scholarly journals High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors.

1994 ◽  
Vol 14 (2) ◽  
pp. 1419-1430 ◽  
Author(s):  
I K Hornstra ◽  
T P Yang
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Cpg Island ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Methylation Analysis ◽  
Kinase Gene ◽  
X Chromosomes ◽  
Cpg Dinucleotides ◽  
Inactive X Chromosome

DNA methylation within GC-rich promoters of constitutively expressed X-linked genes is correlated with transcriptional silencing on the inactive X chromosome in female mammals. For most X-linked genes, X chromosome inactivation results in transcriptionally active and inactive alleles occupying each female nucleus. To examine mechanisms responsible for maintaining this unique system of differential gene expression, we have analyzed the methylation of individual cytosine residues in the 5' CpG island of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Methylation analysis of 142 CpG dinucleotides by genomic sequencing was carried out on purified DNA using the cytosine-specific Maxam and Gilbert DNA sequencing reaction in conjunction with ligation-mediated PCR. These studies demonstrate the 5' CpG islands of active and 5-azacytidine-reactivated alleles are essentially unmethylated while the inactive allele is hypermethylated. The inactive allele is completely methylated at nearly all CpG dinucleotides except in a 68-bp region containing four adjacent GC boxes where most CpG dinucleotides are either unmethylated or partially methylated. Curiously, these GC boxes exhibit in vivo footprints only on the active X chromosome, not on the inactive X. The methylation pattern of the inactive HPRT gene is strikingly different from that reported for the inactive X-linked human phosphoglycerate kinase gene which exhibits methylation at all CpG sites in the 5' CpG island. These results suggest that the position of methylated CpG dinucleotides, the density of methylated CpGs, the length of methylated regions, and/or chromatin structure associated with methylated DNA may have a role in repressing the activity of housekeeping promoters on the inactive X chromosome. The pattern of DNA methylation on the inactive human HPRT gene may also provide insight into the process of inactivating the gene early in female embryogenesis.

scholarly journals High-resolution methylation analysis of the human hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes: correlation with binding sites for transcription factors

1994 ◽  
Vol 14 (2) ◽  
pp. 1419-1430
Author(s):  
I K Hornstra ◽  
T P Yang
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Cpg Island ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Methylation Analysis ◽  
Kinase Gene ◽  
X Chromosomes ◽  
Cpg Dinucleotides ◽  
Inactive X Chromosome

DNA methylation within GC-rich promoters of constitutively expressed X-linked genes is correlated with transcriptional silencing on the inactive X chromosome in female mammals. For most X-linked genes, X chromosome inactivation results in transcriptionally active and inactive alleles occupying each female nucleus. To examine mechanisms responsible for maintaining this unique system of differential gene expression, we have analyzed the methylation of individual cytosine residues in the 5' CpG island of the human hypoxanthine phosphoribosyltransferase (HPRT) gene on the active and inactive X chromosomes. Methylation analysis of 142 CpG dinucleotides by genomic sequencing was carried out on purified DNA using the cytosine-specific Maxam and Gilbert DNA sequencing reaction in conjunction with ligation-mediated PCR. These studies demonstrate the 5' CpG islands of active and 5-azacytidine-reactivated alleles are essentially unmethylated while the inactive allele is hypermethylated. The inactive allele is completely methylated at nearly all CpG dinucleotides except in a 68-bp region containing four adjacent GC boxes where most CpG dinucleotides are either unmethylated or partially methylated. Curiously, these GC boxes exhibit in vivo footprints only on the active X chromosome, not on the inactive X. The methylation pattern of the inactive HPRT gene is strikingly different from that reported for the inactive X-linked human phosphoglycerate kinase gene which exhibits methylation at all CpG sites in the 5' CpG island. These results suggest that the position of methylated CpG dinucleotides, the density of methylated CpGs, the length of methylated regions, and/or chromatin structure associated with methylated DNA may have a role in repressing the activity of housekeeping promoters on the inactive X chromosome. The pattern of DNA methylation on the inactive human HPRT gene may also provide insight into the process of inactivating the gene early in female embryogenesis.

scholarly journals Hemimethylation and hypersensitivity are early events in transcriptional reactivation of human inactive X-linked genes in a hamster x human somatic cell hybrid.

1992 ◽  
Vol 12 (9) ◽  
pp. 3819-3826 ◽  
Author(s):  
T Sasaki ◽  
R S Hansen ◽  
S M Gartler
Keyword(s):  
Somatic Cell ◽  
X Chromosome ◽  
Cell Hybrid ◽  
Somatic Cell Hybrid ◽  
Time Course ◽  
Cpg Island ◽  
Hprt Gene ◽  
Inactive X Chromosome ◽  
Growing Cell ◽  
Transcriptional Reactivation

Reactivation of the hypoxanthine phosphoribosyltransferase (HPRT) gene on an inactive human X chromosome in a somatic cell hybrid was analyzed following exposure to 5-aza-2'-deoxycytidine. Hemimethylation and chromatin hypersensitivity in the 5' CpG island appeared by 6 h after exposure and continued to increase for 24 h in an exponentially growing cell culture. These results imply that the conformation of inactive chromatin requires a symmetrically methylated 5' G+C-rich promoter region. In addition, quantitative analysis of the time course patterns suggest that chromatin sensitivity changes may depend on strand-specific demethylation. Symmetrically demethylated DNA was first detected at 24 h and continued to increase until 48 h. HPRT mRNA was first detected at 24 h and increased in a biphasic pattern until 48 h. These results suggest that hemimethylation permits nuclease attack but not transcription factor binding, which requires symmetrically demethylated DNA. We also show that in G1-arrested cells, 5-aza-2'-deoxycytidine has no effect on methylation, chromatin conformation, or transcription. We conclude that reactivation of the HPRT gene present on the inactive X chromosome of a somatic cell hybrid involves the initial events of DNA hemimethylation and chromatin hypersensitivity at the 5' CpG island, followed by symmetrical demethylation and transcriptional reactivation.

scholarly journals In vivo footprinting and high-resolution methylation analysis of the mouse hypoxanthine phosphoribosyltransferase gene 5' region on the active and inactive X chromosomes.

1996 ◽  
Vol 16 (11) ◽  
pp. 6190-6199 ◽  
Author(s):  
M D Litt ◽  
I K Hornstra ◽  
T P Yang
Keyword(s):  
High Resolution ◽  
X Chromosome ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
Genomic Sequencing ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Methylation Analysis ◽  
In Vivo Footprinting

To investigate potential mechanisms regulating the hypoxanthine phosphoribosyltransferase (HPRT) gene by X-chromosome inactivation, we performed in vivo footprinting and high-resolution DNA methylation analysis on the 5' region of the active and inactive mouse HPRT alleles and compared these results with those from the human HPRT gene. We found multiple footprinted sites on the active mouse HPRT allele and no footprints on the inactive allele. Comparison of the footprint patterns of the mouse and human HPRT genes demonstrated that the in vivo binding of regulatory proteins between these species is generally conserved but not identical. Detailed nucleotide sequence comparison of footprinted regions in the mouse and human genes revealed a novel 9-bp sequence associated with transcription factor binding near the transcription sites of both genes, suggesting the identification of a new conserved initiator element. Ligation-mediated PCR genomic sequencing showed that all CpG dinucleotides examined on the active allele are unmethylated, while the majority of CpGs on the inactive allele are methylated and interspersed with a few hypomethylated sites. This pattern of methylation on the inactive mouse allele is notably different from the unusual methylation pattern of the inactive human gene, which exhibited strong hypomethylation specifically at GC boxes. These studies, in conjunction with other genomic sequencing studies of X-linked genes, demonstrate that (i) the active alleles are essentially unmethylated, (ii) the inactive alleles are hypermethylated, and (iii) the high-resolution methylation patterns of the hypermethylated inactive alleles are not strictly conserved. There is no obvious correlation between the pattern of methylated sites on the inactive alleles and the pattern of binding sites for transcription factors on the active alleles. These results are discussed in relationship to potential mechanisms of transcriptional regulation by X-chromosome inactivation.

CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene

1994 ◽  
Vol 14 (12) ◽  
pp. 7975-7983
Author(s):  
J G Park ◽  
V M Chapman
Keyword(s):  
Sequence Analysis ◽  
Cell Lines ◽  
X Chromosome ◽  
Genomic Dna ◽  
Cell Populations ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Cpg Sites ◽  
Inactive X Chromosome ◽  
Methylation Patterns

Inactive-X-chromosome genes in mammalian females have methylated CpG islands. We have questioned whether there are variable levels of cytosine methylation at different CpG sites within the island that might indicate the presence of primary sites of methylation which may be critical for the maintenance of gene repression and candidate sites for the initiation of inactivation. To address these questions, we have analyzed the methylation patterns of 32 CpG sites of the X-linked hypoxanthine phosphoribosyltransferase (Hprt) gene on the active and inactive X chromosomes of mouse tissues and cell lines, using genomic sequencing of bisulfite-treated genomic DNA. Cytosine is deaminated by bisulfite, but methylcytosine is not affected. Cell lines that were heterozygous for the Hprt deletion mutation (Hprtb-m3) and a functional Hprt allele were selected with 6-thioguanine. The resulting cell populations uniformly carry the intact Hprt allele on the inactive X chromosome. The methylation of these CpG sites was determined either by the direct sequence analysis of bisulfite-treated and amplified DNA or by the sequence analysis of clones derived from the amplified DNA. No CpG methylation was detected on the active Hprt genes from either males or the active X chromosome of females. On average, 22 CpGs were methylated in the other 50% of female DNA, and the level of methylation at individual sites varied from 42 to 100%. Analysis of the inactive Hprt gene in two cell lines showed that averages of 14 and 18 CpGs were methylated and that the frequency of methylation at 32 individual sites ranged from 3 to 100%. The highest frequency of methylation in cell lines coincided with the sequences flanking transcription initiation sites. These results suggest that methylation patterns are heterogeneous within a tissue and even in clonal cell populations and that specific subsets of CpG sites sustain high methylation frequencies which may be critical for the maintenance of X-chromosome inactivation. The bisulfite method identified which CpG sites were methylated on the inactive X chromosome, and it provided a quantitative estimate of the frequency of methylation of these sites in genomic DNA.

Hemimethylation and hypersensitivity are early events in transcriptional reactivation of human inactive X-linked genes in a hamster x human somatic cell hybrid

1992 ◽  
Vol 12 (9) ◽  
pp. 3819-3826
Author(s):  
T Sasaki ◽  
R S Hansen ◽  
S M Gartler
Keyword(s):  
Somatic Cell ◽  
X Chromosome ◽  
Cell Hybrid ◽  
Somatic Cell Hybrid ◽  
Time Course ◽  
Cpg Island ◽  
Hprt Gene ◽  
Inactive X Chromosome ◽  
Growing Cell ◽  
Transcriptional Reactivation

Reactivation of the hypoxanthine phosphoribosyltransferase (HPRT) gene on an inactive human X chromosome in a somatic cell hybrid was analyzed following exposure to 5-aza-2'-deoxycytidine. Hemimethylation and chromatin hypersensitivity in the 5' CpG island appeared by 6 h after exposure and continued to increase for 24 h in an exponentially growing cell culture. These results imply that the conformation of inactive chromatin requires a symmetrically methylated 5' G+C-rich promoter region. In addition, quantitative analysis of the time course patterns suggest that chromatin sensitivity changes may depend on strand-specific demethylation. Symmetrically demethylated DNA was first detected at 24 h and continued to increase until 48 h. HPRT mRNA was first detected at 24 h and increased in a biphasic pattern until 48 h. These results suggest that hemimethylation permits nuclease attack but not transcription factor binding, which requires symmetrically demethylated DNA. We also show that in G1-arrested cells, 5-aza-2'-deoxycytidine has no effect on methylation, chromatin conformation, or transcription. We conclude that reactivation of the HPRT gene present on the inactive X chromosome of a somatic cell hybrid involves the initial events of DNA hemimethylation and chromatin hypersensitivity at the 5' CpG island, followed by symmetrical demethylation and transcriptional reactivation.

scholarly journals Contribution of epigenetic changes to escape from X-chromosome inactivation

2021 ◽  
Author(s):  
Bradley P. Balaton ◽  
Carolyn J. Brown
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Cpg Islands ◽  
X Chromosome Inactivation ◽  
Chromosome Inactivation ◽  
X Chromosomes ◽  
Epigenetic Marks ◽  
Inactive X Chromosome ◽  
Gene Level ◽  
Allelic Differences

AbstractBackgroundX-chromosome inactivation (XCI) is the epigenetic inactivation of one of two X chromosomes in XX eutherian mammals. The facultatively heterochromatic inactive X chromosome acquires many chromatin changes including DNA methylation and histone modifications. Despite these changes, some genes escape or variably escape from inactivation, and to the extent that they have been studied, epigenetic marks correlate with expression.ResultsWe downloaded data from the International Human Epigenome Consortium and compared previous XCI status calls to DNA methylation, H3K4me1, H3K4me3, H3K9me3, H3K27ac, H3K27me3 and H3K36me3. At genes subject to XCI we found heterochromatic marks enriched, and euchromatic marks depleted on the inactive X when compared to the active X. Similar results were seen for genes escaping XCI although with diminished effect with H3K27me3 being most enriched. Using sample-specific XCI status calls made using allelic expression or DNA methylation we also compared differences between samples with opposite XCI statuses at variably escaping genes. We found some marks significantly differed with XCI status, but which marks were significant was not consistent between genes. We trained a model to predict XCI status from these epigenetic marks and obtained over 75% accuracy for genes escaping and over 90% for genes subject to XCI. This model allowed us to make novel XCI status calls for genes without allelic differences or CpG islands required for other XCI status calling methods. Using these calls to examine a domain of variably escaping genes, we saw XCI status vary at the level of individual genes and not at the domain level.ConclusionHere we show that epigenetic marks differ between genes that are escaping and those subject to XCI, and that genes escaping XCI still differ between the active and inactive Xs. We show epigenetic differences at variably escaping genes, between samples escaping and those subject to XCI. Lastly we show gene-level regulation of variably escaping genes within a domain.

scholarly journals Allele-specific non-CG DNA methylation marks domains of active chromatin in female mouse brain

2017 ◽  
Vol 114 (14) ◽  
pp. E2882-E2890 ◽  
Author(s):  
Christopher L. Keown ◽  
Joel B. Berletch ◽  
Rosa Castanon ◽  
Joseph R. Nery ◽  
Christine M. Disteche ◽  
...  
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Transcriptional Repression ◽  
Transcriptional Activity ◽  
Specific Gene ◽  
Epigenetic Mark ◽  
Gene Promoters ◽  
X Chromosomes ◽  
Inactive X Chromosome ◽  
Allele Specific

DNA methylation at gene promoters in a CG context is associated with transcriptional repression, including at genes silenced on the inactive X chromosome in females. Non-CG methylation (mCH) is a distinct feature of the neuronal epigenome that is differentially distributed between males and females on the X chromosome. However, little is known about differences in mCH on the active (Xa) and inactive (Xi) X chromosomes because stochastic X-chromosome inactivation (XCI) confounds allele-specific epigenomic profiling. We used whole-genome bisulfite sequencing in a mouse model with nonrandom XCI to examine allele-specific DNA methylation in frontal cortex. Xi was largely devoid of mCH, whereas Xa contained abundant mCH similar to the male X chromosome and the autosomes. In contrast to the repressive association of DNA methylation at CG dinucleotides (mCG), mCH accumulates on Xi in domains with transcriptional activity, including the bodies of most genes that escape XCI and at the X-inactivation center, validating this epigenetic mark as a signature of transcriptional activity. Escape genes showing CH hypermethylation were the only genes with CG-hypomethylated promoters on Xi, a well-known mark of active transcription. Finally, we found extensive allele-specific mCH and mCG at autosomal imprinted regions, some with a negative correlation between methylation in the two contexts, further supporting their distinct functions. Our findings show that neuronal mCH functions independently of mCG and is a highly dynamic epigenomic correlate of allele-specific gene regulation.

scholarly journals CpG island promoter region methylation patterns of the inactive-X-chromosome hypoxanthine phosphoribosyltransferase (Hprt) gene.

1994 ◽  
Vol 14 (12) ◽  
pp. 7975-7983 ◽  
Author(s):  
J G Park ◽  
V M Chapman
Keyword(s):  
Sequence Analysis ◽  
Cell Lines ◽  
X Chromosome ◽  
Genomic Dna ◽  
Cell Populations ◽  
Hypoxanthine Phosphoribosyltransferase ◽  
Hprt Gene ◽  
Cpg Sites ◽  
Inactive X Chromosome ◽  
Methylation Patterns

Inactive-X-chromosome genes in mammalian females have methylated CpG islands. We have questioned whether there are variable levels of cytosine methylation at different CpG sites within the island that might indicate the presence of primary sites of methylation which may be critical for the maintenance of gene repression and candidate sites for the initiation of inactivation. To address these questions, we have analyzed the methylation patterns of 32 CpG sites of the X-linked hypoxanthine phosphoribosyltransferase (Hprt) gene on the active and inactive X chromosomes of mouse tissues and cell lines, using genomic sequencing of bisulfite-treated genomic DNA. Cytosine is deaminated by bisulfite, but methylcytosine is not affected. Cell lines that were heterozygous for the Hprt deletion mutation (Hprtb-m3) and a functional Hprt allele were selected with 6-thioguanine. The resulting cell populations uniformly carry the intact Hprt allele on the inactive X chromosome. The methylation of these CpG sites was determined either by the direct sequence analysis of bisulfite-treated and amplified DNA or by the sequence analysis of clones derived from the amplified DNA. No CpG methylation was detected on the active Hprt genes from either males or the active X chromosome of females. On average, 22 CpGs were methylated in the other 50% of female DNA, and the level of methylation at individual sites varied from 42 to 100%. Analysis of the inactive Hprt gene in two cell lines showed that averages of 14 and 18 CpGs were methylated and that the frequency of methylation at 32 individual sites ranged from 3 to 100%. The highest frequency of methylation in cell lines coincided with the sequences flanking transcription initiation sites. These results suggest that methylation patterns are heterogeneous within a tissue and even in clonal cell populations and that specific subsets of CpG sites sustain high methylation frequencies which may be critical for the maintenance of X-chromosome inactivation. The bisulfite method identified which CpG sites were methylated on the inactive X chromosome, and it provided a quantitative estimate of the frequency of methylation of these sites in genomic DNA.

scholarly journals Divergent DNA methylation signatures of X chromosome regulation in marsupials and eutherians

2020 ◽  
Author(s):  
Devika Singh ◽  
Dan Sun ◽  
Andrew G. King ◽  
David E. Alquezar-Planas ◽  
Rebecca N. Johnson ◽  
...  
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Specific Expression ◽  
X Chromosomes ◽  
Phascolarctos Cinereus ◽  
Male And Female ◽  
Inactive X Chromosome ◽  
Genome Wide ◽  
Promoter Dna Methylation ◽  
Promoter Dna

AbstractX chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is well characterized in eutherian mammals. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here we examine genome-wide signatures of DNA methylation from methylation maps across fives tissues from a male and female koala (Phascolarctos cinereus) and present the first whole genome, multi-tissue marsupial “methylome atlas.” Using these novel data, we elucidate divergent versus common features of marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in marsupials primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx, the primary lncRNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-(and presumably inactive X chromosome-) specific expression. Finally, we utilize the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5 %) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.

scholarly journals Koala methylomes reveal divergent and conserved DNA methylation signatures of X chromosome regulation

2021 ◽  
Vol 288 (1945) ◽  
pp. 20202244
Author(s):  
Devika Singh ◽  
Dan Sun ◽  
Andrew G. King ◽  
David E. Alquezar-Planas ◽  
Rebecca N. Johnson ◽  
...  
Keyword(s):  
Dna Methylation ◽  
X Chromosome ◽  
Epigenetic Regulation ◽  
Specific Expression ◽  
X Chromosomes ◽  
Phascolarctos Cinereus ◽  
Male And Female ◽  
Inactive X Chromosome ◽  
Promoter Dna ◽  
Female Specific

X chromosome inactivation (XCI) mediated by differential DNA methylation between sexes is an iconic example of epigenetic regulation. Although XCI is shared between eutherians and marsupials, the role of DNA methylation in marsupial XCI remains contested. Here, we examine genome-wide signatures of DNA methylation across fives tissues from a male and female koala ( Phascolarctos cinereus ), and present the first whole-genome, multi-tissue marsupial ‘methylome atlas’. Using these novel data, we elucidate divergent versus common features of representative marsupial and eutherian DNA methylation. First, tissue-specific differential DNA methylation in koalas primarily occurs in gene bodies. Second, females show significant global reduction (hypomethylation) of X chromosome DNA methylation compared to males. We show that this pattern is also observed in eutherians. Third, on average, promoter DNA methylation shows little difference between male and female koala X chromosomes, a pattern distinct from that of eutherians. Fourth, the sex-specific DNA methylation landscape upstream of Rsx , the primary lnc RNA associated with marsupial XCI, is consistent with the epigenetic regulation of female-specific (and presumably inactive X chromosome-specific) expression. Finally, we use the prominent female X chromosome hypomethylation and classify 98 previously unplaced scaffolds as X-linked, contributing an additional 14.6 Mb (21.5%) to genomic data annotated as the koala X chromosome. Our work demonstrates evolutionarily divergent pathways leading to functionally conserved patterns of XCI in two deep branches of mammals.

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