Protocadherin Genes Research Articles

Identification of genetic variants associated with exercise-induced atrial fibrillation in high-performing racehorses

Abstract Background Atrial fibrillation (AF) is the most common cardiac arrhythmia in athletes. Despite a lower burden of classical cardiovascular risk factors, the prevalence of AF in athletes is comparable to that in the general population of similar age (~13%, mean age 65.5), suggesting that exercise-induced AF may have different underlying mechanisms. The prevalence of AF in thoroughbred (TB) and standardbred (STB) racehorses is also relatively high (~5%), suggesting exercise as an independent risk factor. Due to selective breeding and complete control of pedigrees, racehorses are well-suited for genetic studies. Common and rare genetic variation explain a substantial part of AF variance, but no study has identified genetic variants associated with exercise-induced AF in any species. Objective In this project, we aimed to use the racehorse as a model to identify genetic variants associated with exercise-induced AF, and in a future study, possibly replicate the results in human athletes. Here we present preliminary results from the genome-wide association study (GWAS) of AF in racehorses. Methods We collected blood or hair samples from 181 STB and TB racehorses in which 71 had paroxysmal or persistent AF verified by ECG. We extracted DNA and performed whole-genome genotyping using a commercial equine genotyping platform (670k markers). We phased and imputed un-genotyped markers and increased the sample size of the control group, using publicly available equine data from similar breeds from the European Nucleotide Archive. We conducted a GWAS of 7.85 million markers in 71 cases and 296 controls using PLINK2 with covariates adjusted for population stratification, batch and sex. Results We identified one significant locus downstream of the protocadherin gene PCDH18 associated with AF in racehorses. The same locus has been associated with the ECG-trait P-wave terminal force in humans. Changes in P-wave terminal force is associated with increased atrial size, delayed interatrial conduction, left ventricular fibrosis and left atrial function, and considered a good predictor of AF occurrence. Several non-significant but interesting signals were also identified including the genes SLC6A15 (amino acid transport), MYBPH (myosin binding), MYF6 and MYOG (myoblast differentiation). Conclusion In these preliminary results, we have identified a new genetic locus associated with AF in racehorses. In addition, we have identified several candidate genes for further studies to elucidate the genetic background of exercise-induced AF. We aim to replicate our findings in a population of human athletes. Our results may aid the development of risk prediction tools for exercise-induced AF and new drugs for tailored treatment of AF in general.

DNA methylation governs the sensitivity of repeats to restriction by the HUSH-MORC2 corepressor

The human silencing hub (HUSH) complex binds to transcripts of LINE-1 retrotransposons (L1s) and other genomic repeats, recruiting MORC2 and other effectors to remodel chromatin. How HUSH and MORC2 operate alongside DNA methylation, a central epigenetic regulator of repeat transcription, remains largely unknown. Here we interrogate this relationship in human neural progenitor cells (hNPCs), a somatic model of brain development that tolerates removal of DNA methyltransferase DNMT1. Upon loss of MORC2 or HUSH subunit TASOR in hNPCs, L1s remain silenced by robust promoter methylation. However, genome demethylation and activation of evolutionarily-young L1s attracts MORC2 binding, and simultaneous depletion of DNMT1 and MORC2 causes massive accumulation of L1 transcripts. We identify the same mechanistic hierarchy at pericentromeric α-satellites and clustered protocadherin genes, repetitive elements important for chromosome structure and neurodevelopment respectively. Our data delineate the epigenetic control of repeats in somatic cells, with implications for understanding the vital functions of HUSH-MORC2 in hypomethylated contexts throughout human development.

Tuning cohesin trajectories enables differential readout of the Pcdhα cluster across neurons.

Expression of Protocadherin (Pcdh) genes is critical to the generation of neuron identity and wiring of the nervous system. Pcdhα genes are arranged in clusters and exhibit a range of expression profiles, from stochastic to deterministic. Because Pcdhα promoters have high sequence identity and share distal enhancers, how distinct neurons choose which gene to express remains unclear. We show that the interplay between multiple enhancers, epigenetics, and genome folding orchestrates differential readouts of the locus across neurons. The probability of Pcdhα promoter choice depends on enhancer/promoter encounters catalyzed by cohesin, whose extrusion trajectories determine the likelihood that an individual promoter can "escape" heterochromatin-mediated silencing. We propose that tunable locus-specific regulatory elements and cell type-specific cohesin activity underlie the generation of cellular diversity by Pcdh genes.

Abstract 7004: DNA Methylation analysis of African American colorectal cancers reveal race-specific alterations

Abstract African Americans (AAs) have among the highest incidence and mortality rates of colorectal cancer (CRC) in the US. They present with more right-sided, microsatellite stable (MSS) tumors and are diagnosed at earlier ages than non-Hispanic Whites (NHW). While DNA methylation changes and their significance have been previously described in CRC, much less is known about the unique DNA methylation changes that occur in AA CRCs compared to NHW CRCs. In the current study, we analyzed DNA methylation changes in AA patients and compared those changes to data from NHWs. AA patients diagnosed with a MSS colorectal tumor at the Yale New Health System were included in the study. A pathologist reviewed archival slides, and selected areas from CRCs (n=160) and adjacent normal tissue (n=42) were cored (median age=59; 82 females, 78 males). DNA was bisulfite converted and analysis performed using EPIC arrays. The 450K methylation array from NHW CRCs was also analyzed for 217 CRCs and 20 normal tissues obtained from TCGA (median age=68; 103 females, 114 males). Differentially methylated regions (DMRs) with |Δβ|>0.1, FDR corrected p-value <0.05 between tumor and normal tissue were identified using SeSAMe. We identified 4881 DMRs (57% hyper- and 44% hypomethylated) between AA CRC and normal tissue. Of these, 46% were in CpG Islands, 15% in CpG Shores, 4% in CpG shelves, and 36% in open sea. Most DMRs (71%) were in promoter regions, with 18% in gene bodies, 5% in enhancer regions, and 6% in intergenic regions. Next, we performed additional DMR analysis using overlapping probes from both arrays to compare NHW with AA data. We identified 2239 DMRs between AA CRC and normal tissue compared to 4138 in NHW. Of the 2239 AA DMRs, 1419 (66%) were common with NHW DMRs, 790 (34%) were unique to AAs. There were many overlapping pathways and genes; however, the DMRs impacting these pathways differed between AA and NHW CRCs. Hypermethylation of LCK, a tyrosine kinase involved in T-cell receptor signaling, was found in 60% of AA CRCs compared to 5.9% of NHW CRCs. LCK hypermethylation leads to CD4/8 T-cell inactivation and linked to poor outcomes in lung cancer. We observed AA-specific hypermethylation of SSTR1 in 57.2% of AA CRCs compared to 27.6% of NHW CRCs. Somatostasin (SST) suppresses tumor growth by regulating certain factors involved in cell proliferation. Hypermethylation of SSTR1 has been associated with various malignancies including gastric cancer. Lastly, promoter hypermethylation of 3 protocadherin genes was found in 65.1% of AAs compared to 33.1% NHWs. Protocadherins regulate WNT signaling and hypermethylation is associated with poor outcomes in multiple cancers. Taken together, these results show AA-specific CRC methylation changes compared with NHW CRCs. Further work is warranted to investigate the potential functional and clinical implications of these methylation alterations, some of which may represent therapeutic targets and biomarkers. Citation Format: Seeta Rajpara, David N. Buckley, Rosa Xicola, Reger Mikaeel, Mary K. Yagle, Baris Kerimoglu, Curtis Thorne, Dante A. Bellomo, Megha Padi, Xavier Llor, Nathan Ellis, Bodour Salhia. DNA Methylation analysis of African American colorectal cancers reveal race-specific alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7004.

Histone methyltransferase SETD2 is required for proper hippocampal lamination and neuronal maturation.

Proper formation of the hippocampus is crucial for the brain to execute memory and learning functions. However, many questions remain regarding how pyramidal neurons (PNs) of the hippocampus mature and precisely position. Here we revealed that Setd2, the methyltransferase for histone 3 lysine 36 trimethylation (H3K36me3), is essential for the precise localization and maturation of PNs in the hippocampal CA1. The ablation of Setd2 in neural progenitors leads to irregular lamination of the CA1 and increased numbers of PNs in the stratum oriens. Setd2 deletion in postmitotic neurons causes mislocalization and immaturity of CA1 PNs. Transcriptome analyses revealed that SETD2 maintains the expressions of clustered protocadherin (cPcdh) genes. Together, Setd2 is required for proper hippocampal lamination and maturation of CA1 PNs.

Abstract C060: DNA Methylation analysis of African American colorectal cancers reveal race-specific alterations

Abstract African Americans (AAs) have among the highest incidence and mortality rates of colorectal cancer (CRC) in the US. They present with more right-sided, microsatellite stable tumors and are diagnosed at earlier ages than non-Hispanic Whites (NHW). While DNA methylation changes and their significance have been previously described in CRC, much less is known about the unique DNA methylation changes that occur in AA CRCs compared to NHW CRCs. In the current study, we analyzed DNA methylation changes in AA patients and compared those changes to data from NHWs. AA patients diagnosed with a microsatellite-stable colorectal tumor at the Yale New Health System were included in the study. A pathologist reviewed archival slides, and selected areas from tumor (n=160) and adjacent normal tissue (n=42) were cored. The median age of patients was 59 from 82 females and 78 males. Extracted DNA was bisulfite converted and tested using EPIC arrays. DNA methylation (450K) from NHW CRCs was also analyzed for 217 CRCs and 20 normal tissues obtained from TCGA (median age=70; 114 males, 103 females). Differentially methylated regions (DMRs) with |Db|>0.1, FDR corrected p-value <0.05 between tumor and normal tissue were identified using SeSAMe. We identified 4881 DMRs (57% hyper- and 44% hypomethylated) between AA CRC and normal tissue. Of these, 46% were in CpG Islands, 15% in CpG Shores, 4% in CpG shelves, and 36% in open sea. Most DMRs (71%) were in promoter regions, compared with 18% in gene bodies, 5% in enhancer regions, and 6% in intergenic regions. Next, we performed an additional DMR analysis using overlapping probes from the 450K and EPIC arrays to compare TCGA NHW with Yale AA data. We identified 2290 DMRs between AA CRC and normal tissue compared to 4657 in NHW. Of the 2290 AA DMRs, 1405 (61%) were common with NHW DMRs, 885 (39%) were unique to AAs. There were many overlapping pathways and genes involving metabolic functions, DNA damage response, immune response, and WNT signaling. However, the DMRs impacting these pathways differed between AA and NHW CRCs. Several AA-specific DMRs were associated with immune response and included LCK and GPSM3 genes. Hypermethylation of LCK, a tyrosine kinase involved in T-cell receptor signaling, was found in 60% of AA CRCs compared to 8% of NHW CRCs. LCK hypermethylation leads to CD4/8 T-cell inactivation and is linked to poor outcomes in lung cancer. We also observed AA-specific hypermethylation of GPSM3 in 60% of AA CRCs compared to 18% of NHW CRCs. GPSM3 is associated with CD4/8 T-cell infiltration. Lastly, promoter hypermethylation of 6 protocadherin genes was found in 59% of AAs compared to 20% NHWs. Protocadherins play a role in regulating WNT signaling and hypermethylation has been associated with poor outcomes in multiple cancers. Taken together, these results show AA-specific CRC methylation changes compared with NHW CRCs. Further work is warranted to investigate the potential functional and clinical implications of these methylation alterations, some of which may represent therapeutic targets and biomarkers. Citation Format: David N. Buckley, Rosa Xicola, Mikaeel Reger, Mary K. Yagle, Baris Kerimoglu, Curtis Thorne, Dante Bellomo, Megha Padi, Xavier Llor, Nathan Ellis, Bodour Salhia. DNA Methylation analysis of African American colorectal cancers reveal race-specific alterations [abstract]. In: Proceedings of the 16th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2023 Sep 29-Oct 2;Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2023;32(12 Suppl):Abstract nr C060.

D DX41-Low Expression Mimics Germline DDX41-Phenotype in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a complex disease characterized by diverse molecular pathogenesis. Genetic alterations, including germline and somatic variants in the DEAD box helicase 41 gene ( DDX41) located on chromosome 5 play an increasingly recognized role. Recent reports indicate that 5% of intensively treated adult AML patients harbor DDX41 germline mutations ( DDX41MutGL), and their precise impact remains incompletely understood. These studies suggest that DDX41MutGL may define a distinct biological subgroup, associated with e.g. older age, male gender, low blast, and low white blood cell count (WBC). To further elucidate the role of DDX41 in AML, we performed a retrospective analysis of 906 unselected adult AML patients from the AML Cooperative Group (AMLCG) registry (2015-2022), by targeted sequencing. As DDX41MutGL are typically “null mutations”, leading to reduced DDX41 expression, we additionally investigated whether DDX41 gene expression correlates or resembles the observed germline phenotype. Our analysis encompassed >1000 independent gene expression profiles (GSE37642, GSE14468, and GSE106291) of intensively treated adult AML patients. We identified a sub-cohort with low DDX41 ( DDX41-low) expression and re-analyzed them with next-generation sequencing to detect DDX41MutGL. Additionally, we correlated gene expression data with 198 DNA methylation profiles (from patients who had undergone both analyses) to identify potential epigenetic mechanisms underlying DDX41-low expression. Among the 906 patients (median age 61 years; range 18-98 years), we identified 11 unrelated individuals with suspected DDX41MutGL (VAF > 40%). Notably, the overall frequency of DDX41MutGL in the German unselected AML patient population was merely 1%, considerably lower than previously reported (p<0.0001 in comparison to Duployez et al. 2022). Characteristically, DDX41MutGL patients in this cohort were mostly male (73%), with a median age of 67 years displaying low WBC (average 1,3G/l) and a normal karyotype (80%) at initial diagnosis. Remarkably, our analysis of independent large gene expression cohorts revealed a pattern of phenotypic association in patients with decreased DDX41 expression ( DDX41-low) closely resembling those with DDX41MutGL. These associations were consistent across different datasets and included e.g. older age, low WBC, and low blast count. Furthermore, DDX41-low patients had poor overall survival. The DDX41-low subgroup constituted almost 10% of all AML patients, surpassing the reported frequencies of DDX41MutGL. In an exploratory pilot study, we screened 48 patients exhibiting the lowest DDX41 gene expression by sequencing the coding regions for DDX41 mutations but found none, suggesting alternative mechanisms, such as copy number changes, non-coding alterations or aberrant DNA methylation patterns that may replicate the phenotypic effects associated with DDX41MutGL. To further elucidate this mechanism, we correlated the gene expression of DDX41 with all DNA methylation loci (CpG sites covered by the EPIC array) on chromosome 5 using Spearman correlation analysis (GSE106291). We plotted the correlations against the sorted chromosome length, identifying a peak at a specific location corresponding to the PCDH (Protocadherin) cluster (Figure). Thus, DDX41-low was associated with reduced methylation at the PCDH loci and linked to higher PCDH gene expression. Across several data sets, we confirmed the association between higher PCDH cluster gene expression and low DDX41 expression. In summary, our study reveals that the frequency of DDX41MutGL in an unselected population of German AML patients is considerably lower than previously reported. Additionally, we found that apart from DDX41MutGL, DDX41-low exhibits a comparable clinical profile and is linked to specific methylation and gene expression patterns, with a notable emphasis on the PCDH complex located on chromosome 5. Understanding the functional implications of PCDH genes and their interaction with DDX41 in AML may significantly advance our knowledge of AML pathogenesis. Gaining insights into DDX41 alterations may open avenues for personalized therapeutic approaches and further prognostic stratification for AML patients with distinct molecular characteristics.

Identification of quantitative trait loci and associated candidate genes for pregnancy success in Angus–Brahman crossbred heifers

BackgroundIn beef cattle, more than 50% of the energy input to produce a unit of beef is consumed by the female that produced the calf. Development of genomic tools to identify females with high genetic merit for reproductive function could increase the profitability and sustainability of beef production.ResultsGenome-wide association studies (GWAS) were performed using a single-step genomic best linear unbiased prediction approach on pregnancy outcome traits from a population of Angus–Brahman crossbred heifers. Furthermore, a validation GWAS was performed using data from another farm. Heifers were genotyped with the Bovine GGP F250 array that contains 221,077 SNPs. In the discovery population, heifers were bred in winter breeding seasons involving a single round of timed artificial insemination (AI) followed by natural mating for 3 months. Two phenotypes were analyzed: pregnancy outcome to first-service AI (PAI; n = 1,481) and pregnancy status at the end of the breeding season (PEBS; n = 1,725). The heritability was estimated as 0.149 and 0.122 for PAI and PEBS, respectively. In the PAI model, one quantitative trait locus (QTL), located between 52.3 and 52.5 Mb on BTA7, explained about 3% of the genetic variation, in a region containing a cluster of γ-protocadherin genes and SLC25A2. Other QTLs explaining between 0.5% and 1% of the genetic variation were found on BTA12 and 25. In the PEBS model, a large QTL on BTA7 was synonymous with the QTL for PAI, with minor QTLs located on BTA5, 9, 10, 11, 19, and 20. The validation population for pregnancy status at the end of the breeding season were Angus–Brahman crossbred heifers bred by natural mating. In concordance with the discovery population, the large QTL on BTA7 and QTLs on BTA10 and 12 were identified.ConclusionsIn summary, QTLs and candidate SNPs identified were associated with pregnancy outcomes in beef heifers, including a large QTL associated with a group of protocadherin genes. Confirmation of these associations with larger populations could lead to the development of genomic predictions of reproductive function in beef cattle. Moreover, additional research is warranted to study the function of candidate genes associated with QTLs.

Innovative Family-Based Genetically Informed Series of Analyses of Whole-Exome Data Supports Likely Inheritance for Grammar in Children with Specific Language Impairment.

Individuals with specific language impairment (SLI) struggle with language acquisition despite average non-verbal intelligence and otherwise typical development. One SLI account focuses on grammar acquisition delay. The current study aimed to detect novel rare genetic variants associated with performance on a grammar assessment, the Test of Early Grammatical Impairment (TEGI), in English-speaking children. The TEGI was selected due to its sensitivity and specificity, consistently high heritability estimates, and its absence from all but one molecular genetic study. We performed whole exome sequencing (WES) in eight families with SLI (n = 74 total) and follow-up Sanger sequencing in additional unrelated probands (n = 146). We prioritized rare exonic variants shared by individuals with low TEGI performance (n = 34) from at least two families under two filtering workflows: (1) novel and (2) previously reported candidate genes. Candidate variants were observed on six new genes (PDHA2, PCDHB3, FURIN, NOL6, IQGAP3, and BAHCC1), and two genes previously reported for overall language ability (GLI3 and FLNB). We specifically suggest PCDHB3, a protocadherin gene, and NOL6 are critical for ribosome synthesis, as they are important targets of SLI investigation. The proposed SLI candidate genes associated with TEGI performance emphasize the utility of precise phenotyping and family-based genetic study.

PCDHA1 High Expression is Associated With Poor Prognosis and Correlated With Immune Cell Infiltration in Breast Cancer

IntroductionBreast cancer (BC) remains one of the biggest threats to women's health. Protocadherin gene Protocadherin Alpha 1 (PCDHA1) is abnormally highly expressed in breast cancer tissues. However, the biological role of PCDHA1 in breast cancer has not been fully elucidated and the relationship with the immune microenvironment needs to be further studied. Materials and MethodsTCGA-BRCA gene expression profiles were used to characterize PCDHA1. Kaplan-Meier method was used to estimate PCDHA1 prognosis potential. Gene set enrichment analysis (GSEA) analysis was performed to determine the signaling pathways altered by PCDHA1 aberrant expression. The correlations between PCDHA1 and immune cell infiltration levels were analyzed by CIBERSORT. Wilcoxon's rank-sum test was used to identify chemokine and chemokine receptors significantly associated with PCDHA1. The CCK8 assay and the transwell invasion assay were occupied to evaluate the effect of PCDHA1 overexpression on BC cells. ResultsSurvival analysis revealed PCDHA1 overexpression was associated with poor prognosis in BC. Enrichment analysis uncovered several metabolism pathways were activated by PCDHA1 overexpression. Moreover, PCDHA1 was positively correlated with activated NK cells but negatively correlated with resting NK cells infiltration. In addition, chemokines CCL28, CXCL17, and receptor CCR9 expression were associated with PCDHA1 overexpression. The CCK8 assay and the transwell invasion assay proved that PCDHA1 overexpression enhanced MCF-7 and MDA-MB-231 cell proliferation and invasion. ConclusionThis study demonstrated that PCDHA1 effectively predicted BC prognosis. Upregulated PCDHA1 activated metabolism pathways, and promoted NK cells and chemokines. PCDHA1 overexpression enhanced BC cell proliferation and invasion. Therefore, an understanding of PCDHA1’s function in BC may yield insights into the mechanisms of BC development.

The gold-ringed octopus (Amphioctopus fangsiao) genome and cerebral single-nucleus transcriptomes provide insights into the evolution of karyotype and neural novelties

BackgroundColeoid cephalopods have distinctive neural and morphological characteristics compared to other invertebrates. Early studies reported massive genomic rearrangements occurred before the split of octopus and squid lineages (Proc Natl Acad Sci U S A 116:3030-5, 2019), which might be related to the neural innovations of their brain, yet the details remain elusive. Here we combine genomic and single-nucleus transcriptome analyses to investigate the octopod chromosome evolution and cerebral characteristics.ResultsWe present a chromosome-level genome assembly of a gold-ringed octopus, Amphioctopus fangsiao, and a single-nucleus transcriptome of its supra-esophageal brain. Chromosome-level synteny analyses estimate that the chromosomes of the ancestral octopods experienced multiple chromosome fission/fusion and loss/gain events by comparing with the nautilus genome as outgroup, and that a conserved genome organization was detected during the evolutionary process from the last common octopod ancestor to their descendants. Besides, protocadherin, GPCR, and C2H2 ZNF genes are thought to be highly related to the neural innovations in cephalopods (Nature 524:220–4, 2015), and the chromosome analyses pinpointed several collinear modes of these genes on the octopod chromosomes, such as the collinearity between PCDH and C2H2 ZNF, as well as between GPCR and C2H2 ZNF. Phylogenetic analyses show that the expansion of the octopod protocadherin genes is driven by a tandem-duplication mechanism on one single chromosome, including two separate expansions at 65 million years ago (Ma) and 8–14 Ma, respectively. Furthermore, we identify eight cell types (i.e., cholinergic and glutamatergic neurons) in the supra-esophageal brain of A. fangsiao, and the single-cell expression analyses reveal the co-expression of protocadherin and GPCR in specific neural cells, which may contribute to the neural development and signal transductions in the octopod brain.ConclusionsThe octopod genome analyses reveal the dynamic evolutionary history of octopod chromosomes and neural-related gene families. The single-nucleus transcriptomes of the supra-esophageal brain indicate their cellular heterogeneities and functional interactions with other tissues (i.e., gill), which provides a foundation for further octopod cerebral studies.

Correlation between DNA Methylation and Cell Proliferation Identifies New Candidate Predictive Markers in Meningioma.

Meningiomas are the most common primary tumors of the central nervous system. Based on the 2021 WHO classification, they are classified into three grades reflecting recurrence risk and aggressiveness. However, the WHO's histopathological criteria defining these grades are somewhat subjective. Together with reliable immunohistochemical proliferation indices, other molecular markers such as those studied with genome-wide epigenetics promise to revamp the current prognostic classification. In this study, 48 meningiomas of various grades were randomly included and explored for DNA methylation with the Infinium MethylationEPIC microarray over 850k CpG sites. We conducted differential and correlative analyses on grade and several proliferation indices and markers, such as mitotic index and Ki-67 or MCM6 immunohistochemistry. We also set up Cox proportional hazard models for extensive associations between CpG methylation and survival. We identified loci highly correlated with cell growth and a targeted methylation signature of regulatory regions persistently associated with proliferation, grade, and survival. Candidate genes under the control of these regions include SMC4, ESRRG, PAX6, DOK7, VAV2, OTX1, and PCDHA-PCDHB-PCDHG, i.e., the protocadherin gene clusters. This study highlights the crucial role played by epigenetic mechanisms in shaping dysregulated cellular proliferation and provides potential biomarkers bearing prognostic and therapeutic value for the clinical management of meningioma.

The HUSH complex controls brain architecture and protocadherin fidelity

The HUSH (human silencing hub) complex contains the H3K9me3 binding protein M-phase phosphoprotein 8 (MPP8) and recruits the histone methyltransferase SETDB1 as well as Microrchidia CW-type zinc finger protein 2 (MORC2). Functional and mechanistic studies of the HUSH complex have hitherto been centered around SETDB1 while the in vivo functions of MPP8 and MORC2 remain elusive. Here, we show that genetic inactivation of Mphosph8 or Morc2a in the nervous system of mice leads to increased brain size, altered brain architecture, and behavioral changes. Mechanistically, in both mouse brains and human cerebral organoids, MPP8 and MORC2 suppress the repetitive-like protocadherin gene cluster in an H3K9me3-dependent manner. Our data identify MPP8 and MORC2, previously linked to silencing of repetitive elements via the HUSH complex, as key epigenetic regulators of protocadherin expression in the nervous system and thereby brain development and neuronal individuality in mice and humans.

Innovative mouse models for the tumor suppressor activity of Protocadherin-10 isoforms

BackgroundNonclustered mouse protocadherin genes (Pcdh) encode proteins with a typical single ectodomain and a cytoplasmic domain with conserved motifs completely different from those of classic cadherins. Alternative splice isoforms differ in the size of these cytoplasmic domains. In view of the compelling evidence for gene silencing of protocadherins in human tumors, we started investigations on Pcdh functions in mouse cancer models.MethodsFor Pcdh10, we generated two mouse lines: one with floxed exon 1, leading to complete Pcdh10 ablation upon Cre action, and one with floxed exons 2 and 3, leading to ablation of only the long isoforms of Pcdh10. In a mouse medulloblastoma model, we used GFAP-Cre action to locally ablate Pcdh10 in combination with Trp53 and Rb1 ablation. From auricular tumors, that also arose, we obtained tumor-derived cell lines, which were analyzed for malignancy in vitro and in vivo. By lentiviral transduction, we re-expressed Pcdh10 cDNAs. RNA-Seq analyses were performed on these cell families.ResultsSurprisingly, not only medulloblastomas were generated in our model but also tumors of tagged auricles (pinnae). For both tumor types, ablation of either all or only long isoforms of Pcdh10 aggravated the disease. We argued that the perichondrial stem cell compartment is at the origin of the pinnal tumors. Immunohistochemical analysis of these tumors revealed different subtypes. We obtained several pinnal-tumor derived (PTD) cell lines and analyzed these for anchorage-independent growth, invasion into collagen matrices, tumorigenicity in athymic mice. Re-expression of either the short or a long isoform of Pcdh10 in two PTD lines counteracted malignancy in all assays. RNA-Seq analyses of these two PTD lines and their respective Pcdh10-rescued cell lines allowed to identify many interesting differentially expressed genes, which were largely different in the two cell families.ConclusionsA new mouse model was generated allowing for the first time to examine the remarkable tumor suppression activity of protocadherin-10 in vivo. Despite lacking several conserved motifs, the short isoform of Pcdh10 was fully active as tumor suppressor. Our model contributes to scrutinizing the complex molecular mechanisms of tumor initiation and progression upon PCDH10 silencing in many human cancers.

Common deletion variants causing protocadherin-α deficiency contribute to the complex genetics of BAV and left-sided congenital heart disease.

SummaryBicuspid aortic valve (BAV) with ~1%–2% prevalence is the most common congenital heart defect (CHD). It frequently results in valve disease and aorta dilation and is a major cause of adult cardiac surgery. BAV is genetically linked to rare left-heart obstructions (left ventricular outflow tract obstructions [LVOTOs]), including hypoplastic left heart syndrome (HLHS) and coarctation of the aorta (CoA). Mouse and human studies indicate LVOTO is genetically heterogeneous with a complex genetic etiology. Homozygous mutation in the Pcdha protocadherin gene cluster in mice can cause BAV, and also HLHS and other LVOTO phenotypes when accompanied by a second mutation. Here we show two common deletion copy number variants (delCNVs) within the PCDHA gene cluster are associated with LVOTO. Analysis of 1,218 white individuals with LVOTO versus 463 disease-free local control individuals yielded odds ratios (ORs) at 1.47 (95% confidence interval [CI], 1.13–1.92; p = 4.2 × 10−3) for LVOTO, 1.47 (95% CI, 1.10–1.97; p = 0.01) for BAV, 6.13 (95% CI, 2.75–13.7; p = 9.7 × 10−6) for CoA, and 1.49 (95% CI, 1.07–2.08; p = 0.019) for HLHS. Increased OR was observed for all LVOTO phenotypes in homozygous or compound heterozygous PCDHA delCNV genotype comparison versus wild type. Analysis of an independent white cohort (381 affected individuals, 1,352 control individuals) replicated the PCDHA delCNV association with LVOTO. Generalizability of these findings is suggested by similar observations in Black and Chinese individuals with LVOTO. Analysis of Pcdha mutant mice showed reduced PCDHA expression at regions of cell-cell contact in aortic smooth muscle and cushion mesenchyme, suggesting potential mechanisms for BAV pathogenesis and aortopathy. Together, these findings indicate common variants causing PCDHA deficiency play a significant role in the genetic etiology of common and rare LVOTO-CHD.

Mechanism of REST/NRSF regulation of clustered protocadherin α genes.

Repressor element-1 silencing transcription factor (REST) or neuron-restrictive silencer factor (NRSF) is a zinc-finger (ZF) containing transcriptional repressor that recognizes thousands of neuron-restrictive silencer elements (NRSEs) in mammalian genomes. How REST/NRSF regulates gene expression remains incompletely understood. Here, we investigate the binding pattern and regulation mechanism of REST/NRSF in the clustered protocadherin (PCDH) genes. We find that REST/NRSF directionally forms base-specific interactions with NRSEs via tandem ZFs in an anti-parallel manner but with striking conformational changes. In addition, REST/NRSF recruitment to the HS5–1 enhancer leads to the decrease of long-range enhancer-promoter interactions and downregulation of the clustered PCDHα genes. Thus, REST/NRSF represses PCDHα gene expression through directional binding to a repertoire of NRSEs within the distal enhancer and variable target genes.

A developmental lineage-based gene co-expression network for mouse pancreatic β-cells reveals a role for Zfp800 in pancreas development.

To gain a deeper understanding of pancreatic β-cell development, we used iterative weighted gene correlation network analysis to calculate a gene co-expression network (GCN) from 11 temporally and genetically defined murine cell populations. The GCN, which contained 91 distinct modules, was then used to gain three new biological insights. First, we found that the clustered protocadherin genes are differentially expressed during pancreas development. Pcdhγ genes are preferentially expressed in pancreatic endoderm, Pcdhβ genes in nascent islets, and Pcdhα genes in mature β-cells. Second, after extracting sub-networks of transcriptional regulators for each developmental stage, we identified 81 zinc finger protein (ZFP) genes that are preferentially expressed during endocrine specification and β-cell maturation. Third, we used the GCN to select three ZFPs for further analysis by CRISPR mutagenesis of mice. Zfp800 null mice exhibited early postnatal lethality, and at E18.5 their pancreata exhibited a reduced number of pancreatic endocrine cells, alterations in exocrine cell morphology, and marked changes in expression of genes involved in protein translation, hormone secretion and developmental pathways in the pancreas. Together, our results suggest that developmentally oriented GCNs have utility for gaining new insights into gene regulation during organogenesis.

Right Place at the Right Time: How Changes in Protocadherins Affect Synaptic Connections Contributing to the Etiology of Neurodevelopmental Disorders.

During brain development, neurons need to form the correct connections with one another in order to give rise to a functional neuronal circuitry. Mistakes during this process, leading to the formation of improper neuronal connectivity, can result in a number of brain abnormalities and impairments collectively referred to as neurodevelopmental disorders. Cell adhesion molecules (CAMs), present on the cell surface, take part in the neurodevelopmental process regulating migration and recognition of specific cells to form functional neuronal assemblies. Among CAMs, the members of the protocadherin (PCDH) group stand out because they are involved in cell adhesion, neurite initiation and outgrowth, axon pathfinding and fasciculation, and synapse formation and stabilization. Given the critical role of these macromolecules in the major neurodevelopmental processes, it is not surprising that clinical and basic research in the past two decades has identified several PCDH genes as responsible for a large fraction of neurodevelopmental disorders. In the present article, we review these findings with a focus on the non-clustered PCDH sub-group, discussing the proteins implicated in the main neurodevelopmental disorders.

In Colorectal Cancer Cells With Mutant KRAS, SLC25A22-Mediated Glutaminolysis Reduces DNA Demethylation to Increase WNT Signaling, Stemness, and Drug Resistance

Mutant KRAS promotes glutaminolysis, a process that uses steps from the tricarboxylic cycle to convert glutamine to α-ketoglutarate and other molecules via glutaminase and SLC25A22. This results in inhibition of demethylases and epigenetic alterations in cells that increase proliferation and stem cell features. We investigated whether mutant KRAS-mediated glutaminolysis affects the epigenomes and activities of colorectal cancer (CRC) cells. We created ApcminKrasG12D mice with intestine-specific knockout of SLC25A22 (ApcminKrasG12DSLC25A22fl/fl mice). Intestine tissues were collected and analyzed by histology, immunohistochemistry, and DNA methylation assays; organoids were derived and studied for stem cell features, along with organoids derived from 2 human colorectal tumor specimens. Colon epithelial cells (1CT) and CRC cells (DLD1, DKS8, HKE3, and HCT116) that expressed mutant KRAS, with or without knockdown of SLC25A22 or other proteins, were deprived of glutamine or glucose and assayed for proliferation, colony formation, glucose or glutamine consumption, and apoptosis; gene expression patterns were analyzed by RNA sequencing, proteins by immunoblots, and metabolites by liquid chromatography-mass spectrometry, with [U-13C5]-glutamine as a tracer. Cells and organoids with knocked down, knocked out, or overexpressed proteins were analyzed for DNA methylation at CpG sites using arrays. We performed immunohistochemical analyses of colorectal tumor samples from 130 patients in Hong Kong (57 with KRAS mutations) and Kaplan-Meier analyses of survival. We analyzed gene expression levels of colorectal tumor samples in The Cancer Genome Atlas. CRC cells that express activated KRAS required glutamine for survival, and rapidly incorporated it into the tricarboxylic cycle (glutaminolysis); this process required SLC25A22. Cells incubated with succinate and non-essential amino acids could proliferate under glutamine-free conditions. Mutant KRAS cells maintained a low ratio of α-ketoglutarate to succinate, resulting in reduced 5-hydroxymethylcytosine-a marker of DNA demethylation, and hypermethylation at CpG sites. Many of the hypermethylated genes were in the WNT signaling pathway and at the protocadherin gene cluster on chromosome 5q31. CRC cells without mutant KRAS, or with mutant KRAS and knockout of SLC25A22, expressed protocadherin genes (PCDHAC2, PCDHB7, PCDHB15, PCDHGA1, and PCDHGA6)-DNA was not methylated at these loci. Expression of the protocadherin genes reduced WNT signaling to β-catenin and expression of the stem cell marker LGR5. ApcminKrasG12DSLC25A22fl/fl mice developed fewer colon tumors than ApcminKrasG12D mice (P < .01). Organoids from ApcminKrasG12DSLC25A22fl/fl mice had reduced expression of LGR5 and other markers of stemness compared with organoids derived from ApcminKrasG12D mice. Knockdown of SLC25A22 in human colorectal tumor organoids reduced clonogenicity. Knockdown of lysine demethylases, or succinate supplementation, restored expression of LGR5 to SLC25A22-knockout CRC cells. Knockout of SLC25A22 in CRC cells that express mutant KRAS increased their sensitivity to 5-fluorouacil. Level of SLC25A22 correlated with levels of LGR5, nuclear β-catenin, and a stem cell-associated gene expression pattern in human colorectal tumors with mutations in KRAS and reduced survival times of patients. In CRC cells that express activated KRAS, SLC25A22 promotes accumulation of succinate, resulting in increased DNA methylation, activation of WNT signaling to β-catenin, increased expression of LGR5, proliferation, stem cell features, and resistance to 5-fluorouacil. Strategies to disrupt this pathway might be developed for treatment of CRC.

Cognitive Stimulation Induces Differential Gene Expression in Octopus vulgaris: The Key Role of Protocadherins.

Octopuses are unique invertebrates, with sophisticated and flexible behaviors controlled by a high degree of brain plasticity, learning, and memory. Moreover, in Octopus vulgaris, it has been demonstrated that animals housed in an enriched environment show adult neurogenesis in specific brain areas. Firstly, we evaluated the optimal acclimatization period needed for an O. vulgaris before starting a cognitive stimulation experiment. Subsequently, we analyzed differential gene expression in specific brain areas in adult animals kept in tested (enriched environment), wild (naturally enriched environment), and control conditions (unenriched environment). We selected and sequenced three protocadherin genes (PCDHs) involved in the development and maintenance of the nervous system; three Pax genes that control cell specification and tissue differentiation; the Elav gene, an earliest marker for neural cells; and the Zic1 gene, involved in early neural formation in the brain. In this paper, we evaluated gene expression levels in O. vulgaris under different cognitive stimulations. Our data shows that Oct-PCDHs genes are upregulated in the learning and lower motor centers in the brain of both tested and wild animals (higher in the latter). Combining these results with our previous studies on O. vulgaris neurogenesis, we proposed that PCDH genes may be involved in adult neurogenesis processes, and related with their cognitive abilities.