SummaryWhether gene repositioning to the nuclear periphery during differentiation adds another layer of regulation to gene expression remains controversial. Here, we resolve this by manipulating gene positions through targeting the nuclear envelope transmembrane proteins (NETs) that direct their normal repositioning during myogenesis. Combining transcriptomics with high-resolution DamID mapping of nuclear envelope-genome contacts, we show that three muscle-specific NETs, NET39, Tmem38A, and WFS1, direct specific myogenic genes to the nuclear periphery to facilitate their repression. Retargeting a NET39 fragment to nucleoli correspondingly repositioned a target gene, indicating a direct tethering mechanism. Being able to manipulate gene position independently of other changes in differentiation revealed that repositioning contributes ⅓ to ⅔ of a gene’s normal repression in myogenesis. Together, these NETs affect 37% of all genes changing expression during myogenesis, and their combined knockdown almost completely blocks myotube formation. This unequivocally demonstrates that NET-directed gene repositioning is critical for developmental gene regulation.

MYCN amplification drives one in six cases of neuroblastoma. The supernumerary gene copies are commonly found on highly rearranged, extrachromosomal circular DNA (ecDNA). The exact amplicon structure has not been described thus far and the functional relevance of its rearrangements is unknown. Here, we analyze the MYCN amplicon structure using short-read and Nanopore sequencing and its chromatin landscape using ChIP-seq, ATAC-seq and Hi-C. This reveals two distinct classes of amplicons which explain the regulatory requirements for MYCN overexpression. The first class always co-amplifies a proximal enhancer driven by the noradrenergic core regulatory circuit (CRC). The second class of MYCN amplicons is characterized by high structural complexity, lacks key local enhancers, and instead contains distal chromosomal fragments harboring CRC-driven enhancers. Thus, ectopic enhancer hijacking can compensate for the loss of local gene regulatory elements and explains a large component of the structural diversity observed in MYCN amplification.

While analyzing the DNA methylome of multiple myeloma (MM), a plasma cell neoplasm, by whole-genome bisulfite sequencing and high-density arrays, we observed a highly heterogeneous pattern globally characterized by regional DNA hypermethylation embedded in extensive hypomethylation. In contrast to the widely reported DNA hypermethylation of promoter-associated CpG islands (CGIs) in cancer, hypermethylated sites in MM, as opposed to normal plasma cells, were located outside CpG islands and were unexpectedly associated with intronic enhancer regions defined in normal B cells and plasma cells. Both RNA-seq and in vitro reporter assays indicated that enhancer hypermethylation is globally associated with down-regulation of its host genes. ChIP-seq and DNase-seq further revealed that DNA hypermethylation in these regions is related to enhancer decommissioning. Hypermethylated enhancer regions overlapped with binding sites of B cellspecific transcription factors (TFs) and the degree of enhancer methylation inversely correlated with expression levels of these TFs in MM. Furthermore, hypermethylated regions in MM were methylated in stem cells and gradually became demethylated during normal B-cell differentiation, suggesting that MM cells either reacquire epigenetic features of undifferentiated cells or maintain an epigenetic signature of a putative myeloma stem cell progenitor. Overall, we have identified DNA hypermethylation of developmentally regulated enhancers as a new type of epigenetic modification associated with the pathogenesis of MM.

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Long non-coding RNAs (lncRNAs) can be important components in gene-regulatory networks 1 , 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. There has been enormous progress in exploring disease variants in the human genome. Yet, the interpretation of variants in the non-coding genome remains a challenge owing to the myriad mechanisms by which they can potentially cause disease. Besides disrupting cis-regulatory elements, non-coding variants may interfere with the function of non-coding transcripts. Indeed, a substantial fraction of the human genome is transcribed into RNA, although most transcripts lack protein-coding potential and are referred to as non-coding transcripts 2. Characterization of a small number of these RNA molecules has revealed that they may have roles as regulators of gene expression through diverse modes of action 3. However, the identification of functional non-coding transcript loci remains challenging. Thus, annotating non-coding transcript loci and unravelling their function will substantially improve our knowledge about gene regulation and the identification and interpretation of non-coding genetic variants with respect to disease pathogenesis. Non-coding deletions cause limb malformations We identified 27-63-kb non-coding deletions of chromosome 2 in three unrelated individuals (patients 1-3) with a type of limb malformation that, to our knowledge, remains undescribed. Affected individuals had a severe shortening and deformation of the legs and feet, 3/4 syndactyly of the hands, as well as the presence of nails on the palmar side of fingers IV and V (Fig. 1a, Extended Data Fig. 1a, b, Supplementary Note 1). Radiographs showed normal femora but severely shortened tibiae, triangular fibulae and malformed or absent bones in the feet (Fig. 1a, Extended Data Fig. 1a, Supplementary Note 1). Exome s...

In the past 15 years our perception of nuclear envelope function has evolved perhaps nearly as much as the nuclear envelope itself evolved in the last 3 billion years. Historically viewed as little more than a diffusion barrier between the cytoplasm and the nucleoplasm, the nuclear envelope is now known to have roles in the cell cycle, cytoskeletal stability and cell migration, genome architecture, epigenetics, regulation of transcription, splicing, and DNA replication. Here we will review both what is known and what is speculated about the role of the nuclear envelope in genome organization, particularly with respect to the positioning and repositioning of genes and chromosomes within the nucleus during differentiation.

The 3D organization of the genome changes concomitantly with expression changes during hematopoiesis and immune activation. Studies have focused either on lamina-associated domains (LADs) or on topologically associated domains (TADs), defined by preferential local chromatin interactions, and chromosome compartments, defined as higher-order interactions between TADs sharing functionally similar states. However, few studies have investigated how these affect one another. To address this, we mapped LADs using Lamin B1-DamID during Jurkat T-cell activation, finding significant genome reorganization at the nuclear periphery dominated by release of loci frequently important for T-cell function. To assess how these changes at the nuclear periphery influence wider genome organization, our DamID data sets were contrasted with TADs and compartments. Features of specific repositioning events were then tested by fluorescence in situ hybridization during T-cell activation. First, considerable overlap between TADs and LADs was observed with the TAD repositioning as a unit. Second, A1 and A2 subcompartments are segregated in 3D space through differences in proximity to LADs along chromosomes. Third, genes and a putative enhancer in LADs that were released from the periphery during T-cell activation became preferentially associated with A2 subcompartments and were constrained to the relative proximity of the lamina. Thus, lamina associations influence internal nuclear organization, and changes in LADs during T-cell activation may provide an important additional mode of gene regulation.

It is well established that the nuclear envelope has many distinct direct connections to chromatin that contribute to genome organization. The functional consequences of genome organization on gene regulation are less clear. Even less understood is how interactions of lamins and nuclear envelope transmembrane proteins (NETs) with chromatin can produce anchoring tethers that can withstand the physical forces of and on the genome. Chromosomes are the largest molecules in the cell, making megadalton protein structures like the nuclear pore complexes and ribosomes seem small by comparison. Thus to withstand strong forces from chromosome dynamics an anchoring tether is likely to be much more complex than a single protein-protein or protein-DNA interaction. Here we will briefly review known NE-genome interactions that likely contribute to spatial genome organization, postulate in the context of experimental data how these anchoring tethers contribute to gene regulation, and posit several hypotheses for the physical nature of these tethers that need to be investigated experimentally. Significantly, disruption of these anchoring tethers and the subsequent consequences for gene regulation could explain how mutations in nuclear envelope proteins cause diseases ranging from muscular dystrophy to lipodystrophy to premature aging progeroid syndromes. The two favored hypotheses for nuclear envelope protein involvement in disease are (1) weakening nuclear and cellular mechanical stability, and (2) disrupting genome organization and gene regulation. Considerable experimental support has been obtained for both. The integration of both mechanical and gene expression defects in the disruption of anchoring tethers could provide a unifying hypothesis consistent with both.