Distinct Subdomains of Human TAF_II130 Are Required for Interactions with Glutamine-Rich Transcriptional Activators

ABSTRACT

TFIID is a multiprotein complex consisting of the TATA box binding protein and multiple tightly associated proteins (TAF_IIs) that are required for transcription by selected activators. We previously reported cloning and partial characterization of human TAF_II130 (hTAF_II130). The central domain of hTAF_II130 contains four glutamine-rich regions, designated Q1 to Q4, that are involved in interactions with the transcriptional activator Sp1. Mutational analysis has revealed specific regions within the glutamine-rich (Q1 to Q4) central region of hTAF_II130 that are required for interaction with distinct activation domains. We tested amino- and carboxyl-terminal deletions of hTAF_II130 for interaction with Sp1 activation domains A and B (Sp1A and Sp1B) and the N-terminal activation domain of CREB (CREB-N) by using the yeast two-hybrid system. Our results indicate that Sp1B interacts almost exclusively with the Q1 region of hTAF_II130. In contrast, Sp1A makes multiple contacts with Q1 to Q4 of hTAF_II130, while CREB-N interacts primarily with the Q1-Q2 hTAF_II130 subdomain. Consistent with these interaction studies, overexpression of the Q1-to-Q4 region in HeLa cells inhibits Sp1- but not VP16-mediated transcriptional activation. These findings indicate that the Q1-to-Q4 region of hTAF_II130 is required for Sp1-mediated transcriptional enhancement in mammalian cells and that different activation domains target distinct subdomains of hTAF_II130.

ACKNOWLEDGMENTS

We are grateful to Grace Gill of Harvard Medical School for her help and generous gifts of the yeast and mammalian plasmids carrying Sp1 derivatives and to Michael Garabedian of New York University Medical Center for many valuable discussions. We thank Kelly Vogel and Amy Kun for technical assistance, Eileen Rojo-Niersbach and Stavros Giannakopoulos for their help with the project, Muktar Mahajan for advice on the yeast two-hybrid assay, Sobha Pisharody for assistance with cell culture, and David Ron for the gift of the reporter plasmid. Critical reading of the manuscript by Michael Garabedian and Grace Gill was greatly appreciated.

This work was supported by a grant from the National Institutes of Health (R01-BM51314). D.S. was supported by a National Institutes of Health Training Grant (5T32 AI07180), and N.T. was supported in part by The Irma T. Hirschl Trust.