Coordinated transcriptional regulation of hspa1a gene by multiple transcription factors: Crucial roles for HSF-1, NF-Y, NF-κB, and CREB

Binu K. Sasi, Parshuram J. Sonawane, Vinayak Gupta, Bhavani S. Sahu, Nitish R. Mahapatra

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33 Scopus citations

Abstract

Although the transcript level of inducible heat shock protein 70.3 (Hsp70.3, also known as Hspa1a) is altered in various disease states, its transcriptional regulation remains incompletely understood. Here, we systematically analyzed the Hspa1a promoter to identify major cis elements and transcription factors that may govern the constitutive/inducible gene expression. Computational analyses coupled with extensive in vitro (promoter-reporter activity and electrophoretic mobility shift assays) and in vivo (chromatin immunoprecipitation assays) revealed interaction of several transcription factors with Hspa1a promoter motifs: HSF-1 (heat shock factor 1) at - 114/- 97 bp and - 788/- 777 bp, NF-Y (nuclear transcription factor Y) at - 73/- 58 bp, NF-κB (nuclear factor kappa B) at - 133/- 124 bp, and CREB (cAMP response element binding protein) at - 483/- 476 bp. Consistently, siRNA (small interfering RNA)-mediated down-regulation of each of these transcription factors caused substantial reduction of endogenous Hspa1a expression. Heat-shock-induced activation of Hspa1a was coordinately regulated by HSF-1 and NF-Y/NF-κB. The Hspa1a expression was augmented by TNF-α (tumor necrosis factor-alpha) and forskolin in NF-κB and CREB-dependent manners, respectively. NF-κB and CREB also activated Hspa1a transcription in cardiac myoblasts upon exposure to ischemia-like conditions. Taken together, this study discovered previously unknown roles for NF-κB and CREB to regulate Hspa1a expression and a coordinated action by several transcription factors for Hspa1a transactivation under heat-shock/ischemia-like conditions and thereby provided new insights into the mechanism of Hspa1a regulation.

Original languageEnglish (US)
Pages (from-to)116-135
Number of pages20
JournalJournal of Molecular Biology
Volume426
Issue number1
DOIs
StatePublished - Jan 9 2014

Bibliographical note

Funding Information:
This work was supported by a grant ( LSRB/196/BTB/2009 ) from the Defense Research and Development Organization, Government of India , to N.R.M. B.K.S. is thankful to the Council of Scientific and Industrial Research, Government of India, for research fellowships. B.K.S. acknowledges Prasanna Kumar Reddy Allu, Kalyani Ananthamohan, and Lakshmi Subrahmanian, Cardiovascular Genetics Lab, IIT Madras, for their help in this study. The authors would like to acknowledge Dr. S. Mahalingam, Dr. Amal K. Bera, Dr. D. Karunagaran, and Dr. Suresh Kumar Rayala of IIT Madras for providing access to several equipment in their laboratories. The authors are grateful to Dr. Shioko Kimura, National Cancer Institute, Bethesda, for NF-YA, NF-YB, and NF-YC plasmids; to Dr. Roberto Mantovani, University of Milan, for the NF-YA dominant-negative plasmid; to Dr. Richard Voellmy, HSF Pharmaceuticals, Switzerland, for the HSF-1 and dominant-negative HSF-1 plasmid; to Dr. David Ginty, Howard Hughes Medical Institute, for the VP16 CREB plasmid; to Richard H. Goodman, Vollum Institute, Oregon Health Sciences University, Portland, for the K-CREB plasmid; and to Dr. Warner C. Greene, Virology & Immunology, Gladstone Institute, for the p50, p65, and p65 Shr plasmids.

Copyright:
Copyright 2020 Elsevier B.V., All rights reserved.

Keywords

  • gene expression
  • heat shock protein
  • regulatory motifs
  • transcription factors

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