP), a known TSG, in UHRF1-knockdown cells, and this gene is hypermethylated in cervical cancer tissue and cell lines, as indicated by whole-genome methylation analysis. Up-regulation of UHRF1 and decreased TXNIP were further detected in cervical cancer by western blot and immunohistochemistry and confirmed by Oncomine database analysis. Using chromatin immunoprecipitation, we identified the inverted CCAAT domain-containing UHRF1-binding site in the TXNIP promoter and demonstrated UHRF1 knockdown decreases UHRF1 promoter binding and enhances TXNIP expression through demethylation of this region. TXNIP promoter CpG methylation was further confirmed in cervical cancer tissue by pyrosequencing and methylation-specific polymerase chain reaction. Critically, down-regulation of UHRF1 by siRNA or UHRF1 antagonist (thymoquinone) induces cell cycle arrest and apoptosis, and ubiquitin-specific protease 7 (USP7), which stabilises and promotes UHRF1 function, is increased by HPV viral protein E6/E7 overexpression. These results indicate HPV might induce carcinogenesis through UHRF1-mediated TXNIP promoter methylation, thus suggesting a possible link between CpG methylation and cervical cancer.
| S-EPMC8019600 | biostudies-literature