An unanticipated and tremendous amount of the noncoding sequence of the human genome is transcribed. Long noncoding RNAs (lncRNAs) constitute a significant fraction of non-protein-coding transcripts; however, their functions remain enigmatic. We demonstrate that deletions of a small noncoding differentially methylated region at 16q24.1, including lncRNA genes, cause a lethal lung developmental disorder, alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV), with parent-of-origin effects. We identify overlapping deletions 250 kb upstream of FOXF1 in nine patients with ACD/MPV that arose de novo specifically on the maternally inherited chromosome and delete lung-specific lncRNA genes. These deletions define a distant cis-regulatory region that harbors, besides lncRNA genes, also a differentially methylated CpG island, binds GLI2 depending on the methylation status of this CpG island, and physically interacts with and up-regulates the FOXF1 promoter. We suggest that lung-transcribed 16q24.1 lncRNAs may contribute to long-range regulation of FOXF1 by GLI2 and other transcription factors. Perturbation of lncRNA-mediated chromatin interactions may, in general, be responsible for position effect phenomena and potentially cause many disorders of human development.

Reprogramming of DNA methylation patterns during mammalian preimplantation development involves the concurrent maintenance of methylation on differentially methylated domains (DMDs) of imprinted genes and a marked reduction of global (non-DMD) genomic methylation. In the developing mammalian embryo, one allele of a DMD is unmethylated, and the opposite parental allele is methylated, having inherited this methylation from the parental gamete. The maintenance of DMDs is important for monoallelic imprinted gene expression and normal development of the embryo. Because the DNMT1 cytosine methyltransferase governs maintenance methylation in mammals, rearrangements of non-DMD, but not DMD methylation in preimplantation embryos suggest that the preimplantation DNMT1-dependent maintenance mechanism specifically targets DMD sequences. We explored this possibility using an engineered mouse ES cell line to screen for mutant DNMT1 proteins that protect against the loss of DMD and/or global (non-DMD) methylation in the absence of the wild-type endogenous DNMT1 methyltransferase. We identified DNMT1 mutants that were defective in maintenance of either DMD and/or non-DMD methylation. Among these, one mutant maintained non-DMD methylation but not imprinted DMD methylation and another mutant maintained just DMD methylation. The mutated amino acids of these mutants reside in a mammal-specific, disordered region near the amino terminus of DNMT1. These findings suggest that DNMT1 participates in epigenetic reprogramming through its ability to distinguish different categories of methylated sequences.epigenetic ͉ imprinting ͉ methylase ͉ methylation ͉ reprogramming G enomic imprinting is a mammalian epigenetic process that distinguishes maternal and paternal alleles to ensure parent-specific (monoallelic) expression of Ϸ80 imprinted genes (1). The molecular basis of this process is de novo methylation during gametogenesis and maintenance methylation during embryogenesis; this sequence of activities leads to the generation of imprinted DMDs (2). Methylation is placed on DMD sequences in the maternal and paternal germ lines by the DNMT3a cytosine methyltransferase (3, 4). Following fertilization DMD methylation is maintained (inherited) during preimplantation development by the combined action of different isoforms of the DNMT1 cytosine methyltransferase (5-8). The M r 175,000 DNMT1o form is synthesized in the oocyte and maintains methylation during preimplantation development (5, 9), whereas the M r 190,000 DNMT1s form is synthesized both in the oocyte and in the early embryo and this form also functions in preimplantation embryos (6). Along with this maintenance of DMD methylation is a significant reduction in the level of global (non-DMD) methylation (10, 11). The concurrent inheritance of DMD methylation and reduction of global methylation rearranges the genome's methylation patterns just before the formation of pluripotent embryo stem cells (12).Maintenance of DMD methylation in the presence of a reduction in the average level of g...

Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.

Lay Abstract Aside from the sex chromosome, all other locations in the human genome have the general expectation that two copies per location are inherited, one from mother and one from father. Yet, with finer and finer characterization of the genome we now know that this rule can be broken. When the rule is broken, we say the region has a copy number variant or CNV. These CNVs can arise de novo or can be inherited and they can be harmless or increase risk for disease. A particular region of chromosome 15, near the centromere on the long arm (i.e., 15q11.2-q13), is a hotspot for CNVs and it has several breakpoints (BP). Among the rearrangements observed in this region, CNVs from the interval between the common BP1 and BP2 have been reported to be associated with developmental disorders, including autism spectrum disorder (ASD). Yet its effect on risk for ASD, while likely to be small, is not fully characterized and thus poses a challenge to recurrence-risk counseling. We estimated its effect on risk and ASD-related phenotypes in a well-characterized ASD sample, the Simons Simplex Collection. We find that BP1-BP2 CNVs contribute only modestly to risk and have similarly small effect on traits related to ASD. To be consistent with the current American College of Medical Genetics guidelines for interpretation of postnatal CNV, the BP1-BP2 deletion and duplication CNVs would probably best be classified as variants of uncertain significance (VOUS): they appear to have an impact on risk, but one so modest that these CNVs do not merit pathogenic status. Scientific Abstract The proximal region of chromosome 15 is one of the genomic hotspots for copy number variants (CNVs). Among the rearrangements observed in this region, CNVs from the interval between the common breakpoints 1 and 2 (BP1 and BP2) have been reported cosegregating with autism spectrum disorder (ASD). Although evidence supporting an association between BP1-BP2 CNVs and autism accumulates, the magnitude of the effect of BP1-BP2 CNVs remains elusive, posing a great challenge to recurrence-risk counseling. To gain further insight into their pathogenicity for ASD, we estimated the penetrance of the BP1-BP2 CNVs for ASD as well as their effects on ASD related phenotypes in a well-characterized ASD sample (n=2,525 families). TDT revealed significant preferential transmission only for the duplicated chromosome in probands (20T:9NT). The penetrance of the BP1-BP2 CNVs for ASD was low, conferring additional risks of 0.3% (deletion) and 0.8% (duplication). Stepwise regression analyzes suggest a greater effect of the CNVs on ASD related phenotype in males and when maternally inherited. Taken together, the results are consistent with BP1-BP2 CNVs as risk factors for autism. However their effect is modest, more akin to that seen for common variants. To be consistent with the current American College of Medical Genetics guidelines for interpretation of postnatal CNV, the BP1-BP2 deletion and duplication CNVs would probably best be classified as variants of uncertain s...