Chromodomain Helicase DNA-Binding Proteins and Spermatogenesis: Current Advances.

Epileptic encephalopathies (EE) are severe epilepsy syndromes characterized by multiple seizure types, developmental delay and even regression. This class of disorders are increasingly being identified as resulting from de novo genetic mutations including many identified mutations in the family of chromodomain helicase DNA binding (CHD) proteins. In particular, several de novo pathogenic mutations have been identified in the gene encoding chromodomain helicase DNA binding protein 2 (CHD2), a member of the sucrose nonfermenting (SNF-2) protein family of epigenetic regulators. These mutations in the CHD2 gene are causative of early onset epileptic encephalopathy, abnormal brain function, and intellectual disability. Our understanding of the mechanisms by which modification or loss of CHD2 cause this condition remains poorly understood. Here, we review what is known and still to be elucidated as regards the structure and function of CHD2 and how its dysregulation leads to a highly variable range of phenotypic presentations.

Background and aims: In the past decade, epigenetic mechanisms involved in the regulation of tumor-specific gene expression have been elucidated partially, prompting the development of therapeutic approaches in cancer cells. In this study, we used an unbiased screen to investigate therapeutic targets which might be effective combination with DNMT inhibitors in reactivating hypermethylated genes. Methods: We screened a whole genome siRNA library targeting 21360 genes using a colon cancer cell line that harbors a cytomegalovirus (CMV) promoter driven GFP stably integrated and silenced by hypermethylation. GFP-positive cell percentages were measured using Guava EasyCyte Plus flow analyzer software and normalized by Z-score. The primary siRNA library contained SMART pools (Dharmacon) of 4 siRNA duplexes per gene. To validate hits, transfections using individual siRNA were performed, followed by FACS analysis and qRT-PCR. DNA methylation status was evaluated by pyrosequencing analysis and Digital Restriction Enzyme Analysis of Methylation (DREAM), based on next generation sequencing analysis. Results: The screen was conducted in combination with low dose decitabine. Of the 21,630 genes tested, 37 (0.2%) were positive (Z-score > sample mean + 5SD) and proceeded to validation studies. We successfully confirmed top hits by flow cytometry and qRT-PCR. Among them, some genes are known epigenetic regulators such as chromodomain helicase DNA-binding protein 4 (CHD4), activating transcription factor 7 interacting protein (ATF7IP) and DNMT1 itself. Interestingly well-known epigenetic regulators such as other DNMTs, histone deacetylases and histone methylases were generally absent among the top hits. CHD4 depletion in combination with DNMT inhibition synergistically reactivated expression of endogenously methylated genes and inhibited cell growth of colon cancer cell lines with minimal effects on the cell growth of non-cancerous cell lines. Depletion of CHD4 alone also inhibited cell growth of colon cancer cell line but not of non-cancerous cell lines. ATF7IP was previously reported to synergize with DNA methylation to maintain X chromosome inactivation, and here we show that it also plays a role in autosomal gene silencing. Other genes which were not previously known as epigenetic regulators are now under investigation. Conclusions: A whole genome siRNA screen in combination with the DNMT inhibitor decitabine identified many new target genes that might be epigenetic regulators and potential targets for drug development. Citation Format: Yasuyuki Okamoto, Woonbok Chung, Judith Garriga, Jaroslav Jelinek, Jean-Pierre J. Issa. Discovering therapeutic epigenetic targets using whole genome siRNA screening. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2958. doi:10.1158/1538-7445.AM2015-2958

[This corrects the article on p. 234 in vol. 22, PMID: 29302581.].

Male infertility as a window to health

Review on CHD5 (chromodomain helicase DNA binding protein 5), with data on DNA, on the protein encoded, and where the gene is implicated.

Harmine is a natural compound possessing insulin-sensitizing effect in db/db diabetic mice. However its effect on adipose tissue browning is unknown. Here we reveal that harmine antagonizes high fat diet-induced adiposity. Harmine-treated mice gained less weight on a high fat diet and displayed increased energy expenditure and adipose tissue thermogenesis. In vitro, harmine potently induced the expression of thermogenic genes in both brown and white adipocytes, which was largely abolished by inhibition of RAC1/MEK/ERK pathway. Post-transcriptional modification analysis revealed that chromodomain helicase DNA binding protein 4 (CHD4) is a potential downstream target of harmine-mediated ERK activation. CHD4 directly binds the proximal promoter region of Ucp1, which is displaced upon treatment of harmine, thereby serving as a negative modulator of Ucp1. Thus, here we reveal a new application of harmine in combating obesity via this off-target effect in adipocytes.

Growth factor indepdendent 1 (GFI1) is a SNAG-domain, DNA binding transcriptional repressor which controls myeloid differentiation through molecular mechanisms and co-factors that still remain to be clearly identified. Here we show that GFI1 associates with the chromodomain helicase DNA binding protein 4 (CHD4) and other components of the Nucleosome remodeling and deacetylase (NuRD) complex. In granulo-monocytic precursors, GFI1, CHD4 or GFI1/CHD4 complexes occupy sites enriched for histone marks associated with active transcription suggesting that GFI1 recruits the NuRD complex to target genes regulated by active or bivalent promoters and enhancers. GFI1 and GFI1/CHD4 complexes occupy promoters that are either enriched for IRF1 or SPI1 consensus binding sites, respectively. During neutrophil differentiation, chromatin closure and depletion of H3K4me2 occurs at different degrees depending on whether GFI1, CHD4 or both are present, indicating that GFI1 is more efficient in depleting of H3K4me2 and -me1 marks when associated with CHD4. Our data suggest that GFI1/CHD4 complexes regulate histone modifications differentially to enable regulation of target genes affecting immune response, nucleosome organization or cellular metabolic processes and that both the target gene specificity and the activity of GFI1 during myeloid differentiation depends on the presence of chromatin remodeling complexes.

Single-cell analyses reveal increased gene expression variability in human neurodevelopmental conditions.

The genetic complexity of neurodevelopmental disorders (NDD), combined with a heterogeneous clinical presentation, makes accurate assessment of their molecular bases and pathogenic mechanisms challenging. Our purpose is to reveal the pathogenic variant underlying a complex NDD through identification of the "full" spectrum of structural genomic and genetic variants. Therefore, clinical phenotyping and identification of variants by genome and exome sequencing, together with comprehensive assessment of these and affected candidate genes, were carried out. A maternally-inherited familial translocation [t(17;19)(p13.1;p13.3)mat] disrupting the GSG1 like 2 gene (GSG1L2), a 3.2 Mb dup(2)(q14.3q21.1) encompassing the autosomal dominant OMIM phenotype-associated PROC and HS6ST1 gene, and a novel frameshift c.4442del, p.(Gly1481Valfs*21) variant within exon 30 of the Chromodomain helicase DNA binding protein 4 (CHD4) have been identified. Considering the pathogenic potential of each variant and the proband's phenotype, we conclude that this case basically fits the Sifrim-Hitz-Weiss syndrome or CHD4-associated neurodevelopmental phenotype. Finally, our data highlight the need for identification of the "full" spectrum of structural genomic and genetic variants and of reverse comparative phenotyping, including unrelated patients with variants in same genes, for improved genomic healthcare of patients with NDD.

BackgroundProstate cancer (PCa) is one of the most common malignancies in men worldwide, and advanced or metastatic disease remains a major therapeutic challenge. Chromodomain helicase DNA binding protein 1-like (CHD1L) has been implicated as an oncogenic driver in multiple cancer types, yet its role in prostate cancer pathogenesis is not fully defined. The purpose of this study is to investigate the biological significance of CHD1L in prostate cancer and to evaluate the therapeutic potential of its selective inhibitor OTI-611.MethodsBioinformatics analyses were conducted to assess the expression, prognostic significance of CHD1L in PCa patients. In vitro, cell viability, cycle progression, apoptosis, and migration/invasion were evaluated using CCK-8, colony formation, flow cytometry, transwell assays. In vivo treatment potential of OTI-611 was assessed through a nude mouse xenograft model. Protein and mRNA levels were determined by western blot and qPCR, respectively. Synergism of OTI-611 and docetaxel was determined using SynergyFinder 3.0.ResultsWe demonstrated that CHD1L was significantly upregulated in PCa patients and correlates with poor prognosis. Genetic knockdown of CHD1L substantially inhibits PCa cell proliferation and induces apoptosis. Moreover, inhibition of CHD1L by the small molecule OTI-611 significantly suppresses PCa cell proliferation, migration, and invasion, and induces apoptosis both in vitro and in vivo. Mechanistically, inhibition of CHD1L induces the expression of FOXO3 (a classic transcription factor) and its downstream target PUMA (a key apoptosis inducer). Restricting the expression of FOXO3 significantly reverses the anti-tumor effects induced by OTI-611. Furthermore, OTI-611 synergizes with docetaxel to enhance apoptotic cell death, providing a promising strategy to overcome docetaxel resistance.ConclusionsOur study demonstrates that CHD1L is markedly upregulated in prostate cancer and contributes to tumor progression. Pharmacological inhibition of CHD1L with the selective inhibitor OTI-611 significantly suppresses proliferation, migration, and invasion, while inducing apoptosis in vitro and in vivo. Mechanistically, these effects are mediated through activation of the FOXO3–PUMA axis, as FOXO3 suppression abrogates OTI-611–induced apoptosis. Moreover, OTI-611 exhibits strong synergy with docetaxel, enhancing apoptotic cell death and providing a potential strategy to improve therapeutic efficacy in prostate cancer.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12967-025-07529-5.

CHD6 is a chromatin remodeling protein characterized to play a role in transcriptional repression of genes and viruses. It occurs in a nuclear location as a component of a larger complex which associates with RNA Pol II. Mutations in CHD6 are associated with motor coordination defects, and development of cancers following substitutions and translocations.

Mutations in Chromodomain Helicase DNA Binding Protein 7 (CHD7) and Paired Box Gene 4 (PAX4) are critical for normal cartilage development and are implicated through their impact on chondrocyte functions. This study examines how these genetic alterations specifically modulate Tumor protein p53 (p53) expression to affect cellular proliferation and apoptosis, shedding light on potential therapeutic targets for mitigating developmental anomalies in cartilage. Using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)- associated protein 9 (Cas9), specific mutations were introduced into CHD7 and PAX4 in chondrocytes. Subsequent analyses included 5-ethynyl-2'-deoxyuridine (EdU) assay for proliferation, Terminal deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) staining for apoptosis, quantitative real-time polymerase chain reaction (qRTPCR), and Western blot alongside co-immunoprecipitation (Co-IP) to evaluate expression levels and protein interactions. Mutations in CHD7 and PAX4 resulted in decreased proliferation and increased apoptosis in chondrocytes. Notably, these mutations disrupted the interaction between the mutant proteins and p53, leading to altered expression of apoptotic regulators such as Bcl2-associated X protein (Bax), B-cell lymphoma 2 (Bcl2), indicating activation of p53-dependent apoptotic pathways. This study elucidates the core molecular mechanism by which mutations in the CHD7 and PAX4 genes disrupt their interaction with p53, leading to aberrant activation of the p53-dependent apoptotic pathway. These findings provide a new theoretical basis and potential intervention strategies for developing p53 pathwaytargeted therapies to treat related cartilage developmental disorders. Future research should focus on in vivo validation and mechanistic refinement. The study reveals that CHD7 and PAX4 mutations exacerbate the apoptotic pathways in chondrocytes by enhancing the activity of p53, leading to decreased cell proliferation and increased apoptosis. These findings underscore the mutations' profound impact on cartilage cell dynamics and highlight the therapeutic potential of targeting p53 to correct the cellular imbalances caused by these genetic changes in cartilage-related developmental disorders.

BackgroundOur recent studies suggested that the chromodomain helicase DNA binding protein 1-like (CHD1L) gene plays an oncogenic role in human hepatocellular carcinoma. However, the status of CHD1L protein expression in ovarian cancer and its clinical/prognostic significance are obscure.MethodsIn this study, immunohistochemistry (IHC) for CHD1L was performed on a tissue microarray (TMA) containing 102 primary ovarian carcinomas and 44 metastatic lesions (omental metastasis). Receiver-operator curve (ROC) analysis was used to evaluate patients’ survival status.ResultsThere is an augmented tendency of CHD1L expression in ovarian carcinoma metastasis than in primary lesions (P<0.05). A significant association was found between positive expression of CHD1L and tumors histological type (P <0.05). By univariate survival analysis of the ovarian carcinoma cohorts, positive expression of CHD1L was significantly correlated with shortened patient survival (mean 66.7 months versus 97.4 months, P<0.05). Moreover, CHD1L expression was evaluated to be a significant and independent prognostic factor in multivariate analysis (P<0.05).ConclusionsThese findings provide evidence that positive expression of CHD1L protein is significantly correlated with the metastasis proceeding of ovarian carcinoma, and CHD1L protein expression, as examined by IHC, may act as a novel prognostic biomarker for patients with ovarian carcinoma.

Summary CHARGE syndrome, an autosomal dominant inherited multiple malformation syndrome is caused by mutations in the chromodomain helicase DNA binding protein 7 gene (CHD7). In 10% of ‘typical’ CHARGE patients and 40-50% of ‘atypical’ CHARGE patients no mutation in the CHD7 gene is detectable and therefore the molecular cause is still unknown. CHD7 is a nuclear chromatin remodeling protein found in big protein complexes and regulating gene expression of various genes. We suggested that the characterization of CHD7 interacting partners can be useful for understanding the pathogenesis of CHARGE syndrome. Furthermore, for other genetic diseases it was shown that mutations in interacting partners lead to the same or a similar phenotype. Therefore, we hypothesized that CHD7 interacting partners are good candidates, leading to CHARGE syndrome when they are mutated. In the first part of the thesis, CHD8 was identified as an interacting partner of CHD7. The interaction of the CHD7 part (1593 – 2178aa) with the CHD8 part (1789 – 2302aa) was validated by different molecular techniques. Additionally, we could show that the interaction takes place in the nucleoplasm. Furthermore, the influence of 3 known (p.His2096Arg, p.Val2102Ile and p.Gly2108Arg) and one (p.Trp2091Arg) newly identified CHD7 missense mutation on the CHD7-CHD8 interaction was elucidated by direct Yeast two hybrid (Y2H) and co-immunoprecipitation (Co-IP) methods. By Y2H we could demonstrate that the missense mutations p.Trp2091Arg, p.His2096Arg and p.Gly2108Arg disrupt the CHD7-CHD8 interaction, while no influence on the interaction could be seen in Co-IP assasys. Therefore, we suggest that CHD7 and CHD8 interacting directly (shown by Y2H) and indirectly via additional linker proteins which build together with CHD7 and CHD8 a large protein complex (explaining the Co-IP result). In addition, we reported about the sequence analysis of the CHD8 gene in CHD7 mutation negative patients with CHARGE syndrome. In the second part of the thesis, we showed the results of a SILAC and mass-spectrometry approach that identified the hitherto unknown protein FAM124B as a member of CHD7-CHD8 containing complex. The interaction with the CHD8 part (1789 – 2302aa) was validated by direct Y2H and Co-IP methods whereas the interaction with the CHD7 part (1593 – 2178aa) could be only confirmed with the Co-IP assay, demonstrating that FAM124B interacts not directly with the analyzed CHD7 region. Furthermore, we characterized FAM124B. We could show that FAM124B is localized in the nucleoplasm like CHD7 and CHD8. Murine Fam124B is expressed in the organs affected in CHARGE syndrome. High expression could be observed in the developing brain, heart, lung and spinal cord of E12.5 mouse embryos. Recently, CHD8 mutations are shown to be a cause of autism spectrum disorders (ASD) as well as neurodevelopmental disorders (NDD). In conclusion, our results indicate that the hitherto uncharacterized protein FAM124B might be very important for embryonic development and could be involved in the pathogenesis of CHARGE syndrome and ASD/NDDs. Moreover, to analyse the possible role of FAM124B in the pathogenesis of CHARGE and neurocristopathies, we plan to perform a knockdown of FAM124B in Xenopus laevis and test its influence on genes related to neural crest formation. Generation of a knockout mouse model for Fam124B and further characterization of the CHD7 interacting complex members will help to learn more about the molecular mechanism behind CHARGE syndrome and ASD/NDDs.

The minichromosome maintenance complex (MCM) proteins are required for processive DNA replication and are a target of S-phase checkpoints. The eukaryotic MCM complex consists of six proteins (MCM2–7) that form a heterohexameric ring with DNA helicase activity, which is loaded on chromatin to form the pre-replication complex. Upon entry in S phase, the helicase is activated and opens the DNA duplex to recruit DNA polymerases at the replication fork. The MCM complex thus plays a crucial role during DNA replication, but recent work suggests that MCM proteins could also be involved in DNA repair. Here, we employed a combination of stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative proteomics with immunoprecipitation of green fluorescent protein-tagged fusion proteins to identify proteins interacting with the MCM complex, and quantify changes in interactions in response to DNA damage. Interestingly, the MCM complex showed very dynamic changes in interaction with proteins such as Importin7, the histone chaperone ASF1, and the Chromodomain helicase DNA binding protein 3 (CHD3) following DNA damage. These changes in interactions were accompanied by an increase in phosphorylation and ubiquitination on specific sites on the MCM proteins and an increase in the co-localization of the MCM complex with γ-H2AX, confirming the recruitment of these proteins to sites of DNA damage. In summary, our data indicate that the MCM proteins is involved in chromatin remodeling in response to DNA damage.