Use of a modified yeast one-hybrid screen to identify BAF60a interactions with the Vitamin D receptor heterodimer.
A modified yeast one-hybrid screen was used to isolate proteins capable of interacting with the Vitamin D receptor (VDR) heterodimer complex while driving expression from a repressor Vitamin D response element (VDRE). Four of nine independent colonies recovered in the screen coded for full-length BAF60a, a component of the mammalian SWI/SNF complex. Deletion studies in yeast were unable to localize a unique region of BAF60a responsible for interaction with the heterodimer complex, as only the full-length protein would support reporter gene expression. Pull-down analyses revealed that BAF60a displayed strong interactions with either the unliganded or liganded heterodimer complex, but neither individual receptor component alone. Transient transfection analysis in opossum kidney (OK) cells indicated that BAF60a decreased basal transcriptional activity from the negative VDRE, but had no effect on hormone-induced repression. Transcriptional activity from an enhancer VDRE also exhibited decreased basal transcriptional activity, but also augmented hormone-dependent enhancer activity, resulting in an overall increased sensitivity to hormone. In summary, BAF60a has been identified as a factor that specifically interacts with the VDR heterodimer complex using a modified yeast one-hybrid selection strategy. This suggests that BAF60a may be a link between mammalian SWI/SNF-like chromatin remodeling complexes and the VDR heterodimer.
Koszewski
Nicholas J
NJ
Division of Nephrology, Bone and Mineral Metabolism, University of Kentucky Medical Center, 800 Rose Street, Lexington, KY 40536-0298, USA. njhosz0@uky.edu
Henry
Kenneth W
KW
Lubert
Eric J
EJ
Gravatte
Holli
H
Noonan
Daniel J
DJ
D000818:Animals; D002460:Cell Line; D002868:Chromosomal Proteins, Non-Histone; D019281:Dimerization; D017353:Gene Deletion; D017930:Genes, Reporter / Q000235:genetics; D005982:Glutathione Transferase / Q000032:analysis / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000166:cytology; D009893:Opossums; D010281:Parathyroid Hormone / Q000235:genetics; D018167:Receptors, Calcitriol / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018168:Receptors, Retinoic Acid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018411:Spodoptera; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D030181:Vitamin D Response Element / Q000235:genetics; D015003:Yeasts / Q000235:genetics*; D001616:beta-Galactosidase / Q000032:analysis / Q000378:metabolism
D002868:Chromosomal Proteins, Non-Histone; D010281:Parathyroid Hormone; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D011994:Recombinant Proteins; C109786:SMARCD1 protein, human; D014157:Transcription Factors; D005982:Glutathione Transferase; D001616:beta-Galactosidase