Glutamine Rich and Basic Region/Leucine Zipper (bZIP) Domains Stabilize cAMP-response Element-binding Protein (CREB) Binding to Chromatin

Bernhard M Mayr,Ernesto Guzman,Marc Montminy

doi:10.1074/jbc.m414144200

Bernhard M Mayr, Ernesto Guzman + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m414144200

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Abstract

We have examined the dynamics of cAMP-response element-binding protein (CREB) binding to chromatin in live cells using fluorescence recovery after photobleaching (FRAP). CREB was found to bind to target sites with a residence time of 100 s, and exposure to a cAMP agonist had no effect on these kinetics. In addition to the basic region/leucine zipper (bZIP) domain, a glutamine-rich trans-activation domain in CREB called Q2 also appeared to be critical for promoter occupancy. Indeed, mutations in Q2 that reduced residence time by FRAP assay disrupted target gene activation via CREB in cells exposed to a cAMP agonist. Notably, insertion of the glutamine-rich B trans-activation domain of SP1 into a mutant CREB polypeptide lacking Q2 stabilized CREB occupancy and rescued target gene activation. These results suggest a novel mechanism by which the family of glutamine-rich activators promotes cellular gene expression.

Highlights

□S The on-line version of this article contains a summary of fluorescence recovery after photobleaching (FRAP) assays for wild-type and mutant yellow fluorescent protein (YFP)-cAMP-response element-binding protein (CREB) polypeptides in the form of supplemental Table I
We have examined the dynamics of cAMP-response element-binding protein (CREB) binding to chromatin in live cells using fluorescence recovery after photobleaching (FRAP)
To evaluate CREB binding to chromatin, we performed FRAP studies using a full-length CREB polypeptide fused to YFP

Summary

Introduction

□S The on-line version of this article (available at www.jbc.org) contains a summary of FRAP assays for wild-type and mutant YFP-CREB polypeptides in the form of supplemental Table I. Mutations in Q2 that reduced residence time by FRAP assay disrupted target gene activation via CREB in cells exposed to a cAMP agonist.

Results

Conclusion