Chromodomain Helicase DNA Binding Research Articles

Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B

Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with “epidrugs” is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4, lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.

Refractory testicular germ cell tumors are highly sensitive to the targeting of polycomb pathway demethylases KDM6A and KDM6B.

Testicular germ cell tumors (TGCTs) can be treated with cisplatin-based therapy. However, a clinically significant number of cisplatin-resistant patients die from progressive disease as no effective alternatives exist. Curative cisplatin therapy results in acute and life-long toxicities in the young TGCT patient population providing a rationale to decrease cisplatin exposure. In contrast to genetic alterations, recent evidence suggests that epigenetics is a major driving factor for TGCT formation, progression, and response to chemotherapy. Hence, targeting epigenetic pathways with "epidrugs" is one potential relatively unexplored strategy to advance TGCT treatment beyond cisplatin. In this report, we demonstrate for the first time that targeting polycomb demethylases KDM6A and KDM6B with epidrug GSK-J4 can treat both cisplatin-sensitive and -resistant TGCTs. While GSK-J4 had minimal effects alone on TGCT tumor growth in vivo, it dramatically sensitized cisplatin-sensitive and -resistant TGCTs to cisplatin. We validated KDM6A/KDM6B as the target of GSK-J4 since KDM6A/KDM6B genetic depletion had a similar effect to GSK-J4 on cisplatin-mediated anti-tumor activity and transcriptome alterations. Pharmacologic and genetic targeting of KDM6A/KDM6B potentiated or primed the p53-dominant transcriptional response to cisplatin, with also evidence for basal activation of p53. Further, several chromatin modifier genes, including BRD4 , lysine demethylases, chromodomain helicase DNA binding proteins, and lysine methyltransferases, were repressed with cisplatin only in KDM6A/KDM6B-targeted cells, implying that KDM6A/KDM6B inhibition sets the stage for extensive chromatin remodeling of TGCT cells upon cisplatin treatment. Our findings demonstrate that targeting polycomb demethylases is a new potent pharmacologic strategy for treating cisplatin resistant TGCTs that warrants clinical development.

Chromatin remodeller Chd7 is developmentally regulated in the neural crest by tissue-specific transcription factors.

Neurocristopathies such as CHARGE syndrome result from aberrant neural crest development. A large proportion of CHARGE cases are attributed to pathogenic variants in the gene encoding CHD7, chromodomain helicase DNA binding protein 7, which remodels chromatin. While the role for CHD7 in neural crest development is well documented, how this factor is specifically up-regulated in neural crest cells is not understood. Here, we use epigenomic profiling of chick and human neural crest to identify a cohort of enhancers regulating Chd7 expression in neural crest cells and other tissues. We functionally validate upstream transcription factor binding at candidate enhancers, revealing novel epistatic relationships between neural crest master regulators and Chd7, showing tissue-specific regulation of a globally acting chromatin remodeller. Furthermore, we find conserved enhancer features in human embryonic epigenomic data and validate the activity of the human equivalent CHD7 enhancers in the chick embryo. Our findings embed Chd7 in the neural crest gene regulatory network and offer potentially clinically relevant elements for interpreting CHARGE syndrome cases without causative allocation.

NEAT1 promotes genome stability via m6A methylation-dependent regulation of CHD4.

Long noncoding (lnc)RNAs emerge as regulators of genome stability. The nuclear-enriched abundant transcript 1 (NEAT1) is overexpressed in many tumors and is responsive to genotoxic stress. However, the mechanism that links NEAT1 to DNA damage response (DDR) is unclear. Here, we investigate the expression, modification, localization, and structure of NEAT1 in response to DNA double-strand breaks (DSBs). DNA damage increases the levels and N6-methyladenosine (m6A) marks on NEAT1, which promotes alterations in NEAT1 structure, accumulation of hypermethylated NEAT1 at promoter-associated DSBs, and DSB signaling. The depletion of NEAT1 impairs DSB focus formation and elevates DNA damage. The genome-protective role of NEAT1 is mediated by the RNA methyltransferase 3 (METTL3) and involves the release of the chromodomain helicase DNA binding protein 4 (CHD4) from NEAT1 to fine-tune histone acetylation at DSBs. Our data suggest a direct role for NEAT1 in DDR.

Effects of two chd2-knockout strains on the morphology and behavior in zebrafish.

The chromodomain helicase DNA binding domain 2 (CHD2) gene is an ATPase and a member of the SNF2-like family of helicase-related enzymes. CHD2 plays critical roles in human brain development and function, and homozygous mutation of Chd2 in mice results in perinatal lethality. To further elucidate the effects of chd2, we used CRISPR/Cas9 to create two chd2-knockout strains (fdu901, 11,979-11982delGGGT, and fdu902, 27350delG) in zebrafish. We found that the deformity and mortality rates of fdu901 and fdu902 were higher than those of the wild type. Developmental delay was more obvious and embryo mortality was higher in fdu901 than in fdu902. However, the embryo deformity rate in fdu902 was higher than that in fdu901. Although there were no significant differences in behavior between the two knockout zebrafish and wild-type zebrafish at 7days post fertilization (dpf), fdu901 and fdu902 zebrafish showed different alterations. The excitability of fdu902 was higher than that of fdu901. Overall, our data demonstrate that two homozygous chd2 knockout mutations were survivable and could be stably inherited and that fdu901 and fdu902 zebrafish differed in behavior and morphology. These two models might be good tools for understanding the functions of the different domains of chd2.

Abstract We146: CHD8 is Essential for Postnatal Heart Function through Preserving Mitochondrial Integrity

Objective: Epigenetic regulation plays a crucial role in heart development and disease, although its mechanisms remain elusive. Known mutations in epigenetic regulators have been linked to cardiac disease. For instance, the mutation in the chromodomain helicase DNA-binding (CHD) protein 7 gene, which leads to CHARGE syndrome, is known to results in heart defects. However, the role of CHD8, an interacting partner of CHD7, in the heart has not been investigated. This study aims to explore the function of CHD8 in the heart. Methods and Results: We generated a cardiac-specific Chd8 conditional knockout mouse line (Chd8 cKO) by breeding Chd8 fl/fl mice with aMHC Cre mice. Over 60% of Chd8 cKO mice died between 7 and 9 months of age, with increased heart weight to bodyweight ratio and reduced cardiac function, increased fibrosis, and signs of dilated cardiomyopathy. Electron microscopy analysis revealed disordered sarcomeres and escalated mitophagy in CHD8-deficient hearts. Using Genome-wide transcriptome analysis though RNA sequencing, we discovered that genes involved in mitochondrial oxidative phosphorylation and muscle contraction were down-regulated in Chd8 cKO hearts. Proteins in the electron transport chain complexes were down regulated and mitophagy marker LC3 increased in the Chd8 cKO hearts compared with the control mice. Upregulated pathways include inflammatory response suggesting that the innate immune response may contribute to dilated cardiomyopathy. Conclusions: Our findings underscore CHD8 as a key regulator of cardiac function and regeneration by influencing chromatin accessibility of genes involved in mitochondrial oxidative phosphorylation and contractility. Overall, our study provides new insights into the epigenetic regulation of cardiac function and the CHD8’s role in heart development and disease. It implies that activating CHD8 may be a promising therapeutic strategy for treating dilated cardiomyopathy and other heart-related diseases.

Abstract Mo055: CHD4 Interacts With TBX5 to Maintain the Gene Regulatory Network of Postnatal Atrial Cardiomyocytes

Atrial fibrillation (AF) is the most common sustained arrhythmia in humans, leading to increased mortality due to increased incidence of stroke and heart failure. Over 200 genes are associated with AF by GWAS studies, suggesting a nuanced gene regulatory network (GRN) is required to maintain atrial cardiomyocyte (aCM) function and rhythm. Among these genes is the transcription factor TBX5, which we and others have shown is a master regulator of aCM identity that is essential for atrial rhythm homeostasis. We showed that TBX5 is required to maintain the chromatin looping, accessibility, and activity of atrial enhancers. Chromodomain helicase DNA binding protein 4 (CHD4), a chromatin remodeling protein that interacts with TBX5, is canonically thought to repress transcription as a component of the NuRD complex. Here, we investigated the contribution of CHD4 to TBX5 regulation of aCM gene expression. Methods: We inactivated CHD4 specifically within atrial cardiomyocytes using Cre recombinase driven by the aCM-specific Nppa promoter delivered postnatally with AAV9. Atrial remodeling, transcriptomic changes and AF susceptibility were examined. Results: We show that inactivating CHD4 in postnatal aCMs resulted in fibrotic atrial remodeling, spontaneous AF in a subset of mice, and increased AF susceptibility and burden after electrical pacing. Transcriptomic profiling of CHD4 aCM KO atria revealed gene expression changes that were highly correlated to altered gene expression downstream of TBX5 inactivation. Mechanistically, we show TBX5 recruits CHD4 to 33,170 genomic regions that are important for aCM gene regulation, whose expression is concordantly regulated downstream of TBX5 and CHD4. Together, these data reveal that CHD4 is required by TBX5 to promote accessibility of enhancers and activate many TBX5 target genes to maintain atrial rhythm.

Cancer-associated fibroblast-derived colony-stimulating factor 2 confers acquired osimertinib resistance in lung adenocarcinoma via promoting ribosome biosynthesis.

Acquired resistance is a major obstacle to the therapeutic efficacy of osimertinib in lung adenocarcinoma (LUAD), but the underlying mechanisms are still not fully understood. Cancer-associated fibroblasts (CAFs) are the most abundant stromal cell type in LUAD tumor-microenvironment (TME) and have emerged as a key player in chemoresistance. However, the function of CAFs in osimertinib resistance is still unclear. Here, we showed that CAFs derived from osimertinib-resistant LUAD tissues (CAFOR) produced much more colony-stimulating factor 2 (CSF2) than those isolated from osimertinib-sensitive tissues. CAFOR-derived CSF2 activated the Janus kinase 2 (JAK2)/Signal transducer and activator of transcription 3 (STAT3) signaling pathway and upregulated lnc-CSRNP3 in LUAD cells. Lnc-CSRNP3 then promoted the expression of nearby gene CSRNP3 by recruiting chromodomain helicase DNA binding protein 9 (CHD9) and inhibited the phosphatase activity of the serine/threonine protein phosphatase 1 catalytic subunit α (PP1α), thereby induced osimertinib resistance by enhancing ribosome biogenesis. Collectively, our study reveals a critical role for CAFs in the development of osimertinib resistance and identifies the CSF2 pathway as an attractive target for monitoring osimertinib efficacy and overcoming osimertinib resistance in LUAD.

IGF1R and FLT1 in female endothelial cells and CHD2 in male microglia play important roles in Alzheimer's disease based on gender difference analysis

ObjectiveThis study investigated sex-specific pathogenesis mechanisms in Alzheimer's disease (AD) using single-nucleus RNA sequencing (snRNA-seq) data. MethodsData from the Gene Expression Omnibus (GEO) were searched using terms “Alzheimer's Disease”, “single cell”, and “Homo sapiens”. Studies excluding APOE E4 and including comprehensive gender information with 10× sequencing methods were selected, resulting in GSE157827 and GSE174367 datasets from human prefrontal cortex samples. Sex-stratified analyses were conducted on these datasets, and the outcomes of the analysis for GSE157827 were compared with those of GSE174367. The findings were validated using expression profiling from the mouse dataset GSE85162. Furthermore, real-time PCR experiments in mice further confirmed these findings. The Seurat R package was used to identify cell types, and batch effects were mitigated using the Harmony R package. Cell proportions by sex were compared using the Mann-Whitney-Wilcoxon test, and gene expression variability was displayed with an empirical cumulative distribution plot. Differentially expressed genes were identified using the FindMarkers function with the MAST test. Transcription factors were analyzed using the RcisTarget R package. ResultsSeven cell types were identified: astrocytes, endothelial cells, excitatory neurons, inhibitory neurons, microglia, oligodendrocytes, and oligodendrocyte progenitor cells. Additionally, five distinct subpopulations of both endothelial and microglial cells were also identified, respectively. Key findings included: (1) In endothelial cells, genes involved in synapse organization, such as Insulin Like Growth Factor 1 Receptor (IGF1R) and Fms Related Receptor Tyrosine Kinase 1(FLT1), showed higher expression in females with AD. (2) In microglial cells, genes in the ribosome pathway exhibited higher expression in males without AD compared to females (with or without AD) and males with AD. (3) Chromodomain Helicase DNA Binding Protein 2 (CHD2) negatively regulated gene expression in the ribosome pathway in male microglia, suppressing AD, this finding was further validated in mice. (4) Differences between Asians and Caucasians were observed based on sex and disease status stratification. ConclusionsIGF1R and FLT1 in endothelial cells contribute to AD in females, while CHD2 negatively regulates ribosome pathway gene expression in male microglia, suppressing AD in humans and mice.

Evaluation of CHD5, H3K9me3, and H4K12ac in Human Testes with Spermatogenic Maturation Arrest: A Cross-Sectional Study.

Spermatogenic maturation arrest is thought to be caused by epigenetic defects, specifically in chromatin remodeling and histone modification. This study evaluated the status of chromatin remodeling chromodomain helicase DNA binding protein 5 (CHD5) and histone modifications histone 4 lys-12 acetylation (H4K12ac) and histone 3 lys-9 trimethylation (H3K9me3) in human testicular biopsies, based on maturation arrest type. The cross-sectional study utilized 18 Bouin-fixed paraffin-embedded (BFPE) specimens prepared from residual tissue from routine laboratory tests of infertile patients. The expression of CHD5, H4K12ac, and H3K9me3 was examined through immunohistochemistry (IHC). The intensity was measured using ImageJ with IHC Profiler and StarDist plugins. Statistical analysis was performed using Python with Scipy.Stats module. The data were tested with Shapiro- Wilk for normality and Levene test for homogeneity. The differences in the intensity of spermatogenic cells were assessed using Kruskal-Wallis and Mann-Whitney tests. A difference was considered statistically significant if P<0.05. We found three types of maturation arrest, including Sertoli cell only (n=5), spermatocyte arrest (n=4), and spermatid arrest (n=9). CHD5 was positive in spermatogonia and round spermatids but absent in spermatocytes. The mean grey value (MGV) of CHD5 in spermatogonia was generally weak in spermatocyte arrest (157.4 ± 16.6) and spermatid arrest (155.3 ± 16.8), and there was no significant difference between them [P=0.49, 95% confidence interval (CI): (-4.3, 6), effect size (r): 0.02]. Although there was a significant difference in the expression of H3K9me3 and H4K12ac (P<0.001), both histone modifications were found in all observed spermatogenic cells. The expressions of CHD5, H3K9me3, and H4K12ac in different spermatogenic cell types produce similar results, indicating that they cannot be used as markers to determine the type of spermatogenic maturation arrest in humans. The significant finding in this research is the expression of CHD5 in human spermatogonia cells, which requires further study for elaboration.

Single-cell RNA analysis of chromodomain-encoding genes in mesenchymal stromal cells of the mouse dental pulp.

The chromodomain helicase DNA-binding (CHD) and chromobox (CBX) families of proteins play crucial roles in cell fate decisions, differentiation, and cell proliferation in a broad variety of tissues and cell types. CHD proteins are ATP-dependent epigenetic enzymes actively engaged in transcriptional regulation, DNA replication, and DNA damage repair, whereas CBX proteins are transcriptional repressors mainly involved in the formation of heterochromatin. The pleiotropic effects of CHD and CBX proteins are largely dependent on their versatility to interact with other key components of the epigenetic and transcriptional machinery. Although the function and regulatory modes of CHD and CBX factors are well established in many cell types, little is known about their roles during osteogenic differentiation. A single-cell RNA-sequencing analysis of the mouse incisor dental pulp revealed distinct spatiotemporal expression patterns of CHD- and CBX-encoding genes within different clusters of mesenchymal stromal cells (MSCs) representing various stages of osteogenic differentiation. Additionally, genes encoding interaction partners of CHD and CBX proteins, such as subunits of the trithorax-COMPASS and polycomb chromatin remodeling complexes, exhibited differential co-expression behaviors within MSC subpopulations. Thus, CHD- and CBX-encoding genes show partially overlapping but distinct expression patterns in MSCs, suggesting their differential roles in osteogenic cell fate decisions.

CHD9NB Is a Novel Brush Border Marker in the Intestine and Beyond

The public availability of single cell RNA sequencing datasets has accelerated discovery of novel gene markers of specific cell types. A recently published dataset of human small intestine and colon (GSE185224) revealed 14 distinct epithelial clusters, including tuft cell, a rare sensory cell population. We probed this dataset for novel tuft cell-specific makers and investigated the localization of candidate marker proteins. Using dataset GSE185224, Leiden clustering and UMAP visualization were generated, and tuft cell clusters were identified by previously known markers, e.g. POU2F3. Genes were systematically parsed through and CHD9NB (Chromodomain Helicase DNA Binding Protein 9 neighbor) was identified as a candidate novel tuft cell marker gene. CHD9NB encodes a 52 amino acid protein with no published information for function. Antibodies were raised against recombinant human CHD9NB. Paraffn sections of tissues and enteroids from healthy individuals and mice were immunostained against CHD9NB and brush border or cell structural markers. CHD9NB was expressed along apical actin bundles of intestinal tuft cells. Contrary to transcriptional level, which was lower in enterocytes than tuft cells, intense CHD9NB immunostaining was observed at the tips of microvilli along mature enterocytes in human and mouse small intestine and colon. This microvillus tip localization was suggested by the presence of CHD9NB above the ACTG1 staining in brush border, similar to EPS8. Less intense CHD9NB was detected at both the tips and base of microvilli of the developing brush border in intestinal crypts, suggesting a differentiation stage-dependent localization of CHD9NB in the intestinal epithelial cells. Differentiated human enteroids exhibited either villus-like (microvillus tip-specific) or crypt-like (present at both the base and tips) CHD9NB staining, likely suggesting a range of enteroid differentiation states. To determine whether CHD9NB is specific to the intestinal microvilli or more generally expressed in brush borders of other organs, sections of kidney, inner ear, and lung were stained. CHD9NB staining was specific to brush border-containing, OAT1+ proximal tubules of the kidney, and absent in brush border-lacking distal tubules and glomeruli. In contrast to tips of microvilli in enterocytes, CHD9NB was localized to the base of the brush border of proximal tubules. CHD9NB staining in the murine cochlea was specific to the DCLK1+ cells of the stria vascularis, rather than hair cells. In the lung, CHD9NB was detected along the ciliated cells of the human bronchiole. We propose CHD9NB as a novel brush border and intestinal tuft cell marker, with organ-specific localization patterns beyond the intestine. Further studies will explore the function of CHD9NB in enteroids. NSF GRFP; NIH R01DK128190; RC2DK118640. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

CHD4 acts as a prognostic factor and drives radioresistance in HPV negative HNSCC

Despite great efforts in improving existing therapies, the outcome of patients with advanced radioresistant HPV-negative head and neck squamous cell carcinoma (HNSCC) remains poor. The chromatin remodeler Chromodomain helicase DNA binding protein 4 (CHD4) is involved in different DNA-repair mechanisms, but the role and potential in HNSCC has not been explored yet. In the present study, we evaluated the prognostic significance of CHD4 expression using in silico analysis of the pan-cancer dataset. Furthermore, we established a monoclonal HNSCC CHD4 knockdown cell clone utilizing the CRISPR/Cas9 system. Effects of lower CHD4 expression on radiosensitivity after increasing doses of ionizing radiation were characterized using clonogenic assays and cell numbers. The in silico analysis revealed that high CHD4 expression is associated with significant poorer overall survival of HPV-negative HNSCC patients. Additionally, the knockdown of CHD4 significantly increased the radiosensitivity of HNSCC cells. Therefore, CHD4 might be involved in promoting radioresistance in hard-to-treat HPV-negative HNSCC entities. We conclude that CHD4 could serve as a prognostic factor in HPV-negative HNSCC tumors and is a potential target protein overcoming radioresistance in HNSCC. Our results and the newly established cell clone laid the foundation to further characterize the underlying mechanisms and ultimately use CHD4 in HNSCC therapies.

The CHD family chromatin remodeling enzyme, Kismet, promotes both clathrin-mediated and activity-dependent bulk endocytosis.

Chromodomain helicase DNA binding domain (CHD) proteins, including CHD7 and CHD8, remodel chromatin to enable transcriptional programs. Both proteins are important for proper neural development as heterozygous mutations in Chd7 and Chd8 are causative for CHARGE syndrome and correlated with autism spectrum disorders, respectively. Their roles in mature neurons are poorly understood despite influencing the expression of genes required for cell adhesion, neurotransmission, and synaptic plasticity. The Drosophila homolog of CHD7 and CHD8, Kismet (Kis), promotes neurotransmission, endocytosis, and larval locomotion. Endocytosis is essential in neurons for replenishing synaptic vesicles, maintaining protein localization, and preserving the size and composition of the presynaptic membrane. Several forms of endocytosis have been identified including clathrin-mediated endocytosis, which is coupled with neural activity and is the most prevalent form of synaptic endocytosis, and activity-dependent bulk endocytosis, which occurs during periods of intense stimulation. Kis modulates the expression of gene products involved in endocytosis including promoting shaggy/GSK3β expression while restricting PI3K92E. kis mutants electrophysiologically phenocopy a liquid facets mutant in response to paradigms that induce clathrin-mediated endocytosis and activity-dependent bulk endocytosis. Further, kis mutants do not show further reductions in endocytosis when activity-dependent bulk endocytosis or clathrin-mediated endocytosis are pharmacologically inhibited. We find that Kis is important in postsynaptic muscle for proper endocytosis but the ATPase domain of Kis is dispensable for endocytosis. Collectively, our data indicate that Kis promotes both clathrin-mediated endocytosis and activity-dependent bulk endocytosis possibly by promoting transcription of several endocytic genes and maintaining the size of the synaptic vesicle pool.

Abstract 1049 O-GlcNAc crosslinking of Chromodomain Helicase DNA Binding Protein 4 (CHD4) reveals novel functions for the Nucleosome Chromatin Remodeling Complex

Abstract 1049 O-GlcNAc crosslinking of Chromodomain Helicase DNA Binding Protein 4 (CHD4) reveals novel functions for the Nucleosome Chromatin Remodeling Complex

Aspirin-exacerbated respiratory disease is associated with variants in filaggrin, epithelial integrity, and cellular interactions

BackgroundPrevious studies identified upto 6% of patients with Aspirin-Exacerbated Respiratory Disease (AERD) have family history of AERD indicating a possible link with genetic polymorphisms. However, whole exome sequencing (WES) studies of such associations are currently lacking. ObjectivesWe sought to examine whether WES can identify pathogenic variants associated with AERD. MethodsAERD diagnosis was confirmed in patients with nasal polyps and asthma. WES was performed using an Illumina sequencing platform. Human Phenotype Ontology (HPO) terms were used to define the patients’ phenotypes. Exomiser was used to annotate, filter, and prioritize possible disease-causing genetic variants. ResultsAmong thirty-nine patients with AERD, 41% reported a family history of asthma and 5% a family history of AERD. Pathogenic exome variants in the filaggrin gene (FLG) were found in two patients (5%). Other variants not known to be pathogenic were detected in an additional 16 patients (41%) in genes related to epithelial integrity and cellular interactions, including genes encoding desmoglein 3 (DSG3), dynein axonemal heavy chain 9 (DNAH9), collagen type VII alpha 1 chain (COL7A1), collagen type XVII alpha 1 chain (COL17A1), chromodomain helicase DNA binding protein-7 (CHD7), TSC complex subunit 2/tuberous sclerosis-2 protein (TSC2), P-selectin (SELP), and platelet-derived growth factor receptor-alpha (PDGFRA). ConclusionWES identified a monogenic susceptibility to AERD in 5% of patients with FLG pathogenic variants. Other variants, not previously identified as pathogenic, were found in genes relevant to epithelial integrity and cellular interactions and may further reveal genetic factors that contribute to this condition.

Appropriate Primer Selection Improves Molecular Bird Sexing Accuracy

Birds sexing utilize the Polymerase Chain Reaction (PCR) technique is increasingly being used by researchers and breeders. The PCR technique has high sensitivity, but its success is influenced by the specificity of the DNA template with the oligo primer used. This study aimed to evaluate 5 types of PCR primers P2/P8, 2550F/2718R, CHD1F/CHD1R, 1237L/1272H, and CHD1LF/CHD1LR to determine the sex of Phasianidae, Anatidae, Muscicapidae, and Psittacidae families. This research was conducted by tested primers mentioned above to amplify the target gene chromodomain helicase DNA binding 1 (CHD1) on DNA samples of each pair of males and females from four bird families, respectively. The results indicated that CHD1LF/CHD1LR PCR primer gave the best results and was recommended to determine the sex of four families tested. Some of other primers tested in this study failed to amplify targeted gene correctly, it is important to use appropriate primer to increase bird sexing accuracy.

Chromatin gatekeeper and modifier CHD proteins in development, and in autism and other neurological disorders.

Chromatin, a protein-DNA complex, is a dynamic structure that stores genetic information within the nucleus and responds to molecular/cellular changes in its structure, providing conditional access to the genetic machinery. ATP-dependent chromatin modifiers regulate access of transcription factors and RNA polymerases to DNA by either "opening" or "closing" the structure of chromatin, and its aberrant regulation leads to a variety of neurodevelopmental disorders. The chromodomain helicase DNA-binding (CHD) proteins are ATP-dependent chromatin modifiers involved in the organization of chromatin structure, act as gatekeepers of genomic access, and deposit histone variants required for gene regulation. In this review, we first discuss the structural and functional domains of the CHD proteins, and their binding sites, and phosphorylation, acetylation, and methylation sites. The conservation of important amino acids in SWItch/sucrose non-fermenting (SWI/SNF) domains, and their protein and mRNA tissue expression profiles are discussed. Next, we convey the important binding partners of CHD proteins, their protein complexes and activities, and their involvements in epigenetic regulation. We also show the ChIP-seq binding dynamics for CHD1, CHD2, CHD4, and CHD7 proteins at promoter regions of histone genes, as well as several genes that are critical for neurodevelopment. The role of CHD proteins in development is also discussed. Finally, this review provides information about CHD protein mutations reported in autism and neurodevelopmental disorders, and their pathogenicity. Overall, this review provides information on the progress of research into CHD proteins, their structural and functional domains, epigenetics, and their role in stem cell, development, and neurological disorders.

Abstract P1009: Investigating The Effect Of Patient Missense Variants In CHD7 Using An Induced Pluripotent Stem Cell Model

Congenital heart disease (CHD) is a phenotypically diverse and genetically heterogenous disease. While monogenic causes of familial CHD and damaging de novo variants are known contributors to CHD risk, novel variants of uncertain significance (VUS) detected among CHD probands remain under characterized. In particular, the effect of missense variants in CHD7 (chromodomain helicase DNA binding protein 7), remain poorly explored. CHD7 is an established CHD gene with variants associated with CHARGE syndrome (Coloboma, Heart defects, Atresia of the choanae, Retardation of growth and development, Genital/urinary abnormalities, and Ear abnormalities) and incomplete syndromic phenotypes. We hypothesized that some CHD7 missense variants in genetically engineered human-induced pluripotent stem cells (iPSCs) perturb essential cardiac developmental pathways. CHD7 is highly expressed in iPSCs and during differentiation to iPSC-cardiomyocytes. Out of seventy rare patient missense variants in CHD7 reported by the Pediatric Cardiac Genetics Consortium (PCGC), six were chosen for genomic editing. To investigate the functional effects of missense variants in CHD7, we first predicted pathogenicity using AlphaFold, an algorithm which models variant impact on the three-dimensional structure of proteins. We then introduced six patient missense variants (Q161R, R286G, I1500V, T262A, T894A, L2299P) into an iPSC line using adenine base editors. Heterozygous and homozygous loss-of-function CHD7 lines were generated for comparison. RNA expression profiles of edited iPSCs were analyzed using single cell gene expression with multiplexing to assess the impact of the missense variants on transcriptional profiles. Bulk RNA sequencing was performed in parallel to better characterize transcriptional changes. These results further our understanding of how CHD7 missense variants may contribute to CHD risk. In the future, these approaches could be applied to a broader list of genes, as rare patient missense variants in definitive and candidate CHD genes require further investigation to determine pathogenicity. Developing iPSC models to investigate functional effects of missense variants can aid in understanding the complex genetic architecture of CHD.

ESS2 controls prostate cancer progression through recruitment of chromodomain helicase DNA binding protein 1

Molecular targeted therapy using poly (ADP-ribose) polymerase inhibitors has improved survival in patients with castration-resistant prostate cancer (CRPC). However, this approach is only effective in patients with specific genetic mutations, and additional drug discovery targeting epigenetic modulators is required. Here, we evaluated the involvement of the transcriptional coregulator ESS2 in prostate cancer. ESS2-knockdown PC3 cells dramatically inhibited proliferation in tumor xenografts in nude mice. Microarray analysis revealed that ESS2 regulated mRNA levels of chromodomain helicase DNA binding protein 1 (CHD1)-related genes and other cancer-related genes, such as PPAR-γ, WNT5A, and TGF-β, in prostate cancer. ESS2 knockdown reduced nuclear factor (NF)-κB/CHD1 recruitment and histone H3K36me3 levels on the promoters of target genes (TNF and CCL2). In addition, we found that the transcriptional activities of NF-κB, NFAT and SMAD2/3 were enhanced by ESS2. Tamoxifen-inducible Ess2-knockout mice showed delayed prostate development with hypoplasia and disruption of luminal cells in the ventral prostate. Overall, these findings identified ESS2 acts as a transcriptional coregulator in prostate cancer and ESS2 can be novel epigenetic therapeutic target for CRPC.