Mammary gland development during pregnancy is controlled by lactogenic hormones via the JAK2-STAT5 pathway. Gene deletion studies in mice have revealed the crucial roles of both STAT5A and STAT5B in establishing the genetic programs necessary for the development of mammary epithelium and successful lactation. Several hundred single nucleotide polymorphisms (SNPs) have been identified in human STAT5B, although their pathophysiological significance remains largely unknown. The SH2 domain is vital for STAT5B activation, and this study focuses on the impact of two specific missense mutations identified in T cell leukemias, the substitution of tyrosine 665 with either phenylalanine (Y665F) or histidine (Y665H). By introducing these human mutations into the mouse genome, we uncovered distinct and opposite functions. Mice harboring the STAT5BY665H mutation failed to develop functional mammary tissue, resulting in lactation failure, while STAT5BY665F mice exhibited accelerated mammary development during pregnancy. Transcriptomic and epigenomic analyses identified STAT5BY665H as Loss-Of-Function (LOF) mutation, impairing enhancer establishment and alveolar differentiation, whereas STAT5BY665F acted as a Gain-Of-Function (GOF) mutation, elevating enhancer formation. Persistent hormonal stimulation through two pregnancies led to the establishment of enhancer structures, gene expression and successful lactation in STAT5BY665H mice. Lastly, we demonstrate that Olah, a gene known to drive life-threatening viral disease in humans, is regulated by STAT5B through a candidate four-partite super-enhancer. In conclusion, our findings underscore the role of human STAT5B variants in modulating mammary gland homeostasis and their critical impact on lactation.

The impact of single nucleotide polymorphisms (SNP) on physiology is often underestimated. One amino acid change can result in a variety of phenotypes apparent only in response to disease or injury. Even known pathogenic SNPs have widespread effects that are currently unaccounted for. In this study, we investigated the impact of the known activating and pathogenic STAT5B Y665F mutation in a renal injury context in mice carrying this variant. Using ischemia-reperfusion model of acute kidney injury, immunohistochemistry, RNA-seq and ChIP-seq, we establish the protective role of STAT5B activation in renal epithelium and showcase the shifts in transcriptomic landscape in a tissue not associated with the usual human phenotype of the Stat5b Y665F mutation. Our data indicate new links between the JAK/STAT pathway and known kidney injury markers, contribute to the understanding of the sexual dimorphism of renal disease, and provide new potential targets for JAK inhibitor- and amino acid transport modulation-based therapies.

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.

Transcription enhancers are genomic sequences regulating common and tissue-specific genes and their disruption can contribute to human disease development and progression. Klotho, a sexually dimorphic gene specifically expressed in kidney, is well-linked to kidney dysfunction and its deletion from the mouse genome leads to premature aging and death. However, the sexually dimorphic regulation of Klotho is not understood. Here, we characterize two candidate Klotho enhancers using H3K27ac epigenetic marks and transcription factor binding and investigate their functions, individually and combined, through CRISPR-Cas9 genome engineering. We discovered that only the distal (E1), but not the proximal (E2) candidate region constitutes a functional enhancer, with the double deletion not causing Klotho expression to further decrease. E1 activity is dependent on HNF1b transcription factor binding site within the enhancer. Further, E1 controls the sexual dimorphism of Klotho as evidenced by qPCR and RNA-seq. Despite the sharp reduction of Klotho mRNA, unlike germline Klotho knockouts, mutant mice presented normal phenotype, including weight, lifespan, and serum biochemistry. Lastly, only males lacking E1 display more prominent acute, but not chronic kidney injury responses, indicating a remarkable range of potential adaptation to isolated Klotho loss, especially in female E1 knockouts, retaining renoprotection despite over 80% Klotho reduction.

Motivation Circular RNAs (circRNAs) are long non-coding RNAs (lncRNAs) often associated with diseases and considered potential biomarkers for diagnosis and treatment. Among other functions, circRNAs have been shown to act as microRNA (miRNA) sponges, preventing the role of miRNAs that repress their targets. However, there is no pipeline to systematically assess the sponging potential of circRNAs. Results We developed circRNA-sponging, a nextflow pipeline that (1) identifies circRNAs via backsplicing junctions detected in RNA-seq data, (2) quantifies their expression values in relation to their linear counterparts spliced from the same gene, (3) performs differential expression analysis, (4) identifies and quantifies miRNA expression from miRNA-sequencing (miRNA-seq) data, (5) predicts miRNA binding sites on circRNAs, (6) systematically investigates potential circRNA-miRNA sponging events, (7) creates a network of competing endogenous RNAs, and (8) identifies potential circRNA biomarkers. We showed the functionality of the circRNA-sponging pipeline using RNA sequencing data from brain tissues, where we identified two distinct types of circRNAs characterized by a specific ratio of the number of the binding site to the length of the transcript. The circRNA-sponging pipeline is the first end-to-end pipeline to identify circRNAs and their sponging systematically with raw total RNA-seq and miRNA-seq files, allowing us to better indicate the functional impact of circRNAs as a routine aspect in transcriptomic research. Availability https://github.com/biomedbigdata/circRNA-sponging Contact markus.daniel.hoffmann@tum.de; markus.list@tum.de Supplementary Material Supplementary data are available at Bioinformatic Advances online.

Bulk RNA sequencing (RNA-seq) of blood is an established key technology for analyzing gene expression in health and disease. In order to interpret the immune system's behavior, it is necessary to additionally consider complete blood count (CBC) data offering insights into the abundance of immune cells. However, CBC data is frequently unavailable in published data sets. We employ multiple datasets of patients infected with various SARS-CoV-2 variants (in total 240 samples with up to 200 million reads sequencing depth) to showcase that computational cell-type deconvolution methods (e.g., MCP-counter, xCell, EPIC, quanTIseq) could make such data sets more insightful by estimating immune cell abundances. Furthermore, we can observe varying levels of lymphocyte exhaustion and increased neutrophil levels between SARS-CoV-2 variants and disease progression, indicating markers that could be used in everyday clinical practice to estimate the disease severity of a newly admitted patient once the utilization of RNA-seq becomes feasible in clinics. Additionally, we employ the data to screen for B and T cell receptor (BCR/TCR) sequences using the tools MiXCR and TRUST4 to show that - combined with sequence alignments and pBLAST - they could be used to classify a patient's disease. Finally, we investigated the sequencing depth necessary to perform such analyses and concluded that 10 million reads per sample is sufficient. In conclusion, our study reveals that computational cell-type deconvolution and BCR/TCR methods can supplement missing CBC data in bulk RNA-seq analyses and offer insights into immune responses, disease severity, and pathogen-specific immunity, all achievable with a sequencing depth of 10 million reads per sample.

BACKGROUND:Eukaryotic gene expression is controlled by cis-regulatory elements (CREs), including promoters and enhancers, which are bound by transcription factors (TFs). Differential expression of TFs and their binding affinity at putative CREs determine tissue- and developmental-specific transcriptional activity. Consolidating genomic datasets can offer further insights into the accessibility of CREs, TF activity, and, thus, gene regulation. However, the integration and analysis of multimodal datasets are hampered by considerable technical challenges. While methods for highlighting differential TF activity from combined chromatin state data (e.g., chromatin immunoprecipitation [ChIP], ATAC, or DNase sequencing) and RNA sequencing data exist, they do not offer convenient usability, have limited support for large-scale data processing, and provide only minimal functionality for visually interpreting results. RESULTS:We developed TF-Prioritizer, an automated pipeline that prioritizes condition-specific TFs from multimodal data and generates an interactive web report. We demonstrated its potential by identifying known TFs along with their target genes, as well as previously unreported TFs active in lactating mouse mammary glands. Additionally, we studied a variety of ENCODE datasets for cell lines K562 and MCF-7, including 12 histone modification ChIP sequencing as well as ATAC and DNase sequencing datasets, where we observe and discuss assay-specific differences. CONCLUSION:TF-Prioritizer accepts ATAC, DNase, or ChIP sequencing and RNA sequencing data as input and identifies TFs with differential activity, thus offering an understanding of genome-wide gene regulation, potential pathogenesis, and therapeutic targets in biomedical research.

BackgroundPreclinical studies investigating physiological and pathophysiological processes in the kidney are mainly performed in males. This is due to a higher susceptibility to kidney injury and subsequent disease progression in male rodents. In consequence, sexual dimorphism of kidney injury markers like Klotho is often overlooked. Klotho is well-linked to renal health and its deletion in mice results in severe phenotype and premature death. Here, we identified putative Klotho enhancers and investigated their functions in males and females using mice with deletions in the Klotho-associated enhancers. Experimental designWe generated mutant mice carrying deletion of putative Klotho enhancers using CRISPR/Cas9 gene editing. Warm ischemia-reperfusion surgery was performed bilaterally to induce acute kidney injury and unilaterally in a fibrosis model. Using ChIP-seq and RNA-seq, we analyzed chromatin features and gene expression in mouse kidney. ELISA assay was utilized to measure serum FGF23 levels. ResultsWe detected Klotho gene expression being twice as high in males compared to females at baseline. Enhancer deletion decreased Klotho mRNA levels more effectively in female then in male mice (90 vs. 50%). ChIP-seq data suggest additional regulatory elements present only in male mice, as promoter marks remain more pronounced in males even after deletion. Weight, lifespan and fertility of the knockout mice was not impacted. Baseline serum FGF23 level was significantly higher only in female enhancer knockouts (192.4 vs. 599.6pg/ml, p=0.0003). Severe bilateral ischemia resulted in similar creatinine increase in male and female WT and knockout mice, but only male knockout mice displayed higher Havcr1 expression after injury than controls (1048 vs. 231.4, p=0.0016). 28 days after unilateral renal ischemia, fibrosis as measured by Acta2 and Tgfb expression and Masson Trichrome staining was not significantly changed regardless of genotype. ConclusionsOur results demonstrate sexual dimorphism of Klotho gene expression and its enhancer regulation. Despite having a larger effect in female mice, including changes in baseline FGF23 levels, only male knockout mice are more susceptible to acute injury and the deletion has no impact on the fibrosis model. Further dissection of the mechanisms of Klotho regulation is necessary to reexamine its efficacy as a kidney injury marker.

SARS-CoV-2 infections initiate cytokine storms and activate genetic programs leading to progressive hyperinflammation in multiple organs of patients with COVID-19. While it is known that COVID-19 impacts kidney function, leading to increased mortality, cytokine response of renal epithelium has not been studied in detail. Here, we report on the genetic programs activated in human primary proximal tubule (HPPT) cells by interferons and their suppression by ruxolitinib, a Janus kinase (JAK) inhibitor used in COVID-19 treatment. Integration of our data with those from patients with acute kidney injury and COVID-19, as well as other tissues, permitted the identification of kidney-specific interferon responses. Additionally, we investigated the regulation of the recently discovered isoform (dACE2) of the angiotensin-converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. Using ChIP-seq, we identified candidate interferon-activated enhancers controlling the ACE2 locus, including the intronic dACE2 promoter. Taken together, our study provides an in-depth understanding of genetic programs activated in kidney cells.

Recently, a short, interferon-inducible isoform of Angiotensin-Converting Enzyme 2 (ACE2), dACE2 was identified. ACE2 is a SARS-Cov-2 receptor and changes in its renal expression have been linked to several human nephropathies. These changes were never analyzed in context of dACE2, as its expression was not investigated in the kidney. We used Human Primary Proximal Tubule (HPPT) cells to show genome-wide gene expression patterns after cytokine stimulation, with emphasis on the ACE2/dACE2 locus. Putative regulatory elements controlling dACE2 expression were identified using ChIP-seq and RNA-seq. qRT-PCR differentiating between ACE2 and dACE2 revealed 300- and 600-fold upregulation of dACE2 by IFNα and IFNβ, respectively, while full length ACE2 expression was almost unchanged. JAK inhibitor ruxolitinib ablated STAT1 and dACE2 expression after interferon treatment. Finally, with RNA-seq, we identified a set of genes, largely immune-related, induced by cytokine treatment. These gene expression profiles provide new insights into cytokine response of proximal tubule cells.

Background: Severe COVID-19 causes cytokine storm, worsening patient prognosis and contributing to acute kidney injury (AKI) development. Genetic programs activated in the renal epithelium by cytokines like interferon, as well as those ablated by JAK inhibitors, like ruxolitinib, were previously not investigated in detail. Additionally, a short isoform of ACE2, deltaACE2 (dACE2), of unknown function was recently identified as an interferon-stimulated gene, and it's presence, inducibility and regulation in the kidney was not explored. Methods: We treated Human Primary Proximal Tubule (HPPT) renal epithelial cells with IFNa, IFNβ, IFNg, IL-1β and ruxolitinib and used RNA-seq to explore gene expression patterns. We performed GSEA analysis and compared this data to available AKI and renal COVID-19 datasets, as well as to other human interferon-treated tissues. We also measured mRNA expression of both ACE2 isoforms by RT-qPCR before and after cytokine stimulation and identified changes in gene regulatory elements of the ACE2 locus using ChIP-seq. Results: RNA-seq analysis identified genes significantly induced by IFNα (746), IFNβ (1169), IFNγ (1280) and IL-1β (2142), mostly immunity related. We saw an overlap of 162 genes between IFNβ treatment and the post-AKI dataset and of only 35 with severe COVID-19. Comparison of kidney, lung and liver cells treated with IFNβ revealed a shared set of 153 genes and unique 685 renal genes. Using RT-qPCR we show 300-and 600-fold upregulation of dACE2 mRNA by IFNα and IFNβ, respectively, while full length ACE2 expression is almost unchanged. RNA-seq data revealed abundant fragment mapping to exons corresponding to dACE2 compared to rest of the transcript. ChIP-seq analysis showed additional putative regulatory elements in ACE2 locus, including intragenic enhancers and a dACE2 promoter. JAK inhibitor ruxolitinib successfully ablated 79.5% (929) of genes induced by IFNβ in HPPT cells, including dACE2. Conclusions: We generated and made available novel RNA-seq and ChIP-seq datasets for human renal proximal tubule cells stimulated with cytokines. We observed that type I interferons significantly upregulated only the short isoform of SARS-CoV-2 receptor ACE2 and we linked it to JAK/STAT pathway, which may be an important factor in COVID-19 therapies using JAK inhibitors.

The endogenous repair process can result in recovery after acute kidney injury (AKI) with adaptive proliferation of tubular epithelial cells, but repair can also lead to fibrosis and progressive kidney disease. There is currently limited knowledge about transcriptional regulators regulating these repair programs. Herein we establish the enhancer and superenhancer landscape after AKI by ChIP-seq in uninjured and repairing kidneys on day two after ischemia reperfusion injury (IRI). We identify key transcription factors including HNF4A, GR, STAT3 and STAT5, which show specific binding at enhancer and super-enhancer sites, revealing enhancer dynamics and transcriptional changes during kidney repair. Loss of bromodomain-containing protein 4 function before IRI leads to impaired recovery after AKI and increased mortality. Our comprehensive analysis of epigenetic changes after kidney injury in vivo has the potential to identify targets for therapeutic intervention. Importantly, our data also call attention to potential caveats involved in use of BET inhibitors in patients at risk for AKI.

Background Pannexin1 (Panx1), an ATP release channel, is present in most mammalian tissues, but the role of Panx1 in health and disease is not fully understood. Panx1 may serve to modulate AKI; ATP is a precursor to adenosine and may function to block inflammation, or ATP may act as a danger-associated molecular pattern and initiate inflammation.Methods We used pharmacologic and genetic approaches to evaluate the effect of Panx1 on kidney ischemia-reperfusion injury (IRI), a mouse model of AKI.Results Pharmacologic inhibition of gap junctions, including Panx1, by administration of carbenoxolone protected mice from IRI. Furthermore, global deletion of Panx1 preserved kidney function and morphology and diminished the expression of proinflammatory molecules after IRI. Analysis of bone marrow chimeric mice revealed that Panx1 expressed on parenchymal cells is necessary for ischemic injury, and both proximal tubule and vascular endothelial Panx1 tissue-specific knockout mice were protected from IRI. In vitro, Panx1-deficient proximal tubule cells released less and retained more ATP under hypoxic stress.Conclusions Panx1 is involved in regulating ATP release from hypoxic cells, and reducing this ATP release may protect kidneys from AKI.

The nervous and immune systems interact in complex ways to maintain homeostasis and respond to stress or injury, and rapid nerve conduction can provide instantaneous input for modulating inflammation. The inflammatory reflex referred to as the cholinergic antiinflammatory pathway regulates innate and adaptive immunity, and modulation of this reflex by vagus nerve stimulation (VNS) is effective in various inflammatory disease models, such as rheumatoid arthritis and inflammatory bowel disease. Effectiveness of VNS in these models necessitates the integration of neural signals and α7 nicotinic acetylcholine receptors (α7nAChRs) on splenic macrophages. Here, we sought to determine whether electrical stimulation of the vagus nerve attenuates kidney ischemia-reperfusion injury (IRI), which promotes the release of proinflammatory molecules. Stimulation of vagal afferents or efferents in mice 24 hours before IRI markedly attenuated acute kidney injury (AKI) and decreased plasma TNF. Furthermore, this protection was abolished in animals in which splenectomy was performed 7 days before VNS and IRI. In mice lacking α7nAChR, prior VNS did not prevent IRI. Conversely, adoptive transfer of VNS-conditioned α7nAChR splenocytes conferred protection to recipient mice subjected to IRI. Together, these results demonstrate that VNS-mediated attenuation of AKI and systemic inflammation depends on α7nAChR-positive splenocytes.