Repression of myogenin function by TGF-beta 1 is targeted at the basic helix-loop-helix motif and is independent of E2A products.

J.F Martin,L Li,E.N Olson

doi:10.1016/s0021-9258(19)49859-1

J.F Martin, L Li + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(19)49859-1

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Abstract

The muscle-specific helix-loop-helix (HLH) proteins myogenin, MyoD, myf5, and MRF4 form hetero-oligomers with ubiquitous HLH proteins encoded by the E2A gene and activate muscle transcription by binding to a DNA sequence known as an E-box (CANNTG). Transforming growth factor-beta (TGF-beta) can inhibit muscle differentiation by silencing the transcription-activating potential of myogenic HLH proteins without affecting their ability to bind DNA. We show that repression by TGF-beta is directed at the basic-HLH motif of myogenin and is independent of E2A products. Using a series of reporter genes as targets for trans-activation by myogenin, transcriptional repression by TGF-beta is also shown to map to the E-box motif and to not require heterologous DNA sequence elements. These results demonstrate that TGF-beta represses muscle-specific transcription through a post-translational mechanism that renders the basic-HLH regions of the myogenic regulators nonfunctional. The selective repression of myogenic HLH proteins by TGF-beta indicates that the TGF-beta signaling system can discriminate between different classes of HLH proteins and implies that myogenic HLH proteins activate muscle-specific transcription through a unique mechanism.

Highlights

11 Established Investigator of the American Heart Association
These factors shahroemology within a basic helix-loop-helix motif and can each activamteyogenesis when expressed in a variety of nonmyogenic cell types
Products-To investigate the possibility that E2A products pendently of DNA binding [34],we were especially interested (E12/E47), thein vivo partners for myogenin, were function- in whether the transcriptional activation domains (TADs) in ally inactivated by TGF-@,we examined whether TGF-@was the N and C termini might be targets for TGF-@signals

Summary

RESULTS

Using reporter plasmids containingeithera factors, MyoD, myogenin, myf5,and MRF4, were sensitive to complex muscle-specific enhancer or a multimerized E-box repression by TGF-@,we transiently transfected 10T1/2 fimotif, we demonstrate that TGF-@-mediatedrepression broblasts with expression vectors encoding each factor and is directed at theE-box consensus sequence and is independ- assayed for myogenic conversion by immunostaining for MHC ent of surrounding DNA sequence elements These results in the presence and absence of TGF-@.As shown previously suggest that TGF-@-mediated intracellularsignals selectively [13,14,15,16,17], each of the myogenic factors activated myogenesis in silence the activity of myogenic HLH proteins and that this 10T1/2 cells (Table I). 5’ enhancer inserted into the BamHI site 3’ of CAT and the 246-bp MCK basal promoter 5’ of CAT in the vector pSVOCAT This reporter plasmid has been shown to be muscle-specific and to be TABLEI TGF-8 represses myogenic conversion by the MyoD family trans-activated by myogenin and MyoD [39]. Myogenin and myogenin mutant1s0w0ere contained in the expression vector EMSV, which contains the Moloney sarcoma

Myogenic regulator

DISCUSSION