Biomedical subjects

Howard Cedar

Publications and source records attributed to Howard Cedar.

18 recordsLinked to original sources

PubMed2006

Polycomb-mediated methylation on Lys27 of histone H3 pre-marks genes for de novo methylation in cancer.

Many genes associated with CpG islands undergo de novo methylation in cancer. Studies have suggested that the pattern of this modification may be partially determined by an instructive mechanism that recognizes specifically marked regions of the genome. Using chromatin immunoprecipitation analysis, here we show that genes methylated in cancer cells are specifically packaged with nucleosomes containing histone H3 trimethylated on Lys27. This chromatin mark is established on these unmethylated CpG island genes early in development and then maintained in differentiated cell types by the presence of an EZH2-containing Polycomb complex. In cancer cells, as opposed to normal cells, the presence of this complex brings about the recruitment of DNA methyl transferases, leading to de novo methylation. These results suggest that tumor-specific targeting of de novo methylation is pre-programmed by an established epigenetic system that normally has a role in marking embryonic genes for repression.

Caco-2 Cells↗

PubMed2006

Fine tuning of globin gene expression by DNA methylation.

Expression patterns in the globin gene cluster are subject to developmental regulation in vivo. While the gamma(A) and gamma(G) genes are expressed in fetal liver, both are silenced in adult erythrocytes. In order to decipher the role of DNA methylation in this process, we generated a YAC transgenic mouse system that allowed us to control gamma(A) methylation during development. DNA methylation causes a 20-fold repression of gamma(A) both in non-erythroid and adult erythroid cells. In erythroid cells this modification works as a dominant mechanism to repress gamma gene expression, probably through changes in histone acetylation that prevent the binding of erythroid transcription factors to the promoter. These studies demonstrate that DNA methylation serves as an elegant in vivo fine-tuning device for selecting appropriate genes in the globin locus. In addition, our findings provide a mechanism for understanding the high levels of gamma-globin transcription seen in patients with Hereditary Persistence of Fetal Hemoglobin, and help explain why 5azaC and butyrate compounds stimulate gamma-globin expression in patients with beta-hemoglobinopathies.

Acetylation↗

PubMed2006

G9a-mediated irreversible epigenetic inactivation of Oct-3/4 during early embryogenesis.

Oct-3/4 is a POU domain homeobox gene that is expressed during gametogenesis and in early embryonic cells, where it has been shown to be important for maintaining pluripotency. Following implantation, this gene undergoes a novel multi-step programme of inactivation. Transcriptional repression is followed by a pronounced increase in histone H3 methylation on Lys 9 that is mediated by the SET-containing protein, G9a. This step sets the stage for local heterochromatinization via the binding of HP1 and is required for subsequent de novo methylation at the promoter by the enzymes Dnmt3a/3b. Genetic studies show that these epigenetic changes actually have an important role in the inhibition of Oct-3/4 re-expression, thereby preventing reprogramming.

Acetylation↗

PubMed2006

Evidence for an instructive mechanism of de novo methylation in cancer cells.

DNA methylation has a role in the regulation of gene expression during normal mammalian development but can also mediate epigenetic silencing of CpG island genes in cancer and other diseases. Many individual genes (including tumor suppressors) have been shown to undergo de novo methylation in specific tumor types, but the biological logic inherent in this process is not understood. To decipher this mechanism, we have adopted a new approach for detecting CpG island DNA methylation that can be used together with microarray technology. Genome-wide analysis by this technique demonstrated that tumor-specific methylated genes belong to distinct functional categories, have common sequence motifs in their promoters and are found in clusters on chromosomes. In addition, many are already repressed in normal cells. These results are consistent with the hypothesis that cancer-related de novo methylation may come about through an instructive mechanism.

Animals↗

PubMed2005

Silence of the genes--mechanisms of long-term repression.

A large fraction of genes in the mammalian genome is repressed in every cell throughout development. Here, we propose that this long-term silencing is carried out by distinct molecular mechanisms that operate in a global manner and, once established, can be maintained autonomously through DNA replication. Both individually and in combination these mechanisms bring about repression, mainly by lowering gene accessibility through closed chromatin structures.

Animals↗

PubMed2004

Epigenetic ontogeny of the Igk locus during B cell development.

To become accessible for rearrangement, the immunoglobulin kappa locus must undergo a series of epigenetic changes. This begins in pro-B cells with the relocation of both immunoglobulin kappa alleles from the periphery to the center of the nucleus. In pre-B cells, one allele became preferentially packaged into an active chromatin structure characterized by histone acetylation and methylation of histone H3 lysine 4, while the other allele was recruited to heterochromatin, where it was associated with heterochromatin protein-gamma and Ikaros. These events in cis made only one allele accessible to trans-acting factors, such as RelB, which mediated DNA demethylation, to facilitate rearrangement. These results suggest that early B lymphoid epigenetic changes generate differential structures that serve as the basis for allelic exclusion.

Alleles↗

PubMed2004

CTCF elements direct allele-specific undermethylation at the imprinted H19 locus.

The H19 imprinted gene locus is regulated by an upstream 2 kb imprinting control region (ICR) that influences allele-specific expression, DNA methylation, and replication timing. This ICR becomes de novo methylated during late spermatogenesis in the male but emerges from oogenesis in an unmethylated form, and this allele-specific pattern is then maintained throughout early development and in all tissues of the mouse. We have used a genetic approach involving transfection into embryonic stem (ES) cells in order to decipher how the maternal allele is protected from de novo methylation at the time of implantation. Our studies show that CCCTC binding factor (CTCF) boundary elements within the ICR have the ability to prevent de novo methylation on the maternal allele. Since CTCF does not recognize its binding sequence when methylated, this reaction does not occur on the paternal allele, thus preserving the gamete-derived, allele-specific pattern. These results suggest that CTCF may play a general role in the maintenance of differential methylation patterns in vivo.

Alleles↗

PubMed2004

A stepwise epigenetic process controls immunoglobulin allelic exclusion.

During the differentiation of T and B cells, immune-receptor loci in the genome must be made sterically accessible so that they can undergo rearrangement. Here, we discuss how this is carried out by the stepwise removal of epigenetic repression mechanisms - such as later-replication timing, heterochromatization, histone hypo-acetylation and DNA methylation - in a manner that initially favours one allele in each cell. We propose that this mechanism of allelic exclusion might also be the basis for the generation of gene diversity in other systems.

Alleles↗

PubMed2003

A multistep mechanism for the activation of rearrangement in the immune system.

Rearrangement of immune receptor loci is a developmentally controlled process that takes place exclusively in lymphoid cells. We have used a stable transfection system in pre-B cells to show that DNA methylation brings about histone underacetylation, histone H3(K9) methylation, DNaseI resistance, and strong inhibition of both transcription and recombination. Strikingly, this repression is maintained in dividing cells even after removal of the original methyl groups responsible for its establishment, but in this state, rearrangement can now be induced by reacetylation of local histones using the drug Trichostatin A. This same combination of demethylation and histone acetylation is also required to activate germline transcription and recombination from the endogenous kappa locus in vivo. These results indicate that the regulation of rearrangement is carried out by a multilayered synergistic process.

Animals↗

PubMed2003

Biallelic germline transcription at the kappa immunoglobulin locus.

Rearrangement of antigen receptor genes generates a vast array of antigen receptors on lymphocytes. The establishment of allelic exclusion in immunoglobulin genes requires differential treatment of the two sequence identical alleles. In the case of the kappa immunoglobulin locus, changes in chromatin structure, methylation, and replication timing of the two alleles are all potentially involved in regulating rearrangement. Additionally, germline transcription of the kappa locus which precedes rearrangement has been proposed to reflect an opening of the chromatin structure rendering it available for rearrangement. As the initial restriction of rearrangement to one allele is critical to the establishment of allelic exclusion, a key question is whether or not germline transcription at the kappa locus is monoallelic or biallelic. We have used a sensitive reverse transcription-polymerase chain reaction (RT-PCR) assay and an RNA-fluorescence in situ hybridization (FISH) to show that germline transcription of the kappa locus is biallelic in wild-type immature B cells and in recombination activating gene (RAG)-/-, mu+ B cells. Therefore, germline transcription is unlikely to dictate which allele will be rearranged first and rather reflects a general opening on both alleles that must be accompanied by a mechanism allowing one of the two alleles to be rearranged first.

Alleles↗

PubMed2003

Regulation of imprinting: A multi-tiered process.

Although most mammalian genes are expressed from both alleles, there is a small group of special genes which are imprinted so that only one of the parental alleles is actually expressed in target cells. This epigenetic process involves regulation at a number of different stages of development and is very complex. In principle, imprinted gene regions must be marked in cis in the gametes using epigenetic features capable of being maintained through cell division and able to direct multigenic monoallelic expression in differentiated cells of the mature organism. The difference between alleles must be erased during early gametogenesis to allow the imprint to be reset in the mature gametes. In this review we will summarize what is currently known about the molecular mechanisms which mediate these steps.

Alleles↗

PubMed2003

Epigenetic mechanisms that regulate antigen receptor gene expression.

Functional immunoglobulin and T-cell receptor genes are generated from germline V, D and J gene segments by a series of site-specific recombination events. This process is regulated by the availability of recombination machinery and by the ordered accessibility of appropriate target gene segments. Recent studies highlight the importance of chromatin remodelling and locus positioning for controlling antigen receptor gene expression and recombination.

Acetylation↗

PubMed2003

The role of DNA methylation in setting up chromatin structure during development.

DNA methylation inhibits gene expression in animal cells, probably by affecting chromatin structure. Biochemical studies suggest that this process may be mediated by methyl-specific binding proteins that recruit enzymatic machinery capable of locally altering histone modification. To test whether DNA methylation actually has a role in the assembly of chromatin during normal development, we used cell transfection and a transgene construct genetically programmed to be either methylated or unmethylated in all cell types of the mouse. Chromatin immunoprecipitation (ChIP) analysis shows that the presence of DNA methylation brings about the deacetylation of histone H4 and methylation of Lys9 of histone H3 (H3 Lys9) and prevents methylation of Lys4 of histone H3 (H3 Lys4), thus generating a structure identical to that of methylated sequences in the genome. These results indicate that the methylation pattern established in early embryogenesis is profoundly important in setting up the structural profile of the genome.

Acetylation↗

PubMed2003

Replicating by the clock.

The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate at different times during S phase. Active genes are generally associated with early replication, whereas inactive genes replicate late. This expression pattern might be facilitated by the differential restructuring of chromatin at the time of replication in early or late S phase.

Animals↗

PubMed2002

Establishment of transcriptional competence in early and late S phase.

In animal cells, the process of DNA replication takes place in a programmed manner, with each gene region designated to replicate at a fixed time slot in S phase. Housekeeping genes undergo replication in the first half of S phase in all cell types, whereas the replication of many tissue specific genes is developmentally controlled, being late in most tissues but early in the tissue of expression. Here we employ nuclear DNA injection as an experimental system to test whether this phenomenon is due to differences in the ability to set up transcriptional competence during S phase. Our results show that, regardless of sequence, exogenous genes are a better template for transcription when injected into nuclei of cells in early as opposed to late S phase, and this expression state, once initiated, is preserved after cell division. DNA injected in late S phase is apparently repressed because it is packaged into chromatin containing deacetylated histones, and the same is true for late replicating chromosomal DNA. These findings suggest a mechanistic connection between replication timing and gene expression that might help to explain how epigenetic states can be maintained in vivo.

Acetylation↗

PubMed2002

The imprinting mechanism of the Prader-Willi/Angelman regional control center.

The 2 Mb domain on chromosome 15q11-q13 that carries the imprinted genes involved in Prader-Willi (PWS) and Angelman (AS) syndromes is under the control of an imprinting center comprising two regulatory regions, the PWS-SRO located around the SNRPN promoter and the AS-SRO located 35 kb upstream. Here we describe the results of an analysis of the epigenetic features of these two sequences and their interaction. The AS-SRO is sensitive to DNase I, and packaged with acetylated histone H4 and methylated histone H3(K4) only on the maternal allele, and this imprinted epigenetic structure is maintained in dividing cells despite the absence of clearcut differential DNA methylation. Genetic analysis shows that the maternal AS-SRO is essential for setting up the DNA methylation state and closed chromatin structure of the neighboring PWS-SRO. In contrast, the PWS-SRO has no influence on the epigenetic features of the AS-SRO. These results suggest a stepwise, unidirectional program in which structural imprinting at the AS-SRO brings about allele-specific repression of the maternal PWS-SRO, thereby preventing regional activation of genes on this allele.

Base Sequence↗

PubMed2002

Differential accessibility at the kappa chain locus plays a role in allelic exclusion.

Gene rearrangement in the immune system is always preceded by DNA demethylation and increased chromatin accessibility. Using a model system in which rearrangement of the endogenous immunoglobulin kappa locus is prevented, we demonstrate that these epigenetic and chromatin changes actually occur on one allele with a higher probability than the other. It may be this process that, together with feedback inhibition, serves as the basis for allelic exclusion.

Alleles↗

PubMed1998

Analysis of putative RNase sensitivity and protease insensitivity of demethylation activity in extracts from rat myoblasts.

The mechanism for demethylation of DNA in rat myoblasts has recently been studied using a new in vitro system that monitors demethylation in whole cell extracts. Previous investigations using this system had indicated that demethylation is resistant to conditions that are normally assumed to denature or digest proteins. Remarkably, it was reported that the activity appeared to be sensitive to the action of ribonuclease, suggesting a role for RNA in the demethylation of DNA. This manuscript reports that, upon further purification of the extract, demethylation activity has properties that are different. When subjected to more rigorous procedures for digestion of proteins, the demethylase activity disappears. Furthermore, RNase sensitivity of the extract disappears when a quantity of unmethylated competitor DNA is added to the reaction mix or when extracts treated with RNase are subsequently treated with protease. Although a role for RNA cannot be completely discounted, it is unlikely that this demethyl-ation reaction involves RNA cofactors or ribozyme components. These results have important implications for the mechanism of DNA demethylation and they exemplify the potential pitfalls of experiments in which new biological roles for RNA are evaluated using RNase sensitivity experiments.

Animals↗