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

Dhirendra P Singh, E. Kubo, Y. Takamura, T. Shinohara, A. Kumar, Leo T. Chylack, N. Fatma

Research output: Contribution to journalArticle

43 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)379-394
Number of pages16
JournalJournal of Molecular Biology
Volume355
Issue number3
DOIs
StatePublished - Jan 20 2006

Fingerprint

Nuclear Localization Signals
Amino Acids
DNA
Cellular Microenvironment
lens epithelium-derived growth factor
Cell Physiological Phenomena
Response Elements
Nuclear Proteins
Heat-Shock Proteins
Protein Binding
Transcriptional Activation
Genes
Shock
Homeostasis
Hot Temperature
Gene Expression

Keywords

  • DNA-binding domain
  • Lens epithelium-derived growth factor
  • PWWP domain
  • Stress and heat shock elements
  • Transcription-regulation

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

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. / Singh, Dhirendra P; Kubo, E.; Takamura, Y.; Shinohara, T.; Kumar, A.; Chylack, Leo T.; Fatma, N.

In: Journal of Molecular Biology, Vol. 355, No. 3, 20.01.2006, p. 379-394.

Research output: Contribution to journalArticle

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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.",
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