Myogenin Induces the Myocyte-Specific Enhancer Binding Factor MEF-2 Independently of Other Muscle-Specific Gene Products

The molecular characterization of a cardiac determination gene has been an elusive goal for the past several years. Prior to cloning of the skeletal muscle determination factor MyoD, the presence of a dominantly acting skeletal muscle determination factor had been inferred from the observation that the skeletal muscle phenotype was dominant in skeletal muscle-fibroblast heterokaryons (H. M. Blau, G. K. Pavlath, E. C. Hardeman, C.-P. Chiu, L. Siberstein, S. G. Webster, S. C. Miller, and D. Webster, Science 230:758-766, 1985). In these experiments, we have examined cardiac-fibroblast heterokaryons to investigate the existence of a dominantly acting cardiac determination factor. We have employed a novel experimental approach using primary embryonic fibroblasts from transgenic mice as a means of assaying for the activation of a cardiac promoter-luciferase reporter transgene within fibroblast nuclei. This approach provides a potential means of genetic selection for a dominantly acting positive factor and can be generalized to other systems. We have examined the expression of three markers of the cardiac lineage: a myofibrillar protein promoter (MLC2), a secreted protein (ANF), and a transcription factor (MEF2). MEF2 is specific to both cardiac and skeletal muscle cells. Our results indicate that in a majority of heterokaryons with an equal ratio of cardiac to fibroblast nuclei, none of these cardiac markers are expressed, indicating that the cardiac phenotype is not dominant over the embryonic fibroblast phenotype. The distinction from previous results with skeletal muscle is emphasized by our results with MEF2, which is dominantly expressed in skeletal muscle-fibroblast but not cardiac-fibroblast heterokaryons, supporting its divergent regulation in the two cell types.

Section: Resultssupporting

confidence: 85%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Heterokaryons of Cardiac Myocytes and Fibroblasts Reveal the Lack of Dominance of the Cardiac Muscle Phenotype

Evans

Tai

Tan

et al. 1994

“…Some links between the MyoD family and other transcription factors have been suggested. For example, exogenous expression of myogenin induces MEF2 binding activity (19,93), an intact A/T-rich site in the MCK enhancer is required for transactivation of a reporter gene by myogenic HLH proteins (18), and myosin heavy-chain IIB regions that contain MEFi and A/T-rich sites synergistically increase reporter expression when placed together upstream of a basal promoter (78). Since MyoD family members are not expressed in the heart at any time during development (3,44,65,74), the role of E boxes and their putative binding factors in cardiac gene regulation is yet to be established.…”

Section: Discussionmentioning

confidence: 99%

“…The MCK enhancer contains two sites rich in adenine and thymine residues, the MEF2 and A/T-rich sites. The MEF2 site binds the muscle-specific factor MEF2 as well as ubiquitous factors (10,19,28,30,31,93). MEF2 and related sites have been implicated in the regulation of a variety of muscle genes (7,61,62,78,89,95).…”

mentioning

confidence: 99%

Multiple Regulatory Elements Contribute Differentially to Muscle Creatine Kinase Enhancer Activity in Skeletal and Cardiac Muscle

Amacher

Buskin

Hauschka

1993

We have used transient transfections in MM14 skeletal muscle cells, newborn rat primary ventricular myocardiocytes, and nonmuscle cells to characterize regulatory elements of the mouse muscle creatine kinase (MCK) gene. Deletion analysis of MCK 5'-flanking sequence reveals a striated muscle-specific, positive regulatory region between -1256 and -1020. A 206-bp fragment from this region acts as a skeletal muscle enhancer and confers orientation-dependent activity in myocardiocytes. A 110-bp enhancer subfragment confers high-level expression in skeletal myocytes but is inactive in myocardiocytes, indicating that skeletal and cardiac muscle MCK regulatory sites are distinguishable. To further delineate muscle regulatory sequences, we tested six sites within the MCK enhancer for their functional importance. Mutations at five sites decrease expression in skeletal muscle, cardiac muscle, and nonmuscle cells. Mutations at two of these sites, Left E box and MEF2, cause similar decreases in all three cell types. Mutations at three sites have larger effects in muscle than nonmuscle cells; an A/T-rich site mutation has a pronounced effect in both striated muscle types, mutations at the MEF1 (Right E-box) site are relatively specific to expression in skeletal muscle, and mutations at the CArG site are relatively specific to expression in cardiac muscle. Changes at the AP2 site tend to increase expression in muscle cells but decrease it in nonmuscle cells. In contrast to reports involving cotransfection of 10T1/2 cells with plasmids expressing the myogenic determination factor MyoD, we show that the skeletal myocyte activity of multimerized MEF1 sites is 30-fold lower than that of the 206-bp enhancer. Thus, MyoD binding sites alone are not sufficient for high-level expression in skeletal myocytes containing endogenous levels of MyoD and other myogenic determination factors.

FoxP1 Represses MEF2A in Striated Muscle

Steiman,

Miyake,

McDermott

2024

Myocyte Enhancer Factor 2 (MEF2) is a transcription factor complex encoded by genes mef2a-d.MEF2 proteins belong to the MADS-box DNA binding protein superfamily involved in many developmental pathways including myogenesis and the survival of cardiomyocytes. MEF2 proteins interact with myogenic regulatory factors and modulate the expression of muscle-specific genes. Thus, unbiased characterization of the MEF2A interactome would expand our understanding of MEF2 functions. A GFP-nanotrap purification followed by LC-MS/MS proteomic analysis was employed to identify MEF2A interacting proteins. Following gene ontology analysis, we focused on an interaction between MEF2A and its novel interacting protein, FOXP1, in striated muscle. FOXP1 was found in the MEF2A-immunocomplex in muscle cells, and ectopic expression of FOXP1 delays myogenic differentiation. FOXP1 inhibited MEF2A activation on myogenic reporter genes driven by the regulatory regions of creatine kinase muscle and myogenin genes. Additionally, siRNA-mediated deletion of FOXP1 in myoblasts and cardiomyocytes enhances MEF2A transactivation properties. Lastly, various biochemical experiments indicate that FOXP1 antagonizes p38 MAPK activation of MEF2A through Threonine-312 phosphorylation. Collectively, we documented a novel repressive interaction of FOXP1 and MEF2A in proliferating striated muscle cells. iii Acknowledgements I would like to begin by expressing my gratitude to Dr. McDermott for recognizing my potential beyond my math grades and providing me an opportunity to pursue my research ambitions. Your expertise and guidance have laid a strong foundation for my journey in this everevolving field. Dr. Tetsuaki Miyake, I cannot thank you enough for your dedication to helping me design experiments and succeed as a graduate student. Thank you for making me feel smarter than I am is. Catherine, I am very grateful for your patience with my high maintenance tendencies, I will miss our weekly lunches dearly. Xiao Dong (XD), I cannot thank you enough for generously spending your weekends with me at the lab to teach me new techniques. To my fellow lab mates, past and present, thank you for your support and always making me laugh. Thank you, Jonathan Kelebeev, my concert buddy, for training me. Thank you, Gurnoor Brar and Dr. Amira Moustafa, for your help with animal care. Thank you, Anastasia MacKeracher, for always making me laugh, good luck at regionals! Thank you, FATIMA Farhat, for keeping me sane these past 2 years. I would also like to thank my previous research mentors who helped me start my research journey, Dr.