Poly(ADP-Ribose) Polymerase Binds with Transcription Enhancer Factor 1 to MCAT1 Elements To Regulate Muscle-Specific Transcription

Abstract

Striated muscle-specific expression of the cardiac troponin T (cTNT) gene is mediated through two MCAT elements that act via binding of transcription enhancer factor 1 (TEF-1) to the MCAT core motifs and binding of an auxiliary protein to nucleotides flanking the 5′ side of the core motif. Using DNA-protein and protein-protein binding experiments, we identified a 140-kDa polypeptide that bound both the muscle-specific flanking sequences of the most distal MCAT1 element and TEF-1. Screening of an expression library with the MCAT1 element yielded a cDNA encoding a truncated form of poly(ADP-ribose) polymerase (PARP). Endogenous PARP from embryonic tissue nuclear extracts migrated as a 140-kDa protein. Recombinant full-length PARP preferentially bound the wild-type MCAT1 element and was shown to physically interact with TEF-1. In addition, endogenous TEF-1 could be coimmunoprecipitated with PARP from extracts of primary skeletal muscle cells. Recombinant PARP was able to ADP-ribosylate TEF-1 in vitro. Inhibition of the enzymatic activity of PARP repressed expression of an MCAT1-dependent reporter in transiently transfected primary muscle cells. Together, these data implicate PARP as the auxiliary protein that binds with TEF-1 to the MCAT1 element to provide muscle-specific gene transcription.

ACKNOWLEDGMENTS

We are grateful to G. de Murcia for generously providing the baculovirus containing the full-length cDNA for human PARP (20), Eric Olson and Brian Black for the MEF2 and MyoD reporter constructs (pE102MEF2X2CAT and 4R-TKCAT, and Sarah Larkin for providing the series of mutant MCAT1 oligonucleotides. Nina Kostanian provided expert technical assistance. We also thank Arnold Caplan, James Cleaver, and Mark Smulson as well as present and past members of the Ordahl lab for useful discussions and helpful suggestions during the course of this work. We thank Iain Farrance, Sarah Larkin, Axel Thomson, Paul Webb, and members of the Ordahl lab for their comments on the manuscript.

This work was done during the tenure of a research fellowship from the American Heart Association, California Affiliate, to A.J.B. and was supported by NIH grants HL35561 and HL59693 to C.P.O.

ADDENDUM IN PROOF

After this paper was accepted, Shieh et al. (W. M. Shieh, J.-C. Ames, M. V. Wilson, Z.-Q. Wang, D. W. Koh, M. K. Jacobson, and E. L. Jacobson, J. Biol. Chem.273:30069–30072, 1998) reported that cells from PARP null mice synthesize ADP-ribose polymers. This suggests that there may be other pathways of poly(ADP-ribosyl)ation which do not involve PARP.