Epigenetic modification of the oxytocin and glucocorticoid receptor genes is linked to attachment avoidance in young adults

Attachment in the context of intimate pair bonds is most frequently studied in terms of the universal strategy to draw near, or away, from significant others at moments of personal distress. However, important interindividual differences in the quality of attachment exist, usually captured through secure versus insecure – anxious and/or avoidant – attachment orientations. Since Bowlby’s pioneering writings on the theory of attachment, it has been assumed that attachment orientations are influenced by both genetic and social factors – what we would today describe and measure as gene by environment interaction mediated by epigenetic DNA modification – but research in humans on this topic remains extremely limited. We for the first time examined relations between intra-individual differences in attachment and epigenetic modification of the oxytocin receptor (OXTR) and glucocorticoid receptor (NR3C1) gene promoter in 109 young adult human participants. Our results revealed that attachment avoidance was significantly and specifically associated with increased OXTR and NR3C1 promoter methylation. These findings offer first tentative clues on the possible etiology of attachment avoidance in humans by showing epigenetic modification in genes related to both social stress regulation and HPA axis functioning

Sex-dependent gene co-expression in the human body

Many pathophysiological mechanisms in human health and disease are dependent on sex. Systems biology approaches are successfully used to decipher human disease etiology, yet the effect of sex on gene network biology is mostly unknown. To address this, we used RNA-sequencing data of over 700 individuals spanning 24 tissues from the Genotype-Tissue Expression project to generate a whole-body gene co-expression map and quantified the sex differences per tissue. We found that of the 13,787 genes analyzed in 24 tissues, 29.5% of the gene co-expression is influenced by sex. For example, skeletal muscle was predominantly enriched with genes co-expressed stronger in males, whereas thyroid primarily contained genes co-expressed stronger in females. This was accompanied by consistent sex differences in pathway enrichment, including hypoxia, epithelial-to-mesenchymal transition, and inflammation over the human body. Furthermore, multi-organ analyses revealed consistent sex-dependent gene co-expression over numerous tissues which was accompanied by enrichment of transcription factor binding motifs in the promoters of these genes. Finally, we show that many sex-biased genes are associated with sex-biased diseases, such as autoimmunity and cancer, and more often the target of FDA-approved drugs than non-sexbiased genes. Our study suggests that sex affects biological gene networks by differences in gene co-expression and that attention to the effect of sex on biological responses to medical drugs is warranted

Sex differences in the genetic and molecular mechanisms of coronary artery disease

Sex differences in coronary artery disease (CAD) presentation, risk factors and prognosis have been widely studied. Similarly, studies on atherosclerosis have shown prominent sex differences in plaque biology. Our understanding of the underlying genetic and molecular mechanisms that drive these differences remains fragmented and largely understudied. Through reviewing genetic and epigenetic studies, we identified more than 40 sex-differential candidate genes (13 within known CAD loci) that may explain, at least in part, sex differences in vascular remodeling, lipid metabolism and endothelial dysfunction. Studies with transcriptomic and single-cell RNA sequencing data from atherosclerotic plaques highlight potential sex differences in smooth muscle cell and endothelial cell biology. Especially, phenotypic switching of smooth muscle cells seems to play a crucial role in female atherosclerosis. This matches the known sex differences in atherosclerotic phenotypes, with men being more prone to lipid-rich plaques, while women are more likely to develop fibrous plaques with endothelial dysfunction. To unravel the complex mechanisms that drive sex differences in CAD, increased statistical power and adjustments to study designs and analysis strategies are required. This entails increasing inclusion rates of women, performing well-defined sex-stratified analyses and the integration of multi-omics data

Intrinsic transcriptomic sex differences in human endothelial cells at birth and in adults are associated with coronary artery disease targets

Sex differences in endothelial cell (EC) biology may reflect intrinsic differences driven by chromosomes or sex steroid exposure and gender differences accumulated over life. We analysed EC gene expression data from boy-girl twins at birth and in non-twin adults to detect sex differences at different stages of life, and show that 14-25% of the EC transcriptome is sex-biased. By combining data from both stages of life, we identified sex differences that are present at birth and maintained throughout life, and those that are acquired over life. Promisingly, we found that genes that present with an acquired sex difference in ECs are more likely to be targets of sex steroids. Annotating both gene sets with data from multiple genome-wide association studies (GWAS) revealed that genes with an intrinsic sex difference in ECs are enriched for coronary artery disease GWAS hits. This study underscores the need for treating sex as a biological variable

Regulation of a progenitor gene program by SOX4 is essential for mammary tumor proliferation

In breast cancer the transcription factor SOX4 has been shown to be associated with poor survival, increased tumor size and metastasis formation. This has mostly been attributed to the ability of SOX4 to regulate Epithelial-to-Mesenchymal-Transition (EMT). However, SOX4 regulates target gene transcription in a context-dependent manner that is determined by the cellular and epigenetic state. In this study we have investigated the loss of SOX4 in mammary tumor development utilizing organoids derived from a PyMT genetic mouse model of breast cancer. Using CRISPR/Cas9 to abrogate SOX4 expression, we found that SOX4 is required for inhibiting differentiation by regulating a subset of genes that are highly activated in fetal mammary stem cells (fMaSC). In this way, SOX4 re-activates an oncogenic transcriptional program that is regulated in many progenitor cell-types during embryonic development. SOX4-knockout organoids are characterized by the presence of more differentiated cells that exhibit luminal or basal gene expression patterns, but lower expression of cell cycle genes. In agreement, primary tumor growth and metastatic outgrowth in the lungs are impaired in SOX4KO tumors. Finally, SOX4KO tumors show a severe loss in competitive capacity to grow out compared to SOX4-proficient cells in primary tumors. Our study identifies a novel role for SOX4 in maintaining mammary tumors in an undifferentiated and proliferative state. Therapeutic manipulation of SOX4 function could provide a novel strategy for cancer differentiation therapy, which would promote differentiation and inhibit cycling of tumor cells

Transcriptomic and Epigenomic Profiling of Histone Deacetylase Inhibitor Treatment Reveals Distinct Gene Regulation Profiles Leading to Impaired Neutrophil Development

The clinical use of histone deacetylase inhibitors (HDACi) for the treatment of bone marrow failure and hematopoietic malignancies has increased dramatically over the last decades. Nonetheless, their effects on normal myelopoiesis remain poorly evaluated. Here, we treated cord blood derived CD34+ progenitor cells with two chemically distinct HDACi inhibitors MS-275 or SAHA and analyzed their effects on the transcriptome (RNA-seq), epigenome (H3K27ac ChIP-seq) and functional and morphological characteristics during neutrophil development. MS-275 (entinostat) selectively inhibits class I HDACs, with a preference for HDAC1, while SAHA (vorinostat) is a non-selective class I/II HDACi. Treatment with individual HDACi resulted in both overlapping and distinct effects on both transcriptome and epigenome, whereas functional effects were relatively similar. Both HDACi resulted in reduced expansion and increased apoptosis in neutrophil progenitor cells. Morphologically, HDACi disrupted normal neutrophil differentiation what was illustrated by decreased percentages of mature neutrophils. In addition, while SAHA treatment clearly showed a block at the promyelocytic stage, MS-275 treatment was characterized by dysplastic features and skewing towards the monocytic lineage. These effects could be mimicked using shRNA-mediated knockdown of HDAC1. Taken together, our data provide novel insights into the effects of HDAC inhibition on normal hematopoietic cells during neutrophil differentiation. These findings should be taken into account when considering the clinical use of MS-275 and SAHA, and can be potentially utilized to tailor more specific, hematopoietic-directed HDACi in the future

Transcriptome sequencing reveals two subtypes of cortisol-secreting adrenocortical tumours in dogs and identifies CYP26B1 as a potential new therapeutic target

Cushing's syndrome (CS) is a serious endocrine disorder that is relatively common in dogs, but rare in humans. In ~15%–20% of cases, CS is caused by a cortisol-secreting adrenocortical tumour (csACT). To identify differentially expressed genes that can improve prognostic predictions after surgery and represent novel treatment targets, we performed RNA sequencing on csACTs (n = 48) and normal adrenal cortices (NACs; n = 10) of dogs. A gene was declared differentially expressed when the adjusted p-value was 2 or < −2. Between NACs and csACTs, 98 genes were differentially expressed. Based on the principal component analysis (PCA) the csACTs were separated in two groups, of which Group 1 had significantly better survival after adrenalectomy (p =.002) than Group 2. Between csACT Group G1 and Group 2, 77 genes were differentially expressed. One of these, cytochrome P450 26B1 (CYP26B1), was significantly associated with survival in both our canine csACTs and in a publicly available data set of 33 human cortisol-secreting adrenocortical carcinomas. In the validation cohort, CYP26B1 was also expressed significantly higher (p =.012) in canine csACTs compared with NACs. In future studies it would be interesting to determine whether CYP26B1 inhibitors could inhibit csACT growth in both dogs and humans

Developmental programming in human umbilical cord vein endothelial cells following fetal growth restriction

Background: Fetal growth restriction (FGR) is associated with an increased susceptibility for various noncommunicable diseases in adulthood, including cardiovascular and renal disease. During FGR, reduced uteroplacental blood flow, oxygen and nutrient supply to the fetus are hypothesized to detrimentally influence cardiovascular and renal programming. This study examined whether developmental programming profiles, especially related to the cardiovascular and renal system, differ in human umbilical vein endothelial cells (HUVECs) collected from pregnancies complicated by placental insufficiency-induced FGR compared to normal growth pregnancies. Our approach, involving transcriptomic profiling by RNA-sequencing and gene set enrichment analysis focused on cardiovascular and renal gene sets and targeted DNA methylation assays, contributes to the identification of targets underlying long-term cardiovascular and renal diseases. Results: Gene set enrichment analysis showed several downregulated gene sets, most of them involved in immune or inflammatory pathways or cell cycle pathways. seven of the 22 significantly upregulated gene sets related to kidney development and four gene sets involved with cardiovascular health and function were downregulated in FGR (n = 11) versus control (n = 8). Transcriptomic profiling by RNA-sequencing revealed downregulated expression of LGALS1, FPR3 and NRM and upregulation of lincRNA RP5-855F14.1 in FGR compared to controls. DNA methylation was similar for LGALS1 between study groups, but relative hypomethylation of FPR3 and hypermethylation of NRM were present in FGR, especially in male offspring. Absolute differences in methylation were, however, small. Conclusion: This study showed upregulation of gene sets related to renal development in HUVECs collected from pregnancies complicated by FGR compared to control donors. The differentially expressed gene sets related to cardiovascular function and health might be in line with the downregulated expression of NRM and upregulated expression of lincRNA RP5-855F14.1 in FG

Genomic DNA Pooling Strategy for Next-Generation Sequencing-Based Rare Variant Discovery in Abdominal Aortic Aneurysm Regions of Interest—Challenges and Limitations

The costs and efforts for sample preparation of hundreds of individuals, their genomic enrichment for regions of interest, and sufficient deep sequencing bring a significant burden to next-generation sequencing-based experiments. We investigated whether pooling of samples at the level of genomic DNA would be a more versatile strategy for lowering the costs and efforts for common disease-associated rare variant detection in candidate genes or associated loci in a substantial patient cohort. We performed a pilot experiment using five pools of 20 abdominal aortic aneurysm (AAA) patients that were enriched on separate microarrays for the reported 9p21.3 associated locus and 42 additional AAA candidate genes, and sequenced on the SOLiD platform. Here, we discuss challenges and limitations connected to this approach and show that the high number of novel variants detected per pool and allele frequency deviations to the usually highly false positive cut-off region for variant detection in non-pooled samples can be limiting factors for successful variant prioritization and confirmation. We conclude that barcode indexing of individual samples before pooling followed by a multiplexed enrichment strategy should be preferred for detection of rare genetic variants in larger sample sets rather than a genomic DNA pooling strategy

Transcriptome analysis reveals microvascular endothelial cell-dependent pericyte differentiation

Microvascular homeostasis is strictly regulated, requiring close interaction between endothelial cells and pericytes. Here, we aimed to improve our understanding of how microvascular crosstalk affects pericytes. Human-derived pericytes, cultured in absence, or presence of human endothelial cells, were studied by RNA sequencing. Compared with mono-cultured pericytes, a total of 6704 genes were differentially expressed in co-cultured pericytes. Direct endothelial contact induced transcriptome profiles associated with pericyte maturation, suppression of extracellular matrix production, proliferation, and morphological adaptation. In vitro studies confirmed enhanced pericyte proliferation mediated by endothelial-derived PDGFB and pericyte-derived HB-EGF and FGF2. Endothelial-induced PLXNA2 and ACTR3 upregulation also triggered pericyte morphological adaptation. Pathway analysis predicted a key role for TGFβ signaling in endothelial-induced pericyte differentiation, whereas the effect of signaling via gap- and adherens junctions was limited. We demonstrate that endothelial cells have a major impact on the transcriptional profile of pericytes, regulating endothelial-induced maturation, proliferation, and suppression of ECM production