A-form DNA structure is a determinant of transcript levels from the Xenopus gata2 promoter in embryos.
We have previously shown that a critical region of the gata2 promoter contains an inverted CCAAT box and adopts a partial A-form DNA structure in vitro. At gastrula stages of development transcription requires binding of CBTF (CCAAT box transcription factor), a multi-subunit transcription factor, to this region. Xilf3 is one component of CBTF and the double stranded RNA binding domains (dsRBDs) of Xilf3 must be active for both binding to, and transcription from, this promoter. Here we determine the contribution of DNA sequence and structure at the gata2 promoter to transcriptional activity. In all the constructs we tested a CCAAT box was a requirement for full activity. However, base substitutions that increase B-form structure propensity in the sequences flanking the CCAAT box are equally able to decrease activity even if a CCAAT box is present. In contrast, mutations that maintain A-form propensity in these regions also maintain, or increase, transcription factor binding and transcriptional activity. We propose a two-component model for the interaction of CBTF with the gata2 promoter, requiring both a CCAAT sequence and flanking A-form DNA structures. These results support a novel role for dsRBDs in transcriptional regulation and suggest a function for A-form DNA in vivo.
Llewellyn
Katrina J
KJ
Biophysics laboratories, School of Biological Sciences, IBBS, University of Portsmouth, Portsmouth, PO1 2DT, UK.
Cary
Peter D
PD
McClellan
James A
JA
Guille
Matthew J
MJ
Scarlett
Garry P
GP
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D044704:DNA, A-Form / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004625:Embryo, Nonmammalian / Q000196:embryology / Q000378:metabolism*; D050989:GATA2 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D050779:Nuclear Factor 90 Proteins / Q000235:genetics / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D029867:Xenopus Proteins / Q000235:genetics* / Q000378:metabolism; D014982:Xenopus laevis / Q000196:embryology / Q000235:genetics*
D023081:CCAAT-Binding Factor; D044704:DNA, A-Form; D050989:GATA2 Transcription Factor; C494714:GATA2 protein, Xenopus; D050779:Nuclear Factor 90 Proteins; D011993:Recombinant Fusion Proteins; D029867:Xenopus Proteins; D008156:Luciferases