| dc.description.abstract | Investigations detailed in this thesis constitute research work undertaken in our laboratory on Transforming Growth Factor5 (TGF5), with particular reference to determining the gene structure, characterizing the promoter, localizing the mRNA in Xenopus laevis embryos, and studying the regulation of its gene expression.
Transforming Growth Factor (TGF) is a prototype of a large superfamily of growth factors that are potent regulators of cellular proliferation, differentiation, and morphogenesis. Members of the TGF superfamily have been identified in organisms ranging from nematodes to humans. The superfamily comprises over thirty distinct members grouped into subfamilies including TGFs, activins/inhibins, Bone Morphogenetic Proteins (BMPs), the Drosophila decapentaplegic product (dpp), Müllerian Inhibiting Substance, Xenopus laevis Vg1 and Vgr1, and glialcellline derived neurotrophic factor (GDNF).
The bioactive forms of TGFs are homodimers or heterodimers linked by disulfide bonds via the seventh cysteine of each monomer. The monomers are synthesized as Cterminal portions of precursor proteins and are cleaved at clusters of basic residues after dimerization. TGFs signal through two distinct transmembrane serine/threonine kinases.
In vertebrates, the TGF family consists of five distinct genes. Isoforms 1, 2 and 3 are present in mammals; 2, 3 and 4 in birds; 2 and 5 in amphibians; and 2 has also been identified in fishes. All isoforms share >70% aminoacid sequence identity. Most studies on transcriptional regulation have focused on mammalian isoforms 1-3. Their promoters show no significant homology, consistent with differing mechanisms of transcriptional regulation and distinct expression profiles during embryogenesis.
Expression of TGF5 mRNA in Xenopus laevis has been reported from neurula stages onwards. In adults, highest expression is seen in the lung, followed by heart, kidney, stomach and liver. TGF1 and TGF5 have been shown to induce their own transcription, and AP1 mediates TGF1 autoinduction.
To understand mechanisms underlying stagespecific and tissuespecific expression of TGF5, this thesis localizes its mRNA in early Xenopus embryos, characterizes its 5flanking region, and determines its gene structure.
Chapter 1
Summarizes biochemical properties of the TGF family, their expression during mammalian embryogenesis, and their effects on proliferation, differentiation and morphogenesis.
Chapter 2
Describes the molecular and cellbiology techniques used in the study.
Chapter 3 - Genomic Structure and Promoter Characterization
A Xenopus laevis genomic library was screened, yielding five genomic clones (XBG1, XBG2, XBG3, Fg1, Fg2). Clones Fg1 and Fg2 were identical; Fg1 contained 2410 bp of 5flanking region. Clones XBG1-3 contained the exonic regions of the gene.
Key findings:
The 5flanking sequence showed no significant homology to promoters of other TGF isoforms.
Multiple consensus transcriptionfactor binding sites were identified: AP1, AP2, C/EBP, CREBP/JUN, Ets1, ER, GATA1, vMyb, Myc/Max, MyoD, NF1, Nkx2.5, Oct1, Sox5/SRY, Sp1, TR and USF.
The TGF5 gene comprises seven exons; intron-exon boundaries obey the GT-AG rule.
All splice junctions except that of intron 1 correspond precisely to those in mammalian TGF1 and TGF3 genes.
Introns vary in size from 85 bp to ~10 kb; the gene is ~20 kb long.
These analyses confirm that all TGF isoforms share a conserved intron-exon organization.
Chapter 4 - Transcriptional Regulation
Transcription start sites (TSS):
5RACE identified two TSS (tss1 and tss2), located 26 bp and 112 bp upstream of the reported 5 end of the TGF5 cDNA. Different TSS usage may occur at different developmental stages.
Promoter analysis:
Luciferase reporter assays using overlapping promoter fragments showed:
L50 (+83 to 466 bp relative to tss1) had maximal promoter activity - representing the functional core promoter.
L42 (+118 to 1278 bp) showed 3.3fold reduced activity, indicating a possible negative regulatory region upstream of 466 bp.
No promoter activity was detected further upstream.
Regulation in cell lines:
Northern blotting showed high expression of TGF5 mRNA in XTC cells (tadpolederived), but not in A6 cells (kidneyderived). RTPCR confirmed trace expression in A6 cells.
Serum strongly induced TGF5 mRNA in XTC cells:
Induction began by 3 h
Reached plateau by 9 h
Serum responsiveness may involve Nkx2.5 binding sites found in the promoter.
mRNA Localization in Embryos
Wholemount in situ hybridization revealed TGF5 expression predominantly in mesodermal derivatives, including:
somites
branchial arches
heart anlage
tail region
Earliest expression was detected in dorsal mesoderm at stages 12/13 and 14/15. Transient expression was seen in:
cement gland (stage 20)
branchial arches and heart anlage (stage 26)
tail region (stages 22/24 onwards)
end of notochord and adjoining mesoderm (stage 29)
As the embryo entered tailbud stages (26/27), expression increased in posterior somites. This pattern resembles expression of MyoD in Xenopus laevis, suggesting that MyoDbinding sites in the promoter might regulate these developmental patterns.
Conclusion
The expression pattern and promoter analysis strongly suggest important roles for TGF5 during early Xenopus laevis development. Despite considerable homology between mammalian TGF1 and Xenopus TGF5, comparison of their 5flanking regions shows that the two genes are distinct. | |
