DNA binding domains and nuclear localization signal of LEDGF: contribution of two helix-turn-helix (HTH)-like domains and a stretch of 58 amino acids of the N-terminal to the trans-activation potential of LEDGF.

Abstract

Lens epithelium derived growth factor (LEDGF), a nuclear protein, plays a role in regulating the transcription of stress-associated genes such as heat shock proteins by binding to consensus core DNA sequences nAGGn or nGAAn or their repeats, and in doing so helps to provide cyto-protection. However, additional information is required to identify the specific structural features of LEDGF involved in gene transcription. Here we have investigated the functional domains activating and repressing DNA-binding modules, by using a DNA binding assay and trans-activation experiments performed by analyzing proteins prepared from deletion constructs. The results disclosed the DNA-binding domain of N-terminal LEDGF mapped between amino acid residues 5 and 62, a 58 amino acid residue stretch PWWP domain which binds to stress response elements (STRE; A/TGGGGA/T). C-terminal LEDGF contains activation domains, an extensive loop-region (aa 418-530) with two helix-turn-helix (HTH)-like domains, and binds to a heat shock element (HSE; nGAAn). A trans-activation assay using Hsp27 promoter revealed that both HTH domains contribute in a cooperative manner to the trans-activation potential of LEDGF. Interestingly, removal of N-terminal LEDGF (aa 1-187) significantly enhances the gene activation potential of C-terminal LEDGF (aa 199-530); thus the N-terminal domain (aa 5-62), exhibits auto-transcriptional repression activity. It appears that this domain is involved in stabilizing the LEDGF-DNA binding complex. Collectively, our results demonstrate that LEDGF contains three DNA-binding domains, which regulate gene expression depending on cellular microenvironment and thus modify the physiology of cells to maintain cellular homeostasis.

• Authors: Dhirendra P Singh, E Kubo, Y Takamura, T Shinohara, A Kumar, Leo T Chylack Jr, N Fatma
• Journal: Journal of molecular biology (J Mol Biol) Vol. 355 Issue 3 Pg. 379-94 (Jan 20 2006) ISSN: 0022-2836 [Print] Netherlands
• PMID: 16318853 (Publication Type: Journal Article, Research Support, N.I.H., Extramural, Research Support, Non-U.S. Gov't)

Chemical References

• Heat-Shock Proteins
• Intercellular Signaling Peptides and Proteins
• Nuclear Localization Signals
• lens epithelium-derived growth factor
• DNA

Topics

• Animals
• Base Sequence
• Cloning, Molecular
• Consensus Sequence
• DNA (metabolism)
• Heat-Shock Proteins (genetics)
• Helix-Turn-Helix Motifs
• Humans
• Intercellular Signaling Peptides and Proteins (genetics, metabolism)
• Mutagenesis, Site-Directed
• Mutation
• Nuclear Localization Signals (genetics, metabolism)
• Oxidative Stress
• Promoter Regions, Genetic
• Protein Binding
• Protein Structure, Tertiary
• Transcriptional Activation