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CHD7, an ATP-dependent chromatin remodeler, is disrupted in CHARGE syndrome, an autosomal dominant disorder characterized by variably penetrant abnormalities in craniofacial, cardiac, and nervous system tissues. The inner ear is uniquely sensitive to CHD7 levels and is the most commonly affected organ in individuals with CHARGE. Interestingly, upregulation or downregulation of retinoic acid (RA) signaling during embryogenesis also leads to developmental defects similar to those in CHARGE syndrome, suggesting that CHD7 and RA may have common target genes or signaling pathways. Here, we tested three separate potential mechanisms for CHD7 and RA interaction: (a) direct binding of CHD7 with RA receptors, (b) regulation of CHD7 levels by RA, and (c) CHD7 binding and regulation of RA-related genes. We show that CHD7 directly regulates expression of Aldh1a3, the gene encoding the RA synthetic enzyme ALDH1A3 and that loss of Aldh1a3 partially rescues Chd7 mutant mouse inner ear defects. Together, these studies indicate that ALDH1A3 acts with CHD7 in a common genetic pathway to regulate inner ear development, providing insights into how CHD7 and RA regulate gene expression and morphogenesis in the developing embryo.

CHARGE syndrome is a sporadic autosomal-dominant genetic disorder characterized by a complex array of birth defects so named for its cardinal features of ocular coloboma, heart defects, choanal atresia, growth retardation, genital abnormalities, and ear abnormalities. Approximately two-thirds of individuals clinically diagnosed with CHARGE syndrome have heterozygous loss-of-function mutations in the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7), an ATP-dependent chromatin remodeler. To examine the role of Chd7 in development, a zebrafish model was generated through morpholino (MO)-mediated targeting of the zebrafish chd7 transcript. High doses of chd7 MO induce lethality early in embryonic development. However, low dose-injected embryos are viable, and by 4 days post-fertilization, morphant fish display multiple defects in organ systems analogous to those affected in humans with CHARGE syndrome. The chd7 morphants show elevated expression of several potent cell-cycle inhibitors including ink4ab (p16/p15), p21 and p27, accompanied by reduced cell proliferation. We also show that Chd7 is required for proper organization of neural crest-derived craniofacial cartilage structures. Strikingly, MO-mediated knockdown of the jumonji domain-containing histone demethylase fbxl10/kdm2bb, a repressor of ribosomal RNA (rRNA) genes, rescues cell proliferation and cartilage defects in chd7 morphant embryos and can lead to complete rescue of the CHARGE syndrome phenotype. These results indicate that CHARGE-like phenotypes in zebrafish can be mitigated through modulation of fbxl10 levels and implicate FBXL10 as a possible therapeutic target in CHARGE syndrome., (© 2013 Elsevier Inc. All rights reserved.)

CHARGE syndrome is an autosomal dominant multisystem disorder caused by mutation in the CHD7 gene, encoding chromodomain helicase DNA-binding protein 7. Molecular diagnostic testing for CHD7 mutation has been available in a clinical setting since 2005. We report here the results from the first 642 unrelated proband samples submitted for testing. Thirty-two percent (n = 203) of patient samples had a heterozygous pathogenic variant identified. The lower mutation rate than that published for well-characterized clinical samples is likely due to referral bias, as samples submitted for clinical testing may be for "rule-out" diagnoses, rather than solely to confirm clinical suspicion. We identified 159 unique pathogenic mutations, and of these, 134 mutations were each seen in a single individual and 25 mutations were found in two to five individuals (n =69). Of the 203 mutations, only 9 were missense, with 107 nonsense, 69 frameshift, and 15 splice-site mutations likely leading to haploinsufficiency at the cellular level. An additional 72 variations identified in the 642 tested samples (11%) were considered to have unknown clinical significance. Copy number changes (deletion/duplication of the entire gene or one/several exons) were found to account for a very small number of cases (n = 3). This cohort represents the largest CHARGE syndrome sample size to date and is intended to serve as a resource for clinicians, genetic counselors, researchers, and other diagnostic laboratories.

De novo mutation of the gene encoding chromodomain helicase DNA-binding protein 7 (CHD7) is the primary cause of CHARGE syndrome, a complex developmental disorder characterized by the co-occurrence of a specific set of birth defects. Recent studies indicate that CHD7 functions as a transcriptional regulator in the nucleoplasm. Here, we report based on immunofluorescence and western blotting of subcellular fractions that CHD7 is also constitutively localized to the nucleolus, the site of rRNA transcription. Standard chromatin immunoprecipitation (ChIP) assays indicate that CHD7 physically associates with rDNA, a result that is also observable upon alignment of whole-genome CHD7 ChIP coupled with massively parallel DNA sequencing data to the rDNA reference sequence. ChIP-chop analyses demonstrate that CHD7 specifically associates with hypomethylated, active rDNA, suggesting a role as a positive regulator of rRNA synthesis. Consistent with this hypothesis, siRNA-mediated depletion of CHD7 results in hypermethylation of the rDNA promoter and a concomitant reduction of 45S pre-rRNA levels. Accordingly, cells overexpressing CHD7 show increased levels of 45S pre-rRNA compared with control cells. Depletion of CHD7 also reduced cell proliferation and protein synthesis. Lastly, compared with wild-type ES cells, the levels of 45S pre-rRNA are reduced in both Chd7(+/-) and Chd7(-/-) mouse ES cells, as well as in Chd7(-/-) whole mouse embryos and multiple tissues dissected from Chd7(+/-) embryos. Together with previously published studies, these results indicate that CHD7 dually functions as a regulator of both nucleoplasmic and nucleolar genes and provide a novel avenue for investigation into the pathogenesis of CHARGE syndrome.

CHARGE syndrome [coloboma of the eye, heart defects, atresia of the choanae, retardation of growth and/or development, genital and/or urinary abnormalities, and ear abnormalities (including deafness)] is a genetic disorder characterized by a specific and a recognizable pattern of anomalies. De novo mutations in the gene encoding chromodomain helicase DNA binding protein 7 (CHD7) are the major cause of CHARGE syndrome. Here, we review the clinical features of 379 CHARGE patients who tested positive or negative for mutations in CHD7. We found that CHARGE individuals with CHD7 mutations more commonly have ocular colobomas, temporal bone anomalies (semicircular canal hypoplasia/dysplasia), and facial nerve paralysis compared with mutation negative individuals. We also highlight recent genetic and genomic studies that have provided functional insights into CHD7 and the pathogenesis of CHARGE syndrome., ((c) 2010 Wiley-Liss, Inc.)

CHD7 is a member of the chromodomain helicase DNA binding domain family of ATP-dependent chromatin remodeling enzymes. De novo mutation of the CHD7 gene is a major cause of CHARGE syndrome, a genetic disease characterized by a complex constellation of birth defects (Coloboma of the eye, Heart defects, Atresia of the choanae, severe Retardation of growth and development, Genital abnormalities, and Ear abnormalities). To gain insight into the function of CHD7, we mapped the distribution of the CHD7 protein on chromatin using the approach of chromatin immunoprecipitation on tiled microarrays (ChIP-chip). These studies were performed in human colorectal carcinoma cells, human neuroblastoma cells, and mouse embryonic stem (ES) cells before and after differentiation into neural precursor cells. The results indicate that CHD7 localizes to discrete locations along chromatin that are specific to each cell type, and that the cell-specific binding of CHD7 correlates with a subset of histone H3 methylated at lysine 4 (H3K4me). The CHD7 sites change concomitantly with H3K4me patterns during ES cell differentiation, suggesting that H3K4me is part of the epigenetic signature that defines lineage-specific association of CHD7 with specific sites on chromatin. Furthermore, the CHD7 sites are predominantly located distal to transcription start sites, most often contained within DNase hypersensitive sites, frequently conserved, and near genes expressed at relatively high levels. These features are similar to those of gene enhancer elements, raising the possibility that CHD7 functions in enhancer mediated transcription, and that the congenital anomalies in CHARGE syndrome are due to alterations in transcription of tissue-specific genes normally regulated by CHD7 during development.

CHD7 is one of nine members of the chromodomain helicase DNA-binding domain family of ATP-dependent chromatin remodeling enzymes found in mammalian cells. De novo mutation of CHD7 is a major cause of CHARGE syndrome, a genetic condition characterized by multiple congenital anomalies. To gain insights to the function of CHD7, we used the technique of chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-Seq) to map CHD7 sites in mouse ES cells. We identified 10,483 sites on chromatin bound by CHD7 at high confidence. Most of the CHD7 sites show features of gene enhancer elements. Specifically, CHD7 sites are predominantly located distal to transcription start sites, contain high levels of H3K4 mono-methylation, found within open chromatin that is hypersensitive to DNase I digestion, and correlate with ES cell-specific gene expression. Moreover, CHD7 co-localizes with P300, a known enhancer-binding protein and strong predictor of enhancer activity. Correlations with 18 other factors mapped by ChIP-seq in mouse ES cells indicate that CHD7 also co-localizes with ES cell master regulators OCT4, SOX2, and NANOG. Correlations between CHD7 sites and global gene expression profiles obtained from Chd7+/+, Chd7+/-, and Chd7-/- ES cells indicate that CHD7 functions at enhancers as a transcriptional rheostat to modulate, or fine-tune the expression levels of ES-specific genes. CHD7 can modulate genes in either the positive or negative direction, although negative regulation appears to be the more direct effect of CHD7 binding. These data indicate that enhancer-binding proteins can limit gene expression and are not necessarily co-activators. Although ES cells are not likely to be affected in CHARGE syndrome, we propose that enhancer-mediated gene dysregulation contributes to disease pathogenesis and that the critical CHD7 target genes may be subject to positive or negative regulation. [ABSTRACT FROM AUTHOR]