Kathryn Woodfine

G9a Histone Methyltransferase Contributes to Imprinting in the Mouse Placenta

ABSTRACT Whereas DNA methylation is essential for genomic imprinting, the importance of histone methylation in the allelic expression of imprinted genes is unclear. Imprinting control regions (ICRs), however, are marked by histone H3-K9 methylation on their DNA-methylated allele. In the placenta, the paternal silencing along the Kcnq1 domain on distal chromosome 7 also correlates with the presence of H3-K9 methylation, but imprinted repression at these genes is maintained independently of DNA methylation. To explore which histone methyltransferase (HMT) could mediate the allelic H3-K9 methylation on distal chromosome 7, and at ICRs, we generated mouse conceptuses deficient for the SET domain protein G9a. We found that in the embryo and placenta, the differential DNA methylation at ICRs and imprinted genes is maintained in the absence of G9a. Accordingly, in embryos, imprinted gene expression was unchanged at the domains analyzed, in spite of a global loss of H3-K9 dimethylation (H3K9me2). In contrast, the placenta-specific imprinting of genes on distal chromosome 7 is impaired in the absence of G9a, and this correlates with reduced levels of H3K9me2 and H3K9me3. These findings provide the first evidence for the involvement of an HMT and suggest that histone methylation contributes to imprinted gene repression in the trophoblast.

Regulation of alternative polyadenylation by genomic imprinting

Maternally and paternally derived alleles can utilize different promoters, but allele-specific differences in cotranscriptional processes have not been reported. We show that alternative polyadenylation sites at a novel murine imprinted gene (H13) are utilized in an allele-specific manner. A differentially methylated CpG island separates polyA sites utilized on maternal and paternal alleles, and contains an internal promoter. Two genetic systems show that alleles lacking methylation generate truncated H13 transcripts that undergo internal polyadenylation. On methylated alleles, the internal promoter is inactive and elongation proceeds to downstream polyadenylation sites. This demonstrates that epigenetic modifications can influence utilization of alternative polyadenylation sites.

Generation of an epigenetic signature by chronic hypoxia in prostate cells

Increasing levels of tissue hypoxia have been reported as a natural feature of the aging prostate gland and may be a risk factor for the development of prostate cancer. In this study, we have used PwR-1E benign prostate epithelial cells and an equivalently aged hypoxia-adapted PwR-1E sub-line to identify phenotypic and epigenetic consequences of chronic hypoxia in prostate cells. We have identified a significantly altered cellular phenotype in response to chronic hypoxia as characterized by increased receptor-mediated apoptotic resistance, the induction of cellular senescence, increased invasion and the increased secretion of IL-1 beta, IL6, IL8 and TNFalpha cytokines. In association with these phenotypic changes and the absence of HIF-1 alpha protein expression, we have demonstrated significant increases in global levels of DNA methylation and H3K9 histone acetylation in these cells, concomitant with the increased expression of DNA methyltransferase DMNT3b and gene-specific changes in DNA methylation at key imprinting loci. In conclusion, we have demonstrated a genome-wide adjustment of DNA methylation and histone acetylation under chronic hypoxic conditions in the prostate. These epigenetic signatures may represent an additional mechanism to promote and maintain a hypoxic-adapted cellular phenotype with a potential role in tumour development.

Replication timing of human chromosome 6.

Genomic microarrays have been used to assess DNA replication timing in a variety of eukaryotic organisms1-3. A replication timing map of the human genome has already been published at a 1Mb resolution3. Here we describe how the same method can be used to assess the replication timing of chromosome 6 with a greater resolution using an array of overlapping tile path clones. We report the replication timing map of the whole of chromosome 6 in general, and the MHC region in particular. Positive correlations are observed between replication timing and a number of genomic features including GC content, repeat content and transcriptional activity.

Chromosome-wide identification of novel imprinted genes using microarrays and uniparental disomies

Genomic imprinting refers to a specialized form of epigenetic gene regulation whereby the expression of a given allele is dictated by parental origin. Defining the extent and distribution of imprinting across genomes will be crucial for understanding the roles played by imprinting in normal mammalian growth and development. Using mice carrying uniparental disomies or duplications, microarray screening and stringent bioinformatics, we have developed the first large-scale tissue-specific screen for imprinted gene detection. We quantify the stringency of our methodology and relate it to previous non-tissue-specific large-scale studies. We report the identification in mouse of four brain-specific novel paternally expressed transcripts and an additional three genes that show maternal expression in the placenta. The regions of conserved linkage in the human genome are associated with the Prader–Willi Syndrome (PWS) and Beckwith–Wiedemann Syndrome (BWS) where imprinting is known to be a contributing factor. We conclude that large-scale systematic analyses of this genre are necessary for the full impact of genomic imprinting on mammalian gene expression and phenotype to be elucidated.

The Parental Non-Equivalence of Imprinting Control Regions during Mammalian Development and Evolution

In mammals, imprinted gene expression results from the sex-specific methylation of imprinted control regions (ICRs) in the parental germlines. Imprinting is linked to therian reproduction, that is, the placenta and imprinting emerged at roughly the same time and potentially co-evolved. We assessed the transcriptome-wide and ontology effect of maternally versus paternally methylated ICRs at the developmental stage of setting of the chorioallantoic placenta in the mouse (8.5dpc), using two models of imprinting deficiency including completely imprint-free embryos. Paternal and maternal imprints have a similar quantitative impact on the embryonic transcriptome. However, transcriptional effects of maternal ICRs are qualitatively focused on the fetal-maternal interface, while paternal ICRs weakly affect non-convergent biological processes, with little consequence for viability at 8.5dpc. Moreover, genes regulated by maternal ICRs indirectly influence genes regulated by paternal ICRs, while the reverse is not observed. The functional dominance of maternal imprints over early embryonic development is potentially linked to selection pressures favoring methylation-dependent control of maternal over paternal ICRs. We previously hypothesized that the different methylation histories of ICRs in the maternal versus the paternal germlines may have put paternal ICRs under higher mutational pressure to lose CpGs by deamination. Using comparative genomics of 17 extant mammalian species, we show here that, while ICRs in general have been constrained to maintain more CpGs than non-imprinted sequences, the rate of CpG loss at paternal ICRs has indeed been higher than at maternal ICRs during evolution. In fact, maternal ICRs, which have the characteristics of CpG-rich promoters, have gained CpGs compared to non-imprinted CpG-rich promoters. Thus, the numerical and, during early embryonic development, functional dominance of maternal ICRs can be explained as the consequence of two orthogonal evolutionary forces: pressure to tightly regulate genes affecting the fetal-maternal interface and pressure to avoid the mutagenic environment of the paternal germline.

Molecular mechanisms of genomic imprinting and clinical implications for cancer.

Genomic imprinting is an epigenetic marking of genes in the parental germline that ensures the stable transmission of monoallelic gene expression patterns in a parent-of-origin-specific manner. Epigenetic marking systems are thus able to regulate gene activity independently of the underlying DNA sequence. Several imprinted gene products regulate cell proliferation and fetal growth; loss of their imprinted state, which effectively alters their dosage, might promote or suppress tumourigenic processes. Conversely, global epigenetic changes that underlie tumourigenesis might affect imprinted gene expression. Here, we review imprinted genes with regard to their roles in epigenetic predisposition to cancer, and discuss acquired epigenetic changes (DNA methylation, histone modifications and chromatin conformation) either as a result of cancer or as an early event in neoplasia. We also address recent work showing the potential role of noncoding RNA in modifying chromatin and affecting imprinted gene expression, and summarise the effects of loss of imprinting in cancer with regard to the roles that imprinted genes play in regulating growth signalling cascades. Finally, we speculate on the clinical applications of epigenetic drugs in cancer.

Transcript- and tissue-specific imprinting of a tumour suppressor gene

The Bladder Cancer-Associated Protein gene (BLCAP; previously BC10) is a tumour suppressor that limits cell proliferation and stimulates apoptosis. BLCAP protein or message are downregulated or absent in a variety of human cancers. In mouse and human, the first intron of Blcap/BLCAP contains the distinct Neuronatin (Nnat/NNAT) gene. Nnat is an imprinted gene that is exclusively expressed from the paternally inherited allele. Previous studies found no evidence for imprinting of Blcap in mouse or human. Here we show that Blcap is imprinted in mouse and human brain, but not in other mouse tissues. Moreover, Blcap produces multiple distinct transcripts that exhibit reciprocal allele-specific expression in both mouse and human. We propose that the tissue-specific imprinting of Blcap is due to the particularly high transcriptional activity of Nnat in brain, as has been suggested previously for the similarly organized and imprinted murine Commd1/U2af1-rs1 locus. For Commd1/U2af1-rs1, we show that it too produces distinct transcript variants with reciprocal allele-specific expression. The imprinted expression of BLCAP and its interplay with NNAT at the transcriptional level may be relevant to human carcinogenesis.

Genomic imprinting of Dopa decarboxylase in heart and reciprocal allelic expression with neighboring Grb10.

ABSTRACT By combining a tissue-specific microarray screen with mouse uniparental duplications, we have identified a novel imprinted gene, Dopa decarboxylase (Ddc), on chromosome 11. Ddc_exon1a is a 2-kb transcript variant that initiates from an alternative first exon in intron 1 of the canonical Ddc transcript and is paternally expressed in trabecular cardiomyocytes of the embryonic and neonatal heart. Ddc displays tight conserved linkage with the maternally expressed and methylated Grb10 gene, suggesting that these reciprocally imprinted genes may be coordinately regulated. In Dnmt3L mutant embryos that lack maternal germ line methylation imprints, we show that Ddc is overexpressed and Grb10 is silenced. Their imprinting is therefore dependent on maternal germ line methylation, but the mechanism at Ddc does not appear to involve differential methylation of the Ddc_exon1a promoter region and may instead be provided by the oocyte mark at Grb10. Our analysis of Ddc redefines the imprinted Grb10 domain on mouse proximal chromosome 11 and identifies Ddc_exon1a as the first example of a heart-specific imprinted gene.

Evaluation of allelic expression of imprinted genes in adult human blood.

Background Imprinted genes are expressed from only one allele in a parent-of-origin dependent manner. Loss of imprinted (LOI) expression can result in a variety of human disorders and is frequently reported in cancer. Biallelic expression of imprinted genes in adult blood has been suggested as a useful biomarker and is currently being investigated in colorectal cancer. In general, the expression profiles of imprinted genes are well characterised during human and mouse fetal development, but not in human adults. Methodology/Principal Findings We investigated quantitative expression of 36 imprinted genes in adult human peripheral blood leukocytes obtained from healthy individuals. Allelic expression was also investigated in B and T lymphocytes and myeloid cells. We found that 21 genes were essentially undetectable in adult blood. Only six genes were demonstrably monoallelic, and most importantly, we found that nine genes were either biallelic or showed variable expression in different individuals. Separated leukocyte populations showed the same expression patterns as whole blood. Differential methylation at each of the imprinting control loci analysed was maintained, including regions that contained biallelically expressed genes. This suggests in some cases methylation has become uncoupled from its role in regulating gene expression. Conclusions/Significance We conclude that only a limited set of imprinted genes, including IGF2 and SNRPN, may be useful for LOI cancer biomarker studies. In addition, blood is not a good tissue to use for the discovery of new imprinted genes. Finally, lymphocyte DNA methylation status in the adult may not always be a reliable indicator of monoallelic gene expression.