title: Myeloid cell leukemia-1 (Mc1-1) is a candidate target gene of hypoxia-inducible factor-1 (HIF-1) in the testis.
abstract: BACKGROUND
  Spermatic cord torsion can lead to testis ischemia (I) and subsequent ischemia-reperfusion (I/R) causing germ cell-specific apoptosis. Previously, we demonstrated that the hypoxia-inducible factor-1 (HIF-1) transcription factor, a key regulator of physiological responses to hypoxia, is abundant in Leydig cells in normoxic and ischemic testes. We hypothesize that testicular HIF-1 activates the expression of antiapoptotic target genes to protect Leydig cells from apoptosis. In silico analysis of testis genes containing a consensus hypoxia response element (HRE, 5'-RCGTG-3') identified myeloid cell leukemia-1 (Mcl-1) as a potential HIF-1 target gene. The purpose of this study was to determine whether HIF-1 shows DNA-binding activity in normoxic and ischemic testes and whether Mcl-1 is a target gene of testicular HIF-1.
  
  
  METHODS
  The testicular HIF-1 DNA-binding capacity was analyzed in vitro using a quantitative enzyme-linked immunosorbent assay (ELISA) and electrophoretic mobility shift assays (EMSA). MCL-1 protein expression was evaluated by immunoblot analysis and immunohistochemistry. The binding of testicular HIF-1 to the Mcl-1 gene was examined via chromatin immunoprecipitation (ChIP) analysis.
  
  
  RESULTS
  The ELISA and EMSA assays demonstrated that testicular HIF-1 from normoxic and ischemic testes binds DNA equally strongly, suggesting physiological roles for HIF-1 in the normoxic testis, unlike most tissues in which HIF-1 is degraded under normoxic conditions and is only activated by hypoxia. MCL-1 protein was determined to be abundant in both normoxic and ischemic testes and expressed in Leydig cells. In a pattern identical to that of HIF-1 expression, the steady-state levels of MCL-1 were not significantly affected by I or I/R and MCL-1 co-localized with HIF-1α in Leydig cells. Chromatin immunoprecipitation (ChIP) analysis using a HIF-1 antibody revealed sequences enriched for the Mcl-1 promoter.
  
  
  CONCLUSIONS
  The results demonstrated that, unlike what is observed in most tissues, HIF-1 displays DNA-binding activity in both normoxic and ischemic testes, and Mcl-1 may be a key target gene of testicular HIF-1 with potential roles in the antiapoptotic protection of Leydig cells.
authors.affiliation: Department of Biology, Monmouth University, 400 Cedar Avenue, West Long Branch, NJ 07764, USA. mpalladi@monmouth.edu
authors.forename: Michael A
authors.initials: MA
authors.lastname: Palladino
authors.forename: Anoop
authors.initials: A
authors.lastname: Shah
authors.forename: Rebecca
authors.initials: R
authors.lastname: Tyson
authors.forename: Jaclyn
authors.initials: J
authors.lastname: Horvath
authors.forename: Christine
authors.initials: C
authors.lastname: Dugan
authors.forename: Marie
authors.initials: M
authors.lastname: Karpodinis
mesh_terms: D000818:Animals; D017209:Apoptosis; D015687:Cell Hypoxia; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism; D051793:Hypoxia-Inducible Factor 1 / Q000032:analysis* / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000032:analysis; D015151:Immunoblotting; D007150:Immunohistochemistry; D007511:Ischemia / Q000378:metabolism; D007985:Leydig Cells / Q000737:chemistry; D008297:Male; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013737:Testis / Q000098:blood supply / Q000737:chemistry*
chemical_list: D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D019253:Proto-Oncogene Proteins c-bcl-2; D004247:DNA