Noncoding deletions reveal a gene that is critical for intestinal function.

Large-scale genome sequencing is poised to provide a substantial increase in the rate of discovery of disease-associated mutations, but the functional interpretation of such mutations remains challenging. Here we show that deletions of a sequence on human chromosome 16 that we term the intestine-critical region (ICR) cause intractable congenital diarrhoea in infants1,2. Reporter assays in transgenic mice show that the ICR contains a regulatory sequence that activates transcription during the development of the gastrointestinal system. Targeted deletion of the ICR in mice caused symptoms that recapitulated the human condition. Transcriptome analysis revealed that an unannotated open reading frame (Percc1) flanks the regulatory sequence, and the expression of this gene was lost in the developing gut of mice that lacked the ICR. Percc1-knockout mice displayed phenotypes similar to those observed upon ICR deletion in mice and patients, whereas an ICR-driven Percc1 transgene was sufficient to rescue the phenotypes found in mice that lacked the ICR. Together, our results identify a gene that is critical for intestinal function and underscore the need for targeted in vivo studies to interpret the growing number of clinical genetic findings that do not affect known protein-coding genes.

HIC2 regulates isoform switching during maturation of the cardiovascular system.

Physiological changes during embryonic development are associated with changes in the isoform expression of both myocyte sarcomeric proteins and of erythrocyte haemoglobins. Cell type-specific isoform expression of these genes also occurs. Although these changes appear to be coordinated, it is unclear how changes in these disparate cell types may be linked. The transcription factor Hic2 is required for normal cardiac development and the mutant is embryonic lethal. Hic2 embryos exhibit precocious expression of the definitive-lineage haemoglobin Hbb-bt in circulating primitive erythrocytes and of foetal isoforms of cardiomyocyte genes (creatine kinase, Ckm, and eukaryotic elongation factor Eef1a2) as well as ectopic cardiac expression of fast-twitch skeletal muscle troponin isoforms. We propose that HIC2 regulates a switching event within both the contractile machinery of cardiomyocytes and the oxygen carrying systems during the developmental period where demands on cardiac loading change rapidly.

HIC2 is a novel dosage-dependent regulator of cardiac development located within the distal 22q11 deletion syndrome region.

RATIONALE: 22q11 deletion syndrome arises from recombination between low-copy repeats on chromosome 22. Typical deletions result in hemizygosity for TBX1 associated with congenital cardiovascular disease. Deletions distal to the typically deleted region result in a similar cardiac phenotype but lack in extracardiac features of the syndrome, suggesting that a second haploinsufficient gene maps to this interval. OBJECTIVE: The transcription factor HIC2 is lost in most distal deletions, as well as in a minority of typical deletions. We used mouse models to test the hypothesis that HIC2 hemizygosity causes congenital heart disease. METHODS AND RESULTS: We created a genetrap mouse allele of Hic2. The genetrap reporter was expressed in the heart throughout the key stages of cardiac morphogenesis. Homozygosity for the genetrap allele was embryonic lethal before embryonic day E10.5, whereas the heterozygous condition exhibited a partially penetrant late lethality. One third of heterozygous embryos had a cardiac phenotype. MRI demonstrated a ventricular septal defect with over-riding aorta. Conditional targeting indicated a requirement for Hic2 within the Nkx2.5+ and Mesp1+ cardiovascular progenitor lineages. Microarray analysis revealed increased expression of Bmp10. CONCLUSIONS: Our results demonstrate a novel role for Hic2 in cardiac development. Hic2 is the first gene within the distal 22q11 interval to have a demonstrated haploinsufficient cardiac phenotype in mice. Together our data suggest that HIC2 haploinsufficiency likely contributes to the cardiac defects seen in distal 22q11 deletion syndrome.

Chromatin stretch enhancer states drive cell-specific gene regulation and harbor human disease risk variants.

Chromatin-based functional genomic analyses and genomewide association studies (GWASs) together implicate enhancers as critical elements influencing gene expression and risk for common diseases. Here, we performed systematic chromatin and transcriptome profiling in human pancreatic islets. Integrated analysis of islet data with those from nine cell types identified specific and significant enrichment of type 2 diabetes and related quantitative trait GWAS variants in islet enhancers. Our integrated chromatin maps reveal that most enhancers are short (median = 0.8 kb). Each cell type also contains a substantial number of more extended (≥ 3 kb) enhancers. Interestingly, these stretch enhancers are often tissue-specific and overlap locus control regions, suggesting that they are important chromatin regulatory beacons. Indeed, we show that (i) tissue specificity of enhancers and nearby gene expression increase with enhancer length; (ii) neighborhoods containing stretch enhancers are enriched for important cell type-specific genes; and (iii) GWAS variants associated with traits relevant to a particular cell type are more enriched in stretch enhancers compared with short enhancers. Reporter constructs containing stretch enhancer sequences exhibited tissue-specific activity in cell culture experiments and in transgenic mice. These results suggest that stretch enhancers are critical chromatin elements for coordinating cell type-specific regulatory programs and that sequence variation in stretch enhancers affects risk of major common human diseases.

Tbx1 controls cardiac neural crest cell migration during arch artery development by regulating Gbx2 expression in the pharyngeal ectoderm.

Elucidating the gene regulatory networks that govern pharyngeal arch artery (PAA) development is an important goal, as such knowledge can help to identify new genes involved in cardiovascular disease. The transcription factor Tbx1 plays a vital role in PAA development and is a major contributor to cardiovascular disease associated with DiGeorge syndrome. In this report, we used various genetic approaches to reveal part of a signalling network by which Tbx1 controls PAA development in mice. We investigated the crucial role played by the homeobox-containing transcription factor Gbx2 downstream of Tbx1. We found that PAA formation requires the pharyngeal surface ectoderm as a key signalling centre from which Gbx2, in response to Tbx1, triggers essential directional cues to the adjacent cardiac neural crest cells (cNCCs) en route to the caudal PAAs. Abrogation of this signal generates cNCC patterning defects leading to PAA abnormalities. Finally, we showed that the Slit/Robo signalling pathway is activated during cNCC migration and that components of this pathway are affected in Gbx2 and Tbx1 mutant embryos at the time of PAA development. We propose that the spatiotemporal control of this tightly orchestrated network of genes participates in crucial aspects of PAA development.

Hes1 expression is reduced in Tbx1 null cells and is required for the development of structures affected in 22q11 deletion syndrome.

22q11 deletion syndrome (22q11DS) is characterised by aberrant development of the pharyngeal apparatus and the heart with haploinsufficiency of the transcription factor TBX1 being considered the major underlying cause of the disease. Tbx1 mutations in mouse phenocopy the disorder. In order to identify the transcriptional dysregulation in Tbx1-expressing lineages we optimised fluorescent-activated cell sorting of beta-galactosidase expressing cells (FACS-Gal) to compare the expression profile of Df1/Tbx1(lacZ) (effectively Tbx1 null) and Tbx1 heterozygous cells isolated from mouse embryos. Hes1, a major effector of Notch signalling, was identified as downregulated in Tbx1(-)(/)(-) mutants. Hes1 mutant mice exhibited a partially penetrant range of 22q11DS-like defects including pharyngeal arch artery (PAA), outflow tract, craniofacial and thymic abnormalities. Similar to Tbx1 mice, conditional mutagenesis revealed that Hes1 expression in embryonic pharyngeal ectoderm contributes to thymus and pharyngeal arch artery development. These results suggest that Hes1 acts downstream of Tbx1 in the morphogenesis of pharyngeal-derived structures.

Murine embryonic expression of the gene for the UV-responsive protein p15(PAF).

In a screen for genes expressed in the embryonic mouse facial primordia, we identified the gene sequence annotated as KIAA0101, which has previously been shown to encode a novel proliferating cell nuclear antigen (PCNA)-interacting protein named p15(PAF). We have since demonstrated that this protein also interacts in a complex with the tumour suppressor product p33ING1b, and that overexpression results in a decrease in UV-induced cell death. Although available data suggest widespread or ubiquitous expression in the adult, here we report highly restricted expression of the p15(PAF) gene in a spatio-temporal manner during mouse embryogenesis. Major sites of expression include the facial prominences, limbs, somites, brain, spinal cord and hair follicles. Based on the nature of its interacting partners, p15(PAF) is proposed to play a role in tumorigenesis. Our data also suggest a role in embryonic development, consistent with findings that a wide range of tumours result from aberrant activity of key developmental pathways.

Evolutionary conservation and murine embryonic expression of the gene encoding the SERTA domain-containing protein CDCA4 (HEPP).

Cdca4 (Hepp) was originally identified as a gene expressed specifically in hematopoietic progenitor cells as opposed to hematopoietic stem cells. More recently, it has been shown to stimulate p53 activity and also lead to p53-independent growth inhibition when overexpressed. We independently isolated the murine Cdca4 gene in a genomic expression-based screen for genes involved in mammalian craniofacial development, and show that Cdca4 is expressed in a spatio-temporally restricted pattern during mouse embryogenesis. In addition to expression in the facial primordia including the pharyngeal arches, Cdca4 is expressed in the developing limb buds, brain, spinal cord, dorsal root ganglia, teeth, eye and hair follicles. Along with a small number of proteins from a range of species, the predicted CDCA4 protein contains a novel SERTA motif in addition to cyclin A-binding and PHD bromodomain-binding regions of homology. While the function of the SERTA domain is unknown, proteins containing this domain have previously been linked to cell cycle progression and chromatin remodelling. Using in silico database mining we have extended the number of evolutionarily conserved orthologues of known SERTA domain proteins and identified an uncharacterised member of the SERTA domain family, SERTAD4, with orthologues to date in human, mouse, rat, dog, cow, Tetraodon and chicken. Immunolocalisation of transiently and stably transfected epitope-tagged CDCA4 protein in mammalian cells suggests that it resides predominantly in the nucleus throughout all stages of the cell cycle.

Pax9 and Jagged1 act downstream of Gli3 in vertebrate limb development.

From early in limb development the transcription factor Gli3 acts to define boundaries of gene expression along the anterior-posterior (AP) axis, establishing asymmetric patterns required to provide positional information. As limb development proceeds, posterior mesenchyme expression of Sonic hedgehog (Shh) regulates Gli3 transcription and post-translational processing to specify digit number and identity. The molecular cascades dependent on Gli3 at later stages of limb development, which link early patterning events with final digit morphogenesis, remain poorly characterised. By analysing the transcriptional consequences of loss of Gli3 in the anterior margin of the E11.5 and E12.5 limb bud in the polydactylous mouse mutant extra-toes (Gli3(Xt/Xt)), we have identified a number of known and novel transcripts dependent on Gli3 in the limb. In particular, we demonstrated that the genes encoding the paired box transcription factor Pax9, the Notch ligand Jagged1 and the cell surface receptor Cdo are dependent on Gli3 for correct expression in the anterior limb mesenchyme. Analysis of expression in compound Shh;Gli3 mutant mouse embryos and in both in vitro and in vivo Shh signaling assays, further defined the importance of Shh regulated processing of Gli3 in controlling gene expression. In particular Pax9 regulation by Shh and Gli3 was shown to be context dependent, with major differences between the limb and somite revealed by Shh bead implantation experiments in the chick. Jagged1 was shown to be induced by Shh in the chick limb and in a C3H10T1/2 cell based signaling assay, with Shh;Gli3 mutant analysis indicating that expression is dependent on Gli3 derepression. Our data have also revealed that perturbation of early patterning events within the Gli3(Xt/Xt) limb culminates in a specific delay of anterior chondrogenesis which is subsequently realised as extra digits.