Chromodomain Helicase DNA-binding Protein Research Articles

The chromatin-remodeling factor CHD4 coordinates signaling and repair after DNA damage

In response to ionizing radiation (IR), cells delay cell cycle progression and activate DNA repair. Both processes are vital for genome integrity, but the mechanisms involved in their coordination are not fully understood. In a mass spectrometry screen, we identified the adenosine triphosphate-dependent chromatin-remodeling protein CHD4 (chromodomain helicase DNA-binding protein 4) as a factor that becomes transiently immobilized on chromatin after IR. Knockdown of CHD4 triggers enhanced Cdc25A degradation and p21(Cip1) accumulation, which lead to more pronounced cyclin-dependent kinase inhibition and extended cell cycle delay. At DNA double-strand breaks, depletion of CHD4 disrupts the chromatin response at the level of the RNF168 ubiquitin ligase, which in turn impairs local ubiquitylation and BRCA1 assembly. These cell cycle and chromatin defects are accompanied by elevated spontaneous and IR-induced DNA breakage, reduced efficiency of DNA repair, and decreased clonogenic survival. Thus, CHD4 emerges as a novel genome caretaker and a factor that facilitates both checkpoint signaling and repair events after DNA damage.

Regulation of serum response factor activity and smooth muscle cell apoptosis by chromodomain helicase DNA-binding protein 8

Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation, migration, and apoptosis. It is generally accepted that one mechanism by which SRF regulates these diverse functions is through pathway-specific cofactor interactions. A novel SRF cofactor, chromodomain helicase DNA binding protein 8 (CHD8), was isolated from a yeast two-hybrid screen using SRF as bait. CHD8 is highly expressed in adult smooth muscle tissues. Coimmunoprecipitation assays from A10 smooth muscle cells demonstrated binding of endogenous SRF and CHD8. Data from GST-pulldown assays indicate that the NH(2)-terminus of CHD8 can interact directly with the MADS domain of SRF. Adenoviral-mediated knockdown of CHD8 in smooth muscle cells resulted in attenuated expression of SRF-dependent, smooth muscle-specific genes. Knockdown of CHD8, SRF, or CTCF, a previously described binding partner of CHD8, in A10 VSMCs also resulted in a marked induction of apoptosis. Mechanistically, apoptosis induced by CHD8 knockdown was accompanied by attenuated expression of the anti-apoptotic proteins, Birc5, and CARD10, whereas SRF knockdown attenuated expression of CARD10 and Mcl-1, but not Birc5, and CTCF knockdown attenuated expression of Birc5. These data suggest that CHD8 plays a dual role in smooth muscle cells modulating SRF activity toward differentiation genes and promoting cell survival through interactions with both SRF and CTCF to regulate expression of Birc5 and CARD10.

Metastasis Tumor Antigen 2 (MTA2) Is Involved in Proper Imprinted Expression of H19 and Peg3 During Mouse Preimplantation Development1

The epigenetic mechanisms involved in establishing and maintaining genomic imprinting are steadily being unmasked. The nucleosome remodeling and histone deacetylation (NuRD) complex is implicated in regulating DNA methylation and expression of the maternally expressed H19 gene in preimplantation mouse embryos. To dissect further the function of the NuRD complex in genomic imprinting, we employed an RNA interference (RNAi) strategy to deplete the NuRD complex component Metastasis Tumor Antigen 2 (MTA2). We found that Mta2 is the only zygotically expressed Mta gene prior to the blastocyst stage, and that RNAi-mediated knockdown of Mta2 transcript leads to biallelic H19 expression and loss of DNA methylation in the differentially methylated region in blastocysts. In addition, biallelic expression of the paternally expressed Peg3 gene, but not Snrpn, is also observed in blastocysts following Mta2 knockdown. Loss of MTA2 protein does not result in a decrease in abundance of other NuRD components, including methyl-binding-CpG-binding domain protein 3 (MBD3), histone deacetylases 1 and 2 (HDACs 1 and 2), and chromodomain helicase DNA-binding protein 4 (CHD4). Taken together, our results support a role for MTA2 within the NuRD complex in genomic imprinting.

Regulation of DNA-damage responses and cell-cycle progression by the chromatin remodelling factor CHD4.

The chromatin remodelling factor chromodomain helicase DNA-binding protein 4 (CHD4) is a catalytic subunit of the NuRD transcriptional repressor complex. Here, we reveal novel functions for CHD4 in the DNA-damage response (DDR) and cell-cycle control. We show that CHD4 mediates rapid poly(ADP-ribose)-dependent recruitment of the NuRD complex to DNA-damage sites, and we identify CHD4 as a phosphorylation target for the apical DDR kinase ataxia-telangiectasia mutated. Functionally, we show that CHD4 promotes repair of DNA double-strand breaks and cell survival after DNA damage. In addition, we show that CHD4 acts as an important regulator of the G1/S cell-cycle transition by controlling p53 deacetylation. These results provide new insights into how the chromatin remodelling complex NuRD contributes to maintaining genome stability.

Chromatin Remodeling in Development and Disease: Focus on CHD7

A central goal of developmental biology is to understand the mechanisms whereby undifferentiated, pluripotent cells differentiate into mature, organized cells and tissues. Recent advances in mouse genetics, embryonic stem cell manipulation, RNA inhibition, and genomics have now made it possible to explore these mechanisms with an unprecedented level of resolution. One major class of proteins, the chromodomain helicase DNA-binding (CHD) family of ATP-dependent chromatin remodelers, has emerged as important regulators of cellular differentiation. CHD proteins are thought to function in the nucleus via binding to DNA and regulating gene transcription. CHD7, a member of the CHD family, encodes a protein mutated in human CHARGE syndrome, a multiple anomaly disorder that affects hearing, vision, and cardiac, craniofacial, and nervous system development [1], [2]. Previous reports have shown tissue and developmental stage specific expression of CHD7 [3], [4], and there is evidence that CHD7 binds thousands of sites in the genome [5]. It is thus a major challenge to identify specific mechanisms of CHD7 action in pluripotency versus differentiation.

Novel CHD7 mutations contributing to the mutation spectrum in patients with CHARGE syndrome

CHARGE syndrome is an autosomal dominant inherited multiple malformation disorder typically characterized by coloboma, choanal atresia, hypoplastic semicircular canal, cranial nerve defects, cardiovascular malformations and ear abnormalities. Mutations in the chromodomain helicase DNA-binding protein 7 ( CHD7) gene are the major cause of CHARGE syndrome. Mutation analysis was performed in 18 patients with firm or tentative clinical diagnosis of CHARGE syndrome. In this study eight mutations distributed across the gene were found. Five novel mutations – one missense (c.2936T > C), one nonsense (c.8093C > A) and three frameshift mutations (c.804_805insAT, c.1757_1770del14, c.1793delA) – were identified. As far as familial data were available these mutations were found to have arisen de novo. Comparison of the clinical features of patients with the same mutation demonstrates that expression of the phenotype is highly variable. The mutation detection rate in this study was 44.4% in patients with a clinically established or suspected diagnosis of CHARGE syndrome.

CHD7 functions in the nucleolus as a positive regulator of ribosomal RNA biogenesis.

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.

Clinical genetics of Kallmann syndrome

The Kallmann syndrome (KS) combines hypogonadotropic hypogonadism (HH) with anosmia. This is a clinically and genetically heterogeneous disease. KAL1, encoding the extracellular glycoprotein anosmin-1, is responsible for the X chromosome-linked recessive form of the disease (KAL1). Mutations in FGFR1 or FGF8, encoding fibroblast growth factor receptor-1 and fibroblast growth factor-8, respectively, underlie an autosomal dominant form with incomplete penetrance (KAL2). Mutations in PROKR2 and PROK2, encoding prokineticin receptor-2 and prokineticin-2, have been found in heterozygous, homozygous, and compound heterozygous states. These two genes are likely to be involved both in autosomal recessive monogenic (KAL3) and digenic/oligogenic KS transmission modes. Mutations in any of the above-mentioned KS genes have been found in less than 30% of the KS patients, which indicates that other genes involved in the disease remain to be discovered. Notably, KS may also be part of pleiotropic developmental diseases including CHARGE syndrome; this disease results in most cases from neomutations in CHD7 that encodes a chromodomain helicase DNA-binding protein.

Regulation of Androgen-Responsive Transcription by the Chromatin Remodeling Factor CHD8

The androgen receptor (AR) mediates the effect of androgens through its transcriptional function during both normal prostate development and in the emergence and progression of prostate cancer. AR is known to assemble coactivator complexes at target promoters to facilitate transcriptional activation in response to androgens. Here we identify the ATP-dependent chromatin remodeling factor chromodomain helicase DNA-binding protein 8 (CHD8) as a novel coregulator of androgen-responsive transcription. We demonstrate that CHD8 directly associates with AR and that CHD8 and AR simultaneously localize to the TMPRSS2 enhancer after androgen treatment. In the LNCaP cell line, reduction of CHD8 levels by small interfering RNA treatment severely diminishes androgen-dependent activation of the TMPRSS2 gene. We demonstrate that the recruitment of AR to the TMPRSS2 promoter in response to androgen treatment requires CHD8. Finally, CHD8 facilitates androgen-stimulated proliferation of LNCaP cells, emphasizing the physiological importance of CHD8. Taken together, we present evidence of a functional role for CHD8 in AR-mediated transcriptional regulation of target genes.

Molecular and phenotypic aspects of CHD7 mutation in 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.

CHARGE syndrome

The characteristic phenotype of CHARGE syndrome includes: coloboma, congenital heart defect, choanal atresia, retarded growth and development, genital abnormalities, ear anomalies with or without hearing loss, which give the name (an acronym) to this condition. The molecular cause in 60% of the cases are mutations in the chromodomain helicase DNAbinding protein gene (CHD7), with an estimated frequency of 1 in 10,000 live born infants. We describe 3, not related patients with a clinical diagnosis of CHARGE syndrome and each of them with a different mutation in the CHD7 gene sequence.

Successful cord blood transplantation for a CHARGE syndrome with CHD7 mutation showing DiGeorge sequence including hypoparathyroidism

It is rare that coloboma, heart anomalies, choanal atresia, retarded growth and development, and genital and ear anomalies (CHARGE) syndrome patients have DiGeorge sequence showing severe immunodeficiency due to the defect of the thymus. Although the only treatment to achieve immunological recovery for these patients in countries where thymic transplantation is not ethically approved would be hematopoietic cell transplantation, long-term survival has not been obtained in most patients. On the other hand, it is still not clarified whether hypoparathyroidism is one of the manifestations of CHARGE syndrome. We observed a CHARGE syndrome patient with chromodomain helicase DNA-binding protein 7 mutation showing DiGeorge sequence including the defect of T cells accompanied with the aplasia of the thymus, severe hypoparathyroidism, and conotruncal cardiac anomaly. He received unrelated cord blood transplantation without conditioning at 4 months of age. Recovery of T cell number and of proliferative response against mitogens was achieved by peripheral expansion of mature T cells in cord blood without thymic output. Although he is still suffering from severe hypoparathyroidism, he is alive without serious infections for 10 months.

Binding of the CHD4 PHD2 Finger to Histone H3 is Modulated by Covalent Modifications

CHD4 (chromodomain helicase DNA-binding protein 4) ATPase is a major subunit of the repressive NuRD (nucleosome remodeling and deacetylase) complex, which is involved in transcriptional regulation and development. CHD4 contains two plant homeodomain (PHD) fingers of unknown function. Here we show that the second PHD finger (PHD2) of CHD4 recognizes the amino-terminus of histone H3 and that this interaction is facilitated by acetylation or methylation of Lys9 (H3K9ac and H3K9me, respectively) but is inhibited by methylation of Lys4 (H3K4me) or acetylation of Ala1 (H3A1ac). An 18 μM binding affinity toward unmodified H3 rises to 0.6 μM for H3K9ac and to 0.9 μM for H3K9me3, while dropping to 2.0 mM for H3K4me3, as measured by tryptophan fluorescence and NMR. A peptide library screen further shows that phosphorylation of Thr3, Thr6 or Ser10 abolishes this interaction. A model of the PHD2-H3 complex, generated using a combination of NMR, data-driven docking and mutagenesis data, reveals an elongated site on the PHD2 surface where the H3 peptide is bound. Together our findings suggest that the PHD2 finger plays a role in targeting of the CHD4/NuRD complex to chromatin.

CHD5 is down-regulated through promoter hypermethylation in gastric cancer

BackgroundNonhistone chromosomal proteins in concert with histones play important roles in the replication and repair of DNA and in the regulation of gene expression. The deregulation of these proteins can contribute to the development of a variety of diseases such as cancer. As a nonhistone chromosomal protein, chromodomain helicase DNA binding protein 5 (CHD5) has recently been identified as the product of a novel tumor suppressor gene (TSG), promoting the transcription of p19ink4a and p16arf. The inactivation of CHD5 was achieved partly through genetic deletion since it is located in 1p36, a region frequently deleted in human tumors. In this study, we aim to study the involvement of CHD5 in gastric cancer, the second most common cancer worldwide.MethodsCHD5 expression in a panel of gastric cancer cells were determined by quantitative RT-PCR. The methylation of CHD5 was evaluated by methylation specific PCR and bisulfite genome sequencing. The effect of CHD5 on growth of gastric cancer cells was tested by colony formation assay.ResultsCHD5 expression was down-regulated in all of gastric cancer cell lines used (100%, 7/7) and significantly restored after pharmacological demethylation. Methylation of CHD5 promoter was detected in all of seven gastric cancer cell lines and in the majority of primary gastric carcinoma tissues examined (73%, 11/15). Finally, ectopic expression of CHD5 in gastric cancer cells led to a significant growth inhibition.ConclusionCHD5 was a TSG epigenetically down-regulated in gastric cancer.

Study of smell and reproductive organs in a mouse model for CHARGE syndrome

CHARGE syndrome is a multiple congenital anomaly syndrome characterised by Coloboma, Heart defects, Atresia of choanae, Retardation of growth and/or development, Genital hypoplasia, and Ear anomalies often associated with deafness. It is caused by heterozygous mutations in the CHD7 gene and shows a highly variable phenotype. Anosmia and hypogonadotropic hypogonadism occur in the majority of the CHARGE patients, but the underlying pathogenesis is unknown. Therefore, we studied the ability to smell and aspects of the reproductive system (reproductive performance, gonadotropin-releasing hormone (GnRH) neurons and anatomy of testes and uteri) in a mouse model for CHARGE syndrome, the whirligig mouse (Chd7Whi/+). We showed that Chromodomain Helicase DNA-binding protein 7 (Chd7) is expressed in brain areas involved in olfaction and reproduction during embryonic development. We observed poorer performance in the smell test in adult Chd7Whi/+ mice, secondary either to olfactory dysfunction or to balance disturbances. Olfactory bulb and reproductive organ abnormalities were observed in a proportion of Chd7Whi/+ mice. Hypothalamic GnRH neurons were slightly reduced in Chd7Whi/+ females and reproductive performance was slightly less in Chd7Whi/+ mice. This study shows that the penetrance of anosmia and hypogonadotropic hypogonadism is lower in Chd7Whi/+ mice than in CHARGE patients. Interestingly, many phenotypic features of the Chd7 mutation showed incomplete penetrance in our model mice, despite the use of inbred, genetically identical mice. This supports the theory that the extreme variability of the CHARGE phenotype in both humans and mice might be attributed to variations in the fetal microenvironment or to purely stochastic events.

Binding of the CHD4 PHD2 finger to histone H3 is modulated by covalent modifications

CHD4 (chromodomain helicase DNA-binding protein 4) ATPase is a major subunit of the repressive NuRD (nucleosome remodelling and deacetylase) complex, which is involved in transcriptional regulation and development. CHD4 contains two PHD (plant homeodomain) fingers of unknown function. Here we show that the second PHD finger (PHD2) of CHD4 recognizes the N-terminus of histone H3 and that this interaction is facilitated by acetylation or methylation of Lys9 (H3K9ac and H3K9me respectively) but is inhibited by methylation of Lys4 (H3K4me) or acetylation of Ala1 (H3A1ac). An 18 microM binding affinity toward unmodified H3 rises to 0.6 microM for H3K9ac and to 0.9 microM for H3K9me3, whereas it drops to 2.0 mM for H3K4me3, as measured by tryptophan fluorescence and NMR. A peptide library screen further shows that phosphorylation of Thr3, Thr6 or Ser10 abolishes this interaction. A model of the PHD2-H3 complex, generated using a combination of NMR, data-driven docking and mutagenesis data, reveals an elongated site on the PHD2 surface where the H3 peptide is bound. Together our findings suggest that the PHD2 finger plays a role in targeting of the CHD4/NuRD complex to chromatin.

Cardiac phenotyping in ex vivo murine embryos using µMRI

Microscopic MRI (microMRI) is an emerging technique for high-throughput phenotyping of transgenic mouse embryos, and is capable of visualising abnormalities in cardiac development. To identify cardiac defects in embryos, we have optimised embryo preparation and MR acquisition parameters to maximise image quality and assess the phenotypic changes in chromodomain helicase DNA-binding protein 7 (Chd7) transgenic mice. microMRI methods rely on tissue penetration with a gadolinium chelate contrast agent to reduce tissue T(1), thus improving signal-to-noise ratio (SNR) in rapid gradient echo sequences. We investigated 15.5 days post coitum (dpc) wild-type CD-1 embryos fixed in gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) solutions for either 3 days (2 and 4 mM) or 2 weeks (2, 4, 8 and 16 mM). To assess penetration of the contrast agent into heart tissue and enable image contrast simulations, T(1) and T(*) (2) were measured in heart and background agarose. Compared to 3-day, 2-week fixation showed reduced mean T(1) in the heart at both 2 and 4 mM concentrations (p < 0.0001), resulting in calculated signal gains of 23% (2 mM) and 29% (4 mM). Using T(1) and T(*) (2) values from 2-week concentrations, computer simulation of heart and background signal, and ex vivo 3D gradient echo imaging, we demonstrated that 2-week fixed embryos in 8 mM Gd-DTPA in combination with optimised parameters (TE/TR/alpha/number of averages: 9 ms/20 ms/60 degrees /7) produced the largest SNR in the heart (23.2 +/- 1.0) and heart chamber contrast-to-noise ratio (CNR) (27.1 +/- 1.6). These optimised parameters were then applied to an MRI screen of embryos heterozygous for the gene Chd7, implicated in coloboma of the eye, heart defects, atresia of the choanae, retardation of growth, genital/urinary abnormalities, ear abnormalities and deafness (CHARGE) syndrome (a condition partly characterised by cardiovascular birth defects in humans). A ventricular septal defect was readily identified in the screen, consistent with the human phenotype.

Role of chromodomain helicase DNA-binding protein 2 in DNA damage response signaling and tumorigenesis

The chromodomain helicase DNA binding proteins (CHDs) are known to affect transcription via their ability to remodel chromatin and modulate histone deacetylation. In an effort to understand the functional role of the chromodomain helicase DNA binding protein 2 (CHD2) in mammals, we have generated Chd2 mutant mouse model. Remarkably, the Chd2 protein appears to play a critical role in development, hematopoiesis, and tumor suppression. The Chd2 heterozygous mutant mice exhibit increased extra-medullary hematopoiesis and susceptibility to lymphomas. At the cellular level, Chd2 mutants are defective in hematopoietic stem cell differentiation, accumulate higher levels of the chromatin associated DNA damage response mediator, γH2AX, and exhibit an aberrant DNA damage response after X-ray irradiation. Our data suggest a direct role for the chromatin remodeling protein in DNA damage signaling and genome stability maintenance.

Chromatin remodeling factor CHD5 is silenced by promoter CpG island hypermethylation in human cancer

Changes in chromatin structure are an essential mechanism for gene regulation (transcription, replication, DNA repair and recombination). ATP-dependent chromatin-remodeling enzymes have previously been implicated in maintaining and regulating chromatin structure. These enzymes are divided into several groups including the SWI/SNF, ISWI and CHD families. Although the Chromodomain Helicase DNA-binding protein (CHD) family members are involved in key cellular processes, the disrupted regulation of their expression has not been fully assessed. An impairment of chromatin remodeling activity, mediated by promoter CpG island hypermethylation of the described candidate genes, could be key in cancer pathology. Herein, we examine the DNA methylation profiles of CHD family members (CHD1-9) in different tumor types. For all CHDs, CpG island hypermethylation was only observed at the CHD5 promoter in human cancer cell lines and primary tumors, particularly gliomas and colon and breast carcinomas. RT-qPCR analyses correlated CHD5 loss of expression with hypermethylation of the promoter, and restoration of CHD5 mRNA levels upon treatment with a DNA demethylating agent. These results underpin the epigenetic inactivation of the chromating remodeling factor CHD5 as one contributor for the aberrant structural changes of chromatin throughout the genome of the cancer cell.

Disruption of the TCF4 gene in a girl with mental retardation but without the classical Pitt–Hopkins syndrome

We have characterized a de novo balanced translocation t(18;20)(q21.1;q11.2) in a female patient with mild to moderate mental retardation (MR) and minor facial anomalies. Breakpoint-mapping by fluorescence in situ hybridization indicated that on chromosome 18, the basic helix-loop-helix transcription factor TCF4 gene is disrupted by the breakpoint. TCF4 plays a role in cell fate determination and differentiation. Only recently, mutations in this gene have been shown to result in Pitt-Hopkins syndrome (PHS), defined by severe MR, epilepsy, mild growth retardation, microcephaly, daily bouts of hyperventilation starting in infancy, and distinctive facial features with deep-set eyes, broad nasal bridge, and wide mouth with widely spaced teeth. Breakpoint mapping on the derivative chromosome 20 indicated that here the rearrangement disrupted the chromodomain helicase DNA binding protein 6 (CHD6) gene. To date, there is no indication that CHD6 is involved in disease. Our study indicates that TCF4 gene mutations are not always associated with classical PHS but can give rise to a much milder clinical phenotype. Thus, the possibility exists that more patients with a less severe encephalopathy carry a mutation in this gene.