scholarly journals Challenges in Quantifying Cytosine Methylation in the HIV Provirus

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sarah A. LaMere ◽  
Antoine Chaillon ◽  
Christina Huynh ◽  
Davey M. Smith ◽  
Sara Gianella
Keyword(s):  
Dna Methylation ◽  
Transcriptional Repression ◽  
Cytosine Methylation ◽  
Pcr Amplification ◽  
Sampling Bias ◽  
Epigenetic Mechanism ◽  
Persons Living With Hiv ◽  
Integration Sites

ABSTRACT DNA methylation is an epigenetic mechanism most commonly associated with transcriptional repression. While it is clear that DNA methylation can silence HIV proviral expression in in vitro latency models, its correlation with HIV persistence and expression in vivo is ambiguous, particularly in persons living with HIV (PLWH) receiving antiretroviral therapy (ART). Several factors potentially contribute to discrepancies between results in the literature, including differences in integration sites, functional proviral load, sampling bias, and stochastic PCR amplification. Recent studies into genomic features of cytosine methylation sites in mammalian genes offer potentially significant insights into this mechanism. Here, we discuss the importance of these factors in the context of the HIV.

scholarly journals Differential regulation of DNA methylation versus histone acetylation in cardiomyocytes during HHcy in vitro and in vivo: an epigenetic mechanism

2014 ◽  
Vol 46 (7) ◽  
pp. 245-255 ◽  
Author(s):  
Pankaj Chaturvedi ◽  
Anuradha Kalani ◽  
Srikanth Givvimani ◽  
Pradip Kumar Kamat ◽  
Anastasia Familtseva ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone Acetylation ◽  
Dna Methyltransferase ◽  
Epigenetic Mechanism ◽  
Dependent Manner ◽  
Histone Deacetylase 1 ◽  
H3k9 Acetylation ◽  
Dose Dependent

The mechanisms of homocysteine-mediated cardiac threats are poorly understood. Homocysteine, being the precursor to S-adenosyl methionine (a methyl donor) through methionine, is indirectly involved in methylation phenomena for DNA, RNA, and protein. We reported previously that cardiac-specific deletion of N-methyl-d-aspartate receptor-1 (NMDAR1) ameliorates homocysteine-posed cardiac threats, and in this study, we aim to explore the role of NMDAR1 in epigenetic mechanisms of heart failure, using cardiomyocytes during hyperhomocysteinemia (HHcy). High homocysteine levels activate NMDAR1, which consequently leads to abnormal DNA methylation vs. histone acetylation through modulation of DNA methyltransferase 1 (DNMT1), HDAC1, miRNAs, and MMP9 in cardiomyocytes. HL-1 cardiomyocytes cultured in Claycomb media were treated with 100 μM homocysteine in a dose-dependent manner. NMDAR1 antagonist (MK801) was added in the absence and presence of homocysteine at 10 μM in a dose-dependent manner. The expression of DNMT1, histone deacetylase 1 (HDAC1), NMDAR1, microRNA (miR)-133a, and miR-499 was assessed by real-time PCR as well as Western blotting. Methylation and acetylation levels were determined by checking 5′-methylcytosine DNA methylation and chromatin immunoprecipitation. Hyperhomocysteinemic mouse models (CBS+/−) were used to confirm the results in vivo. In HHcy, the expression of NMDAR1, DNMT1, and matrix metalloproteinase 9 increased with increase in H3K9 acetylation, while HDAC1, miR-133a, and miR-499 decreased in cardiomyocytes. Similar results were obtained in heart tissue of CBS+/− mouse. High homocysteine levels instigate cardiovascular remodeling through NMDAR1, miR-133a, miR-499, and DNMT1. A decrease in HDAC1 and an increase in H3K9 acetylation and DNA methylation are suggestive of chromatin remodeling in HHcy.

scholarly journals Inhibition of DNA Methyltransferases Blocks Mutant Huntingtin-Induced Neurotoxicity

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Yanchun Pan ◽  
Takuji Daito ◽  
Yo Sasaki ◽  
Yong Hee Chung ◽  
Xiaoyun Xing ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cortical Neurons ◽  
Transcriptional Repression ◽  
Cytosine Methylation ◽  
Critical Role ◽  
Dna Methyltransferases ◽  
Mrna Levels ◽  
Mutant Huntingtin ◽  
Striatal Neurons

Abstract Although epigenetic abnormalities have been described in Huntington’s disease (HD), the causal epigenetic mechanisms driving neurodegeneration in HD cortex and striatum remain undefined. Using an epigenetic pathway-targeted drug screen, we report that inhibitors of DNA methyltransferases (DNMTs), decitabine and FdCyd, block mutant huntingtin (Htt)-induced toxicity in primary cortical and striatal neurons. In addition, knockdown of DNMT3A or DNMT1 protected neurons against mutant Htt-induced toxicity, together demonstrating a requirement for DNMTs in mutant Htt-triggered neuronal death and suggesting a neurodegenerative mechanism based on DNA methylation-mediated transcriptional repression. Inhibition of DNMTs in HD model primary cortical or striatal neurons restored the expression of several key genes, including Bdnf, an important neurotrophic factor implicated in HD. Accordingly, the Bdnf promoter exhibited aberrant cytosine methylation in mutant Htt-expressing cortical neurons. In vivo, pharmacological inhibition of DNMTs in HD mouse brains restored the mRNA levels of key striatal genes known to be downregulated in HD. Thus, disturbances in DNA methylation play a critical role in mutant Htt-induced neuronal dysfunction and death, raising the possibility that epigenetic strategies targeting abnormal DNA methylation may have therapeutic utility in HD.

SPARCL1 exhibits different expressions in left- and right-sided colon cancer and is downregulated via DNA methylation

Epigenomics ◽  
2021 ◽  
Vol 13 (16) ◽  
pp. 1269-1282
Author(s):  
Hanguang Hu ◽  
Dehao Wu ◽  
Xibo Liu ◽  
Haifeng Yu ◽  
Junxi Xu ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Colon Cancer ◽  
Cancer Metastasis ◽  
Epigenetic Mechanism ◽  
Tissue Array ◽  
Cellular Motility ◽  
Colorectal Cancer Metastasis ◽  
Left And Right

Aim: The authors previously found that SPARCL1 functions to suppress colorectal cancer metastasis. Here, the epigenetic mechanism of SPARCL1 regulation and its relationship with clinicopathological features in colon cancer were investigated. Materials & methods: SPARCL1 expression was evaluated by immunohistochemistry staining in a tissue array containing 271 left-sided colon cancer samples and 257 right-sided colon cancer samples. In vivo and in vitro DNA methylation states were measured by biochemical sulfide potential assay. The transcription and DNA methylation states in cells were altered by siRNA or decitabine treatment, respectively. Cellular motility properties were compared through transwell assay. Results & conclusion: SPARCL1, mediated by its DNA methylation, may arrest colorectal carcinoma motility. Furthermore, SPARCL1 expression is higher and may have a specific prognostic value in left-sided colon cancer.

scholarly journals Epigenetic modulation of intestinal Na+/H+ exchanger-3 expression

2018 ◽  
Vol 314 (3) ◽  
pp. G309-G318 ◽  
Author(s):  
Anoop Kumar ◽  
Pooja Malhotra ◽  
Hayley Coffing ◽  
Shubha Priyamvada ◽  
Arivarasu N. Anbazhagan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Dna Demethylation ◽  
Epigenetic Mechanism ◽  
Mrna Levels ◽  
In Vivo Models ◽  
Epigenetic Modulation

Na+/H+ exchanger-3 (NHE3) is crucial for intestinal Na+ absorption, and its reduction has been implicated in infectious and inflammatory bowel diseases (IBD)-associated diarrhea. Epigenetic mechanisms such as DNA methylation are involved in the pathophysiology of IBD. Whether changes in DNA methylation are involved in modulating intestinal NHE3 gene expression is not known. Caco-2 and HuTu 80 cells were used as models of human intestinal epithelial cells. Normal C57/BL6, wild-type, or growth arrest and DNA damage-inducible 45b (GADD45b) knockout (KO) mice were used as in vivo models. NHE3 gene DNA methylation levels were assessed by MBDCap (MethyMiner) assays. Results demonstrated that in vitro methylation of NHE3 promoter construct (p-1509/+127) cloned into a cytosine guanine dinucleotide-free lucia vector decreased the promoter activity in Caco-2 cells. DNA methyltransferase inhibitor 5-azacytidine (10 μM, 24 h) caused a significant decrease in DNA methylation of the NHE3 gene and concomitantly increased NHE3 expression in Caco-2 cells. Similarly, 5-azacytidine treatment increased NHE3 mRNA levels in HuTu 80 cells. 5-Azacytidine treatment for 3 wk (10 mg/kg body wt ip, 3 times/wk) also resulted in an increase in NHE3 expression in the mouse ileum and colon. Small-interfering RNA knockdown of GADD45b (protein involved in DNA demethylation) in Caco-2 cells decreased NHE3 mRNA expression. Furthermore, there was a significant decrease in NHE3 mRNA and protein expression in the ileum and colon of GADD45b KO mice. Our findings demonstrate that NHE3 gene expression is regulated by changes in its DNA methylation. NEW & NOTEWORTHY Our studies for the first time demonstrate that Na+/H+ exchanger-3 gene expression is regulated by an epigenetic mechanism involving DNA methylation.

scholarly journals SBA1 Encodes a Yeast Hsp90 Cochaperone That Is Homologous to Vertebrate p23 Proteins

1998 ◽  
Vol 18 (7) ◽  
pp. 3727-3734 ◽  
Author(s):  
Yifang Fang ◽  
Albert E. Fliss ◽  
Jie Rao ◽  
Avrom J. Caplan
Keyword(s):  
Hormone Receptors ◽  
Pcr Amplification ◽  
Steroid Hormone Receptors ◽  
Loss Of Function ◽  
Vitro Assay ◽  
Growth Defects ◽  
Double Deletion ◽  
Affinity Isolation

ABSTRACT The Saccharomyces cerevisiae SBA1 gene was cloned by PCR amplification from yeast genomic DNA following its identification as encoding an ortholog of human p23, an Hsp90 cochaperone. TheSBA1 gene product is constitutively expressed and nonessential, although a disruption mutant grew more slowly than the wild type at both 18 and 37°C. A double deletion of SBA1and STI1, encoding an Hsp90 cochaperone, displayed synthetic growth defects. Affinity isolation of histidine-tagged Sba1p (Sba1His6) after expression in yeast led to coisolation of Hsp90 and the cyclophilin homolog Cpr6. Using an in vitro assembly assay, purified Sba1His6 bound to Hsp90 only in the presence of adenosine 5′-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. Furthermore, interaction between purified Sba1His6 and Hsp90 in yeast extracts was inhibited by the benzoquinoid ansamycins geldanamycin and macbecin. The in vitro assay was also used to identify residues in Hsp90 that are important for complex formation with Sba1His6, and residues in both the N-terminal nucleotide binding domain and C-terminal half were characterized. In vivo analysis of known Hsp90 substrate proteins revealed that Sba1 loss of function had only a mild effect on the activity of the tyrosine kinase v-Src and steroid hormone receptors.

scholarly journals PGC7 suppresses TET3 for protecting DNA methylation

2013 ◽  
Vol 42 (5) ◽  
pp. 2893-2905 ◽  
Author(s):  
Chunjing Bian ◽  
Xiaochun Yu
Keyword(s):  
Dna Methylation ◽  
Molecular Mechanism ◽  
Biological Process ◽  
Cpg Islands ◽  
Binding Motifs ◽  
Genome Wide Analysis ◽  
Dna Motif ◽  
Genome Wide

Abstract Ten-eleven translocation (TET) family enzymes convert 5-methylcytosine to 5-hydroxylmethylcytosine. However, the molecular mechanism that regulates this biological process is not clear. Here, we show the evidence that PGC7 (also known as Dppa3 or Stella) interacts with TET2 and TET3 both in vitro and in vivo to suppress the enzymatic activity of TET2 and TET3. Moreover, lacking PGC7 induces the loss of DNA methylation at imprinting loci. Genome-wide analysis of PGC7 reveals a consensus DNA motif that is recognized by PGC7. The CpG islands surrounding the PGC7-binding motifs are hypermethylated. Taken together, our study demonstrates a molecular mechanism by which PGC7 protects DNA methylation from TET family enzyme-dependent oxidation.

TMOD-02. GENERATION OF A NOVEL MOUSE MODEL FOR BRAIN TUMORS OF THE DNA METHYLATION CLASS “GBM MYCN”

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi215-vi216
Author(s):  
Melanie Schoof ◽  
Carolin Göbel ◽  
Dörthe Holdhof ◽  
Sina Al-Kershi ◽  
Ulrich Schüller
Keyword(s):  
Dna Methylation ◽  
Brain Tumors ◽  
Molecular Mechanisms ◽  
Treatment Options ◽  
Tumor Development ◽  
Model Systems ◽  
Preclinical Research ◽  
Human Gbm

Abstract DNA methylation based classification of brain tumors has revealed a high heterogeneity between tumors and led to the description of multiple distinct subclasses. The increasing subdivision of tumors can help to understand molecular mechanisms of tumor development and to improve therapy if appropriate model systems for preclinical research are available. Multiple recent publications have described a subgroup of pediatric glioblastoma which is clearly separable from other pediatric and adult glioblastoma in its DNA methylation profile (GBM MYCN). Many cases in this group are driven by MYCN amplifications and harbor TP53 mutations. These tumors almost exclusively occur in children and were further described as highly aggressive with a median overall survival of only 14 months. In order to further investigate the biology and treatment options of these tumors, we generated hGFAP-cre::TP53 Fl/Fl ::lsl-MYCN mice. These mice carry a loss of TP53 and show aberrant MYCN expression in neural precursors of the central nervous system. The animals develop large forebrain tumors within the first 80 days of life with 100 % penetrance. These tumors resemble human GBM MYCN tumors histologically and are sensitive to AURKA and ATR inhibitors in vitro. We believe that further characterization of the model and in vivo treatment studies will pave the way to improve treatment of patients with these highly aggressive tumors.

Interspersion of an unusual GCN4 activation site with a complex transcriptional repression site in Ty2 elements of Saccharomyces cerevisiae

1993 ◽  
Vol 13 (4) ◽  
pp. 2091-2103
Author(s):  
S Türkel ◽  
P J Farabaugh
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Transcriptional Activation ◽  
Binding Sites ◽  
Transcriptional Repression ◽  
Physiological Control ◽  
Reading Frame ◽  
Negative Region ◽  
Activation Site

Transcription of the Ty2-917 retrotransposon of Saccharomyces cerevisiae is modulated by a complex set of positive and negative elements, including a negative region located within the first open reading frame, TYA2. The negative region includes three downstream repression sites (DRSI, DRSII, and DRSIII). In addition, the negative region includes at least two downstream activation sites (DASs). This paper concerns the characterization of DASI. A 36-bp DASI oligonucleotide acts as an autonomous transcriptional activation site and includes two sequence elements which are both required for activation. We show that these sites bind in vitro the transcriptional activation protein GCN4 and that their activity in vivo responds to the level of GCN4 in the cell. We have termed the two sites GCN4 binding sites (GBS1 and GBS2). GBS1 is a high-affinity GCN4 binding site (dissociation constant, approximately 25 nM at 30 degrees C), binding GCN4 with about the affinity of a consensus UASGCN4, this though GBS1 includes two differences from the right half of the palindromic consensus site. GBS2 is more diverged from the consensus and binds GCN4 with about 20-fold-lower affinity. Nucleotides 13 to 36 of DASI overlap DRSII. Since DRSII is a transcriptional repression site, we tested whether DASI includes repression elements. We identify two sites flanking GBS2, both of which repress transcription activated by the consensus GCN4-specific upstream activation site (UASGCN4). One of these is repeated in the 12 bp immediately adjacent to DASI. Thus, in a 48-bp region of Ty2-917 are interspersed two positive and three negative transcriptional regulators. The net effect of the region must depend on the interaction of the proteins bound at these sites, which may include their competing for binding sites, and on the physiological control of the activity of these proteins.

scholarly journals DNA methylation specifies chromosomal localization of MeCP2.

1996 ◽  
Vol 16 (1) ◽  
pp. 414-421 ◽  
Author(s):  
X Nan ◽  
P Tate ◽  
E Li ◽  
A Bird
Keyword(s):  
Dna Methylation ◽  
Cultured Cells ◽  
Centromeric Heterochromatin ◽  
Intact Protein ◽  
Chromosomal Protein ◽  
Mouse Cells ◽  
Low Levels ◽  
Mutant Cells

MeCP2 is a chromosomal protein that is concentrated in the centromeric heterochromatin of mouse cells. In vitro, the protein binds preferentially to DNA containing a single symmetrically methylated CpG. To find out whether the heterochromatic localization of MeCP2 depended on DNA methylation, we transiently expressed MeCP2-LacZ fusion proteins in cultured cells. Intact protein was targeted to heterochromatin in wild-type cells but was inefficiently localized in mutant cells with low levels of genomic DNA methylation. Deletions within MeCP2 showed that localization to heterochromatin required the 85-amino-acid methyl-CpG binding domain but not the remainder of the protein. Thus MeCP2 is a methyl-CpG-binding protein in vivo and is likely to be a major mediator of downstream consequences of DNA methylation.

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