Genomic occupancy of Runx2 with global expression profiling identifies a novel dimension to control of osteoblastogenesis

BACKGROUND: Osteogenesis is a highly regulated developmental process and continues during the turnover and repair of mature bone. Runx2, the master regulator of osteoblastogenesis, directs a transcriptional program essential for bone formation through genetic and epigenetic mechanisms. While individual Runx2 gene targets have been identified, further insights into the broad spectrum of Runx2 functions required for osteogenesis are needed. RESULTS: By performing genome-wide characterization of Runx2 binding at the three major stages of osteoblast differentiation--proliferation, matrix deposition and mineralization--we identify Runx2-dependent regulatory networks driving bone formation. Using chromatin immunoprecipitation followed by high-throughput sequencing over the course of these stages, we identify approximately 80,000 significantly enriched regions of Runx2 binding throughout the mouse genome. These binding events exhibit distinct patterns during osteogenesis, and are associated with proximal promoters and also non-promoter regions: upstream, introns, exons, transcription termination site regions, and intergenic regions. These peaks were partitioned into clusters that are associated with genes in complex biological processes that support bone formation. Using Affymetrix expression profiling of differentiating osteoblasts depleted of Runx2, we identify novel Runx2 targets including Ezh2, a critical epigenetic regulator; Crabp2, a retinoic acid signaling component; Adamts4 and Tnfrsf19, two remodelers of the extracellular matrix. We demonstrate by luciferase assays that these novel biological targets are regulated by Runx2 occupancy at non-promoter regions. CONCLUSIONS: Our data establish that Runx2 interactions with chromatin across the genome reveal novel genes, pathways and transcriptional mechanisms that contribute to the regulation of osteoblastogenesis

Protein arginine methyltransferases PRMT1, PRMT4/CARM1 and PRMT5 have distinct functions in control of osteoblast differentiation

Osteogenic differentiation of mesenchymal cells is controlled by epigenetic enzymes that regulate post-translational modifications of histones. Compared to acetyl or methyltransferases, the physiological functions of protein arginine methyltransferases (PRMTs) in osteoblast differentiation remain minimally understood. Therefore, we surveyed the expression and function of all nine mammalian PRMT members during osteoblast differentiation. RNA-seq gene expression profiling shows that Prmt1, Prmt4/Carm1 and Prmt5 represent the most prominently expressed PRMT subtypes in mouse calvarial bone and MC3T3 osteoblasts as well as human musculoskeletal tissues and mesenchymal stromal cells (MSCs). Based on effects of siRNA depletion, it appears that PRMT members have different functional effects: (i) loss of Prmt1 stimulates and (ii) loss of Prmt5 decreases calcium deposition of mouse MC3T3 osteoblasts, while (iii) loss of Carm1 is inconsequential for calcium deposition. Decreased Prmt5 suppresses expression of multiple genes involved in mineralization (e.g., Alpl, Ibsp, Phospho1) consistent with a positive role in osteogenesis. Depletion of Prmt1, Carm1 and Prmt5 has intricate but modest time-dependent effects on the expression of a panel of osteoblast differentiation and proliferation markers but does not change mRNA levels for select epigenetic regulators (e.g., Ezh1, Ezh2, Brd2 and Brd4). Treatment with the Class I PRMT inhibitor GSK715 enhances extracellular matrix mineralization of MC3T3 cells, while blocking formation of H3R17me2a but not H4R3me2a marks. In sum, Prmt1, Carm1 and Prmt5 have distinct biological roles during osteoblast differentiation, and different types histone H3 and H4 arginine methylation may contribute to the chromatin landscape during osteoblast differentiation.</p

Meta-analysis of Genome-Wide Association Studies for Extraversion: Findings from the Genetics of Personality Consortium

Extraversion is a relatively stable and heritable personality trait associated with numerous psychosocial, lifestyle and health outcomes. Despite its substantial heritability, no genetic variants have been detected in previous genome-wide association (GWA) studies, which may be due to relatively small sample sizes of those studies. Here, we report on a large meta-analysis of GWA studies for extraversion in 63,030 subjects in 29 cohorts. Extraversion item data from multiple personality inventories were harmonized across inventories and cohorts. No genome-wide significant associations were found at the single nucleotide polymorphism (SNP) level but there was one significant hit at the gene level for a long non-coding RNA site (LOC101928162). Genome-wide complex trait analysis in two large cohorts showed that the additive variance explained by common SNPs was not significantly different from zero, but polygenic risk scores, weighted using linkage information, significantly predicted extraversion scores in an independent cohort. These results show that extraversion is a highly polygenic personality trait, with an architecture possibly different from other complex human traits, including other personality traits. Future studies are required to further determine which genetic variants, by what modes of gene action, constitute the heritable nature of extraversion

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p&lt;0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p&lt;0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p&lt;0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP &gt;5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Epigenetic regulators controlling osteogenic lineage commitment and bone formation

Bone formation and homeostasis are controlled by environmental factors and endocrine regulatory cues that initiate intracellular signaling pathways capable of modulating gene expression in the nucleus. Bone-related gene expression is controlled by nucleosome-based chromatin architecture that limits the accessibility of lineage-specific gene regulatory DNA sequences and sequence-specific transcription factors. From a developmental perspective, bone-specific gene expression must be suppressed during the early stages of embryogenesis to prevent the premature mineralization of skeletal elements during fetal growth in utero. Hence, bone formation is initially inhibited by gene suppressive epigenetic regulators, while other epigenetic regulators actively support osteoblast differentiation. Prominent epigenetic regulators that stimulate or attenuate osteogenesis include lysine methyl transferases (e.g., EZH2, SMYD2, SUV420H2), lysine deacetylases (e.g., HDAC1, HDAC3, HDAC4, HDAC7, SIRT1, SIRT3), arginine methyl transferases (e.g., PRMT1, PRMT4/CARM1, PRMT5), dioxygenases (e.g., TET2), bromodomain proteins (e.g., BRD2, BRD4) and chromodomain proteins (e.g., CBX1, CBX2, CBX5). This narrative review provides a broad overview of the covalent modifications of DNA and histone proteins that involve hundreds of enzymes that add, read, or delete these epigenetic modifications that are relevant for self-renewal and differentiation of mesenchymal stem cells, skeletal stem cells and osteoblasts during osteogenesis.</p

Epigenetic landscape during osteoblastogenesis defines a differentiation-dependent Runx2 promoter region

Runx2 is a developmentally regulated gene in vertebrates and is essential for bone formation and skeletal homeostasis. The induction of runx2-P1 isoform transcripts is a hallmark of early osteoblastogenesis. Although previous in vitro studies have defined a minimal Runx2-P1 promoter sequence with well-characterized functional elements, several lines of evidence suggest that transcription of the Runx2-P1 isoform relies on elements that extend beyond the previously defined P1 promoter boundaries. In this study, we examined Runx2-P1 transcriptional regulation in a cellular in vivo context during early osteoblastogenesis of MC3T3-E1 cultures and BMSCs induced towards the bone lineage by multi-layered analysis of the Runx2-P1 gene promoter using the following methodologies: 1) sequence homology among several mammalian species, 2) DNaseI hypersensitivity coupled with massively parallel sequencing (DNase-seq), and 3) chromatin immunoprecipitation of activating histone modifications coupled with massively parallel sequencing (ChIP-seq). These epigenetic features have allowed the demarcation of boundaries that redefine the minimal Runx2-P1 promoter to include a 336-bp sequence that mediates responsiveness to osteoblast differentiation. We also find that an additional level of control is contributed by a regulatory region in the 5\u27-UTR of Runx2-P1

DRD2 C957T polymorphism is associated with improved 6-month verbal learning following traumatic brain injury

Traumatic brain injury (TBI) often leads to heterogeneous clinical outcomes, which may be influenced by genetic variation. A single-nucleotide polymorphism (SNP) in the dopamine D2 receptor (DRD2) may influence cognitive deficits following TBI. However, part of the association with DRD2 has been attributed to genetic variability within the adjacent ankyrin repeat and kinase domain containing 1 protein (ANKK1). Here, we utilize the Transforming Research and Clinical Knowledge in Traumatic Brain Injury Pilot (TRACK-TBI Pilot) study to investigate whether a novel DRD2 C957T polymorphism (rs6277) influences outcome on a cognitive battery at 6 months following TBI—California Verbal Learning Test (CVLT-II), Wechsler Adult Intelligence Test Processing Speed Index Composite Score (WAIS-PSI), and Trail Making Test (TMT). Results in 128 Caucasian subjects show that the rs6277 T-allele associates with better verbal learning and recall on CVLT-II Trials 1–5 (T-allele carrier 52.8 ± 1.3 points, C/C 47.9 ± 1.7 points; mean increase 4.9 points, 95% confidence interval [0.9 to 8.8]; p = 0.018), Short-Delay Free Recall (T-carrier 10.9 ± 0.4 points, C/C 9.7 ± 0.5 points; mean increase 1.2 points [0.1 to 2.5]; p = 0.046), and Long-Delay Free Recall (T-carrier 11.5 ± 0.4 points, C/C 10.2 ± 0.5 points; mean increase 1.3 points [0.1 to 2.5]; p = 0.041) after adjusting for age, education years, Glasgow Coma Scale, presence of acute intracranial pathology on head computed tomography scan, and genotype of the ANKK1 SNP rs1800497 using multivariable regression. No association was found between DRD2 C947T and non-verbal processing speed (WAIS-PSI) or mental flexibility (TMT) at 6 months. Hence, DRD2 C947T (rs6277) may be associated with better performance on select cognitive domains independent of ANKK1 following TBI