The imprinted antisense RNA at the Igf2r locus overlaps but does not imprint Mas1

The gene encoding the insulin-like growth-factor type-2 receptor (Igf2r) is maternally expressed and imprinted. A CpG island in Igf2r intron 2 that carries a maternal-specific methylation imprint was shown in a transgenic model to be essential for Igf2r imprinting and for the production of an antisense RNA from the paternal allele. We report here that the endogenous region2 is the promoter for this antisense RNA (named Air, for antisense Igf2r RNA) and that the 3' end lies 107,796 bp distant in an intron of the flanking, but non-imprinted, gene Mas1.     

Interaction between differentially methylated regions partitions the imprinted genes Igf2 and H19 into parent-specific chromatin loops

Imprinted genes are expressed from only one of the parental alleles and are marked epigenetically by DNA methylation and histone modifications. The paternally expressed gene insulin-like growth-factor 2 (Igf2) is separated by approximately 100 kb from the maternally expressed noncoding gene H19 on mouse distal chromosome 7. Differentially methylated regions in Igf2 and H19 contain chromatin boundaries, silencers and activators and regulate the reciprocal expression of the two genes in a methylation-sensitive manner by allowing them exclusive access to a shared set of enhancers. Various chromatin models have been proposed that separate Igf2 and H19 into active and silent domains. Here we used a GAL4 knock-in approach as well as the chromosome conformation capture technique to show that the differentially methylated regions in the imprinted genes Igf2 and H19 interact in mice. These interactions are epigenetically regulated and partition maternal and paternal chromatin into distinct loops. This generates a simple epigenetic switch for Igf2 through which it moves between an active and a silent chromatin domain.     

Maintenance of genomic methylation requires a SWI2/SNF2-like protein.

Altering cytosine methylation by genetic means leads to a variety of developmental defects in mice, plants and fungi. Deregulation of cytosine methylation also has a role in human carcinogenesis. In some cases, these defects have been tied to the inheritance of epigenetic alterations (such as chromatin imprints and DNA methylation patterns) that do not involve changes in DNA sequence. Using a forward genetic screen, we identified a gene (DDM1, decrease in DNA methylation) from the flowering plant Arabidopsis thaliana required to maintain normal cytosine methylation patterns. Additional ddm1 alleles (som4, 5, 6, 7, 8) were isolated in a selection for mutations that relieved transgene silencing (E.J.R., unpublished data). Loss of DDM1 function causes a 70% reduction of genomic cytosine methylation, with most of the immediate hypomethylation occurring in repeated sequences. In contrast, many low-copy sequences initially retain their methylation in ddm1 homozygotes, but lose methylation over time as the mutants are propagated through multiple generations by self-pollination. The progressive effect of ddm1 mutations on low-copy sequence methylation suggests that ddm1 mutations compromise the efficiency of methylation of newly incorporated cytosines after DNA replication. In parallel with the slow decay of methylation during inbreeding, ddm1 mutants accumulate heritable alterations (mutations or stable epialleles) at dispersed sites in the genome that lead to morphological abnormalities. Here we report that DDM1 encodes a SWI2/SNF2-like protein, implicating chromatin remodelling as an important process for maintenance of DNA methylation and genome integrity.     

Antagonism between DNA hypermethylation and enhancer-blocking activity at the H19 DMD is uncovered by CpG mutations

Imprinted expression at the H19-Igf2 locus depends on a differentially methylated domain (DMD) that acts both as a maternal-specific, methylation-sensitive insulator and as a paternal-specific site of hypermethylation. Four repeats in the DMD bind CCCTC-binding factor (CTCF) on the maternal allele and have been proposed to attract methylation on the paternal allele. We introduced point mutations into the DMD to deplete the repeats of CpGs while retaining CTCF-binding and enhancer-blocking activity. Maternal inheritance of the mutations left H19 expression and Igf2 imprinting intact, consistent with the idea that the DMD acts as an insulator. Conversely, paternal inheritance of these mutations disrupted maintenance of DMD methylation, resulting in biallelic H19 expression. Furthermore, an insulator was established on the paternally inherited mutated allele in vivo, reducing Igf2 expression and resulting in a 40% reduction in size of newborn offspring. Thus, the nine CpG mutations in the DMD showed that the two parental-specific roles of the H19 DMD, methylation maintenance and insulator assembly, are antagonistic.     

A functional switch from lung cancer resistance to susceptibility at the Pas1 locus in Kras2LA2 mice

Pulmonary adenoma susceptibility 1 (Pas1) is the major mouse lung cancer susceptibility locus on chromosome 6 (ref. 1). Kras2 is a common target of somatic mutation in chemically induced mouse lung tumors and is a candidate Pas1 gene. M. spretus mice (SPRET/Ei) carry a Pas1 resistance haplotype for chemically induced lung tumors. We demonstrate that the SPRET/Ei Pas1 allele is switched from resistance to susceptibility by fixation of the parental origin of the mutant Kras2 allele. This switch correlates with low expression of endogenous Kras2 in SPRET/Ei. We propose that the Pas1 modifier effect is due to Kras2, and that a sensitive balance between the expression levels of wild-type and mutant alleles determines lung tumor susceptibility. These data demonstrate that cancer predisposition should also be considered in the context of somatic events and could have major implications for the design of human association studies to identify cancer susceptibility genes.     

CTCF-binding sites flank CTG/CAG repeats and form a methylation-sensitive insulator at the DM1 locus

An expansion of a CTG repeat at the DM1 locus causes myotonic dystrophy (DM) by altering the expression of the two adjacent genes, DMPK and SIX5, and through a toxic effect of the repeat-containing RNA. Here we identify two CTCF-binding sites that flank the CTG repeat and form an insulator element between DMPK and SIX5. Methylation of these sites prevents binding of CTCF, indicating that the DM1 locus methylation in congenital DM would disrupt insulator function. Furthermore, CTCF-binding sites are associated with CTG/CAG repeats at several other loci. We suggest a general role for CTG/CAG repeats as components of insulator elements at multiple sites in the human genome.     

Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann syndrome

The overgrowth- and tumor-associated Beckwith-Wiedemann syndrome results from dysregulation of imprinted genes on chromosome 11p15.5. Here we show that inherited microdeletions in the H19 differentially methylated region (DMR) that abolish two CTCF target sites cause this disease. Maternal transmission of the deletions results in hypermethylation of the H19 DMR, biallelic IGF2 expression, H19 silencing and Beckwith-Wiedemann syndrome, indicative of loss of function of the IGF2-H19 imprinting control element.     

A common variant at the TERT-CLPTM1L locus is associated with estrogen receptor-negative breast cancer

Estrogen receptor (ER)-negative breast cancer shows a higher incidence in women of African ancestry compared to women of European ancestry. In search of common risk alleles for ER-negative breast cancer, we combined genome-wide association study (GWAS) data from women of African ancestry (1,004 ER-negative cases and 2,745 controls) and European ancestry (1,718 ER-negative cases and 3,670 controls), with replication testing conducted in an additional 2,292 ER-negative cases and 16,901 controls of European ancestry. We identified a common risk variant for ER-negative breast cancer at the TERT-CLPTM1L locus on chromosome 5p15 (rs10069690: per-allele odds ratio (OR) = 1.18 per allele, P = 1.0 × 10(-10)). The variant was also significantly associated with triple-negative (ER-negative, progesterone receptor (PR)-negative and human epidermal growth factor-2 (HER2)-negative) breast cancer (OR = 1.25, P = 1.1 × 10(-9)), particularly in younger women (<50 years of age) (OR = 1.48, P = 1.9 × 10(-9)). Our results identify a genetic locus associated with estrogen receptor negative breast cancer subtypes in multiple populations.     

Common genetic variants at the CRAC1 (HMPS) locus on chromosome 15q13.3 influence colorectal cancer risk

We mapped a high-penetrance gene (CRAC1; also known as HMPS) associated with colorectal cancer (CRC) in the Ashkenazi population to a 0.6-Mb region on chromosome 15 containing SCG5 (also known as SGNE1), GREM1 and FMN1. We hypothesized that the CRAC1 locus harbored low-penetrance variants that increased CRC risk in the general population. In a large series of colorectal cancer cases and controls, SNPs near GREM1 and SCG5 were strongly associated with increased CRC risk (for rs4779584, P = 4.44 x 10(-14)).