Genetic Approaches To Understanding Organ Development and Function

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The COVID-19 pandemic led to a pause of our experiments with genetically engineered mice in early 2020. All lines were cryopreserved and only maintenance breeding was conducted. Projects were restarted in the fall of 2021 and the mouse colony was built up over the past months. Investigations on the genetic regulation in mammary tissue during pregnancy and lactation were commenced. Objective 1 Several hundred genes are uniquely expressed in mammary tissue and activated by pregnancy and lactation hormones through the tyrosine kinase JAK2 and the transcription factor Signal Transducer and Activator of Transcription (STAT) 5. STAT5 is activated by prolactin, and it controls mammary alveolar development during pregnancy and the activation of genetic programs resulting in lactation. Enhancers are transcription component platforms that control the location, timing and intensity of gene expression. While current approaches, such as the ChIP-seq and physical contact studies, are useful in identifying candidate enhancers, their biological predictions are limited and validation through genetic experiments is needed. Enhancers are occupied by multiple transcription factors (TFs) that might bind directly to DNA through their respective recognition motifs or indirectly through tethering. Since experimental genetic studies generally ablate the entire enhancer, the structural and functional contribution of individual TFs remains to be understood. We have continued investigations on the regulation of a complex locus encoding genes that are induced several hundred-fold during pregnancy. Using mutational analyses and mouse genetics, we have identified a super-enhancer and several enhancers that are activated by the transcription factor STAT5 during pregnancy. This is the first study investigating the regulation a complex multigene locus in mammary tissue using genetic tools. Our study provides comprehensive insight into the anatomy and biology of regulatory elements that employ the JAK/STAT signaling pathway and preferentially activate gene expression during lactation. Objective 2 Mutations in the transcription factor STAT5B have been associated with various human T cell leukemias. Specifically, mutations in the Sh2 domain, a sequence required for the dimerization and activation of STAT5, have been identified. It has been proposed that such mutations confer growth advantages to cells, but the exact molecular mechanism remains obscure. To clarify the exact molecular consequences in vivo, i.e. in an intact organism, we have generated mice that carry two distinct mutations, Y665H and Y665F, that had been identified in large granular leukocytic leukemia. Current studies are focused on identifying and understanding the defects bestowed on cells by these mutations. Objective 3 Candidate transcription enhancers have been identified in kidney tissue using ChIP-seq experiments that identify the binding of transcription factors to the genome and the presence of activating chromatin marks. Mutations disrupting these candidate enhancers have been introduced into the mouse genome and we are currently investigating the biological and molecular consequences.