DNA Methyltransferase Probing of Chromatin Structure Within Populations and on Single Molecules

2009 ◽  
pp. 41-65 ◽  
Author(s):  
Carolina Pardo ◽  
Scott A. Hoose ◽  
Santhi Pondugula ◽  
Michael P. Kladde
Keyword(s):  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Single Molecules

Introduction of a DNA methyltransferase into Drosophila to probe chromatin structure in vivo

Chromosoma ◽  
1996 ◽  
Vol 104 (5) ◽  
pp. 332-340 ◽  
Author(s):  
Debora R. Wines ◽  
Paul B. Talbert ◽  
Denise V. Clark ◽  
Steven Henikoff
Keyword(s):  
Chromatin Structure ◽  
Dna Methyltransferase

Introduction of a DNA methyltransferase into Drosophila to probe chromatin structure in vivo

Chromosoma ◽  
1996 ◽  
Vol 104 (5) ◽  
pp. 332-340 ◽  
Author(s):  
Debora R. Wines ◽  
Paul B. Talbert ◽  
Denise V. Clark ◽  
Steven Henikoff
Keyword(s):  
Chromatin Structure ◽  
Dna Methyltransferase

scholarly journals Methylation-related chromatin structure is associated with exclusion of transcription factors from and suppressed expression of the O-6-methylguanine DNA methyltransferase gene in human glioma cell lines.

1994 ◽  
Vol 14 (10) ◽  
pp. 6515-6521 ◽  
Author(s):  
J F Costello ◽  
B W Futscher ◽  
R A Kroes ◽  
R O Pieper
Keyword(s):  
Transcription Factors ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Glioma Cell ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Mgmt Expression ◽  
Mgmt Gene ◽  
Glioma Cell Lines

There is considerable interest in identifying factors responsible for expression of the O-6-methylguanine DNA methyltransferase (MGMT) gene, as MGMT is a major determinant in the response of glioma cells to the chemotherapeutic agent 1,3 bis(2-chloroethyl)-1-nitrosourea. Recently we have shown that MGMT expression is correlated in a direct, graded fashion with methylation in the body of the MGMT gene and in an inverse, graded fashion with promoter methylation in human glioma cell lines. To determine if promoter methylation is an important component of MGMT expression, this study addressed the complex interactions between methylation, chromatin structure, and in vivo transcription factor occupancy in the MGMT promoter of glioma cell lines with different levels of MGMT expression. Our results show that the basal promoter in MGMT-expressing glioma cell lines, which is 100% unmethylated, was very accessible to restriction enzymes at all sites tested, suggesting that this region may be nucleosome free. The basal promoter in glioma cells with minimal MGMT expression, however, which is 75% unmethylated, was much less accessible, and the basal promoter in nonexpressing cells, which is 50% unmethylated, was entirely inaccessible to restriction enzymes. Despite the presence of the relevant transcription factors in all cell lines examined, in vivo footprinting showed DNA-protein interactions at six Sp1 binding sites and one novel binding site in MGMT-expressing cell lines but no such interactions in nonexpressors. We conclude that in contrast to findings of previous in vitro studies, Sp1 is an important component of MGMT transcription. These correlations also strongly suggest that methylation and chromatin structure, by determining whether Sp1 and other transcription factors can access the MGMT promoter, set the transcriptional state of the MGMT gene.

scholarly journals A Methyltransferase Targeting Assay Reveals Silencer-Telomere Interactions in Budding Yeast

2003 ◽  
Vol 23 (5) ◽  
pp. 1498-1508 ◽  
Author(s):  
Eleonore Lebrun ◽  
Geneviève Fourel ◽  
Pierre-Antoine Defossez ◽  
Eric Gilson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Subtelomeric Region ◽  
Yeast Genome ◽  
Dna Binding Domain ◽  
Bacterial Dna ◽  
Intervening Sequences ◽  
Identification Technique

ABSTRACT We have designed a modified version of the Dam identification technique and used it to probe higher-order chromatin structure in Saccharomyces cerevisiae. We fused the bacterial DNA methyltransferase Dam to the DNA-binding domain of TetR and targeted the resulting chimera to Tet operators inserted in the yeast genome at the repressed locus HML. We then monitored the methylation status of HML and other sequences by a quantitative technique combining methylation-sensitive restriction and real-time PCR. As expected, we found that TetR-Dam efficiently methylated HML in cis. More strikingly, when TetR-Dam was present at HML, we observed increased methylation in the III-L subtelomeric region but not in intervening sequences. This effect was lost when the HML silencers were inactivated by mutations. When the HM silencers and the Tet operators were transferred to a plasmid, strong methylation was clearly observed not only in the III-L subtelomeric region but also at other telomeres. These data indicate that HM silencers can specifically associate with telomeres, even those located on different chromosomes.

scholarly journals Evaluation in mammalian oocytes of gene transcripts linked to epigenetic reprogramming

Reproduction ◽  
2007 ◽  
Vol 134 (4) ◽  
pp. 549-558 ◽  
Author(s):  
Roberto S Oliveri ◽  
Mark Kalisz ◽  
Charlotte Karlskov Schjerling ◽  
Claus Yding Andersen ◽  
Rehannah Borup ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Histone Deacetylases ◽  
Mammalian Cells ◽  
Dna Methyltransferase ◽  
Germinal Vesicle ◽  
Epigenetic Reprogramming ◽  
Sperm Chromatin ◽  
Somatic Nucleus ◽  
Covalent Modifications

The mature mammalian metaphase II (MII) oocyte has a unique ability to reprogram sperm chromatin and support early embryonic development. This feature even extends to the epigenetic reprogramming of a terminally differentiated cell nucleus as observed in connection with somatic cell nuclear transfer. Epigenetic nuclear reprogramming is highly linked to chromatin structure and includes covalent modifications of DNA and core histone proteins as well as reorganization of higher-order chromatin structure. A group of conserved enzymes mediating DNA methylation, methyl-CpG-binding protein (MeCP), histone acetylation and methylation, and chromatin remodeling are extensively involved in epigenetic reprogramming in mammalian cells. Using the oligonucleotide microarray technique, the present study compared the expression levels of 86 genes associated with epigenetic reprogramming in murine in vivo matured MII oocytes with that of germinal vesicle oocytes. Correlation between biological replicates was high. A total of 57 genes with potential reprogramming effect were detected. In MII oocytes, four genes were significant up-regulated, whereas 18 were down-regulated and 35 unchanged. The significantly regulated genes were validated by real-time quantitative RT-PCR. For example, MII oocytes showed a significant down-regulation of oocyte-specific maintenance DNA methyltransferase, Dnmt1o, and up-regulation of MeCP transcript, methyl-CpG binding domain protein 2. Furthermore, histone acetyltransferases were proportionally overrepresented when compared with histone deacetylases. These data elucidate for the first time some of the mechanisms that the oocyte may employ to reprogram a foreign genome either in form of a spermatozoa or a somatic nucleus and may thus be of importance for advancing the fields of stem cell research and regenerative medicine.

scholarly journals In Vivo Chromatin Accessibility Correlates With Gene Silencing in Drosophila

Genetics ◽  
1998 ◽  
Vol 150 (4) ◽  
pp. 1539-1549 ◽  
Author(s):  
Antoine Boivin ◽  
Jean-Maurice Dura
Keyword(s):  
Gene Silencing ◽  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Target Genes ◽  
Position Effect ◽  
Chromatin Accessibility ◽  
Polycomb Group ◽  
Position Effect Variegation ◽  
Direct Support

Abstract Gene silencing by heterochromatin is a well-known phenomenon that, in Drosophila, is called position effect variegation (PEV). The long-held hypothesis that this gene silencing is associated with an altered chromatin structure received direct support only recently. Another gene-silencing phenomenon in Drosophila, although similar in its phenotype of variegation, has been shown to be associated with euchromatic sequences and is dependent on developmental regulators of the Polycomb group (Pc-G) of gene products. One model proposes that the Pc-G products may cause a local heterochromatinization that maintains a repressed state of transcription of their target genes. Here, we test these models by measuring the accessibility of white or miniwhite sequences, in different contexts, to the Escherichia coli dam DNA methyltransferase in vivo. We present evidence that PEV and Pc-G-mediated repression mechanisms, although based on different protein factors, may indeed involve similar higher-order chromatin structure.

scholarly journals Epigenetic Mechanisms in DNA Double Strand Break Repair: A Clinical Review

2021 ◽  
Vol 8 ◽  
Author(s):  
Alejandra Fernandez ◽  
Connor O’Leary ◽  
Kenneth J O’Byrne ◽  
Joshua Burgess ◽  
Derek J Richard ◽  
...  
Keyword(s):  
Dna Damage ◽  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Strand Break ◽  
Dna Methyltransferases ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Epigenetic Mechanisms ◽  
Dna Double Strand Break ◽  
Break Repair

Upon the induction of DNA damage, the chromatin structure unwinds to allow access to enzymes to catalyse the repair. The regulation of the winding and unwinding of chromatin occurs via epigenetic modifications, which can alter gene expression without changing the DNA sequence. Epigenetic mechanisms such as histone acetylation and DNA methylation are known to be reversible and have been indicated to play different roles in the repair of DNA. More importantly, the inhibition of such mechanisms has been reported to play a role in the repair of double strand breaks, the most detrimental type of DNA damage. This occurs by manipulating the chromatin structure and the expression of essential proteins that are critical for homologous recombination and non-homologous end joining repair pathways. Inhibitors of histone deacetylases and DNA methyltransferases have demonstrated efficacy in the clinic and represent a promising approach for cancer therapy. The aims of this review are to summarise the role of histone deacetylase and DNA methyltransferase inhibitors involved in DNA double strand break repair and explore their current and future independent use in combination with other DNA repair inhibitors or pre-existing therapies in the clinic.

5-Azacytidine and G-CSF Derepressed Chromatin Structure of PU.1 and Its Targets Cebpa and Cbfb In Myelodysplastic Syndrome (MDS)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 124-124
Author(s):  
Tomas Stopka ◽  
Pavel Burda ◽  
Petra Basova ◽  
Karin Vargova ◽  
Nikola Curik ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Myelodysplastic Syndrome ◽  
Cell Lines ◽  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Conflicts Of Interest ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Epigenetic Therapy ◽  
Number Of Patients

Abstract Abstract 124 The myelodysplastic syndrome (MDS) represents a heterogeneous disorder characterized by ineffective hematopoiesis and evolution to acute myelogenous leukemia that is strikingly refractory to current therapeutic approaches. Novel epigenetic drugs including DNA-methyltransferase inhibitor 5-Azacitidine (5-AZA, Vidaza) are currently considered to improve clinical response in patients with MDS. MDS is characterized by abnormal differentiation and blocked maturation responsive to 5-AZA, therefore we studied major regulator of hematopoietic differentiation, transcription factor PU.1 as a candidate target of the epigenetic therapy. Transcription factor PU.1 represents very important myelo-lymphoid regulator of differentiation. PU.1 expression is regulated by Upstream Regulatory Element (URE) and its deletion in mouse caused downregulation of PU.1 leading to acute leukemia (Rosenbauer 2004). Our laboratory recently demonstrated that PU.1 in murine acute leukemic cells binds and promotes derepression of CCAAT/enhancer binding protein (C/EBP) alpha (Cebpa) and Core-binding factor, beta subunit (Cbfb) (Burda 2009) that encode two key hematopoietic transcription factors involved in myeloid differentiation. Furthermore, transcriptional regulation through PU.1 binding sites of Cebpa and Cbfb loci involves quantitative increases in a transcriptionally active chromatin mark: acetylation of histone H3 lysine K9. Others reported that Cebpa expression is augmented by G-CSF (Dahl 2003). To determine if 5-AZA regulates PU.1 and its targets we determined their expression and chromatin structure following the 5-AZA treatment in MDS patient-derived blasts and in cell lines derived from MDS (MOLM-13, OCI-M2, SKM-1) and AML (K562). Our data provide evidence that in the chosen cell lines and in so far limited number of patients-derived cells (N=4) the gene expression of PU.1 and its direct targets Cebpa and Cbfb is stimulated by 5-AZA and this effect is further enhanced by G-CSF. Furthermore, marks of activated chromatin structure including histone H3K9 hyperacetylation and H3K4 hypermethylation are increased at the URE of the PU.1 gene again documenting its transcriptional activation. Conversely, levels of H3K9 methylation at URE are significantly reduced upon 5-AZA treatment documenting 5-AZA stimulates loss of repressive chromatin structure near PU.1 gene. These observations are currently compared with responsiveness of the patients to 5-AZA in vivo and expanded to larger set of patients. Our data collectively supports importance of the chromatin structure upstream of PU.1 gene and of its direct targets Cebpa and Cbfb in patients with MDS that may add to better understanding of effectiveness of epigenetic therapy in MDS. (Grants # IGA 10310-3, MSMT 2B06077, SVV-2010-254260507, MPO FR-TI2/509, GAUK 251135 82210). Disclosures: No relevant conflicts of interest to declare.

Abstract 5371: Effect of DNA methyltransferase inhibitor 5-azacitidine on 3D chromatin structure measured by Hi-C

2017 ◽  
Author(s):  
Yuchen Vincent Bai ◽  
John Whitaker ◽  
Emanuele Palescandolo ◽  
Vinod Krishna ◽  
Vipul Bhargava ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Dna Methyltransferase Inhibitor ◽  
3D Chromatin Structure

scholarly journals Inhibitors of Histone Deacetylase and DNA Methyltransferase Synergistically Activate the Methylated Metallothionein I Promoter by Activating the Transcription Factor MTF-1 and Forming an Open Chromatin Structure

2002 ◽  
Vol 22 (23) ◽  
pp. 8302-8319 ◽  
Author(s):  
Kalpana Ghoshal ◽  
Jharna Datta ◽  
Sarmila Majumder ◽  
Shoumei Bai ◽  
Xiaocheng Dong ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Histone Deacetylase ◽  
Chromatin Structure ◽  
Dna Methyltransferase ◽  
Combined Treatment ◽  
Hdac Inhibitors ◽  
Prolonged Treatment ◽  
Open Chromatin ◽  
Chip Assay

ABSTRACT Inhibitors of DNA methyltransferase (Dnmt) and histone deacetylases (HDAC) synergistically activate the methylated metallothionein I gene (MT-I) promoter in mouse lymphosarcoma cells. The cooperative effect of these two classes of inhibitors on MT-I promoter activity was robust following demethylation of only a few CpG dinucleotides by brief exposure to 5-azacytidine (5-AzaC) but persisted even after prolonged treatment with the nucleoside analog. HDAC inhibitors (trichostatin A [TSA] and depsipeptide) either alone or in combination with 5-AzaC did not facilitate demethylation of the MT-I promoter. Treatment of cells with HDAC inhibitors increased accumulation of multiply acetylated forms of H3 and H4 histones that remained unaffected after treatment with 5-AzaC. Chromatin immunoprecipitation (ChIP) assay showed increased association of acetylated histone H4 and lysine 9 (K9)-acetyl H3 with the MT-I promoter after treatment with TSA, which was not affected following treatment with 5-AzaC. In contrast, the association of K9-methyl histone H3 with the MT-I promoter decreased significantly after treatment with 5-AzaC and TSA. ChIP assay with antibodies specific for methyl-CpG binding proteins (MBDs) demonstrated that only methyl-CpG binding protein 2 (MeCP2) was associated with the MT-I promoter, which was significantly enhanced after TSA treatment. Association of histone deacetylase 1 (HDAC1) with the promoter decreased after treatment with TSA or 5-AzaC and was abolished after treatment with both inhibitors. Among the DNA methyltransferases, both Dnmt1 and Dnmt3a were associated with the MT-I promoter in the lymphosarcoma cells, and association of Dnmt1 decreased with time after treatment with 5-AzaC. Treatment of these cells with HDAC inhibitors also increased expression of the MTF-1 (metal transcription factor-1) gene as well as its DNA binding activity. In vivo genomic footprinting studies demonstrated increased occupancy of MTF-1 to metal response elements of the MT-I promoter after treatment with both inhibitors. Analysis of the promoter by mapping with restriction enzymes in vivo showed that the MT-I promoter attained a more open chromatin structure after combined treatment with 5-AzaC and TSA as opposed to treatment with either agent alone. These results implicate involvement of multifarious factors including modified histones, MBDs, and Dnmts in silencing the methylated MT-I promoter in lymphosarcoma cells. The synergistic activation of this promoter by these two types of inhibitors is due to demethylation of the promoter and altered association of different factors that leads to reorganization of the chromatin and the resultant increase in accessibility of the promoter to the activated transcription factor MTF-1.

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