Abstract
Purpose: :
Eukaryotic transcriptional activator, Lens Epithelial Derived Growth Factor, (LEDGF) protects cells by activating small heat shock proteins (hsps). Previously, we have shown that LEDGF binds to the trimeric Heat Shock Factor 1 (HSF1) and cooperatively transactivates hsps to provide protection during heat stress. Herein, we identified the interacting domain of HSF1-LEDGF and tested the ability to activate hsps and cell survival.
Methods: :
A full length HSF1 cDNA cloned into PGEX-2T/pEGFP was used to generate partial constructs, peptides 1/429 amino acid (aa), 1/378aa,1/315aa and 1-212aa, by PCR. N- or C-terminal constructs of LEDGF were similarly prepared. Human lens epithelial cells (hLECs) were transfected with the constructs linked to pEGFP, and fluorescent microscopy was used to ascertain localization pattern. Pulldown and coprecipitation assays tested the interaction of LEDGF and HSF1. Western blot and qPCR monitored expression of HSF1, LEDGF and hsps. MTS and TUNEL assays defined effects of over- and under-expression or coexpression of LEDGF and HSF1 on cell viability. Gel mobility assay assessed DNA binding affinity of LEDGF and HSF1 or their partial constructs to HSE. CAT-ELISA monitored transcriptional activity. LEDGF-SiRNA experiments and hsf1-/- LECs were used to validate results.
Results: :
Fluorescent microscopy showed that pEGFP-HSF1and its partial constructs 1/429,1/378 and 1/315aa are localized in the nucleus. Cells survived under heat stress and had elevated expression of hsp27 and αB-crystallin mRNA and protein with promoter activity similar to full HSF1, while 1/212aa failed to do so. Cells with reduced expression of LEDGF did not activate hsps transcription even with HSF1. C-terminal LEDGF (170/530aa) with integrase binding domain bound to HSF1 and its N-terminal partial peptide 1/429, 1/378, 1/315 but not 1/212aa during stress. This interaction involved DNA binding domain (DBD, 16/120aa) and trimerization domain (TR, 130/203aa). An active form of ΔHSF1, lacking regulatory domain (RD) 221 to 315aa, bound to LEDGF, indicating that a stretch of eight amino acid residues (213LNDSGSAH220) lying between TD and RD is responsible for interaction.
Conclusions: :
A previously unidentified interaction between LEDGF and HSF1 is necessary for synergistic activation of hsps transcription in LECs under stress. The identified amino acids could confer the specificity of the action on HSF1 in diverse biological processes by recruiting its partner factor.
Keywords: transcription factors • gene/expression • protective mechanisms