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Anorexia nervosa (AN) is a complex and heritable eating disorder characterized by dangerously low body weight. Neither candidate gene studies nor an initial genome-wide association study (GWAS) have yielded significant and replicated results. We performed a GWAS in 2907 cases with AN from 14 countries (15 sites) and 14 860 ancestrally matched controls as part of the Genetic Consortium for AN (GCAN) and the Wellcome Trust Case Control Consortium 3 (WTCCC3). Individual association analyses were conducted in each stratum and meta-analyzed across all 15 discovery data sets. Seventy-six (72 independent) single nucleotide polymorphisms were taken forward for in silico (two data sets) or de novo (13 data sets) replication genotyping in 2677 independent AN cases and 8629 European ancestry controls along with 458 AN cases and 421 controls from Japan. The final global meta-analysis across discovery and replication data sets comprised 5551 AN cases and 21 080 controls. AN subtype analyses (1606 AN restricting; 1445 AN binge–purge) were performed. No findings reached genome-wide significance. Two intronic variants were suggestively associated: rs9839776 ( P =3.01 × 10 −7 ) in SOX2OT and rs17030795 ( P =5.84 × 10 −6 ) in PPP3CA . Two additional signals were specific to Europeans: rs1523921 ( P =5.76 × 10 − 6 ) between CUL3 and FAM124B and rs1886797 ( P =8.05 × 10 − 6 ) near SPATA13 . Comparing discovery with replication results, 76% of the effects were in the same direction, an observation highly unlikely to be due to chance ( P =4 × 10 −6 ), strongly suggesting that true findings exist but our sample, the largest yet reported, was underpowered for their detection. The accrual of large genotyped AN case-control samples should be an immediate priority for the field.

The nuclear lamina plays an important role in the regulation of chromatin organization and gene positioning in animals. CROWDED NUCLEI (CRWN) is a strong candidate for the plant nuclear lamina protein in Arabidopsis thaliana but its biological function was largely unknown. Here, we show that CRWNs localize at the nuclear lamina and build the meshwork structure. Fluorescence in situ hybridization and RNA-seq analyses revealed that CRWNs regulate chromatin distribution and gene expression. More than 2000 differentially expressed genes were identified in the crwn1crwn4 double mutant. Copper-associated ( CA ) genes that form a gene cluster on chromosome 5 were among the downregulated genes in the double mutant exhibiting low tolerance to excess copper. Our analyses showed this low tolerance to copper was associated with the suppression of CA gene expression and that CRWN1 interacts with the CA gene locus, enabling the locus to localize at the nuclear lamina under excess copper conditions. The nuclear lamina regulates chromatin organization and gene positioning. Here the authors show that CROWDED NUCLEI proteins contribute to the meshwork lamina structure in Arabidopsis nuclei and regulate copper tolerance by promoting lamina association and expression of copper response genes.

Jens Lykke-Andersen, Frank Baas, Joseph Gleeson and colleagues report that mutations in the 3′ exonuclease TOE1 cause pontocerebellar hypoplasia type 7. They further show that these mutations result in the accumulation of incompletely processed small nuclear RNAs, leading to severe, early-onset neurodegeneration. Deadenylases are best known for degrading the poly(A) tail during mRNA decay. The deadenylase family has expanded throughout evolution and, in mammals, consists of 12 Mg 2+ -dependent 3′-end RNases with substrate specificity that is mostly unknown 1 . Pontocerebellar hypoplasia type 7 (PCH7) is a unique recessive syndrome characterized by neurodegeneration and ambiguous genitalia 2 . We studied 12 human families with PCH7, uncovering biallelic, loss-of-function mutations in TOE1 , which encodes an unconventional deadenylase 3 , 4 . toe1 -morphant zebrafish displayed midbrain and hindbrain degeneration, modeling PCH-like structural defects in vivo . Surprisingly, we found that TOE1 associated with small nuclear RNAs (snRNAs) incompletely processed spliceosomal. These pre-snRNAs contained 3′ genome-encoded tails often followed by post-transcriptionally added adenosines. Human cells with reduced levels of TOE1 accumulated 3′-end-extended pre-snRNAs, and the immunoisolated TOE1 complex was sufficient for 3′-end maturation of snRNAs. Our findings identify the cause of a neurodegenerative syndrome linked to snRNA maturation and uncover a key factor involved in the processing of snRNA 3′ ends.

A new system to monitor the effects of nucleosome depletion in Xenopus egg extracts reveals that nucleosomes are required for spindle assembly and for recruitment of nuclear pore complex (NPC) components to the nuclear envelope for NPC formation. Nucleosomes are the fundamental unit of chromatin, but analysis of transcription-independent nucleosome functions has been complicated by the gene-expression changes resulting from histone manipulation. Here we solve this dilemma by developing Xenopus laevis egg extracts deficient for nucleosome formation and by analyzing the proteomic landscape and behavior of nucleosomal chromatin and nucleosome-free DNA. We show that although nucleosome-free DNA can recruit nuclear-envelope membranes, nucleosomes are required for spindle assembly and for formation of the lamina and of nuclear pore complexes (NPCs). We show that, in addition to the Ran G-nucleotide exchange factor RCC1, ELYS, the initiator of NPC formation, fails to associate with naked DNA but directly binds histone H2A–H2B dimers and nucleosomes. Tethering ELYS and RCC1 to DNA bypasses the requirement for nucleosomes in NPC formation in a synergistic manner. Thus, the minimal essential function of nucleosomes in NPC formation is to recruit RCC1 and ELYS.

ObjectiveRecent studies have found aristaless-related homeobox gene (ARX)/pancreatic and duodenal homeobox 1 (PDX1), alpha-thalassemia/mental retardation X-linked (ATRX)/death domain-associated protein (DAXX) and alternative lengthening of telomeres (ALT) to be promising prognostic biomarkers for non-functional pancreatic neuroendocrine tumours (NF-PanNETs). However, they have not been comprehensively evaluated, especially among small NF-PanNETs (≤2.0 cm). Moreover, their status in neuroendocrine tumours (NETs) from other sites remains unknown.DesignAn international cohort of 1322 NETs was evaluated by immunolabelling for ARX/PDX1 and ATRX/DAXX, and telomere-specific fluorescence in situ hybridisation for ALT. This cohort included 561 primary NF-PanNETs, 107 NF-PanNET metastases and 654 primary, non-pancreatic non-functional NETs and NET metastases. The results were correlated with numerous clinicopathological features including relapse-free survival (RFS).ResultsATRX/DAXX loss and ALT were associated with several adverse prognostic findings and distant metastasis/recurrence (p<0.001). The 5-year RFS rates for patients with ATRX/DAXX-negative and ALT-positive NF-PanNETs were 40% and 42% as compared with 85% and 86% for wild-type NF-PanNETs (p<0.001 and p<0.001). Shorter 5-year RFS rates for ≤2.0 cm NF-PanNETs patients were also seen with ATRX/DAXX loss (65% vs 92%, p=0.003) and ALT (60% vs 93%, p<0.001). By multivariate analysis, ATRX/DAXX and ALT status were independent prognostic factors for RFS. Conversely, classifying NF-PanNETs by ARX/PDX1 expression did not independently correlate with RFS. Except for 4% of pulmonary carcinoids, ATRX/DAXX loss and ALT were only identified in primary (25% and 29%) and NF-PanNET metastases (62% and 71%).ConclusionsATRX/DAXX and ALT should be considered in the prognostic evaluation of NF-PanNETs including ≤2.0 cm tumours, and are highly specific for pancreatic origin among NET metastases of unknown primary.

Topologically associating domains (TADs) are fundamental elements of the eukaryotic genomic structure. However, recent studies suggest that the insulating complexes, CTCF/cohesin, present at TAD borders in mammals are absent from those in Drosophila melanogaster , raising the possibility that border elements are not conserved among metazoans. Using in situ Hi-C with sub-kb resolution, here we show that the D . melanogaster genome is almost completely partitioned into >4000 TADs, nearly sevenfold more than previously identified. The overwhelming majority of these TADs are demarcated by the insulator complexes, BEAF-32/CP190, or BEAF-32/Chromator, indicating that these proteins may play an analogous role in flies as that of CTCF/cohesin in mammals. Moreover, extended regions previously thought to be unstructured are shown to consist of small contiguous TADs, a property also observed in mammals upon re-examination. Altogether, our work demonstrates that fundamental features associated with the higher-order folding of the genome are conserved from insects to mammals. Topologically associating domain (TAD) boundaries in flies seem to be different from those in mammals. Here, the authors use Hi-C with sub-kb resolution to identify about 4000 TADs in flies, most demarcated by the insulator complexes BEAF-32/CP190 or BEAF-32/Chromator like CTCF/cohesin in mammals.

Ivona Aksentijevich and colleagues identify heterozygous loss-of-function mutations in TNFAIP3 (encoding A20) in six unrelated families with early-onset systemic inflammation. Affected individuals exhibit increased expression of NF-κB–mediated proinflammatory cytokines, consistent with the established role of A20 as a potent inhibitor of the NF-κB signaling pathway. Systemic autoinflammatory diseases are driven by abnormal activation of innate immunity 1 . Herein we describe a new disease caused by high-penetrance heterozygous germline mutations in TNFAIP3 , which encodes the NF-κB regulatory protein A20, in six unrelated families with early-onset systemic inflammation. The disorder resembles Behçet's disease, which is typically considered a polygenic disorder with onset in early adulthood 2 . A20 is a potent inhibitor of the NF-κB signaling pathway 3 . Mutant, truncated A20 proteins are likely to act through haploinsufficiency because they do not exert a dominant-negative effect in overexpression experiments. Patient-derived cells show increased degradation of IκBα and nuclear translocation of the NF-κB p65 subunit together with increased expression of NF-κB–mediated proinflammatory cytokines. A20 restricts NF-κB signals via its deubiquitinase activity. In cells expressing mutant A20 protein, there is defective removal of Lys63-linked ubiquitin from TRAF6, NEMO and RIP1 after stimulation with tumor necrosis factor (TNF). NF-κB–dependent proinflammatory cytokines are potential therapeutic targets for the patients with this disease.

The cGAS-STING cytoplasmic-DNA-sensing pathway is activated by accumulation of extrachromosomal telomere repeats, and this proliferation-inhibition mechanism is defective in ALT cancer cells. Extrachromosomal telomere repeat (ECTR) DNA is unique to cancer cells that maintain telomeres through the alternative lengthening of telomeres (ALT) pathway, but the role of ECTRs in ALT development remains elusive. We found that induction of ECTRs in normal human fibroblasts activated the cGAS-STING-TBK1-IRF3 signaling axis to trigger IFNβ production and a type I interferon response, resulting in cell-proliferation defects. In contrast, ALT cancer cells are commonly defective in sensing cytosolic DNA. We found that STING expression was inhibited in ALT cancer cell lines and transformed ALT cells. Notably, the ALT suppressors histone H3.3 and the ATRX–Daxx histone chaperone complex were also required to activate the DNA-sensing pathway. Collectively, our data suggest that the loss of the cGAS-STING pathway may be required to evade ECTR-induced anti-proliferation effects and permit ALT development, and this requirement may be exploited for treatments specific to cancers utilizing the ALT pathway.

The commonly mutated genes in pancreatic neuroendocrine tumors (PanNETs) are ATRX , DAXX , and MEN1 . We genotyped 64 PanNETs and found 58% carry ATRX , DAXX , and MEN1 mutations (A-D-M mutant PanNETs) and this correlates with a worse clinical outcome than tumors carrying the wild-type alleles of all three genes (A-D-M WT PanNETs). We performed RNA sequencing and DNA-methylation analysis to reveal two distinct subgroups with one consisting entirely of A-D-M mutant PanNETs. Two genes differentiating A-D-M mutant from A-D-M WT PanNETs were high ARX and low PDX1 gene expression with PDX1 promoter hyper-methylation in the A-D-M mutant PanNETs. Moreover, A-D-M mutant PanNETs had a gene expression signature related to that of alpha-cells (FDR q -value < 0.009) of pancreatic islets including increased expression of HNF1A and its transcriptional target genes. This gene expression profile suggests that A-D-M mutant PanNETs originate from or transdifferentiate into a distinct cell type similar to alpha cells. In pancreatic neuroendocrine tumors (PanNETs) ATRX, DAXX, and MEN1 are commonly mutated (A-D-M mutant PanNETs). Here, the authors find in a cohort of PanNETS 58% are A-D-M mutant PanNETs, with a worse clinical outcome and differences in gene expression and methylation compared to A-D-M wild type cases- these gene expression differences suggest that A-D-M mutant PanNETs potentially originate from a cell type similar to alpha cells.

In a subset of poorly differentiated and highly aggressive carcinoma, a chromosomal translocation, t(15;19)(q13;p13), results in an in‐frame fusion of the double bromodomain protein, BRD4, with a testis‐specific protein of unknown function, NUT (nuclear protein in testis). In this study, we show that, after binding to acetylated chromatin through BRD4 bromodomains, the NUT moiety of the fusion protein strongly interacts with and recruits p300, stimulates its catalytic activity, initiating cycles of BRD4–NUT/p300 recruitment and creating transcriptionally inactive hyperacetylated chromatin domains. Using a patient‐derived cell line, we show that p300 sequestration into the BRD4–NUT foci is the principal oncogenic mechanism leading to p53 inactivation. Knockdown of BRD4–NUT released p300 and restored p53‐dependent regulatory mechanisms leading to cell differentiation and apoptosis. This study demonstrates how the off‐context activity of a testis‐specific factor could markedly alter vital cellular functions and significantly contribute to malignant cell transformation. The BRD4–NUT fusion protein, associated with an aggressive form of midline carcinoma, induces a feedforward loop involving the massive recruitment of p300 to acetylated chromatin foci, in turn leading to the sequestration and inactivation of p53.