Transcriptional regulation of the muscle-specific troponin I gene

Haishan Lin, Purdue University

Abstract

The quail fast skeletal troponin I (TnI) gene is transcribed specifically in differentiated skeletal muscle cells. TnI gene expression is under the control of an internal regulatory element (IRE) in the first intron which functions as a muscle-specific enhancer. My studies have demonstrated that the muscle regulatory factors MyoD, myogenin, Myf-5, and MRF4, in the presence of the ubiquitous helix-loop-helix transcription factor E12, interact with identical nucleotides within the E-box sequence of the TnI IRE in vitro. Utilizing a novel analysis strategy involving protein cross-linking and two-dimensional gel electrophoresis, I found that the muscle regulatory factors must form heterodimers with E12 to bind efficiently to the TnI E-box. The TnI E-box-binding proteins in vivo are restricted to skeletal muscle cells and are antigenically related to MyoD and myogenin. The full enhancer activity of the IRE requires two additional cis-acting elements, Site I and Site II, which bind to nuclear proteins from both muscle and nonmuscle cells. Point mutations in either Site I or Site II abolish the respective protein binding abilities and greatly reduce the enhancer function of the TnI IRE. These results demonstrate that muscle-specific TnI gene transcription relies on interactions between muscle regulatory factors and ubiquitously expressed proteins. To isolate cDNA clones encoding Site I-binding proteins, $\lambda$gt11 expression libraries were screened using the TnI Site I DNA as a probe. Several cDNA clones obtained from the library screenings encode the human nonhistone chromosomal high mobility group protein (HMG)-I and its isoform HMG-Y. An additional human cDNA clone, referred to as clone E, encodes a ${\sim}60$ kDa protein which also binds to Site I. Sequence analyses suggest that the E cDNA clone encodes a novel DNA-binding protein. Taken together, these results suggest that muscle-specific regulation of TnI gene expression requires a mechanism in which the IRE cis-acting elements, including E-box, Site I, and Site II, each interact with multiple muscle-specific and/or ubiquitous proteins to form an active transcriptional complex.

Degree

Ph.D.

Advisors

Konieczny, Purdue University.

Subject Area

Molecular biology|Genetics

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