Network medicine-based epistasis detection in complex diseases: ready for quantum computing.

Most heritable diseases are polygenic. To comprehend the underlying genetic architecture, it is crucial to discover the clinically relevant epistatic interactions (EIs) between genomic single nucleotide polymorphisms (SNPs) (1-3). Existing statistical computational methods for EI detection are mostly limited to pairs of SNPs due to the combinatorial explosion of higher-order EIs. With NeEDL (network-based epistasis detection via local search), we leverage network medicine to inform the selection of EIs that are an order of magnitude more statistically significant compared to existing tools and consist, on average, of five SNPs. We further show that this computationally demanding task can be substantially accelerated once quantum computing hardware becomes available. We apply NeEDL to eight different diseases and discover genes (affected by EIs of SNPs) that are partly known to affect the disease, additionally, these results are reproducible across independent cohorts. EIs for these eight diseases can be interactively explored in the Epistasis Disease Atlas (https://epistasis-disease-atlas.com). In summary, NeEDL demonstrates the potential of seamlessly integrated quantum computing techniques to accelerate biomedical research. Our network medicine approach detects higher-order EIs with unprecedented statistical and biological evidence, yielding unique insights into polygenic diseases and providing a basis for the development of improved risk scores and combination therapies.

The transcription factor STAT5 is critical in dendritic cells for the development of TH2 but not TH1 responses.

Dendritic cells (DCs) are critical in immune responses, linking innate and adaptive immunity. We found here that DC-specific deletion of the transcription factor STAT5 was not critical for development but was required for T helper type 2 (TH2), but not TH1, allergic responses in both the skin and lungs. Loss of STAT5 in DCs led to the inability to respond to thymic stromal lymphopoietin (TSLP). STAT5 was required for TSLP-dependent DC activation, including upregulation of the expression of costimulatory molecules and chemokine production. Furthermore, TH2 responses in mice with DC-specific loss of STAT5 resembled those seen in mice deficient in the receptor for TSLP. Our results show that the TSLP-STAT5 axis in DCs is a critical component for the promotion of type 2 immunity at barrier surfaces.

Sex-biased genetic programs in liver metabolism and liver fibrosis are controlled by EZH1 and EZH2.

Sex differences in the incidence and progression of many liver diseases, including liver fibrosis and hepatocellular carcinoma, are associated with sex-biased hepatic expression of hundreds of genes. This sexual dimorphism is largely determined by the sex-specific pattern of pituitary growth hormone secretion, which controls a transcriptional regulatory network operative in the context of sex-biased and growth hormone-regulated chromatin states. Histone H3K27-trimethylation yields a major sex-biased repressive chromatin mark deposited at many strongly female-biased genes in male mouse liver, but not at male-biased genes in female liver, and is catalyzed by polycomb repressive complex-2 through its homologous catalytic subunits, Ezh1 and Ezh2. Here, we used Ezh1-knockout mice with a hepatocyte-specific knockout of Ezh2 to investigate the sex bias of liver H3K27-trimethylation and its functional role in regulating sex-differences in the liver. Combined hepatic Ezh1/Ezh2 deficiency led to a significant loss of sex-biased gene expression, particularly in male liver, where many female-biased genes were increased in expression while male-biased genes showed decreased expression. The associated loss of H3K27me3 marks, and increases in the active enhancer marks H3K27ac and H3K4me1, were also more pronounced in male liver. Further, Ezh1/Ezh2 deficiency in male liver, and to a lesser extent in female liver, led to up regulation of many genes linked to liver fibrosis and liver cancer, which may contribute to the observed liver pathologies and the increased sensitivity of these mice to hepatotoxin exposure. Thus, Ezh1/Ezh2-catalyzed H3K27-trimethyation regulates sex-dependent genetic programs in liver metabolism and liver fibrosis through its sex-dependent effects on the epigenome, and may thereby determine the sex-bias in liver disease susceptibility.

Signaling by intrathymic cytokines, not T cell antigen receptors, specifies CD8 lineage choice and promotes the differentiation of cytotoxic-lineage T cells.

Immature CD4(+)CD8(+) (double-positive (DP)) thymocytes are signaled via T cell antigen receptors (TCRs) to undergo positive selection and become responsive to intrathymic cytokines such as interleukin 7 (IL-7). We report here that cytokine signaling is required for positively selected thymocytes to express the transcription factor Runx3, specify CD8 lineage choice and differentiate into cytotoxic-lineage T cells. In DP thymocytes genetically engineered to be cytokine responsive, IL-7 signaling induced TCR-unsignaled DP thymocytes to express Runx3 and to differentiate into mature CD8(+) T cells, completely circumventing positive selection. We conclude that TCR-mediated positive selection converts DP cells into cytokine-responsive thymocytes, but it is subsequent signaling by intrathymic cytokines that specifies CD8 lineage choice and promotes differentiation into cytotoxic-lineage T cells.

Interleukin 7 signaling in dendritic cells regulates the homeostatic proliferation and niche size of CD4+ T cells.

Interleukin 7 (IL-7) and T cell antigen receptor signals have been proposed to be the main drivers of homeostatic T cell proliferation. However, it is not known why CD4(+) T cells undergo less-efficient homeostatic proliferation than CD8(+) T cells do. Here we show that systemic IL-7 concentrations increased during lymphopenia because of diminished use of IL-7 but that IL-7 signaling on IL-7 receptor-alpha-positive (IL-7Ralpha(+)) dendritic cells (DCs) in lymphopenic settings paradoxically diminished the homeostatic proliferation of CD4(+) T cells. This effect was mediated at least in part by IL-7-mediated downregulation of the expression of major histocompatibility complex class II on IL-7Ralpha(+) DCs. Our results indicate that IL-7Ralpha(+) DCs are regulators of the peripheral CD4(+) T cell niche and that IL-7 signals in DCs prevent uncontrolled CD4(+) T cell population expansion in vivo.

Progressing super-enhancer landscape during mammary differentiation controls tissue-specific gene regulation.

The mammary luminal lineage relies on the common cytokine-sensing transcription factor STAT5 to establish super-enhancers during pregnancy and initiate a genetic program that activates milk production. As pups grow, the greatly increasing demand for milk requires progressive differentiation of mammary cells with advancing lactation. Here we investigate how persistent hormonal exposure during lactation shapes an evolving enhancer landscape and impacts the biology of mammary cells. Employing ChIP-seq, we uncover a changing transcription factor occupancy at mammary enhancers, suggesting that their activities evolve with advancing differentiation. Using mouse genetics, we demonstrate that the functions of individual enhancers within the Wap super-enhancer evolve as lactation progresses. Most profoundly, a seed enhancer, which is mandatory for the activation of the Wap super-enhancer during pregnancy, is not required during lactation, suggesting compensatory flexibility. Combinatorial deletions of structurally equivalent constituent enhancers demonstrated differentiation-specific compensatory activities during lactation. We also demonstrate that the Wap super-enhancer, which is built on STAT5 and other common transcription factors, retains its exquisite mammary specificity when placed into globally permissive chromatin, suggesting a limited role of chromatin in controlling cell specificity. Our studies unveil a previously unrecognized progressive enhancer landscape where structurally equivalent components serve unique and differentiation-specific functions.

Octopus-toolkit: a workflow to automate mining of public epigenomic and transcriptomic next-generation sequencing data.

Octopus-toolkit is a stand-alone application for retrieving and processing large sets of next-generation sequencing (NGS) data with a single step. Octopus-toolkit is an automated set-up-and-analysis pipeline utilizing the Aspera, SRA Toolkit, FastQC, Trimmomatic, HISAT2, STAR, Samtools, and HOMER applications. All the applications are installed on the user's computer when the program starts. Upon the installation, it can automatically retrieve original files of various epigenomic and transcriptomic data sets, including ChIP-seq, ATAC-seq, DNase-seq, MeDIP-seq, MNase-seq and RNA-seq, from the gene expression omnibus data repository. The downloaded files can then be sequentially processed to generate BAM and BigWig files, which are used for advanced analyses and visualization. Currently, it can process NGS data from popular model genomes such as, human (Homo sapiens), mouse (Mus musculus), dog (Canis lupus familiaris), plant (Arabidopsis thaliana), zebrafish (Danio rerio), fruit fly (Drosophila melanogaster), worm (Caenorhabditis elegans), and budding yeast (Saccharomyces cerevisiae) genomes. With the processed files from Octopus-toolkit, the meta-analysis of various data sets, motif searches for DNA-binding proteins, and the identification of differentially expressed genes and/or protein-binding sites can be easily conducted with few commands by users. Overall, Octopus-toolkit facilitates the systematic and integrative analysis of available epigenomic and transcriptomic NGS big data.

Dissecting Tissue-Specific Super-Enhancers by Integrating Genome-Wide Analyses and CRISPR/Cas9 Genome Editing.

Recent advances in genome-wide sequencing technologies have provided researchers with unprecedented opportunities to discover the genomic structures of gene regulatory units in living organisms. In particular, the integration of ChIP-seq, RNA-seq, and DNase-seq techniques has facilitated the mapping of a new class of regulatory elements. These elements, called super-enhancers, can regulate cell-type-specific gene sets and even fine-tune gene expression regulation in response to external stimuli, and have become a hot topic in genome biology. However, there is scant genetic evidence demonstrating their unique biological relevance and the mechanisms underlying these biological functions. In this review, we describe a robust genome-wide strategy for mapping cell-type-specific enhancers or super-enhancers in the mammary genome. In this strategy, genome-wide screening of active enhancer clusters that are co-occupied by mammary-enriched transcription factors, co-factors, and active enhancer marks is used to identify bona fide mammary tissue-specific super-enhancers. The in vivo function of these super-enhancers and their associated regulatory elements may then be investigated in various ways using the advanced CRISPR/Cas9 genome-editing technology. Based on our experience targeting various mammary genomic sites using CRISPR/Cas9 in mice, we comprehensively discuss the molecular consequences of the different targeting methods, such as the number of gRNAs and the dependence on their simultaneous or sequential injections. We also mention the considerations that are essential for obtaining accurate results and shed light on recent progress that has been made in developing modified CRISPR/Cas9 genome-editing techniques. In the future, the coupling of advanced genome-wide sequencing and genome-editing technologies could provide new insights into the complex genetic regulatory networks involved in mammary-gland development.

STAT5-Driven Enhancers Tightly Control Temporal Expression of Mammary-Specific Genes.

The de novo formation of milk-secreting mammary epithelium during pregnancy is regulated by prolactin through activation of the transcription factor STAT5, which stimulates the expression of several hundred mammary-specific genes. In addition to its key role in activating gene expression in mammary tissue, STAT5, which is ubiquitously expressed in most cell types, implements T cell-specific programs controlled by interleukins. However, the mechanisms by which STAT5 controls cell-specific genetic programs activated by distinct cytokines remain relatively unknown. Integration of data from genome-wide surveys of chromatin markers and transcription factor binding at regulatory elements may shed light on the mechanisms that drive cell-specific programs. Here, we have illustrated how STAT5 controls cell-specific gene expression through its concentration and an auto-regulatory enhancer supporting its high levels in mammary tissue. The unique genomic features of STAT5-driven enhancers or super-enhancers that regulate mammary-specific genes and their dynamic remodeling in response to pregnancy hormone levels are described. We have further provided biological evidence supporting the in vivo function of a STAT5-driven super-enhancer with the aid of CRISPR/Cas9 genome editing. Finally, we discuss how the functions of mammary-specific super-enhancers are confined by the zinc finger protein, CTCF, to allow exclusive activation of mammary-specific genes without affecting common neighboring genes. This review comprehensively summarizes the molecular pathways underlying differential control of cell-specific gene sets by STAT5 and provides novel insights into STAT5-dependent mammary physiology.

Functional assessment of CTCF sites at cytokine-sensing mammary enhancers using CRISPR/Cas9 gene editing in mice.

The zinc finger protein CTCF has been invoked in establishing boundaries between genes, thereby controlling spatial and temporal enhancer activities. However, there is limited genetic evidence to support the concept that these boundaries restrict the search space of enhancers. We have addressed this question in the casein locus containing five mammary and two non-mammary genes under the control of at least seven putative enhancers. We have identified two CTCF binding sites flanking the locus and two associated with a super-enhancer. Individual deletion of these sites from the mouse genome did not alter expression of any of the genes. However, deletion of the border CTCF site separating the Csn1s1 mammary enhancer from neighboring genes resulted in the activation of Sult1d1 at a distance of more than 95 kb but not the more proximal and silent Sult1e1 gene. Loss of this CTCF site led to de novo interactions between the Sult1d1 promoter and several enhancers in the casein locus. Our study demonstrates that only one out of the four CTCF sites in the casein locus had a measurable in vivo activity. Studies on additional loci are needed to determine the biological role of CTCF sites associated with enhancers.

Differential cytokine sensitivities of STAT5-dependent enhancers rely on Stat5 autoregulation.

Cytokines utilize the transcription factor STAT5 to control cell-specific genes at a larger scale than universal genes, with a mechanistic explanation yet to be supplied. Genome-wide studies have identified putative STAT5-based mammary-specific and universal enhancers, an opportunity to investigate mechanisms underlying their differential response to cytokines. We have now interrogated the integrity and function of both categories of regulatory elements using biological and genetic approaches. During lactation, STAT5 occupies mammary-specific and universal cytokine-responsive elements. Following lactation, prolactin levels decline and mammary-specific STAT5-dependent enhancers are decommissioned within 24 h, while universal regulatory complexes remain intact. These differential sensitivities are linked to STAT5 concentrations and the mammary-specific Stat5 autoregulatory enhancer. In its absence, mammary-specific enhancers, but not universal elements, fail to be fully established. Upon termination of lactation STAT5 binding to a subset of mammary enhancers is substituted by STAT3. No STAT3 binding was observed at the most sensitive STAT5 enhancers suggesting that upon hormone withdrawal their chromatin becomes inaccessible. Lastly, we demonstrate that the mammary-enriched transcription factors GR, ELF5 and NFIB associate with STAT5 at sites lacking bona fide binding motifs. This study provides, for the first time, molecular insight into the differential sensitivities of mammary-specific and universal cytokine-sensing enhancers.

An autoregulatory enhancer controls mammary-specific STAT5 functions.

Signal Transducers and Activators of Transcription (STATs) are principal transcription factors downstream of cytokine receptors. Although STAT5A is expressed in most tissues it remains to be understood why its premier, non-redundant functions are restricted to prolactin-induced mammary gland development and function. We report that the ubiquitously expressed Stat5a/b locus is subject to additional lineage-specific transcriptional control in mammary epithelium. Genome-wide surveys of epigenetic status and transcription factor occupancy uncovered a putative mammary-specific enhancer within the intergenic sequences separating the two Stat5 genes. This region exhibited several hallmarks of genomic enhancers, including DNaseI hypersensitivity, H3K27 acetylation and binding by GR, NFIB, ELF5 and MED1. Mammary-specific STAT5 binding was obtained at two canonical STAT5 binding motifs. CRISPR/Cas9-mediated genome editing was used to delete these sites in mice and determine their biological function. Mutant animals exhibited an 80% reduction of Stat5 levels in mammary epithelium and a concomitant reduction of STAT5-dependent gene expression. Transcriptome analysis identified a class of mammary-restricted genes that was particularly dependent on high STAT5 levels as a result of the intergenic enhancer. Taken together, the mammary-specific enhancer enables a positive feedback circuit that contributes to the remarkable abundance of STAT5 and, in turn, to the efficacy of STAT5-dependent mammary physiology.

Signal transducer and activator of transcription 5 plays a crucial role in hepatic lipid metabolism through regulation of CD36 expression.

AIM: Liver-specific signal transducer and activator of transcription (STAT)5-deficient mice (STAT5KO) show lipid accumulation in the liver. We investigated the role of hepatic STAT5 in lipid metabolism in vitro and in vivo. METHODS AND RESULTS: High expression of CD36, one of the receptors for free fatty acids, is associated with a high concentration of hepatic triglyceride (TG) in STAT5KO mice. Peroxisome proliferator-activated receptor (PPAR)γ, one of the regulatory factors of CD36, was upregulated and microRNA (miR)-20b was downregulated in STAT5KO mice. Reporter assays revealed direct regulation involving miR-20b and the 3'-untranslated region of CD36 mRNA. Treatment with free fatty acids enhanced accumulation of TG in STAT5-deleted hepatoma cells, and this was partially canceled by introduction of siRNA for PPARγ and/or pre-miR-20b through inhibition of CD36 expression. In vivo, STAT5/CD36 double knockout mice displayed hepatic TG was decreased compared to STAT5KO mice and it was also reduced by treatment with PPARγ antagonists, GW9662, and/or pre-miR-20b. CONCLUSIONS: Signal transducer and activator of transcription 5 plays an important role in hepatic fat metabolism through regulation of CD36, and is a potential therapeutic candidate for liver steatosis.

Muscle-specific deletion of signal transducer and activator of transcription 5 augments lipid accumulation in skeletal muscle and liver of mice in response to high-fat diet.

PURPOSE: Growth hormone (GH) controls liver metabolism through the transcription factor signal transducer and activator of transcription 5 (STAT5). However, it remains to be fully understood to what extent other GH/STAT5 target tissues contribute to lipid and glucose metabolism. This question was now addressed in muscle-specific STAT5 knockout (STAT5 MKO) mice model. METHODS: Changes in lipid and glucose metabolism were investigated at physiological and molecular levels in muscle and liver tissues of STAT5 MKO mice under normal diet or high-fat diet (HFD) conditions. RESULTS: STAT5 MKO mice exhibited an increased intramyocellular lipid (IMCL) accumulation in the quadriceps in HFD group. Decreased lipolytic hormone-sensitive lipase transcript levels may contribute to the increased IMCL accumulation in STAT5 MKO mice. STAT5 MKO induced hepatic lipid accumulation without deregulated STAT5 signaling. The upregulation of lipoprotein lipase and Cd36 mRNA levels, an increased trend of very low-density lipoprotein receptor mRNA levels, and elevated circulating concentrations of free fatty acid, triglyceride, and total cholesterol support the increase in hepatic lipid accumulation. CONCLUSIONS: STAT5 MKO in conjunction with a HFD deregulated both lipid and glucose metabolism in skeletal muscle, and this deregulation induced hepatic fat accumulation via increased circulating glucose, FFA, and TG concentrations. Our study emphasizes that muscle-specific STAT5 signaling is important for balancing lipid and glucose metabolism in peripheral tissues, including muscle and liver and that the deregulation of local STAT5 signaling augments HFD-induced lipid accumulation in both muscle and liver.

Loss of EZH2 results in precocious mammary gland development and activation of STAT5-dependent genes.

Establishment and differentiation of mammary alveoli during pregnancy are controlled by prolactin through the transcription factors STAT5A and STAT5B (STAT5), which also regulate temporal activation of mammary signature genes. This study addressed the question whether the methyltransferase and transcriptional co-activator EZH2 controls the differentiation clock of mammary epithelium. Ablation of Ezh2 from mammary stem cells resulted in precocious differentiation of alveolar epithelium during pregnancy and the activation of mammary-specific STAT5 target genes. This coincided with enhanced occupancy of these loci by STAT5, EZH1 and RNA Pol II. Limited activation of differentiation-specific genes was observed in mammary epithelium lacking both EZH2 and STAT5, suggesting a modulating but not mandatory role for STAT5. Loss of EZH2 did not result in overt changes in genome-wide and gene-specific H3K27me3 profiles, suggesting compensation through enhanced EZH1 recruitment. Differentiated mammary epithelia did not form in the combined absence of EZH1 and EZH2. Transplantation experiments failed to demonstrate a role for EZH2 in the activity of mammary stem and progenitor cells. In summary, while EZH1 and EZH2 serve redundant functions in the establishment of H3K27me3 marks and the formation of mammary alveoli, the presence of EZH2 is required to control progressive differentiation of milk secreting epithelium during pregnancy.

Development of mammary luminal progenitor cells is controlled by the transcription factor STAT5A.

Mammary alveologenesis is abrogated in the absence of the transcription factors STAT5A/5B, which mediate cytokine signaling. To reveal the underlying causes for this developmental block, we studied mammary stem and progenitor cells. While loss of STAT5A/5B did not affect the stem cell population and its ability to form mammary ducts, luminal progenitors were greatly reduced and unable to form alveoli during pregnancy. Temporally controlled expression of transgenic STAT5A in mammary epithelium lacking STAT5A/5B restored the luminal progenitor population and rescued alveologenesis in a reversible fashion in vivo. Thus, STAT5A is necessary and sufficient for the establishment of luminal progenitor cells.

Absence of STAT1 in donor-derived plasmacytoid dendritic cells results in increased STAT3 and attenuates murine GVHD.

Selective targeting of non-T cells, including antigen-presenting cells (APCs), is a potential strategy to prevent graft-versus-host-disease (GVHD) but to maintain graft-versus-tumor (GVT) effects. Because type I and II interferons signal through signal transducer and activator of transcription-1 (STAT1), and contribute to activation of APCs after allogeneic bone marrow transplant (alloBMT), we examined whether the absence of STAT1 in donor APCs could prevent GVHD while preserving immune competence. Transplantation of STAT1(-/-) bone marrow (BM) prevented GVHD induced by STAT1(+/+) T cells, leading to expansion of B220(+) cells and regulatory T cells. STAT1(-/-) BM also preserved GVT activity and enhanced overall survival of tumor-challenged mice in the setting of GVHD. Furthermore, recipients of allogeneic STAT1(-/-) BM demonstrated increased CD9(-)Siglec H(hi) plasmacytoid dendritic cells (pDCs), and depletion of pDCs after STAT1(-/-) BM transplantation prevented GVHD resistance. STAT1(-/-) pDCs were found to produce decreased free radicals, IFNα, and interleukin (IL)-12, and increased IL-10. Additionally, STAT1(-/-) pDCs that were isolated after alloBMT showed increased gene expression of S100A8 and S100A9, and transplantation of S100A9(-/-) BM reduced GVHD-free survival. Finally, elevated STAT3 was found in STAT1(-/-) pDCs isolated after alloBMT. We conclude that interfering with interferon signaling in APCs such as pDCs provides a novel approach to regulate the GVHD/GVT axis.

The methyltransferases enhancer of zeste homolog (EZH) 1 and EZH2 control hepatocyte homeostasis and regeneration.

To investigate the role of enhancer of zeste homolog (EZH) 1 and EZH2 in liver homeostasis, mice were generated that carried Ezh1(-/-) and EZH2(fl/fl) alleles and an Alb-Cre transgene. Only the combined loss of EZH1 and EZH2 in mouse hepatocytes caused a depletion of global trimethylation on Lys 27 of histone H3 (H3K27me3) marks and the specific loss of over ∼1900 genes at 3 mo of age. Ezh1(-/-),Ezh2(fl/fl)Alb-Cre mice exhibited progressive liver abnormalities manifested by the development of regenerative nodules and concomitant periportal fibrosis, inflammatory infiltration, and activation of A6-positive hepatic progenitor cells at 8 mo of age. In response to chronic treatment with carbon tetrachloride, all experimental mice, but none of the controls (n = 27 each), showed increased hepatic degeneration associated with liver dysfunction and reduced ability to proliferate. After two-thirds partial hepatectomy, mutant mice (n = 5) displayed increased liver injury and a blunted regenerative response. Genome-wide analyses at 3 mo of age identified 51 genes that had lost H3K27me3 marks, and their expression was significantly increased. These genes were involved in regulation of cell survival, fibrosis, and proliferation. H3K27me3 levels and liver physiology were unaffected in mice lacking either EZH1 globally or EZH2 specifically in hepatocytes. This work demonstrates a critical redundancy of EZH1 and EZH2 in maintaining hepatic homeostasis and regeneration.