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 The Effects of Cadmium Toxicity

2020 ◽  
Vol 17 (11) ◽  
pp. 3782 ◽  
Author(s):  
Giuseppe Genchi ◽  
Maria Stefania Sinicropi ◽  
Graziantonio Lauria ◽  
Alessia Carocci ◽  
Alessia Catalano
Keyword(s):  
Mammalian Cells ◽  
Dna Methyltransferase ◽  
Kidney Diseases ◽  
Cadmium Toxicity ◽  
Indian Mustard ◽  
Eucalyptus Camaldulensis ◽  
Micro Rna ◽  
Epigenetic Changes

Cadmium (Cd) is a toxic non-essential transition metal that poses a health risk for both humans and animals. It is naturally occurring in the environment as a pollutant that is derived from agricultural and industrial sources. Exposure to cadmium primarily occurs through the ingestion of contaminated food and water and, to a significant extent, through inhalation and cigarette smoking. Cadmium accumulates in plants and animals with a long half-life of about 25–30 years. Epidemiological data suggest that occupational and environmental cadmium exposure may be related to various types of cancer, including breast, lung, prostate, nasopharynx, pancreas, and kidney cancers. It has been also demonstrated that environmental cadmium may be a risk factor for osteoporosis. The liver and kidneys are extremely sensitive to cadmium’s toxic effects. This may be due to the ability of these tissues to synthesize metallothioneins (MT), which are Cd-inducible proteins that protect the cell by tightly binding the toxic cadmium ions. The oxidative stress induced by this xenobiotic may be one of the mechanisms responsible for several liver and kidney diseases. Mitochondria damage is highly plausible given that these organelles play a crucial role in the formation of ROS (reactive oxygen species) and are known to be among the key intracellular targets for cadmium. When mitochondria become dysfunctional after exposure to Cd, they produce less energy (ATP) and more ROS. Recent studies show that cadmium induces various epigenetic changes in mammalian cells, both in vivo and in vitro, causing pathogenic risks and the development of various types of cancers. The epigenetics present themselves as chemical modifications of DNA and histones that alter the chromatin without changing the sequence of the DNA nucleotide. DNA methyltransferase, histone acetyltransferase, histone deacetylase and histone methyltransferase, and micro RNA are involved in the epigenetic changes. Recently, investigations of the capability of sunflower (Helianthus annuus L.), Indian mustard (Brassica juncea), and river red gum (Eucalyptus camaldulensis) to remove cadmium from polluted soil and water have been carried out. Moreover, nanoparticles of TiO2 and Al2O3 have been used to efficiently remove cadmium from wastewater and soil. Finally, microbial fermentation has been studied as a promising method for removing cadmium from food. This review provides an update on the effects of Cd exposure on human health, focusing on the cellular and molecular alterations involved.

Chromatin structure and function: the heretical path to an RNA transcription factor

1996 ◽  
Vol 74 (5) ◽  
pp. 623-632 ◽  
Author(s):  
Margarida O. Krause
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
T Antigen ◽  
Temperature Sensitive ◽  
Mobility Shift ◽  
Cell Extracts ◽  
Myc Gene ◽  
And Function

This review represents a synthesis of the work of the author and her collaborators through 40 years of research aimed at an understanding of chromatin composition and functional arrangement. It describes the progressive experimental stages, starting with autoradiography and protein analysis and continuing on to a more functional approach testing the template properties of intact nuclei, as well as nuclei depleted of, or reconstituted with, defined fractions extracted from the chromatin of other cell lines or tissues. As new questions were raised at each phase of these studies, the investigation was shifted from chromosomal proteins to the role of a small RNA that coextracted with one protein fraction and whose properties suggested a transcription-activating function. The active RNA was identified as a class in RNA, designated as 7 SK. Its properties suggested a role in the activation of two oncogenes, the SV 40 T-antigen and the mammalian c-myc gene. A detailed analysis of the c-myc gene expression during transformation induction in temperature-sensitive mammalian cells finally culminated in in vivo evidence for a role of 7 SK in c-myc deregulation, using cells transfected with antisense oligonucleotides to block 7 SK activity. This was followed by an investigation of promoter targeting by 7 SK RNP using electrophoretic mobility shift assays with whole or 7 SK-depleted cell extracts. Taken together, these studies indicate that 7 SK RNP participates in transformation-dependent deregulation of the c-myc gene by activation of two c-myc minor promoters. The implications of these findings are discussed.Key words: chromatin structure, histones, nonhistones, 7 SK RNA, the c-myc gene, transcription regulation, SV 40, transformation.

scholarly journals In Vivo HP1 Targeting Causes Large-Scale Chromatin Condensation and Enhanced Histone Lysine Methylation

2005 ◽  
Vol 25 (11) ◽  
pp. 4552-4564 ◽  
Author(s):  
Pernette J. Verschure ◽  
Ineke van der Kraan ◽  
Wim de Leeuw ◽  
Johan van der Vlag ◽  
Anne E. Carpenter ◽  
...  
Keyword(s):  
Chromatin Structure ◽  
Mammalian Cells ◽  
Large Scale ◽  
Chromosome Region ◽  
Chromatin Condensation ◽  
Regulation Of Gene Expression ◽  
Lac Repressor ◽  
Homologous Proteins ◽  
Lac Operator

ABSTRACT Changes in chromatin structure are a key aspect in the epigenetic regulation of gene expression. We have used a lac operator array system to visualize by light microscopy the effect of heterochromatin protein 1 (HP1) α (HP1α) and HP1β on large-scale chromatin structure in living mammalian cells. The structure of HP1, containing a chromodomain, a chromoshadow domain, and a hinge domain, allows it to bind to a variety of proteins. In vivo targeting of an enhanced green fluorescent protein-tagged HP1-lac repressor fusion to a lac operator-containing, gene-amplified chromosome region causes local condensation of the higher-order chromatin structure, recruitment of the histone methyltransferase SETDB1, and enhanced trimethylation of histone H3 lysine 9. Polycomb group proteins of both the HPC/HPH and the EED/EZH2 complexes, which are involved in the heritable repression of gene activity, are not recruited to the amplified chromosome region by HP1α and HP1β in vivo targeting. HP1α targeting causes the recruitment of endogenous HP1β to the chromatin region and vice versa, indicating a direct interaction between the two HP1 homologous proteins. Our findings indicate that HP1α and HP1β targeting is sufficient to induce heterochromatin formation.

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 INSM1 functions as a transcriptional repressor of the neuroD/β2 gene through the recruitment of cyclin D1 and histone deacetylases

2006 ◽  
Vol 397 (1) ◽  
pp. 169-177 ◽  
Author(s):  
Wei-Dong Liu ◽  
Hong-Wei Wang ◽  
Michelle Muguira ◽  
Mary B. Breslin ◽  
Michael S. Lan
Keyword(s):  
Cyclin D1 ◽  
Chromatin Immunoprecipitation ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Mammalian Cells ◽  
Transcriptional Repressor ◽  
Chromatin Immunoprecipitation Assay ◽  
Immunoprecipitation Assay ◽  
Repressor Activity

INSM1/IA-1 (insulinoma-associated 1) is a developmentally regulated zinc-finger transcription factor, exclusively expressed in the foetal pancreas and nervous system, and in tumours of neuroendocrine origin. We have identified an INSM1 binding site in the neuroD/β2 promoter and demonstrated transcriptional repressor activity of INSM1 by transient transfection assay. A chromatin immunoprecipitation assay confirmed that in vivo INSM1 is situated on the promoter region of the neuroD/β2 gene. In an attempt to elucidate the molecular mechanism of transcriptional repression by the INSM1 gene, cyclin D1 was identified as an interacting protein by using a 45-day-old human foetal brain cDNA library and a yeast two-hybrid screen. The physical association between INSM1 and cyclin D1 was confirmed by in vitro and in vivo pull-down assay. Cyclin D1 co-operates with INSM1 and suppresses neuroD/β2 promoter activity. Co-immunoprecipitation of INSM1, cyclin D1 and HDACs (histone deacetylases) in mammalian cells revealed that INSM1 interacts with HDAC-1 and -3 and that this interaction is mediated through cyclin D1. Overexpression of cyclin D1 and HDAC-3 significantly enhanced the transcriptional repression activity of INSM1 on the neuroD/β2 promoter. A further chromatin immunoprecipitation assay confirmed that HDAC-3 occupies this same region of the neuroD/β2 promoter, by forming a transcription complex with INSM1. Thus we conclude that INSM1 recruits cyclin D1 and HDACs, which confer transcriptional repressor activity.

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 LSH Cooperates with DNA Methyltransferases To Repress Transcription

2007 ◽  
Vol 28 (1) ◽  
pp. 215-226 ◽  
Author(s):  
Kevin Myant ◽  
Irina Stancheva
Keyword(s):  
Dna Methylation ◽  
Chromatin Remodeling ◽  
Transcriptional Repression ◽  
Histone Deacetylases ◽  
Mammalian Cells ◽  
Dna Methyltransferase ◽  
Dna Methyltransferases ◽  
Large Protein ◽  
Large Protein Complex ◽  
Nuclear Extracts

ABSTRACT LSH, a protein related to the SNF2 family of chromatin-remodeling ATPases, is required for efficient DNA methylation in mammals. How LSH functions to support DNA methylation and whether it associates with a large protein complex containing DNA methyltransferase (DNMT) enzymes is currently unclear. Here we show that, unlike many other chromatin-remodeling ATPases, native LSH is present mostly as a monomeric protein in nuclear extracts of mammalian cells and cannot be detected in a large multisubunit complex. However, when targeted to a promoter of a reporter gene, LSH acts as an efficient transcriptional repressor. Using this as an assay to identify proteins that are required for LSH-mediated repression we found that LSH cooperates with the DNMTs DNMT1 and DNMT3B and with the histone deacetylases (HDACs) HDAC1 and HDAC2 to silence transcription. We show that transcriptional repression by LSH and interactions with HDACs are lost in DNMT1 and DNMT3B knockout cells but that the enzymatic activities of DNMTs are not required for LSH-mediated silencing. Our data suggest that LSH serves as a recruiting factor for DNMTs and HDACs to establish transcriptionally repressive chromatin which is perhaps further stabilized by DNA methylation at targeted loci.

scholarly journals p70S6K Controls Selective mRNA Translation during Oocyte Maturation and Early Embryogenesis inXenopus laevis

1999 ◽  
Vol 19 (4) ◽  
pp. 2485-2494 ◽  
Author(s):  
Markus S. Schwab ◽  
Sang H. Kim ◽  
Naohiro Terada ◽  
Catarina Edfjäll ◽  
Sara C. Kozma ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
Translational Control ◽  
Mammalian Cells ◽  
Germinal Vesicle ◽  
Mrna Translation ◽  
Early Embryogenesis ◽  
Entry Site ◽  
Germinal Vesicle Breakdown ◽  
Rapamycin Treatment

ABSTRACT In mammalian cells, p70S6K plays a key role in translational control of cell proliferation in response to growth factors. Because of the reliance on translational control in early vertebrate development, we cloned a Xenopus homolog of p70S6K and investigated the activity profile of p70S6K during Xenopus oocyte maturation and early embryogenesis. p70S6K activity is high in resting oocytes and decreases to background levels upon stimulation of maturation with progesterone. During embryonic development, three peaks of activity were observed: immediately after fertilization, shortly before the midblastula transition, and during gastrulation. Rapamycin, an inhibitor of p70S6K activation, caused oocytes to undergo germinal vesicle breakdown earlier than control oocytes, and sensitivity to progesterone was increased. Injection of a rapamycin-insensitive, constitutively active mutant of p70S6K reversed the effects of rapamycin. However, increases in S6 phosphorylation were not significantly affected by rapamycin during maturation. mosmRNA, which does not contain a 5′-terminal oligopyrimidine tract (5′-TOP), was translated earlier, and a larger amount of Mos protein was produced in rapamycin-treated oocytes. In fertilized eggs rapamycin treatment increased the translation of the Cdc25A phosphatase, which lacks a 5′-TOP. Translation assays in vivo using both DNA and RNA reporter constructs with the 5′-TOP from elongation factor 2 showed decreased translational activity with rapamycin, whereas constructs without a 5′-TOP or with an internal ribosome entry site were translated more efficiently upon rapamycin treatment. These results suggest that changes in p70S6K activity during oocyte maturation and early embryogenesis selectively alter the translational capacity available for mRNAs lacking a 5′-TOP region.

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.

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