Identification of the human HEX1/hExo1 gene promoter and characterization of elements responsible for promoter activity

Paula D. Ladd, David M. Wilson, Mark Kelley, David G. Skalnik

Research output: Contribution to journalArticle

3 Citations (Scopus)

Abstract

HEX1/hExo1 is a Class III nuclease of the RAD2 family with 5′ to 3′ exonuclease and flap structure-specific endonuclease activities. HEX1/hExo1 is expressed at low levels in a wide variety of tissues, but at higher levels in fetal liver and adult bone marrow, suggesting HEX1/hExo1 is important for hematopoietic stem cell development. A putative HEX1/hExo1 promoter fragment extending from -6240 to +1600bp exhibits cell-type specific activity in transient transfection assays. This fragment directs high luciferase reporter gene expression in the hematopoietic cell line K562, chronic myelogenous leukemia cells, but low luciferase expression in the non-hematopoietic cell line HeLa, human cervical carcinoma cells. Deletion studies identified a fragment spanning -688 to +1600bp that exhibits full transcriptional activity while a slightly shorter fragment from -658 to +1600bp exhibits significantly decreased promoter activity. In vitro binding assays revealed DNA-binding activities that interact with -687 to -681bp and -665 to -658bp elements. Oligonucleotide competition and antibody disruption studies determined that the transcription factor CREB-1 recognizes the -687 to -681bp element, while transcription factors Sp1 and Sp3 recognize the -665 to -658bp element. Mutation of either the CREB-1 or Sp1/Sp3 binding sites dramatically reduces HEX1/hExo1 promoter activity and elimination of both elements abolishes promoter activity.

Original languageEnglish
Pages (from-to)187-198
Number of pages12
JournalDNA Repair
Volume2
Issue number2
DOIs
StatePublished - Feb 3 2003

Fingerprint

Forensic Anthropology
spleen exonuclease
Genes
Cells
Luciferases
Assays
Sp3 Transcription Factor
Sp1 Transcription Factor
Cell Line
Exonucleases
Endonucleases
Leukemia, Myelogenous, Chronic, BCR-ABL Positive
Hematopoietic Stem Cells
Stem cells
Reporter Genes
Gene expression
Oligonucleotides
Liver
Transfection
Bone

Keywords

  • DNA recombination and repair
  • Electrophoretic mobility shift assay
  • Hematopoiesis
  • HEX1/hExo1
  • RAD2 nucleases
  • Transient transfection

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Identification of the human HEX1/hExo1 gene promoter and characterization of elements responsible for promoter activity. / Ladd, Paula D.; Wilson, David M.; Kelley, Mark; Skalnik, David G.

In: DNA Repair, Vol. 2, No. 2, 03.02.2003, p. 187-198.

Research output: Contribution to journalArticle

Ladd, Paula D. ; Wilson, David M. ; Kelley, Mark ; Skalnik, David G. / Identification of the human HEX1/hExo1 gene promoter and characterization of elements responsible for promoter activity. In: DNA Repair. 2003 ; Vol. 2, No. 2. pp. 187-198.
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