Identification of direct T-box target genes in the developing zebrafish mesoderm.
The zebrafish genes spadetail (spt) and no tail (ntl) encode T-box transcription factors that are important for early mesoderm development. Although much has been done to characterize these genes, the identity and location of target regulatory elements remain largely unknown. Here, we survey the genome for downstream target genes of the Spt and Ntl T-box transcription factors. We find evidence for extensive additive interactions towards gene activation and limited evidence for combinatorial and antagonistic interactions between the two factors. Using in vitro binding selection assays to define Spt- and Ntl-binding motifs, we searched for target regulatory sequence via a combination of binding motif searches and comparative genomics. We identified regulatory elements for tbx6 and deltaD, and, using chromatin immunoprecipitation, in vitro DNA binding assays and transgenic methods, we provide evidence that both are directly regulated by T-box transcription factors. We also find that deltaD is directly activated by T-box factors in the tail bud, where it has been implicated in starting the segmentation clock, suggesting that spt and ntl act upstream of this process.
Garnett
Aaron T
AT
Department of Molecular and Cell Biology and Center for Integrative Genomics, University of California, Berkeley, CA 94720, USA.
Han
Tina M
TM
Gilchrist
Michael J
MJ
Smith
James C
JC
Eisen
Michael B
MB
Wardle
Fiona C
FC
Amacher
Sharon L
SL
D000818:Animals; D030801:Animals, Genetically Modified; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism; D005326:Fetal Proteins; D018507:Gene Expression Regulation, Developmental; D008648:Mesoderm / Q000196:embryology / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional; D012689:Sequence Homology, Nucleic Acid; D015398:Signal Transduction; D020825:T-Box Domain Proteins / Q000235:genetics* / Q000378:metabolism; D013623:Tail / Q000196:embryology / Q000378:metabolism; D051153:Wnt Proteins / Q000235:genetics / Q000378:metabolism; D015027:Zebrafish / Q000196:embryology* / Q000235:genetics* / Q000378:metabolism; D029961:Zebrafish Proteins / Q000235:genetics* / Q000378:metabolism; D000098908:Brachyury Protein
D004247:DNA; D005326:Fetal Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D020825:T-Box Domain Proteins; D051153:Wnt Proteins; D029961:Zebrafish Proteins; D000098908:Brachyury Protein; C468126:dld protein, zebrafish; C112539:tbx16 protein, zebrafish; C491180:tbx6 protein, zebrafish