Epigenetic regulation of HYAL-1 hyaluronidase expression. identification of HYAL-1 promoter.
HYAL-1 (hyaluronoglucosaminidase-1) belongs to the hyaluronidase family of enzymes that degrade hyaluronic acid. HYAL-1 is a marker for cancer diagnosis and a molecular determinant of tumor growth, invasion, and angiogenesis. The regulation of HYAL-1 expression is unknown. Real time reverse transcription-PCR using 11 bladder and prostate cancer cells and 69 bladder tissues showed that HYAL-1 mRNA levels are elevated 10-30-fold in cells/tissues that express high hyaluronidase activity. Although multiple transcription start sites (TSS) for HYAL-1 mRNA were detected in various tissues, the major TSS in many tissues, including bladder and prostate, was at nucleotide 27274 in the cosmid clone LUCA13 (AC002455). By analyzing the 1532 base sequence 5' to this TSS, using cloning and luciferase reporter assays, we identified a TACAAA sequence at position -31 and the minimal promoter region between nucleotides -93 and -38. Mutational analysis identified that nucleotides -73 to -50 (which include overlapping binding consensus sites for SP1, Egr-1, and AP-2), bases C(-71) and C(-59), and an NFkappaB-binding site (at position -15) are necessary for promoter activity. The chromatin immunoprecipitation assay identified that Egr-1, AP-2, and NFkappaB bind to the promoter in HYAL-1-expressing cells, whereas SP1 binds to the promoter in non-HYAL-1-expressing cells. 5-Aza-2'-deoxycytidine treatment, bisulfite DNA sequencing, and methylation-specific PCR revealed that HYAL-1 expression is regulated by methylation at C(-71) and C(-59); both Cs are part of the SP1/Egr-1-binding sites. Thus, HYAL-1 expression is epigenetically regulated by the binding of different transcription factors to the methylated and unmethylated HYAL-1 promoter.
Lokeshwar
Vinata B
VB
Department of Urology, University of Miami Miller School of Medicine, Miami, Florida 33101, USA. vlokeshw@med.miami.edu
Gomez
Pablo
P
Kramer
Mario
M
Knapp
Judith
J
McCornack
Melissa A
MA
Lopez
Luis E
LE
Fregien
Nevis
N
Dhir
Neetika
N
Scherer
Steve
S
Klumpp
David J
DJ
Manoharan
Murugesan
M
Soloway
Mark S
MS
Lokeshwar
Bal L
BL
D001665:Binding Sites; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D044127:Epigenesis, Genetic; D006801:Humans; D006821:Hyaluronoglucosaminidase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000502:physiology*; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D014018:Tissue Distribution
D017931:DNA Primers; D016328:NF-kappa B; D012333:RNA, Messenger; D004247:DNA; D008156:Luciferases; D006821:Hyaluronoglucosaminidase