title: 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.affiliation: Department of Ophthalmology, and Visual Sciences, University of Nebraska Medical Center, Omaha, NE 68198, USA. dpsingh@unmc.edu
authors.forename: Dhirendra P
authors.initials: DP
authors.lastname: Singh
authors.forename: E
authors.initials: E
authors.lastname: Kubo
authors.forename: Y
authors.initials: Y
authors.lastname: Takamura
authors.forename: T
authors.initials: T
authors.lastname: Shinohara
authors.forename: A
authors.initials: A
authors.lastname: Kumar
authors.forename: Leo T
authors.initials: LT
authors.lastname: Chylack
authors.forename: N
authors.initials: N
authors.lastname: Fatma
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016384:Consensus Sequence; D004247:DNA / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000235:genetics; D019077:Helix-Turn-Helix Motifs; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D019913:Nuclear Localization Signals / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D015533:Transcriptional Activation
chemical_list: D006360:Heat-Shock Proteins; D036341:Intercellular Signaling Peptides and Proteins; D019913:Nuclear Localization Signals; C119380:lens epithelium-derived growth factor; D004247:DNA