Epigenetic Mechanisms Regulate Stem Cell Expressed Genes Pou5f1 and Gfra1 in a Male Germ Cell Line

Male fertility is declining and an underlying cause may be due to environment-epigenetic interactions in developing sperm, yet nothing is known of how the epigenome controls gene expression in sperm development. Histone methylation and acetylation are dynamically regulated in spermatogenesis and are sensitive to the environment. Our objectives were to determine how histone H3 methylation and acetylation contribute to the regulation of key genes in spermatogenesis. A germ cell line, GC-1, was exposed to either the control, or the chromatin modifying drugs tranylcypromine (T), an inhibitor of the histone H3 demethylase KDM1 (lysine specific demethylase 1), or trichostatin (TSA), an inhibitor of histone deacetylases, (HDAC). Quantitative PCR (qPCR) was used to identify genes that were sensitive to treatment. As a control for specificity the Myod1 (myogenic differentiation 1) gene was analyzed. Chromatin immunoprecipitation (ChIP) followed by qPCR was used to measure histone H3 methylation and acetylation at the promoters of target genes and the control, Myod1. Remarkably, the chromatin modifying treatment specifically induced the expression of spermatogonia expressed genes Pou5f1 and Gfra1. ChIP-qPCR revealed that induction of gene expression was associated with a gain in gene activating histone H3 methylation and acetylation in Pou5f1 and Gfra1 promoters, whereas CpG DNA methylation was not affected. Our data implicate a critical role for histone H3 methylation and acetylation in the regulation of genes expressed by spermatogonia – here, predominantly mediated by HDAC-containing protein complexes

Rational drug repurposing using sscMap analysis in a HOX-TALE model of leukemia

Drug discovery and development are often hampered by lack of target identification and clinical tractability. Repurposing of approved drugs to life-threatening diseases such as leukemia is emerging as a promising alternative approach. Connectivity mapping systems link approved drugs with disease-related gene signatures. Relevant preclinical models provide essential tools for system validation and proof-of-concept studies. Herein we describe procedures aimed at generating disease-based gene signatures and applying them to established cross-referencing databases of potential candidate drugs. As a proof of principle, we present the identification of Entinostat as a candidate drug for the treatment of HOX TALE-related leukemia

Liver disintegration in the mouse embryo by deficiency in RNA editing enzyme ADAR1

ADAR1 (adenosine deaminase acting on RNA-1) is widely expressed in mammals, but its biological role is unknown. We show here by gene targeting that ADAR1 selectively edits in vivo two of five closely spaced adenosines in the serotonin 5-hydroxytryptamine subtype 2C receptor pre-mRNA of nervous tissue; and hence, site-selective adenosine-to-inosine editing is indeed a function of ADAR1. Remarkably, homozygosity for two different null alleles of ADAR1 caused a consistent embryonic phenotype appearing early at embryonic day 11 and leading to death between embryonic days 11.5 and 12.5. This phenotype manifests a rapidly disintegrating liver structure, along with severe defects in definitive hematopoiesis, encompassing both erythroid and myeloid/granuloid progenitors as well as spleen colony-forming activity from the aorta-gonad-mesonephros region and fetal liver. Probably as a consequence of these developmental impairments, ADAR1-deficient embryonic stem cells failed to contribute to liver, bone marrow, spleen, thymus, and blood in adult chimeric mice. Thus, ADAR1 subserves critical steps in developing non-nervous tissue, which are likely to include transcript editing

Liver disintegration in the mouse embryo by deficiency in RNA editing enzyme ADAR1

Adenosine deaminase acting on RNA 1 (ADAR1) is widely expressed in the mammal, but its biological role is unknown. We show here by gene targeting that ADAR1 selectively edits in vivo two of five close−spaced adenosines in serotonin 5−HT2C receptor pre−mRNA of nervous tissue and hence, site−selective A−to−I editing is indeed a function of ADAR1. Remarkably, homozygosity for two different null alleles of ADAR1 caused a consistent embryonic phenotype appearing at early E11 and leading to death between E11.5 and E12.5. This phenotype manifests a rapidly disintegrating liver structure, along with severe defects in definitive hematopoiesis, which encompass both erythroid and myeloid−granuloid progenitors as well as spleen colony−forming activity from aorta−gonad−mesonephros and fetal liver. Probably as a consequence of these developmental impairments, ADAR1−deficient embryonic stem cells failed to contribute to liver, bone marrow, spleen, thymus and blood in adult chimeric mice. Thus, ADAR1 subserves critical steps in developing non−nervous tissue, which are likely to include transcript editing

Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase Cgamma2 that specifically increases external Ca2+ entry.

The identification of specific genetic loci that contribute to inflammatory and autoimmune diseases has proved difficult due to the contribution of multiple interacting genes, the inherent genetic heterogeneity present in human populations, and a lack of new mouse mutants. By using N-ethyl-N-nitrosourea (ENU) mutagenesis to discover new immune regulators, we identified a point mutation in the murine phospholipase Cg2 (Plcg2) gene that leads to severe spontaneous inflammation and autoimmunity. The disease is composed of an autoimmune component mediated by autoantibody immune complexes and B and T cell independent inflammation. The underlying mechanism is a gain-of-function mutation in Plcg2, which leads to hyperreactive external calcium entry in B cells and expansion of innate inflammatory cells. This mutant identifies Plcg2 as a key regulator in an autoimmune and inflammatory disease mediated by B cells and non-B, non-T haematopoietic cells and emphasizes that by distinct genetic modulation, a single point mutation can lead to a complex immunological phenotype