Transcriptional regulation during embryogenesis
Final Report Abstract
In this project we have studied the targets of TATA binding protein in zebrafish development using various techniques. We have described a previously uncovered role for TBP in regulating the degradation of maternal mRNAs during the maternal to zygotic transition in zebrafish embryo. We have shown that TBP acts in the miR-430 dependent mRNA degradation pathway, but does not act directly on miR-430 synthesis. It has thus been suggested that TBP acts at the level where the zygotic transcription of a yet unknown mRNA degradation component is required in miR-430 dependent mRNA degradation. To ask questions about the transcriptional role of TBP on developmentally expressed gene promoters we have embarked on the identification of transcriptional start sites at a large scale paralleled by the isolation of zebrafish protein coding gene promoters. We have generated a library of 40 zebrafish promoters and we have studied the direct transcriptional effects of TBP loss on these promoters by Morpholino knock down and subsequent analysis of fluorescent reporter gene expression driven by the selected promoters in transgenic zebrafish. We have identified a negative gene regulatory role of TBP on a small set of promoters which are stimulated upon TBP loss. The promoter for the zebrafish tbp gene itself is among the negatively regulated promoters and this negative regulation appears to be direct when assayed by immunoprecipitation to detect TBP occupancy. We have further analysed the role of TBP in developmental gene regulation through the analysis of enhancer and core promoter interactions in a system biology approach and identified the striking diversity of interactions dependent on core promoter sequence. We have further analysed the core promoter sequence on zebrafish promoter to better understand the relationship between core promoter and TBP function. We have identified 4000 zebrafish promoters based on transcriptional start site analysis, verified these core promoters by chromatin immunoprecipitation with an antibody recognizing the promoter mark H4k4me3 and analysed TBP occupancy on a selected set of these promoters. Taken together, this project highlighted the differential regulatory function of TBP during development thus confirming the notion that general transcription factors such as members of the TBP family, previously though to act ubiquitously, are regulating only subsets of genes and are proposed to be integral part of the regulatory pathways which act during the generation of organismal complexity. Bioinformatic, biochemical (ChIP) and functional analysis (reporters in transgenic fish) of core promoters in this project have contributed to the discovery of diversity of core promoters with sequence dependent features, which define TBP dependence, and the capacity of the core promoter sequence to differentially interact with developmental cis-regulatory elements located away from the core promoter in the zebrafish model system.
Publications
- (2007). New Problems in RNA Polymerase II Transcription Initiation: Matching the Diversity of Core Promoters with a Variety of Promoter Recognition Factors. J. Biol. Chem. 282(20):14685-9
Müller, F., Demény, M.A., Tora, L.
- (2007). The TATA binding protein (TBP) regulates maternal mRNA degradation and differential zygotic gene activation in the zebrafish embryo. EMBO J. 26(17) 3945-56
Ferg, M., Sanges, R., Gehrig, J., Lovas, A., Bauer, M., Kiss, J., Szabo, M., Olasz, F., Pankratz, M., Stupka, E., and Müller, F.
- Automated high-throughput mapping of promoter-enhancer interactions in zebrafish embryos. (2009), Nature Methods, 6(12):911-6
Gehrig J, Reischl M, Kalmár E, Ferg M, Hadzhiev Y, Zaucker A, Song C, Schindler S, Liebel U. and Müller F.
