Testing for Combinatorial Activity of 5′ and 3′ MyoD Binding Regions in Mouse Acta1 Gene Expression
Authors: Esaian Sevan , Gonsalez Carla Macleini , Vu Ha , DeSalvo Gilberto , Morales Juan , Manjurr Ghalib , Wold Barbara , Sharp Sandra 
Published: November 2023

Myogenesis is the process by which pre‐muscle cells (myoblasts) differentiate into mature muscle. The basic‐helix‐loop‐helix myogenic regulatory transcription factors, which include MyoD, myogenin, MRF4, and Myf5, bind to specific E‐box (CANNTG) sequences in the DNA to regulate transcription in pre‐muscle and muscle cells. One of the genes regulated by MyoD is the Acta1 gene, which codes for skeletal muscle actin mRNA. The Acta1 gene is specifically induced during the differentiation of myoblasts to mature muscle, when the muscle contractile apparatus is being assembled. Through ChIP‐Seq (chromatin immunoprecipitation followed by sequencing), we and others have found that MyoD is bound in specific regions of the DNA both upstream and downstream of the Acta1 gene, the upstream region within ~2400 bp of the transcription start site, and the downstream region over 9000 bp from the transcription start site. We hypothesize that the 5′ and 3′ regions of MyoD binding participate in a combinatorial regulation of Acta1 gene transcription. To test this hypothesis, we have made luciferase reporter constructs with the mouse 5′ and 3′ Acta1 MyoD binding regions inserted upstream and downstream, respectively, of the reporter gene, and are using these constructs in transient transfection assays in C2C12 myogenic cells. Plasmid constructs with 5′ insertions only include: a minimal Acta1 promoter region that includes no upstream MyoD binding regions, 5′ insertion of the WT Acta1 5′ region representing the sequence from −2397 to +316 relative to the Acta1 transcription start site in the genome; the same 5′ region, but with a key MyoD binding site disrupted; and the same 5′ region, but with a key MEF2 (Myogenesis enhancing factor 2) binding site disrupted. Both the minimal and WT 5′ regions have also been combined with downstream insertion of two 3′ MyoD binding regions, one a length of 3284 bp and the other an 898bp subfragment that includes only one of the 3′ MyoD binding sites that are present in the longer fragment. Luciferase reporter assays have thus far shown that the 5′ MyoD binding region functions to upregulate the Acta1 gene in a differentiation‐specific manner, and that upregulation does not occur when either the E‐box or the MEF2 binding site has been mutated, suggesting that these binding sites are required for upregulation during myogenesis. Furthermore, in combination with the WT 5′ region, the 3′ region appears to have both inhibitory and activating effects. We are preparing combinations of both the longer and shorter 3′ fragments with the mutated 5′ regions to further test the dependence upon the 5′ MyoD and MEF2 binding sites for differentiation‐specific gene activation. A demonstration of combinatorial regulation at the Acta1 gene will raise the possibility that other muscle specific genes are regulated by MyoD binding regions at locations other than the well‐known regions that have been found located 5′ or within the transcribed regions of muscle specific genes.Support or Funding InformationNIH NIGMS TWD RISE award R25 GM061331 to C. Gutierrez. NSF RUI #MCB‐99831‐40 and #MCB‐9604289 and associated REU supplemental funding to S. B. Sharp. NIH Bridges to the Future GM 49001 to L. M. Tunstad. External funding to B Wold, Caltech.