Proposal Project to Caltech for SURF
Identification of transcription factors that recognize the RGGNCAG and GCTGGAA sites
Fatih Kocabas
Dorsal is one of the major maternal regulatory gradients and threshold outputs of gene expression that result from the graded distribution of this transcription factor. Dorsal target genes and enhancers represent one of the most extensive gene networks known for any developmental process like dorsoventral patterning in the Drosophila early embryo, which is controlled by the sequence specific transcription factor Dorsal (Stathopoulos A., and Levine M., 2002).
Because some transcription factors bind to specific sequences of DNA and many bind both to DNA as well as to other transcription factors, in this project we want to isolate and study transcription factors that work together with Dorsal to specify and control dorsoventral gene expression in Drosophila embryo.
While studying the dorsoventral patterning in the Drosophila early embryo in Stathopoulos’s laboratory, 18 dorsal-dependent enhancers were isolated for genes expressed along the dorsoventral axis using the microarray screen. Several motifs were identified that seem to correlate with different expression patterns along the dorsoventral axis. For example, both the Neu4 and sog enhancers contain a series of evenly spaced, optimal Dorsal binding sites and share a novel sequence motif GCTGGAA. Some unknown transcription factors may interact with GCTGGAA motif and function together with Dorsal so low level of Dorsal will be enough for activation of gene expression in dorsal regions of the neurogenic ectoderm. Another sequence motif was identified is RGGNCAG (where R= A and G) in the vnd enhancer, the rhomboid NEE, and Mes3 enhancer. This new motif helps identify putative mesoderm enhancers, which are often activated by degenerate, low-affinity Dorsal binding sites that are difficult to discern (Stathopoulos A. et al., 2002).
To identify and isolate these transcription factors that recognize the RGGNCAG (where R= A and G) and GCTGGAA sites, DNA affinity chromatography and one-hybrid screens will be used.
Transcription factors are extremely diverse; any one factor corresponds to only a small fraction of the protein molecules - about 0.001% - present in the cell (Kadonaga J. T., and Tjian R., 1986). Therefore, sequence specific DNA affinity chromatography procedure is necessary and it is simply like this:
Previously, two new motifs have been identified by bioinformatics methods among the Dorsal target enhancers and we know that transcriptional factors binds sequence specific to enhancer part of DNA -in our study, to specific motifs such as GCTGGAA and RGGNCAG-(Stathopoulos A. et al.,2002). Because we already identified DNA motifs, we will synthesize a segment of DNA containing the sequence and we will attach this DNA segments to beads of an inert, solid medium (the matrix). After that we will pour a partially purified extract containing the desired transcription factor obtained from early Drosophila embryo to the column. We expect that only molecules specific for the DNA sequence motifs — in this case, transcription factors that recognize the RGGNCAG and GCTGGAA sites— will bind to the beads. After irrelevant protein molecules have passed through the column, we will wash the beads with a suitable buffer that will release the transcription factors so they can be studied further.
After DNA affinity chromatography procedure, we may do one-hybrid screen to detect whether Dorsal is partner of these isolated transcription factors or not.
Result of this project will be identification of transcription factors that recognize the RGGNCAG (where R= A and G) and GCTGGAA sites, and this will help identify putative mesoderm enhancers and it will help understand regulation of gene expression in dorsal regions of the neurogenic ectoderm further (Stathopoulos A. et al., 2002).
Since I have a keen interest in embryology, this project will be very attractive and suitable for me. Moreover, because as life, formation of an organism from only a cell –zygote- and organ formation, cell and tissue differentiation from only one cell is so attractive for me, I believe it will be exciting to study. In addition, we will do this project together with some other undergrads that will be working in the laboratory. To be ready for this project, this semester I will take “Developmental biology” and “Animal Cell Culture Techniques” courses as Technical Elective courses. Besides, I will continue studying Prof. Dr. Akkaya’s Laboratory. Furthermore, I will learn and -if possible- perform DNA affinity chromatography and one-hybrid screen in accordance with Prof. Dr. Akkaya’s studies.
REFERENCES:
Kadonaga J. T., and Tjian R. (1986). Affinity Purification of Sequence-Specific DNA-Binding Proteins. Proceedings of the National Academy of Sciences, USA 83, 5889-5893.
Markstein M., Zinzen R., Markstein P., Yee K., Erives A., Stathopoulos A., Levine M. (2004). A regulatory code for neurogenic gene expression in the Drosophila embryo. Development 131, 2387-2394.
Stathopoulos A., Drenth M.V., Erives A., Markstein M., Levine M. (2002). Whole-Genome Analysis of Dorsal-Ventral Patterning in Drosophila embryo. Cell 11, 687-701.
Stathopoulos A., and Levine M. (2002). Review: Dorsal Gradient Networks in the Drosophila embryo. Developmental Biology, 246, 57-67.