Non-coding deletions identify Maenli lncRNA as a limb-specific En1 regulator.

Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks1, but the exact nature and extent of their involvement in human Mendelian disease is largely unknown. Here we show that genetic ablation of a lncRNA locus on human chromosome 2 causes a severe congenital limb malformation. We identified homozygous 27-63-kilobase deletions located 300 kilobases upstream of the engrailed-1 gene (EN1) in patients with a complex limb malformation featuring mesomelic shortening, syndactyly and ventral nails (dorsal dimelia). Re-engineering of the human deletions in mice resulted in a complete loss of En1 expression in the limb and a double dorsal-limb phenotype that recapitulates the human disease phenotype. Genome-wide transcriptome analysis in the developing mouse limb revealed a four-exon-long non-coding transcript within the deleted region, which we named Maenli. Functional dissection of the Maenli locus showed that its transcriptional activity is required for limb-specific En1 activation in cis, thereby fine-tuning the gene-regulatory networks controlling dorso-ventral polarity in the developing limb bud. Its loss results in the En1-related dorsal ventral limb phenotype, a subset of the full En1-associated phenotype. Our findings demonstrate that mutations involving lncRNA loci can result in human Mendelian disease.

Polycomb-mediated genome architecture enables long-range spreading of H3K27 methylation.

Polycomb-group proteins play critical roles in gene silencing through the deposition of histone H3 lysine 27 trimethylation (H3K27me3) and chromatin compaction. This process is essential for embryonic stem cell (ESC) pluripotency, differentiation, and development. Polycomb repressive complex 2 (PRC2) can both read and write H3K27me3, enabling progressive spreading of H3K27me3 on the linear genome. Long-range Polycomb-associated DNA contacts have also been described, but their regulation and role in gene silencing remain unclear. Here, we apply H3K27me3 HiChIP, a protein-directed chromosome conformation method, and optical reconstruction of chromatin architecture to profile long-range Polycomb-associated DNA loops that span tens to hundreds of megabases across multiple topological associated domains in mouse ESCs and human induced pluripotent stem cells. We find that H3K27me3 loop anchors are enriched for Polycomb nucleation points and coincide with key developmental genes. Genetic deletion of H3K27me3 loop anchors results in disruption of spatial contact between distant loci and altered H3K27me3 in cis, both locally and megabases away on the same chromosome. In mouse embryos, loop anchor deletion leads to ectopic activation of the partner gene, suggesting that Polycomb-associated loops control gene silencing during development. Further, we find that alterations in PRC2 occupancy resulting from an RNA binding­deficient EZH2 mutant are accompanied by loss of Polycomb-associated DNA looping. Together, these results suggest PRC2 uses RNA binding to enhance long-range chromosome folding and H3K27me3 spreading. Developmental gene loci have unique roles in Polycomb spreading, emerging as important architectural elements of the epigenome.

Serial genomic inversions induce tissue-specific architectural stripes, gene misexpression and congenital malformations.

Balanced chromosomal rearrangements such as inversions and translocations can cause congenital disease or cancer by inappropriately rewiring promoter-enhancer contacts1,2. To study the potentially pathogenic consequences of balanced chromosomal rearrangements, we generated a series of genomic inversions by placing an active limb enhancer cluster from the Epha4 regulatory domain at different positions within a neighbouring gene-dense region and investigated their effects on gene regulation in vivo in mice. Expression studies and high-throughput chromosome conformation capture from embryonic limb buds showed that the enhancer cluster activated several genes downstream that are located within asymmetric regions of contact, the so-called architectural stripes3. The ectopic activation of genes led to a limb phenotype that could be rescued by deleting the CCCTC-binding factor (CTCF) anchor of the stripe. Architectural stripes appear to be driven by enhancer activity, because they do not form in mouse embryonic stem cells. Furthermore, we show that architectural stripes are a frequent feature of developmental three-dimensional genome architecture often associated with active enhancers. Therefore, balanced chromosomal rearrangements can induce ectopic gene expression and the formation of asymmetric chromatin contact patterns that are dependent on CTCF anchors and enhancer activity.