Bioinformatics-based analysis of potential candidates chromatin regulators for immune infiltration in osteoarthritis.

BACKGROUND: Through the bioinformatics analysis to screen out the potential chromatin regulators (CRs) under the immune infiltration of osteoarthritis (OA), thus providing some theoretical support for future studies of epigenetic mechanisms under OA immune infiltration. METHODS: By integrating CRs and the OA gene expression matrix, we performed weighted gene co-expression network analysis (WGCNA), differential analysis, and further screened Hub genes by protein-protein interaction (PPI) analysis. Using the OA gene expression matrix, immune infiltration extraction and quantification were performed to analyze the correlations and differences between immune infiltrating cells and their functions. By virtue of these Hub genes, Hub gene association analysis was completed and their upstream miRNAs were predicted by the FunRich software. Moreover, a risk model was established to analyze the risk probability of associated CRs in OA, and the confidence of the results was validated by the validation dataset. RESULTS: This research acquired a total of 32 overlapping genes, and 10 Hub genes were further identified. The strongest positive correlation between dendritic cells and mast cells and the strongest negative correlation between parainflammation and Type I IFN reponse. In the OA group DCs, iDCs, macrophages, MCs, APC co-inhibition, and CCR were significantly increased, whereas B cells, NK cells, Th2 cells, TIL, and T cell co-stimulation were significantly decreased. The risk model results revealed that BRD1 might be an independent risk factor for OA, and the validation dataset results are consistent with it. 60 upstream miRNAs of OA-related CRs were predicted by the FunRich software. CONCLUSION: This study identified certain potential CRs and miRNAs that could regulate OA immunity, thus providing certain theoretical supports for future epigenetic mechanism studies on the immune infiltration of OA.

Phenotypic Variability in Phelan-McDermid Syndrome and Its Putative Link to Environmental Factors.

Phelan-McDermid syndrome (PMS) is a multi-systemic disorder characterized by both genetic and phenotypic variability. Genetic abnormalities causing PMS span from pathogenic variants of the SHANK3 gene to chromosomal rearrangements affecting the 22q13 region and leading to the loss of up to over nine megabases. The clinical presentation of individuals with PMS includes intellectual disability, neonatal hypotonia, delayed or absent speech, developmental delay, and minor dysmorphic facial features. Several other features may present with differences in age of onset and/or severity: seizures, autism, regression, sleep disorders, gastrointestinal problems, renal disorders, dysplastic toenails, and disrupted thermoregulation. Among the causes of this phenotypic variability, the size of the 22q13 deletion has effects that may be influenced by environmental factors interacting with haploinsufficiency or hemizygous variants of certain genes. Another mechanism linking environmental factors and phenotypic variability in PMS involves the loss of one copy of genes like BRD1 or CYP2D6, located at 22q13 and involved in the regulation of genomic methylation or pharmacokinetics, which are also influenced by external agents, such as diet and drugs. Overall, several non-mutually exclusive genetic and epigenetic mechanisms interact with environmental factors and may contribute to the clinical variability observed in individuals with PMS. Characterization of such factors will help to better manage this disorder.

Inactivation of the Schizophrenia-associated BRD1 gene in Brain Causes Failure-to-thrive, Seizure Susceptibility and Abnormal Histone H3 Acetylation and N-tail Clipping.

Genetic studies have repeatedly shown that the Bromodomain containing 1 gene, BRD1, is involved in determining mental health, and the importance of the BRD1 protein for normal brain function has been studied in both cell models and constitutive haploinsufficient Brd1+/- mice. Homozygosity for inactivated Brd1 alleles is lethal during embryonic development in mice. In order to further characterize the molecular functions of BRD1 in the brain, we have developed a novel Brd1 knockout mouse model (Brd1-/-) with bi-allelic conditional inactivation of Brd1 in the central nervous system. Brd1-/- mice were viable but smaller and with reduced muscle strength. They showed reduced exploratory behavior and increased sensitivity to pentylenetetrazole-induced seizures supporting the previously described GABAergic dysfunction in constitutive Brd1+/- mice. Because BRD1 takes part in protein complexes with histone binding and modifying functions, we investigated the effect of BRD1 depletion on the global histone modification pattern in mouse brain by mass spectrometry. We found decreased levels of histone H3 acetylation (H3K9ac, H3K14ac, and H3K18ac) and increased N-tail clipping in consequence of BRD1 depletion. Collectively, the presented results support that BRD1 controls gene expression at the epigenetic level by regulating histone H3 proteoforms in the brain.

Transplanted pluripotent stem cell-derived photoreceptor precursors elicit conventional and unusual light responses in mice with advanced retinal degeneration.

Retinal dystrophies often lead to blindness. Developing therapeutic interventions to restore vision is therefore of paramount importance. Here we demonstrate the ability of pluripotent stem cell-derived cone precursors to engraft and restore light responses in the Pde6brd1 mouse, an end-stage photoreceptor degeneration model. Our data show that up to 1.5% of precursors integrate into the host retina, differentiate into cones, and engraft in close apposition to the host bipolar cells. Half of the transplanted mice exhibited visual behavior and of these 33% showed binocular light sensitivity. The majority of retinal ganglion cells exhibited contrast-sensitive ON, OFF or ON-OFF light responses and even motion sensitivity; however, quite a few exhibited unusual responses (eg, light-induced suppression), presumably reflecting remodeling of the neural retina. Our data indicate that despite relatively low engraftment yield, pluripotent stem cell-derived cone precursors can elicit light responsiveness even at advanced degeneration stages. Further work is needed to improve engraftment yield and counteract retinal remodeling to achieve useful clinical applications.

DNA methylation epi-signature is associated with two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

BACKGROUND: Phelan-McDermid syndrome is characterized by a range of neurodevelopmental phenotypes with incomplete penetrance and variable expressivity. It is caused by a variable size and breakpoint microdeletions in the distal long arm of chromosome 22, referred to as 22q13.3 deletion syndrome, including the SHANK3 gene. Genetic defects in a growing number of neurodevelopmental genes have been shown to cause genome-wide disruptions in epigenomic profiles referred to as epi-signatures in affected individuals. RESULTS: In this study we assessed genome-wide DNA methylation profiles in a cohort of 22 individuals with Phelan-McDermid syndrome, including 11 individuals with large (2 to 5.8 Mb) 22q13.3 deletions, 10 with small deletions (< 1 Mb) or intragenic variants in SHANK3 and one mosaic case. We describe a novel genome-wide DNA methylation epi-signature in a subset of individuals with Phelan-McDermid syndrome. CONCLUSION: We identified the critical region including the BRD1 gene as responsible for the Phelan-McDermid syndrome epi-signature. Metabolomic profiles of individuals with the DNA methylation epi-signature showed significantly different metabolomic profiles indicating evidence of two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

Meiotic Double-Strand Break Processing and Crossover Patterning Are Regulated in a Sex-Specific Manner by BRCA1-BARD1 in Caenorhabditis elegans.

Meiosis is regulated in a sex-specific manner to produce two distinct gametes, sperm and oocytes, for sexual reproduction. To determine how meiotic recombination is regulated in spermatogenesis, we analyzed the meiotic phenotypes of mutants in the tumor suppressor E3 ubiquitin ligase BRC-1-BRD-1 complex in Caenorhabditis elegans male meiosis. Unlike in mammals, this complex is not required for meiotic sex chromosome inactivation, the process whereby hemizygous sex chromosomes are transcriptionally silenced. Interestingly, brc-1 and brd-1 mutants show meiotic recombination phenotypes that are largely opposing to those previously reported for female meiosis. Fewer meiotic recombination intermediates marked by the recombinase RAD-51 were observed in brc-1 and brd-1 mutants, and the reduction in RAD-51 foci could be suppressed by mutation of nonhomologous-end-joining proteins. Analysis of GFP::RPA-1 revealed fewer foci in the brc-1brd-1 mutant and concentration of BRC-1-BRD-1 to sites of meiotic recombination was dependent on DNA end resection, suggesting that the complex regulates the processing of meiotic double-strand breaks to promote repair by homologous recombination. Further, BRC-1-BRD-1 is important to promote progeny viability when male meiosis is perturbed by mutations that block the pairing and synapsis of different chromosome pairs, although the complex is not required to stabilize the RAD-51 filament as in female meiosis under the same conditions. Analyses of crossover designation and formation revealed that BRC-1-BRD-1 inhibits supernumerary COs when meiosis is perturbed. Together, our findings suggest that BRC-1-BRD-1 regulates different aspects of meiotic recombination in male and female meiosis.

Deciphering structure, function and mechanism of lysine acetyltransferase HBO1 in protein acetylation, transcription regulation, DNA replication and its oncogenic properties in cancer.

HBO1 complexes are major acetyltransferase responsible for histone H4 acetylation in vivo, which belongs to the MYST family. As the core catalytic subunit, HBO1 consists of an N-terminal domain and a C-terminal MYST domain that are in charge of acetyl-CoA binding and acetylation reaction. HBO1 complexes are multimeric and normally consist of two native subunits MEAF6, ING4 or ING5 and two kinds of cofactors as chromatin reader: Jade-1/2/3 and BRPF1/2/3. The choices of subunits to form the HBO1 complexes provide a regulatory switch to potentiate its activity between histone H4 and H3 tails. Thus, HBO1 complexes present multiple functions in histone acetylation, gene transcription, DNA replication, protein ubiquitination, and immune regulation, etc. HBO1 is a co-activator for CDT1 to facilitate chromatin loading of MCM complexes and promotes DNA replication licensing. This process is regulated by mitotic kinases such as CDK1 and PLK1 by phosphorylating HBO1 and modulating its acetyltransferase activity, therefore, connecting histone acetylation to regulations of cell cycle and DNA replication. In addition, both gene amplification and protein overexpression of HBO1 confirmed its oncogenic role in cancers. In this paper, we review the recent advances and discuss our understanding of the multiple functions, activity regulation, and disease relationship of HBO1.

CRX Expression in Pluripotent Stem Cell-Derived Photoreceptors Marks a Transplantable Subpopulation of Early Cones.

Death of photoreceptors is a common cause of age-related and inherited retinal dystrophies, and thus their replenishment from renewable stem cell sources is a highly desirable therapeutic goal. Human pluripotent stem cells provide a useful cell source in view of their limitless self-renewal capacity and potential to not only differentiate into cells of the retina but also self-organize into tissue with structure akin to the human retina as part of three-dimensional retinal organoids. Photoreceptor precursors have been isolated from differentiating human pluripotent stem cells through application of cell surface markers or fluorescent reporter approaches and shown to have a similar transcriptome to fetal photoreceptors. In this study, we investigated the transcriptional profile of CRX-expressing photoreceptor precursors derived from human pluripotent stem cells and their engraftment capacity in an animal model of retinitis pigmentosa (Pde6brd1), which is characterized by rapid photoreceptor degeneration. Single cell RNA-Seq analysis revealed the presence of a dominant cell cluster comprising 72% of the cells, which displayed the hallmarks of early cone photoreceptor expression. When transplanted subretinally into the Pde6brd1 mice, the CRX+ cells settled next to the inner nuclear layer and made connections with the inner neurons of the host retina, and approximately one-third of them expressed the pan cone marker, Arrestin 3, indicating further maturation upon integration into the host retina. Together, our data provide valuable molecular insights into the transcriptional profile of human pluripotent stem cells-derived CRX+ photoreceptor precursors and indicate their usefulness as a source of transplantable cone photoreceptors. Stem Cells 2019;37:609-622.

BRCA1 and BARD1 mediate apoptotic resistance but not longevity upon mitochondrial stress in Caenorhabditis elegans.

Interventions that promote healthy aging are typically associated with increased stress resistance. Paradoxically, reducing the activity of core biological processes such as mitochondrial or insulin metabolism promotes the expression of adaptive responses, which in turn increase animal longevity and resistance to stress. In this study, we investigated the relation between the extended Caenorhabditis elegans lifespan elicited by reduction in mitochondrial functionality and resistance to genotoxic stress. We find that reducing mitochondrial activity during development confers germline resistance to DNA damage-induced cell cycle arrest and apoptosis in a cell-non-autonomous manner. We identified the C. elegans homologs of the BRCA1/BARD1 tumor suppressor genes, brc-1/brd-1, as mediators of the anti-apoptotic effect but dispensable for lifespan extension upon mitochondrial stress. Unexpectedly, while reduced mitochondrial activity only in the soma was not sufficient to promote longevity, its reduction only in the germline or in germline-less strains still prolonged lifespan. Thus, in animals with partial reduction in mitochondrial functionality, the mechanisms activated during development to safeguard the germline against genotoxic stress are uncoupled from those required for somatic robustness and animal longevity.

The Schizophrenia-Associated BRD1 Gene Regulates Behavior, Neurotransmission, and Expression of Schizophrenia Risk Enriched Gene Sets in Mice.

BACKGROUND: The schizophrenia-associated BRD1 gene encodes a transcriptional regulator whose comprehensive chromatin interactome is enriched with schizophrenia risk genes. However, the biology underlying the disease association of BRD1 remains speculative. METHODS: This study assessed the transcriptional drive of a schizophrenia-associated BRD1 risk variant in vitro. Accordingly, to examine the effects of reduced Brd1 expression, we generated a genetically modified Brd1+/- mouse and subjected it to behavioral, electrophysiological, molecular, and integrative genomic analyses with focus on schizophrenia-relevant parameters. RESULTS: Brd1+/- mice displayed cerebral histone H3K14 hypoacetylation and a broad range of behavioral changes with translational relevance to schizophrenia. These behaviors were accompanied by striatal dopamine/serotonin abnormalities and cortical excitation-inhibition imbalances involving loss of parvalbumin immunoreactive interneurons. RNA-sequencing analyses of cortical and striatal micropunches from Brd1+/- and wild-type mice revealed differential expression of genes enriched for schizophrenia risk, including several schizophrenia genome-wide association study risk genes (e.g., calcium channel subunits [Cacna1c and Cacnb2], cholinergic muscarinic receptor 4 [Chrm4)], dopamine receptor D2 [Drd2], and transcription factor 4 [Tcf4]). Integrative analyses further found differentially expressed genes to cluster in functional networks and canonical pathways associated with mental illness and molecular signaling processes (e.g., glutamatergic, monoaminergic, calcium, cyclic adenosine monophosphate [cAMP], dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa [DARPP-32], and cAMP responsive element binding protein signaling [CREB]). CONCLUSIONS: Our study bridges the gap between genetic association and pathogenic effects and yields novel insights into the unfolding molecular changes in the brain of a new schizophrenia model that incorporates genetic risk at three levels: allelic, chromatin interactomic, and brain transcriptomic.

GSK6853, a Chemical Probe for Inhibition of the BRPF1 Bromodomain.

The BRPF (Bromodomain and PHD Finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. A selective benzimidazolone BRPF1 inhibitor showing micromolar activity in a cellular target engagement assay was recently described. Herein, we report the optimization of this series leading to the identification of a superior BRPF1 inhibitor suitable for in vivo studies.

1,3-Dimethyl Benzimidazolones Are Potent, Selective Inhibitors of the BRPF1 Bromodomain.

The BRPF (bromodomain and PHD finger-containing) protein family are important scaffolding proteins for assembly of MYST histone acetyltransferase complexes. Here, we report the discovery, binding mode, and structure-activity relationship (SAR) of the first potent, selective series of inhibitors of the BRPF1 bromodomain.