Gene repression paradigms in animal cells.
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Animals↗
Biomedical subjects
H Cedar
Publications and source records attributed to H Cedar.
Asynchronous replication and allelic exclusion in the immune system.
The development of mature B cells involves a series of molecular decisions which culminate in the expression of a single light-chain and heavy-chain antigen receptor on the cell surface. There are two alleles for each receptor locus, so the ultimate choice of one receptor type must involve a process of allelic exclusion. One way to do this is with a feedback mechanism that downregulates rearrangement after the generation of a productive receptor molecule, but recent work suggests that monoallelic epigenetic changes may also take place even before rearrangement. To better understand the basis for distinguishing between alleles, we have analysed DNA replication timing. Here we show that all of the B-cell-receptor loci (mu, kappa and lambda) and the TCRbeta locus replicate asynchronously. This pattern, which is established randomly in each cell early in development and maintained by cloning, represents an epigenetic mark for allelic exclusion, because it is almost always the early-replicating allele which is initially selected to undergo rearrangement in B cells. These results indicate that allelic exclusion in the immune system may be very similar to the process of X chromosome inactivation.
Alleles↗
Developmental regulation of DNA replication timing at the human beta globin locus.
The human beta globin locus replicates late in most cell types, but becomes early replicating in erythroid cells. Using FISH to map DNA replication timing around the endogenous beta globin locus and by applying a genetic approach in transgenic mice, we have demonstrated that both the late and early replication states are controlled by regulatory elements within the locus control region. These results also show that the pattern of replication timing is set up by mechanisms that work independently of gene transcription.
Animals↗
An upstream repressor element plays a role in Igf2 imprinting.
The imprinted Igf2 gene is associated with a small upstream region that is differentially methylated on the active paternal allele. We have identified a repressor element within this sequence and shown that repression is probably mediated through a trans- acting factor, GCF2. DNA methylation of this site abrogates both protein binding and repressor activity. Targeting experiments demonstrate that this element plays a role in the repression of the maternal Igf2 gene in vivo.
Animals↗
Epigenetic crosstalk.
In the November 15 issue of Nature, report that disruption of histone methylation in the fungus Neurospora crassa results in the elimination of DNA methylation. This demonstrates that chromatin structure can pattern DNA methylation and suggests that different epigenetic layers engage in complex crosstalk.
Animals↗
Allele-specific expression patterns of interleukin-2 and Pax-5 revealed by a sensitive single-cell RT-PCR analysis.
Autosomal genes that are subject to random allelic inactivation (RAI), like imprinted genes [1] and genes subject to X-inactivation [2], require mechanisms that dictate the differential transcriptional regulation of two sequence-identical alleles. RAI genes include olfactory receptor genes [3], and the various genes encoding antigen receptors on lymphocytes (immunoglobulin genes, T cell receptor genes and NK receptor genes [4] [5] [6] [7]). These observations raise the possibility that other genes might be similarly regulated. Moreover, an interesting possibility is that certain genes might be monoallelically expressed in some cells and biallelically expressed in others. Recently, reports of monoallelic expression of interleukin-2 (IL-2) [8] [9] and IL-4 [10] [11] have raised the possibility that the cytokine gene family may be subject to monoallelic expression. Another report suggests that the gene encoding the transcription factor Pax-5, which is involved in B-cell (and cerebellar) development [12] [13], is also subject to monoallelic expression [14]. Using a novel single-cell reverse transcription-polymerase chain reaction (RT-PCR) approach, we have analyzed the IL-2 and Pax-5 genes in mice. We found that IL-2 is monoallelically transcribed in some T cells and biallelically transcribed in others, raising interesting questions regarding cytokine gene regulation. Additionally, our analyses suggest that Pax-5 is consistently biallelically transcribed. Thus, the IL-2 gene and other cytokine genes may be regulated in a stochastic manner that results in 0, 1 or 2 alleles of a given cytokine gene expressed in each T cell. This type of regulation could account for the wide variety of different combinations of cytokine genes expressed in individual T cells and therefore plays a role in the generation of T cells with a range of different effector functions.
Alleles↗
Imprinting: focusing on the center.
Recent studies have focused on the identification of imprinting centers and on the elucidation of the mechanisms by which they control imprinting. These studies begin to shed light on the means by which imprinting marks are established in the gametes and on the various molecular strategies utilized to execute differential expression of the two parental alleles.
Alleles↗
The imprinting box of the Prader-Willi/Angelman syndrome domain.
A subset of mammalian genes is monoallelically expressed in a parent-of-origin manner. These genes are subject to an imprinting process that epigenetically marks alleles according to their parental origin during gametogenesis. Imprinted genes can be organized in clusters as exemplified by the 2-Mb domain on human chromosome 15q11-q13 and its mouse orthologue on chromosome 7c (ref. 1). Loss of this 2-Mb domain on the paternal or maternal allele results in two neurogenetic disorders, Prader-Willi syndrome (PWS) or Angelman syndrome (AS), respectively. Microdeletions on the paternal allele share a 4.3-kb short region of overlap (SRO), which includes the SNRPN promoter/exon1, cause PWS and silence paternally expressed genes. Microdeletions on the maternal allele share a 0.88-kb SRO located 35 kb upstream to the SNRPN promoter, cause AS and alleviate repression of genes on the maternal allele. Individuals carrying both AS and PWS deletions on the paternal allele show a PWS phenotype and genotype. These observations suggest that cis elements within the AS-SRO and PWS-SRO constitute an imprinting box that regulates the entire domain on both chromosomes. Here we show that a minitransgene composed of a 200-bp Snrpn promoter/exon1 and a 1-kb sequence located approximately 35 kb upstream to the SNRPN promoter confer imprinting as judged by differential methylation, parent-of-origin-specific transcription and asynchronous replication.
Alleles↗
Asynchronous replication of imprinted genes is established in the gametes and maintained during development.
Genomic imprinting is characterized by allele-specific expression of multiple genes within large chromosomal domains that undergo DNA replication asynchronously during S phase. Here we show, using both fluorescence in situ hybridization analysis and S-phase fractionation techniques, that differential replication timing is associated with imprinted genes in a variety of cell types, and is already present in the pre-implantation embryo soon after fertilization. This pattern is erased before meiosis in the germ line, and parent-specific replication timing is then reset in late gametogenesis in both the male and female. Thus, asynchronous replication timing is established in the gametes and maintained throughout development, indicating that it may function as a primary epigenetic marker for distinguishing between the parental alleles.
Alleles↗
Gene expression. The amazing demethylase.
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Catalysis↗
Developmental regulation of immune system gene rearrangement.
Rearrangement of antigen receptors in the immune system is mediated through the action of complex enhancers which function in both a developmentally stage-specific and a cell-type-specific manner to demethylate DNA, open chromatin structure and tether the recombination machinery to one preferred allele at each locus.
Animals↗
DNA methylation represses transcription in vivo.
DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established in the animal through a series of developmental events. In the mouse blastula, most DNA is unmethylated, but after implantation a wave of de novo methylation modifies most of the genome, excluding the majority of CpG islands, which are mainly associated with housekeeping genes. This genomic methylation pattern is broadly maintained during the life of the organism by maintenance methylation, and generally correlates with gene expression. Experiments both in vitro and in vivo indicate that methylation inhibits transcription. It has not yet been possible, however, to determine the role of DNA methylation on specific sequences during normal development. Cis-acting regulatory elements and trans-acting factors appear to be involved in both stage- and tissue-specific demethylation processes. Sp1-like elements have a key role in protecting the CpG island of Aprt (encoding adenine phosphoribosyltransferase) from de novo methylation, and when these elements are specifically mutated, the Aprt CpG island becomes methylated in transgenic mice. We have now characterized an embryo-specific element from the CpG island sequence upstream of Aprt that can protect itself from de novo methylation in transgenic mice as well as reduce methylation of flanking sequences. We placed this element on a removable cassette adjacent to a human HBB (encoding beta-globin) reporter and generated a transgene whose methylation pattern can be switched in vivo. Analysis of globin transcription in this system showed that methylation in cis inhibits gene expression in a variety of tissues, indicating that DNA modification may serve as a global genomic repressor.
Adenine Phosphoribosyltransferase↗
Demethylation and the establishment of kappa allelic exclusion.
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Alleles↗
DNA methylation models histone acetylation.
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Acetylation↗
Kappa chain monoallelic demethylation and the establishment of allelic exclusion.
Allelic exclusion in kappa light-chain synthesis is thought to result from a feedback mechanism by which the expression of a functional kappa light chain on the surface of the B cell leads to an intracellular signal that down-regulates the V(D)J recombinase, thus precluding rearrangement of the other allele. Whereas such a feedback mechanism clearly plays a role in the maintenance of allelic exclusion, here we provide evidence suggesting that the initial establishment of allelic exclusion involves differential availability of the two kappa alleles for rearrangement. Analysis of kappa+ B-cell populations and of individual kappa+ B cells that have rearranged only one allele demonstrates that in these cells, critical sites on the rearranged allele are unmethylated, whereas the nonrearranged allele remains methylated. This pattern is apparently generated by demethylation that is initiated at the small pre-B cell stage, on a single allele, in a process that occurs prior to rearrangement and requires the presence in cis of both the intronic and 3' kappa enhancers. Taken together with data demonstrating that undermethylation is required for rearrangement, these results indicate that demethylation may actually underly the process of allelic exclusion by directing the initial choice of a single kappa allele for rearrangement.
Alleles↗
DNA methylation: a molecular lock.
In mammals, the promoters of expressed genes are generally unmethylated, whereas those of genes that are not expressed are methylated. Two recent papers help to explain the mechanism by which methylation modulates gene expression.
Animals↗
The role of DNA demethylation during development.
The somatic genomic methylation pattern which plays a role in the suppression of basal gene activity is established anew in each generation through developmentally regulated de novo and demethylation steps. Demethylation appears to be carried out by a nucleotide exchange reaction which may involve RNA molecules, and is directed to specific loci in the genome through interactions between cis acting elements and trans acting factors.
Animals↗
DNA demethylation in vitro: involvement of RNA.
An in vitro system for studying DNA demethylation has been established using extracts from tissue culture cells. This reaction, which is unusually resistant to proteinase K, takes place through the removal of a 5-methylcytosine nucleotide unit from the DNA substrate and its conversion to an RNase-sensitive form. It is likely that this represents the in vivo mechanism, as well, since extracts from L8 myoblasts specifically demethylate an alpha-actin gene, while extracts from F9 teratocarcinoma cells specifically demodify the Aprt CpG island. After pretreatment with proteinase K, these extracts demethylate both genes equally, suggesting that gene specificity may be controlled by protein factors.
Actins↗