DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established ... more 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.
Genomic imprinting is characterized by allele-specific expression of multiple genes within large ... more 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.
The development of mature B cells involves a series of molecular decisions which culminate in the... more 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.
Immune receptor gene expression is regulated by a series of developmental events that modify thei... more Immune receptor gene expression is regulated by a series of developmental events that modify their accessibility in a locus, cell type, stage and allele-specific manner. This is carried out by a programmed combination of many different molecular mechanisms, including region-wide replication timing, changes in nuclear localization, chromatin contraction, histone modification, nucleosome positioning and DNA methylation. These modalities ultimately work by controlling steric interactions between receptor loci and the recombination machinery.
After erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at... more After erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at implantation. We have identified a demethylation pathway in mouse embryonic cells that uses hydroxymethylation (Tet1), deamination (Aid), glycosylation (Mbd4) and excision repair (Gadd45a) genes. Surprisingly, this demethylation system is not necessary for generating the overall bimodal methylation pattern but does appear to be involved in resetting methylation patterns during somatic-cell reprogramming.
CpG island-like sequences are commonly thought to provide the sole signals for designating consti... more CpG island-like sequences are commonly thought to provide the sole signals for designating constitutively unmethylated regions in the genome, thus generating open chromatin domains within a sea of global repression. Using a new database obtained from comprehensive microarray analysis, we show that unmethylated regions (UMRs) seem to be formed during early embryogenesis, not as a result of CpG-ness, but rather through the recognition of specific sequence motifs closely associated with transcription start sites. This same system probably brings about the resetting of pluripotency genes during somatic cell reprogramming. The data also reveal a new class of nonpromoter UMRs that become de novo methylated in a tissue-specific manner during development, and this process may be involved in gene regulation. In short, we show that UMRs are an important aspect of genome structure that have a dynamic role in development.
The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate a... more 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.
An initial strategy for the systematic identification of functional elements in the human genome ... more An initial strategy for the systematic identification of functional elements in the human genome by low-redundancy comparative sequencing. Proc. Natl Acad. Sci. USA 102, 4795-4800 (2005). 5. Ostrander, E. A. & Giniger, E. Semper fidelis: what man's best friend can teach us about human biology and disease.
Protein component of chromatin that is involved in regulation of gene expression. Two of each of ... more Protein component of chromatin that is involved in regulation of gene expression. Two of each of the core histones, H2A, H2B, H3 or H4, make up an octameric nucleosome, around which DNA winds. N-terminal tails of histones can be subject to covalent modification, including methylation and acetylation. CpG island A sequence of at least 200 bp with a greater number of CpG sites than expected given the average GC content of the genome. These regions are typically undermethylated and are found upstream of many mammalian genes.
Mechanisms that confer heritable, but potentially reversible, states of gene activity that are im... more Mechanisms that confer heritable, but potentially reversible, states of gene activity that are imposed by the structure of chromatin or covalent modifications of DNA and histones.
Oct-3/4 is a POU domain homeobox gene that is expressed during gametogenesis and in early embryon... more Oct-3/4 is a POU domain homeobox gene that is expressed during gametogenesis and in early embryonic cells 1,2 , where it has been shown to be important for maintaining pluripotency 3. Following implantation, this gene undergoes a novel multistep 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 reexpression, thereby preventing reprogramming.
In animal cells, the process of DNA replication takes place in a programmed manner, with each gen... more 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.
Previews 933 twists the DNA into yet another conformation, a loop the recruitment of SL-1, which ... more Previews 933 twists the DNA into yet another conformation, a loop the recruitment of SL-1, which itself is subject to phosthat can involve the formation of a full (360Њ) circle. This phorylation by Cdc2/cyclin B (Heix et al., 1998). The unusual DNA-protein complex is called an enhancelatter event decreases the affinity of SL-1 for UBF and some and is thought to interact with coactivator proserves as a negative regulatory input. Nor is it the first teins, such as TBP-TAF1, and corepressors, such as the time ERK1/2 have been implicated in events that impact retinoblastoma protein (see references in Stefanovsky protein translation. Early on, these kinases were identiet al. 2001), to regulate expression of PolI target genes. fied as activators of ribosomal protein S6 kinase (Rsk), Other mechanisms exist to regulate the genes encoda kinase that most likely has less to do with modification ing rRNA. Histone architecture affected by acetylation of ribosomal proteins than its name suggests (Sturgill or deacetylation controls output of the rRNA promoter. et al., 1988). ERK1/2 may instead have their greatest For example, the CREB Binding Protein (CBP), a polyeffects on growth factor-regulated translation through peptide with histone acetylase activity, is recruited to their actions on ribosomal protein transcription. the rRNA promoter by interaction with HMG box 1 and 2 of UBF (Pelletier et al., 2000). This interaction also Ewen D. Gallagher and Melanie H. Cobb leads to acetylation of UBF itself. A "flip-flop" model Department of Pharmacology has been proposed in which the relative level of UBF University of Texas Southwestern Medical School acetylation is regulated by the mutually exclusive bind-5323 Harry Hines Boulevard ing of either CBP or Rb. Methylation of the rRNA pro-Dallas, Texas 75390 moter at nucleotide position Ϫ133 also influences transcription, chiefly by serving to dampen expression by Selected Reading impairing the binding of UBF (Santoro and Grummt,
A technique for the isolation and characterization of newly transcribed murine leukaemia virus RN... more A technique for the isolation and characterization of newly transcribed murine leukaemia virus RNA in chronically infected cells has been developed. Cellular RNA was pulse labelled with aH-uridine and virus-specific sequences were annealed with an excess of mercurated complementary DNA. Based on the affinity between mercurated cDNA and sulphydryl-Sepharose, the hybrid was specifically selected by affinity column chromatography. The specificity of this method was dependent on the purity of the cDNA and it was necessary to remove non-viral sequences from the cDNA in order to isolate virus-specific RNA. Between 0"5 and 0.8 % of the labelled RNA in Moloney MuLV-infected rat cells and 1.5 ~o of the labelled RNA in Moloney MuLV-infected NIH Swiss mouse cells were virusspecific. Using this methodology, the effect of the cell cycle on the transcriptional activity ofproviral genes was investigated. Cultures of Moloney MuLV-infected rat cells arrested in Go phase of the cell cycle released reduced quantities of virus, but continued to synthesize virus RNA. The pools of virus RNA and p3o antigen in the G0-arrested cells equalled the pools in actively dividing cells. These results suggested that post-transcriptional events controlled virus production in the G0arrested cells.
DNA in somatic tissue is characterized by a bimodal pattern of methylation, which is established ... more 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.
Genomic imprinting is characterized by allele-specific expression of multiple genes within large ... more 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.
The development of mature B cells involves a series of molecular decisions which culminate in the... more 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.
Immune receptor gene expression is regulated by a series of developmental events that modify thei... more Immune receptor gene expression is regulated by a series of developmental events that modify their accessibility in a locus, cell type, stage and allele-specific manner. This is carried out by a programmed combination of many different molecular mechanisms, including region-wide replication timing, changes in nuclear localization, chromatin contraction, histone modification, nucleosome positioning and DNA methylation. These modalities ultimately work by controlling steric interactions between receptor loci and the recombination machinery.
After erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at... more After erasure in the early animal embryo, a new bimodal DNA methylation pattern is regenerated at implantation. We have identified a demethylation pathway in mouse embryonic cells that uses hydroxymethylation (Tet1), deamination (Aid), glycosylation (Mbd4) and excision repair (Gadd45a) genes. Surprisingly, this demethylation system is not necessary for generating the overall bimodal methylation pattern but does appear to be involved in resetting methylation patterns during somatic-cell reprogramming.
CpG island-like sequences are commonly thought to provide the sole signals for designating consti... more CpG island-like sequences are commonly thought to provide the sole signals for designating constitutively unmethylated regions in the genome, thus generating open chromatin domains within a sea of global repression. Using a new database obtained from comprehensive microarray analysis, we show that unmethylated regions (UMRs) seem to be formed during early embryogenesis, not as a result of CpG-ness, but rather through the recognition of specific sequence motifs closely associated with transcription start sites. This same system probably brings about the resetting of pluripotency genes during somatic cell reprogramming. The data also reveal a new class of nonpromoter UMRs that become de novo methylated in a tissue-specific manner during development, and this process may be involved in gene regulation. In short, we show that UMRs are an important aspect of genome structure that have a dynamic role in development.
The eukaryotic genome is divided into well-defined DNA regions that are programmed to replicate a... more 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.
An initial strategy for the systematic identification of functional elements in the human genome ... more An initial strategy for the systematic identification of functional elements in the human genome by low-redundancy comparative sequencing. Proc. Natl Acad. Sci. USA 102, 4795-4800 (2005). 5. Ostrander, E. A. & Giniger, E. Semper fidelis: what man's best friend can teach us about human biology and disease.
Protein component of chromatin that is involved in regulation of gene expression. Two of each of ... more Protein component of chromatin that is involved in regulation of gene expression. Two of each of the core histones, H2A, H2B, H3 or H4, make up an octameric nucleosome, around which DNA winds. N-terminal tails of histones can be subject to covalent modification, including methylation and acetylation. CpG island A sequence of at least 200 bp with a greater number of CpG sites than expected given the average GC content of the genome. These regions are typically undermethylated and are found upstream of many mammalian genes.
Mechanisms that confer heritable, but potentially reversible, states of gene activity that are im... more Mechanisms that confer heritable, but potentially reversible, states of gene activity that are imposed by the structure of chromatin or covalent modifications of DNA and histones.
Oct-3/4 is a POU domain homeobox gene that is expressed during gametogenesis and in early embryon... more Oct-3/4 is a POU domain homeobox gene that is expressed during gametogenesis and in early embryonic cells 1,2 , where it has been shown to be important for maintaining pluripotency 3. Following implantation, this gene undergoes a novel multistep 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 reexpression, thereby preventing reprogramming.
In animal cells, the process of DNA replication takes place in a programmed manner, with each gen... more 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.
Previews 933 twists the DNA into yet another conformation, a loop the recruitment of SL-1, which ... more Previews 933 twists the DNA into yet another conformation, a loop the recruitment of SL-1, which itself is subject to phosthat can involve the formation of a full (360Њ) circle. This phorylation by Cdc2/cyclin B (Heix et al., 1998). The unusual DNA-protein complex is called an enhancelatter event decreases the affinity of SL-1 for UBF and some and is thought to interact with coactivator proserves as a negative regulatory input. Nor is it the first teins, such as TBP-TAF1, and corepressors, such as the time ERK1/2 have been implicated in events that impact retinoblastoma protein (see references in Stefanovsky protein translation. Early on, these kinases were identiet al. 2001), to regulate expression of PolI target genes. fied as activators of ribosomal protein S6 kinase (Rsk), Other mechanisms exist to regulate the genes encoda kinase that most likely has less to do with modification ing rRNA. Histone architecture affected by acetylation of ribosomal proteins than its name suggests (Sturgill or deacetylation controls output of the rRNA promoter. et al., 1988). ERK1/2 may instead have their greatest For example, the CREB Binding Protein (CBP), a polyeffects on growth factor-regulated translation through peptide with histone acetylase activity, is recruited to their actions on ribosomal protein transcription. the rRNA promoter by interaction with HMG box 1 and 2 of UBF (Pelletier et al., 2000). This interaction also Ewen D. Gallagher and Melanie H. Cobb leads to acetylation of UBF itself. A "flip-flop" model Department of Pharmacology has been proposed in which the relative level of UBF University of Texas Southwestern Medical School acetylation is regulated by the mutually exclusive bind-5323 Harry Hines Boulevard ing of either CBP or Rb. Methylation of the rRNA pro-Dallas, Texas 75390 moter at nucleotide position Ϫ133 also influences transcription, chiefly by serving to dampen expression by Selected Reading impairing the binding of UBF (Santoro and Grummt,
A technique for the isolation and characterization of newly transcribed murine leukaemia virus RN... more A technique for the isolation and characterization of newly transcribed murine leukaemia virus RNA in chronically infected cells has been developed. Cellular RNA was pulse labelled with aH-uridine and virus-specific sequences were annealed with an excess of mercurated complementary DNA. Based on the affinity between mercurated cDNA and sulphydryl-Sepharose, the hybrid was specifically selected by affinity column chromatography. The specificity of this method was dependent on the purity of the cDNA and it was necessary to remove non-viral sequences from the cDNA in order to isolate virus-specific RNA. Between 0"5 and 0.8 % of the labelled RNA in Moloney MuLV-infected rat cells and 1.5 ~o of the labelled RNA in Moloney MuLV-infected NIH Swiss mouse cells were virusspecific. Using this methodology, the effect of the cell cycle on the transcriptional activity ofproviral genes was investigated. Cultures of Moloney MuLV-infected rat cells arrested in Go phase of the cell cycle released reduced quantities of virus, but continued to synthesize virus RNA. The pools of virus RNA and p3o antigen in the G0-arrested cells equalled the pools in actively dividing cells. These results suggested that post-transcriptional events controlled virus production in the G0arrested cells.
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