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Repetitive elements in differentiated cells are usually silenced. Genome-wide analyses in early mouse development show that repetitive-element expression decreases during development accompanied by the loss of active chromatin marks. LINE-1 and IAP retrotransposons become reactivated after fertilization, and LINE-1 transcription is regulated by short LINE-1 RNAs, which suggests that repetitive elements may be regulated through RNA during the earliest developmental stages. How a more plastic chromatin state is maintained and reversed during development is unknown. Heterochromatin-mediated silencing of repetitive elements occurs in differentiated cells. Here, we used repetitive elements, including retrotransposons, as model loci to address how and when heterochromatin forms during development. RNA sequencing throughout early mouse embryogenesis revealed that repetitive-element expression is dynamic and stage specific, with most repetitive elements becoming repressed before implantation. We show that LINE-1 and IAP retrotransposons become reactivated from both parental genomes after fertilization. Chromatin immunoprecipitation for H3K4me3 and H3K9me3 in 2- and 8-cell embryos indicates that their developmental silencing follows loss of activating marks rather than acquisition of conventional heterochromatic marks. Furthermore, short LINE-1 RNAs regulate LINE-1 transcription in vivo . Our data indicate that reprogramming after mammalian fertilization comprises a robust transcriptional activation of retrotransposons and that repetitive elements are initially regulated through RNA.

Myotonic dystrophy (DM) is caused by the expression of mutant RNAs containing expanded CUG repeats that sequester muscleblind-like (MBNL) proteins, leading to alternative splicing changes. Cardiac alterations, characterized by conduction delays and arrhythmia, are the second most common cause of death in DM. Using RNA sequencing, here we identify novel splicing alterations in DM heart samples, including a switch from adult exon 6B towards fetal exon 6A in the cardiac sodium channel, SCN5A . We find that MBNL1 regulates alternative splicing of SCN5A mRNA and that the splicing variant of SCN5A produced in DM presents a reduced excitability compared with the control adult isoform. Importantly, reproducing splicing alteration of Scn5a in mice is sufficient to promote heart arrhythmia and cardiac-conduction delay, two predominant features of myotonic dystrophy. In conclusion, misregulation of the alternative splicing of SCN5A may contribute to a subset of the cardiac dysfunctions observed in myotonic dystrophy. Patients with myotonic dystrophy (MD) suffer from severe cardiac issues of unknown aetiology. Freyermuth et al . show that fatal changes in cardiac electrophysiological properties in humans and mice with MD may arise from misregulation of the alternative splicing of the cardiac Na + channel SCN5A transcript, resulting in expression of its fetal form.

Background Intellectual disability (ID) is characterised by an extreme genetic heterogeneity. Several hundred genes have been associated to monogenic forms of ID, considerably complicating molecular diagnostics. Trio-exome sequencing was recently proposed as a diagnostic approach, yet remains costly for a general implementation. Methods We report the alternative strategy of targeted high-throughput sequencing of 217 genes in which mutations had been reported in patients with ID or autism as the major clinical concern. We analysed 106 patients with ID of unknown aetiology following array-CGH analysis and other genetic investigations. Ninety per cent of these patients were males, and 75% sporadic cases. Results We identified 26 causative mutations: 16 in X-linked genes (ATRX, CUL4B, DMD, FMR1, HCFC1, IL1RAPL1, IQSEC2, KDM5C, MAOA, MECP2, SLC9A6, SLC16A2, PHF8) and 10 de novo in autosomal-dominant genes (DYRK1A, GRIN1, MED13L, TCF4, RAI1, SHANK3, SLC2A1, SYNGAP1). We also detected four possibly causative mutations (eg, in NLGN3) requiring further investigations. We present detailed reasoning for assigning causality for each mutation, and associated patients’ clinical information. Some genes were hit more than once in our cohort, suggesting they correspond to more frequent ID-associated conditions (KDM5C, MECP2, DYRK1A, TCF4). We highlight some unexpected genotype to phenotype correlations, with causative mutations being identified in genes associated to defined syndromes in patients deviating from the classic phenotype (DMD, TCF4, MECP2). We also bring additional supportive (HCFC1, MED13L) or unsupportive (SHROOM4, SRPX2) evidences for the implication of previous candidate genes or mutations in cognitive disorders. Conclusions With a diagnostic yield of 25% targeted sequencing appears relevant as a first intention test for the diagnosis of ID, but importantly will also contribute to a better understanding regarding the specific contribution of the many genes implicated in ID and autism.

Squamous cell carcinoma (SCC) of the lung is a frequent and aggressive cancer type. Gene amplifications, a known activating mechanism of oncogenes, target the 3q26-qter region as one of the most frequently gained/amplified genomic sites in SCC of various types. Here, we used array comparative genomic hybridization to delineate the consensus region of 3q26.3 amplifications in lung SCC. Recurrent amplifications occur in 20% of lung SCC (136 tumors in total) and map to a core region of 2 Mb (Megabases) that encompasses SOX2, a transcription factor gene. Intense SOX2 immunostaining is frequent in nuclei of lung SCC, indicating potential active transcriptional regulation by SOX2. Analyses of the transcriptome of lung SCC, SOX2-overexpressing lung epithelial cells and embryonic stem cells (ESCs) reveal that SOX2 contributes to activate ESC-like phenotypes and provide clues pertaining to the deregulated genes involved in the malignant phenotype. In cell culture experiments, overexpression of SOX2 stimulates cellular migration and anchorage-independent growth while SOX2 knockdown impairs cell growth. Finally, SOX2 over-expression in non-tumorigenic human lung bronchial epithelial cells is tumorigenic in immunocompromised mice. These results indicate that the SOX2 transcription factor, a major regulator of stem cell function, is also an oncogene and a driver gene for the recurrent 3q26.33 amplifications in lung SCC.

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism.

Background Bardet-Biedl syndrome (BBS) is a pleiotropic recessive disorder that belongs to the rapidly growing family of ciliopathies. It shares phenotypic traits with other ciliopathies, such as Alström syndrome (ALMS), nephronophthisis (NPHP) or Joubert syndrome. BBS mutations have been detected in 16 different genes (BBS1-BBS16) without clear genotype-to-phenotype correlation. This extensive genetic heterogeneity is a major concern for molecular diagnosis and genetic counselling. While various strategies have been recently proposed to optimise mutation detection, they either fail to detect mutations in a majority of patients or are time consuming and costly. Method We tested a targeted exon-capture strategy coupled with multiplexing and high-throughput sequencing on 52 patients: 14 with known mutations as proof-of-principle and 38 with no previously detected mutation. Thirty genes were targeted in total including the 16 BBS genes, the 12 known NPHP genes, the single ALMS gene ALMS1 and the proposed modifier CCDC28B. Results This strategy allowed the reliable detection of causative mutations (including homozygous/heterozygous exon deletions) in 68% of BBS patients without previous molecular diagnosis and in all proof-of-principle samples. Three probands carried homozygous truncating mutations in ALMS1 confirming the major phenotypic overlap between both disorders. The efficiency of detecting mutations in patients was positively correlated with their compliance with the classical BBS phenotype (mutations were identified in 81% of ‘classical’ BBS patients) suggesting that only a few true BBS genes remain to be identified. We illustrate some interpretation problems encountered due to the multiplicity of identified variants. Conclusion This strategy is highly efficient and cost effective for diseases with high genetic heterogeneity, and guarantees a quality of coverage in coding sequences of target genes suited for diagnosis purposes.

Fragile-X syndrome (FXS) is a frequent genetic form of intellectual disability (ID). The main recurrent mutagenic mechanism causing FXS is the expansion of a CGG repeat sequence in the 5'-UTR of the FMR1 gene, therefore, routinely tested in ID patients. We report here three FMR1 intragenic pathogenic variants not affecting this sequence, identified using high-throughput sequencing (HTS): a previously reported hemizygous deletion encompassing the last exon of FMR1, too small to be detected by array-CGH and inducing decreased expression of a truncated form of FMRP protein, in three brothers with ID (family 1) and two splice variants in boys with sporadic ID: a de novo variant c.990+1G>A (family 2) and a maternally inherited c.420-8A>G variant (family 3). After clinical reevaluation, the five patients presented features consistent with FXS (mean Hagerman's scores=15). We conducted a systematic review of all rare non-synonymous variants previously reported in FMR1 in ID patients and showed that six of them are convincing pathogenic variants. This study suggests that intragenic FMR1 variants, although much less frequent than CGG expansions, are a significant mutational mechanism leading to FXS and demonstrates the interest of HTS approaches to detect them in ID patients with a negative standard work-up.

Inherited neuromuscular disorders (NMD) are chronic genetic diseases posing a significant burden on patients and the health care system. Despite tremendous research and clinical efforts, the molecular causes remain unknown for nearly half of the patients, due to genetic heterogeneity and conventional molecular diagnosis based on a gene-by-gene approach. We aimed to test next generation sequencing (NGS) as an efficient and cost-effective strategy to accelerate patient diagnosis. We designed a capture library to target the coding and splice site sequences of all known NMD genes and used NGS and DNA multiplexing to retrieve the pathogenic mutations in patients with heterogeneous NMD with or without known mutations. We retrieved all known mutations, including point mutations and small indels, intronic and exonic mutations, and a large deletion in a patient with Duchenne muscular dystrophy, validating the sensitivity and reproducibility of this strategy on a heterogeneous subset of NMD with different genetic inheritance. Most pathogenic mutations were ranked on top in our blind bioinformatic pipeline. Following the same strategy, we characterized probable TTN , RYR1 and COL6A3 mutations in several patients without previous molecular diagnosis. The cost was less than conventional testing for a single large gene. With appropriate adaptations, this strategy could be implemented into a routine genetic diagnosis set-up as a first screening approach to detect most kind of mutations, potentially before the need of more invasive and specific clinical investigations. An earlier genetic diagnosis should provide improved disease management and higher quality genetic counseling, and ease access to therapy or inclusion into therapeutic trials.

Background CREB and CREM are closely related factors that regulate transcription in response to various stress, metabolic and developmental signals. The CREMτ activator isoform is selectively expressed in haploid spermatids and plays an essential role in murine spermiogenesis. Results We have used chromatin immunoprecipitation coupled to sequencing (ChIP-seq) to map CREM and CREB target loci in round spermatids from adult mouse testis and spermatogonia derived GC1-spg cells respectively. We identify more than 9000 genomic loci most of which are cell-specifically occupied. Despite the fact that round spermatids correspond to a highly specialised differentiated state, our results show that they have a remarkably accessible chromatin environment as CREM occupies more than 6700 target loci corresponding not only to the promoters of genes selectively expressed in spermiogenesis, but also of genes involved in functions specific to other cell types. The expression of only a small subset of these target genes are affected in the round spermatids of CREM knockout animals. We also identify a set of intergenic binding loci some of which are associated with H3K4 trimethylation and elongating RNA polymerase II suggesting the existence of novel CREB and CREM regulated transcripts. Conclusions We demonstrate that CREM and CREB occupy a large number of promoters in highly cell specific manner. This is the first study of CREM target promoters directly in a physiologically relevant tissue in vivo and represents the most comprehensive experimental analysis of CREB/CREM regulatory potential to date.

Laminins are major constituents of basement membranes and are essential for tissue homeostasis. Laminin-511 is highly expressed in the intestine and its absence causes severe malformation of the intestine and embryonic lethality. To understand the mechanistic role of laminin-511 in tissue homeostasis, we used RNA profiling of embryonic intestinal tissue of lama5 knockout mice and identified a lama5 specific gene expression signature. By combining cell culture experiments with mediated knockdown approaches, we provide a mechanistic link between laminin α5 gene deficiency and the physiological phenotype. We show that laminin α5 plays a crucial role in both epithelial and mesenchymal cell behavior by inhibiting Wnt and activating PI3K signaling. We conclude that conflicting signals are elicited in the absence of lama5, which alter cell adhesion, migration as well as epithelial and muscle differentiation. Conversely, adhesion to laminin-511 may serve as a potent regulator of known interconnected PI3K/Akt and Wnt signaling pathways. Thus deregulated adhesion to laminin-511 may be instrumental in diseases such as human pathologies of the gut where laminin-511 is abnormally expressed as it is shown here.