Construction of luciferase reporter plasmids to study contributions of 5’ and 3’ MyoD binding regions to Acta1 gene transcription
Authors:
Gilberto deSalvo, Juan Morales, Sandra Sharp, Ha Vu, Barbara WoldMentor:
Sandra Sharp, Professor of Biological Sciences, California State University Los AngelesThe stages in myogenesis, or muscle cell development, have been extensively described, but the regulatory regions of DNA and the transcription regulatory factors that direct each stage are still under investigation for their respective contributions. MyoD is a muscle-specific transcription factor that plays an early role in the specification of multipotent cells to become pre-muscle cells or myoblasts. Later, MyoD directs differentiation, the process of maturation to muscle. As part of this process, it directs transcription of muscle-specific genes. Acta1 is one such gene. MyoD has been localized by ChIP-Seq to several DNA binding regions within a few kilobases both upstream and downstream of the transcribed region of the gene. We have hypothesized that both the upstream and downstream regions contribute to the transcriptional efficiency of this gene during myogenesis. By using luciferase reporter vectors in transient transfection assays with myoblast cells, functional activity has already been attributed to the upstream regions and the downstream regions independent of each other. Here we describe the construction of a reporter plasmid with a multiple cloning site added downstream of the reporter gene. We show how this construct was used to make reporter plasmids that will be used to test the transcriptional activity of the MyoD binding regions downstream of the Acta 1 gene in combination with the upstream regions. To our knowledge, this is the first time a putative combinatorial transcriptional regulatory action between MyoD binding regions located upstream and downstream of a muscle-specific gene will have been tested. Funded by a CSUPERB JV grant to SB Sharp and BJ Wold and funding from the NIH to BJ Wold.