Genetic Approaches To Understanding Organ Development and Function

National Institute Of Diabetes And Digestive And Kidney Diseases

Investigators

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Abstract

Our research is based on a multidisciplinary approach, using tools of biochemistry, genetics and computational science. In this reporting period, our studies on genetic control mechanisms executed by the JAK/STAT signaling pathway resulted in six publications. One extended study investigated the physiological and pathological impact of a specific human mutations that had been identified in the transcription factor STAT5B in human leukemias. A second study focused on an in depth understanding of a human mutation in the transcription factor STAT3 that had been identified in an individual with Job’s syndrome. A third study used state-of-the-art computational tools and data-driven computational protocols to explore and predict the impact of mutations in candidate regulatory regions within the genome. Lastly, we used computational tools to interrogate human germline and somatic mutations reported in large databases Objective 1 Impact of human STAT5B mutation in immune homeostasis, mammary function and kidney injury STAT5B is a vital transcription factor for lymphocytes. In this study we interrogated the function of two STAT5B mutations from human T-cell leukemias: one substituting tyrosine 665 with phenylalanine (STAT5B Y665F) and the other with histidine (STAT5B Y665H) (PMID: 40228864). In silico modeling predicted divergent energetic effects on homodimerization with a range of pathogenicity. In primary T cells in vitro, STAT5B Y665F showed gain-of-function, whereas STAT5B Y665H demonstrated loss-of-function. Introducing the mutation into the mouse genome illustrated that the gain-of function Stat5b Y665F mutation resulted in accumulation of CD8+ effector and memory and CD4+ regulatory T cells, altering CD8+/CD4+ ratios. In contrast, STAT5B Y665H "knock-in" mice showed diminished CD8+ effector and memory and CD4+ regulatory T cells. In contrast to WT STAT5B, the STAT5B Y665F variant displayed greater STAT5 phosphorylation, DNA binding, and transcriptional activity after cytokine activation, whereas the STAT5BY665H variant resembled a null. The work exemplifies how joining in silico and in vivo studies of single nucleotides deepens our understanding of disease-associated variants, revealing structural determinants of altered function, defining mechanistic roles, and, specifically here, identifying a gain-of-function variant that does not directly induce hematopoietic malignancy. In contrast to the immune system, these mutations had a significantly different impact in mammary (PMID: 40163145) and kidney tissues (PMID: 40841451). 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 focused 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 STAT5B Y665H mutation failed to develop functional mammary tissue, resulting in lactation failure, while STAT5B Y665F mice exhibited accelerated mammary development during pregnancy. Transcriptomic and epigenomic analyses identified STAT5B Y665H 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 Stat5bnY665F mutation in a renal injury context in mice carrying this variant (PMID: 40841451). 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. Objective 2 Understanding human STAT3 mutations in Job’s syndrome STAT3 mutations are commonly observed in human pathology yet have no uniform patient presentation. Their effects range from cancer and autoimmunity to primary immunodeficiencies and bone deformity. Designing animal models of those mutations can help researchers identify their direct effects to better inform the clinical setting. In this study, we investigated a mouse model harboring the same mutation as an autosomal-dominant hyper-IgE syndrome (AD-HIES) patient reported in the literature (PMID: 40868996). Surprisingly, while the deletion of five amino acids in the SH2 domain of STAT3 did result in frequency changes in several immune populations as measured by complete blood count and flow cytometry analysis, it did not yield the expected phenotype of AD-HIES, with no increase in serum IgE or eosinophil count. We additionally provide structural analysis of the STAT3 G656_M660del deletion, visualizing changes in protein architecture and potential effects on the neighboring Y705 phosphorylation site. Our model showcases the sexually dimorphic immune dysregulation caused by a STAT3 mutation and highlights that predicted gain- and loss-of-function mutations can yield unexpected phenotypes. Objective 3 Data-driven projections of candidate enhancer-activating mutations in immune regulation Millions of single nucleotide polymorphisms (SNPs) have been identified in humans, but the functionality of almost all SNPs remains unclear. While current research focuses primarily on SNPs altering one amino acid to another one, most SNPs are in intergenic spaces. Some of these SNPs can be found in candidate cis-regulatory elements (CREs) such as promoters and enhancers, potentially destroying or creating DNA-binding motifs for transcription factors (TFs) and, hence, deregulating the expression of nearby genes. These aspects are understudied due to the sheer number of SNPs and TF binding motifs, making it challenging to identify SNPs that yield phenotypic changes or altered gene expression. We developed a data-driven computational protocol to prioritize high-potential SNPs informed from former knowledge for experimental validation (PMID: 40011812). We evaluated the protocol by investigating SNPs in CREs in the Janus kinase (JAK) - Signal Transducer and Activator of Transcription (-STAT) signaling pathway, which is activated by a plethora of cytokines and crucial in controlling immune responses and has been implicated in diseases like cancer, autoimmune disorders, and responses to viral infections. The protocol involves scanning the entire human genome (hg38) to pinpoint DNA sequences that deviate by only one nucleotide from the canonical binding sites (TTCnnnGAA) for STAT TFs. We narrowed down from an initial pool of 3,301,512 SNPs across 17,039,967 nearly complete STAT motifs and identified six potential gain-of-function SNPs in regions likely to influence regulation within the JAK-STAT pathway. This selection was guided by publicly available open chromatin and gene expression data and further refined by filtering for proximity to immune response genes and conservation between the mouse and human genomes. Our findings highlight the value of combining genomic, epigenomic, and cross-species conservation data to effectively narrow down millions of SNPs to a smaller number with a high potential to induce interferon regulation of nearby genes. These SNPs can finally be reviewed manually, laying the groundwork for a more focused and efficient exploration of regulatory SNPs in an experimental setting. Objective 4 Database approach to understand the impact of somatic and germline mutationsThe JAK-STAT pathway is central to cytokine signaling and controls normal physiology and disease. Aberrant activation via mutations that change amino acids in proteins of the pathway can result in diseases. While disease-centric databases like COSMIC catalog mutations in cancer, their prevalence in healthy populations remains underexplored. We systematically studied such mutations in the JAK-STAT genes by comparing COSMIC and the population-focused All of Us database (PMID: 39979591). Our study revealed frequent mutations in all JAK and STAT domains, particularly among white females. We further identified three categories: Mutations uniquely found in All of Us that were associated with cancer in the literature but could not be found in COSMIC, underscoring COSMIC's limitations. Mutations unique to COSMIC underline their potential as drivers of cancer due to their absence in the general population. Mutations present in both databases, e.g., JAK2 Val617Phe/V617F - widely recognized as a cancer driver in hematopoietic cells, but without disease associations in All of Us, raising the possibility that combinatorial SNPs might be responsible for disease development. These findings illustrate the complementarity of both databases for understanding mutation impacts and underscore the need for multi-mutation analyses to uncover genetic factors underlying complex diseases and advance personalized medicine.