For the past 20 years our laboratory has explored gene expression including transcription of laminin genes in glomerular cells. Laminins, major constituents of basement membrane and extracellular matrix, exist as heterotrimers composed of a, p, and y chains. The y1 chain, found in ten of the sixteen known trimeric laminin isoforms, is the most widely expressed laminin chain. Until now we have focused our studies on laminin y1 (Lamd) chain gene using reporter gene constructs. We have cloned the Lamd gene promoter, identified transcriptional elements and cloned factors that regulate its transcription. The recent publications of human, mouse and rat genome sequences provided critical insights into gene structure and presented important new opportunities to study their expression. It revealed that laminin gene structures are conserved and that the are generally several times longer than an average gene. The size of these genes and the complexity of their regulation pose a challenge for studies based on reporter gene constructs. To enhance the ability to decipher transcriptional and chromatin processes at multiple sites and loci in their natural genomic environment, we developed powerful high-throughput chromatin immunoprecipitation (ChIP) strategies and computational tools to study several events simultaneously. Using these strategies we defined transcription factors, RNA polymerase II (Pol II) and chromatin state along the 125kb-long Lamd gene. The current application represents logical experimental progressionof our work and is aimed to define chromatin and transcriptional processes that control co-expression of specific laminin chains in mesangial cells. We will use the high-throughput strategies to test a hypothesis that in mesangial cells histone modifications control whether transcription of agiven laminin gene is """"""""ON""""""""or """"""""OFF"""""""", and for those laminin genes that are """"""""ON"""""""", the rate of transcription is fine-tuned by a synergistic action of factors recruited to laminin promoters. The following Aims are proposed.
Aim # 1. To estimate transcription rates of laminin genes in rat mesangial cells by examining mRNA levels andstability, andPolII recruitment along these genes.
Aim# 2. Toidentify histone modifications along laminin genes that control theirco-expression.
Aim# 3. Toidentify factors recruited to laminin genes promoters that fine-tune their expression. Laminin accumulates within glomeruli during the progression of glomerular disease with aberrant laminin heterotrimeric composition. The proposed studies will allow us to define the mechanisms and factors that silence some of the laminin chains while facilitating expressionof others inmesangial cells. Once these are known we will define histone-modifying enzymes and factors responsible for disordered expressionof laminin chains in animal models of glomerulonephropathy.
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