Identification of an Sp factor-dependent promoter in GCET, a gene expressed at high levels in germinal center B cells.
Antigen-stimulated B lymphocytes undergo genetic and phenotypic changes in germinal centers (GCs), including affinity maturation of immunoglobulin (Ig) genes and Ig heavy chain isotype switching. Expression of the Germinal Center Expressed Transcript (GCET) gene is up-regulated in murine GC B cells. The human homolog of GCET, HGAL/GCET2, is an important prognostic marker for staging lymphomas derived from GCs. To identify mechanisms that control cell type-specific transcription of GCET, we localized promoter sequences using S1 nuclease protection and functional assays. Sequences comprising a TATA-less promoter were localized to a short region upstream of multiple mRNA start sites. In functional assays, the promoter is active in cells irrespectively of endogenous GCET gene expression. In vitro binding assays identified a non-consensus binding site for Sp factors near sites of transcriptional initiation. The site binds Spl and Sp3 in nuclear extracts and recombinant Spl in vitro, and is required for full promoter function in transient promoter assays. Activation of the promoter by Spl or Sp3 in Spl/3-deficient cells was largely dependent on the Sp site. Together, these data provide the first analysis of regulatory modules necessary for GCET expression, a model for GC B cell-specific transcription.
Allergy Division, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
John W
JW
Steinke
Wendy
W
Hodsdon
Sarah
S
Parenti
Rachel
R
Ostraat
Ryan
R
Lutz
Larry
L
Borish
James
J
Hagman
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000502:physiology; D018858:Germinal Center / Q000166:cytology*; D051379:Mice; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D016329:Sp1 Transcription Factor / Q000502:physiology*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics* / Q000502:physiology; D024363:Transcription Initiation Site
D004268:DNA-Binding Proteins; C494709:Gcet2 protein, mouse; D012333:RNA, Messenger; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; D014157:Transcription Factors; D051705:Sp3 Transcription Factor