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John M Greally

Publications and source records attributed to John M Greally.

13 recordsLinked to original sources
PubMed2007

Genome-wide and locus-specific DNA hypomethylation in G9a deficient mouse embryonic stem cells.

In the mammalian genome, numerous CpG-rich loci define tissue-dependent and differentially methylated regions (T-DMRs). Euchromatin from different cell types differs in terms of its tissue-specific DNA methylation profile as defined by these T-DMRs. G9a is a euchromatin-localized histone methyltransferase (HMT) and catalyzes methylation of histone H3 at lysines 9 and 27 (H3-K9 and -K27). To test whether HMT activity influences euchromatic cytosine methylation, we analyzed the DNA methylation status of approximately 2000 CpG-rich loci, which are predicted in silico, in G9a(-/-) embryonic stem cells by restriction landmark genomic scanning (RLGS). While the RLGS profile of wild-type cells contained about 1300 spots, 32 new spots indicating DNA demethylation were seen in the profile of G9a(-/-) cells. Virtual-image RLGS (Vi-RLGS) allowed us to identify the genomic source of ten of these spots. These were confirmed to be cytosine demethylated, not just at the Not I site detected by the RLGS but extending over several kilobase pairs in cis. Chromatin immunoprecipitation (ChIP) confirmed these loci to be targets of G9a, with decreased H3-K9 and/or -K27 dimethylation in the G9a(-/-) cells. These data indicate that G9a site-selectively contributes to DNA methylation.

Animals↗
PubMed2006

Comparative isoschizomer profiling of cytosine methylation: the HELP assay.

The distribution of cytosine methylation in 6.2 Mb of the mouse genome was tested using cohybridization of genomic representations from a methylation-sensitive restriction enzyme and its methylation-insensitive isoschizomer. This assay, termed HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR), allows both intragenomic profiling and intergenomic comparisons of cytosine methylation. The intragenomic profile shows most of the genome to be contiguous methylated sequence with occasional clusters of hypomethylated loci, usually but not exclusively at promoters and CpG islands. Intergenomic comparison found marked differences in cytosine methylation between spermatogenic and brain cells, identifying 223 new candidate tissue-specific differentially methylated regions (T-DMRs). Bisulfite pyrosequencing confirmed the four candidates tested to be T-DMRs, while quantitative RT-PCR for two genes with T-DMRs located at their promoters showed the HELP data to be correlated with gene activity at these loci. The HELP assay is robust, quantitative, and accurate and is providing new insights into the distribution and dynamic nature of cytosine methylation in the genome.

Animals↗
PubMed2005

Genetic mapping of putative Chrna7 and Luzp2 neuronal transcriptional enhancers due to impact of a transgene-insertion and 6.8 Mb deletion in a mouse model of Prader-Willi and Angelman syndromes.

BACKGROUND: Prader-Willi and Angelman syndrome (PWS and AS) patients typically have an approximately 5 Mb deletion of human chromosome 15q11-q13, of opposite parental origin. A mouse model of PWS and AS has a transgenic insertion-deletion (TgPWS/TgAS) of chromosome 7B/C subsequent to paternal or maternal inheritance, respectively. In this study, we define the deletion endpoints and examine the impact on expression of flanking genes. RESULTS: Using molecular and cytological methods we demonstrate that 13 imprinted and 11 non-imprinted genes are included in the TgPWS/TgAS deletion. Normal expression levels were found in TgPWS brain for genes extending 9.1- or 5.6-Mb centromeric or telomeric of the deletion, respectively. Our molecular cytological studies map the proximal deletion breakpoint between the Luzp2 and Siglec-H loci, and we show that overall mRNA levels of Luzp2 in TgPWS and TgAS brain are significantly reduced by 17%. Intriguingly, 5' Chrna7 shows 1.7-fold decreased levels in TgPWS and TgAS brain whereas there is a > or =15-fold increase in expression in neonatal liver and spleen of these mouse models. By isolating a Chrna7-Tg fusion transcript from TgAS mice, we mapped the telomeric deletion breakpoint in Chrna7 intron 4. CONCLUSION: Based on the extent of the deletion, TgPWS/TgAS mice are models for PWS/AS class I deletions. Other than for the first gene promoters immediately outside the deletion, since genes extending 5.6-9.1 Mb away from each end of the deletion show normal expression levels in TgPWS brain, this indicates that the transgene array does not induce silencing and there are no additional linked rearrangements. Using gene expression, non-coding conserved sequence (NCCS) and synteny data, we have genetically mapped a putative Luzp2 neuronal enhancer responsible for approximately 33% of allelic transcriptional activity. The Chrna7 results are explained by hypothesizing loss of an essential neuronal transcriptional enhancer required for approximately 80% of allelic Chrna7 promoter activity, while the Chrna7 promoter is upregulated in B lymphocytes by the transgene immunoglobulin enhancer. The mapping of a putative Chrna7 neuronal enhancer inside the deletion has significant implications for understanding the transcriptional regulation of this schizophrenia-susceptibility candidate gene.

Alleles↗
PubMed2004

Comparative sequence and x-inactivation analyses of a domain of escape in human xp11.2 and the conserved segment in mouse.

We have performed X-inactivation and sequence analyses on 350 kb of sequence from human Xp11.2, a region shown previously to contain a cluster of genes that escape X inactivation, and we compared this region with the region of conserved synteny in mouse. We identified several new transcripts from this region in human and in mouse, which defined the full extent of the domain escaping X inactivation in both species. In human, escape from X inactivation involves an uninterrupted 235-kb domain of multiple genes. Despite highly conserved gene content and order between the two species, Smcx is the only mouse gene from the conserved segment that escapes inactivation. As repetitive sequences are believed to facilitate spreading of X inactivation along the chromosome, we compared the repetitive sequence composition of this region between the two species. We found that long terminal repeats (LTRs) were decreased in the human domain of escape, but not in the majority of the conserved mouse region adjacent to Smcx in which genes were subject to X inactivation, suggesting that these repeats might be excluded from escape domains to prevent spreading of silencing. Our findings indicate that genomic context, as well as gene-specific regulatory elements, interact to determine expression of a gene from the inactive X-chromosome.

Actins↗
PubMed2003

Homez, a homeobox leucine zipper gene specific to the vertebrate lineage.

This work describes a vertebrate homeobox gene, designated Homez (homeodomain leucine zipper-encoding gene), that encodes a protein with an unusual structural organization. There are several regions within Homez, including three atypical homeodomains, two leucine zipper-like motifs, and an acidic domain. The gene is ubiquitously expressed in human and murine tissues, although the expression pattern is more restricted during mouse development. Genomic analysis revealed that human and mouse genes are located at 14q11.2 and 14C, respectively, and are composed of two exons. The zebrafish and pufferfish homologs share high similarity to mammalian sequences, particularly within the homeodomain sequences. Based on homology of homeodomains and on the similarity in overall protein structure, we delineate Homez and members of ZHX family of zinc finger homeodomain factors as a subset within the superfamily of homeobox-containing proteins. The type and composition of homeodomains in the Homez subfamily are vertebrate-specific. Phylogenetic analysis indicates that Homez lineage was separated from related genes >400 million years ago before separation of ray- and lobe-finned fishes. We apply a duplication-degeneration-complementation model to explain how this family of genes has evolved.

Alternative Splicing↗
PubMed2003

Mammalian linker-histone subtypes differentially affect gene expression in vivo.

Posttranslational modifications and remodeling of nucleosomes are critical factors in the regulation of transcription. Higher-order folding of chromatin also is likely to contribute to the control of gene expression, but the absence of a detailed description of the structure of the chromatin fiber has impaired progress in this area. Mammalian somatic cells contain a set of H1 linker-histone subtypes, H1 (0) and H1a to H1e, that bind to nucleosome core particles and to the linker DNA between nucleosomes. To determine whether the H1 histone subtypes play differential roles in the regulation of gene expression, we combined mice lacking specific H1 histone subtypes with mice carrying transgenes subject to position effects. Because position effects result from the unique chromatin structure created by the juxtaposition of regulatory elements in the transgene and at the site of integration, transgenes can serve as exquisitely sensitive indicators of chromatin structure. We report that some, but not all, linker histones can attenuate or accentuate position effects. The results suggest that the linker-histone subtypes play differential roles in the control of gene expression and that the sequential arrangement of the linker histones on the chromatin fiber might regulate higher-order chromatin structure and fine-tune expression levels.

Anemia, Hemolytic↗
PubMed2003

Epigenetic abnormalities associated with a chromosome 18(q21-q22) inversion and a Gilles de la Tourette syndrome phenotype.

Gilles de la Tourette syndrome (GTS) is a potentially debilitating neuropsychiatric disorder defined by the presence of both vocal and motor tics. Despite evidence that this and a related phenotypic spectrum, including chronic tics (CT) and Obsessive Compulsive Disorder (OCD), are genetically mediated, no gene involved in disease etiology has been identified. Chromosomal abnormalities have long been proposed to play a causative role in isolated cases of GTS spectrum phenomena, but confirmation of this hypothesis has yet to be forthcoming. We describe an i(18q21.1-q22.2) inversion in a patient with CT and OCD. We have fine mapped the telomeric aspect of the rearrangement to within 1 Mb of a previously reported 18q22 breakpoint that cosegregated in a family with GTS and related phenotypes. A comprehensive characterization of this genomic interval led to the identification of two transcripts, neither of which was found to be structurally disrupted. Analysis of the epigenetic characteristics of the region demonstrated a significant increase in replication asynchrony in the patient compared to controls, with the inverted chromosome showing delayed replication timing across at least a 500-kb interval. These findings are consistent with long-range functional dysregulation of one or more genes in the region. Our data support a link between chromosomal aberrations and epigenetic mechanisms in GTS and suggest that the study of the functional consequences of balanced chromosomal rearrangements is warranted in patients with phenotypes of interest, irrespective of the findings regarding structurally disrupted transcripts.

Child↗
PubMed2003

A stain upon the silence: genes escaping X inactivation.

X-chromosome inactivation is a remarkable epigenetic event in mammalian females that results in the transcriptional silencing of one of the pair of X chromosomes. However, not all X-linked genes are subject to inactivation, and in humans, the proportion of genes on the X chromosome that escapes inactivation is more than 15%. Here we examine the causes and consequences of failure to silence the entire X chromosome. We discuss the impact of the evolutionary history of the X (and Y) chromosome, and the bioinformatic approaches that promise to provide new insights into the genomic architecture of genes or regions that escape X-chromosome inactivation.

Animals↗
PubMed2002

Identification of a candidate regulatory region in the human CD8 gene complex by colocalization of DNase I hypersensitive sites and matrix attachment regions which bind SATB1 and GATA-3.

To locate elements regulating the human CD8 gene complex, we mapped nuclear matrix attachment regions (MARs) and DNase I hypersensitive (HS) sites over a 100-kb region that included the CD8B gene, the intergenic region, and the CD8A gene. MARs facilitate long-range chromatin remodeling required for enhancer activity and have been found closely linked to several lymphoid enhancers. Within the human CD8 gene complex, we identified six DNase HS clusters, four strong MARs, and several weaker MARs. Three of the strong MARs were closely linked to two tissue-specific DNase HS clusters (III and IV) at the 3' end of the CD8B gene. To further establish the importance of this region, we obtained 19 kb of sequence and screened for potential binding sites for the MAR-binding protein, SATB1, and for GATA-3, both of which are critical for T cell development. By gel shift analysis we identified two strong SATB1 binding sites, located 4.5 kb apart, in strong MARs. We also detected strong GATA-3 binding to an oligonucleotide containing two GATA-3 motifs located at an HS site in cluster IV. This clustering of DNase HS sites and MARs capable of binding SATB1 and GATA-3 at the 3' end of the CD8B gene suggests that this region is an epigenetic regulator of CD8 expression.

Base Sequence↗
PubMed2002

MEN1 tumor-suppressor protein localizes to telomeres during meiosis.

Multiple endocrine neoplasia type 1 is an autosomal dominant cancer predisposition syndrome caused by mutations in the tumor-suppressor gene MEN1. The gene encodes a nuclear protein, menin, with no recognized functional motifs. Menin has been shown negatively to regulate transcriptional activation mediated by JunD, although the significance of this interaction in normal cell physiology and how the absence of menin leads to tumorigenesis are unknown. Menin is highly expressed in testes. We used immunocytochemistry to explore its role in meiosis and found that it localizes exclusively at telomeres. JunD was not found at telomeres in meiotic cells. In view of elevated telomerase activity or abnormal telomere structure in virtually all malignancies, regulation of telomere function would be an appealing role for a tumor suppressor. However, menin does not specifically associate with telomeres in somatic cells, as indicated by lack of co-localization with the known telomeric protein TRF2. Cells overexpressing menin had normal telomerase activity, and tumors with homozygous MEN1 mutations showed no aberrations in telomere length, indicating that menin does not directly regulate telomerase activity. The role of menin at meiotic telomeres appears to be independent of JunD and may not have a counterpart in somatic cells. These results suggest that menin may play different roles in different tissues through interactions with different proteins.

Genes, Tumor Suppressor↗
PubMed2002

Genomic organization of the genes Gtf2ird1, Gtf2i, and Ncf1 at the mouse chromosome 5 region syntenic to the human chromosome 7q11.23 Williams syndrome critical region.

We have recently isolated a mouse ortholog of human GTF2IRD1 that is related to GTF2I. GTF2IRD1 and GTF2I proteins are characterized by the presence of multiple helix-loop-helix domains and a leucine zipper motif. Both paralogs are closely linked and deleted hemizygously in individuals with Williams syndrome, a dominant genetic condition characterized by unique neurocognitive and behavioral features. We have isolated and analyzed the sequence of bacterial artificial chromosome clones from the syntenic mouse chromosome 5 region that contains Gtf2ird1 and Gtf2i as well as a neighboring gene, Ncf1. Gtf2ird1 is composed of 31 exons spanning >100 kb on mouse chromosome 5 and is located between Cyln2 and Gtf2i. Gtf2i is composed of 34 exons spanning about 77 kb. Ncf1, located downstream of Gtf2i, consists of 11 exons that extend over 8 kb. The gene organization of Gtf2ird1, Gtf2i, and Ncf1 is conserved in mice and humans, although the intronic regions are more compact in the mouse genome. The helix-loop-helix repeats of Gtf2ird1 and Gtf2i are encoded separately on adjacent exons and were generated by independent genomic rearrangements. These studies contribute to our knowledge of transcription factor defects and their pathogenesis in haploinsufficiency conditions.

Amino Acid Sequence↗
PubMed2001

Short interspersed transposable elements (SINEs) are excluded from imprinted regions in the human genome.

To test whether regions undergoing genomic imprinting have unique genomic characteristics, imprinted and nonimprinted human loci were compared for nucleotide and retroelement composition. Maternally and paternally expressed subgroups of imprinted genes were found to differ in terms of guanine and cytosine, CpG, and retroelement content, indicating a segregation into distinct genomic compartments. Imprinted regions have been normally permissive to L1 long interspersed transposable element retroposition during mammalian evolution but universally and significantly lack short interspersed transposable elements (SINEs). The primate-specific Alu SINEs, as well as the more ancient mammalian-wide interspersed repeat SINEs, are found at significantly low densities in imprinted regions. The latter paleogenomic signature indicates that the sequence characteristics of currently imprinted regions existed before the mammalian radiation. Transitions from imprinted to nonimprinted genomic regions in cis are characterized by a sharp inflection in SINE content, demonstrating that this genomic characteristic can help predict the presence and extent of regions undergoing imprinting. During primate evolution, SINE accumulation in imprinted regions occurred at a decreased rate compared with control loci. The constraint on SINE accumulation in imprinted regions may be mediated by an active selection process. This selection could be because of SINEs attracting and spreading methylation, as has been found at other loci. Methylation-induced silencing could lead to deleterious consequences at imprinted loci, where inactivation of one allele is already established, and expression is often essential for embryonic growth and survival.

CpG Islands↗