Silencing subdomains of v-ErbA interact cooperatively with corepressors: involvement of helices 5/6.
Members of the thyroid hormone receptor (TR) family act on vertebrate development and homeostasis by activating or repressing transcription of specific target genes in a ligand-dependent way. Repression by TR in the absence of ligand is mediated by an active silencing mechanism. The oncogene v-ErbA is a variant form of TR unable to bind hormone and thus acts as a constitutive repressor. Functional studies and mutation analysis revealed that the TR/v-ErbA silencing domain is composed of three silencing subdomains (SSD1-3) which, although nonfunctional individually, synergize such that silencing activity is restored when they are combined in a heteromeric complex. Here we demonstrate, using protein interaction assays in vitro and in vivo, that the inactive v-ErbA point mutant L489R within helix 5/6 in SSD2 fails to interact with the two corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptor). Furthermore, mutants in SSD1 and SSD3 exhibit a reduced corepressor recruitment corresponding to their weak residual silencing activity. In mammalian two-hybrid assays, only the combination of all three silencing subdomains, SSD1-3, leads to a cooperative binding to the corepressors N-CoR or SMRT comparable to that of the full-length v-ErbA repression domain. In conclusion, full silencing activity requires corepressor interaction with all three silencing subdomains, SSD1-3. Among these, SSD2 is a new target for N-CoR and SMRT and is essential for corepressor binding and function.
Busch
K
K
Laboratoire de Biologie Moléculaire et de Génie Génétique, Institut de Chimie, Université de Liège, Belgium.
Martin
B
B
Baniahmad
A
A
Martial
J A
JA
Renkawitz
R
R
Muller
M
M
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020868:Gene Silencing; D005816:Genetic Complementation Test; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D056971:Nuclear Receptor Co-Repressor 1; D056985:Nuclear Receptor Co-Repressor 2; D018774:Oncogene Proteins v-erbA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D056971:Nuclear Receptor Co-Repressor 1; D056985:Nuclear Receptor Co-Repressor 2; D018774:Oncogene Proteins v-erbA; D011994:Recombinant Proteins; D012097:Repressor Proteins