vHNF1 BINDING SITES DETECTION IN THE REGULATORY REGION OF FGF3
Advisor: Enrique Blanco García
E-mail: eblanco@imim.es
1. Background (by Dr. Cristina Pujades)
vHNF1 (variant Hepatocyte Nuclear Factor 1, also called HNF1/TCF2) is an
homeodomain-containing transcription factor. vHnf1 deficient mouse embryos
die shortly after implantation due to abnormal extraembryonic visceral
endoderm formation (Barbacci et al, 1999). It has been recently identified as an
early hindbrain gene required for caudal hindbrain specification in zebrafish. We
have explored this genetic network in the chick, inquiring in addition which are
the signaling pathways involved in caudal rhombomere identity control. We
show that misexpression of vHnf1 confers caudal identity to more anterior
regions of the hindbrain without hampering segment formation. The ectopic
expression of mvHnf1 results in activation of Fgf3 along the hindbrain in a cellautonomous and non-autonomous manner and ectopic induction of MafB and
Krox20, and down-regulation of Hoxb1. However, ectopic vHnf1 is not able to
activate all caudal hindbrain markers. Blockade of FGF signals correlates with a
selective loss of MafB and Krox20 but not vHnf1, Fgf3 and Hoxb1 in the
hindbrain. Overexpression of vHnf1 and Fgf3 together caused a dramatic
upregulation of MafB expression. Based on those observations, we propose
that vHnf1 promotes caudal hindbrain identity either in cooperation or in synergy
with FGFs signals activating MafB and Krox20 expression (Aragón et al., 2005).
Aims
In order to determine whether Fgf3 is a direct target of vHnf1, we want to follow
two approaches:
1. To amplify the cFgf3 transcripts (by RT-PCR) of neural tubes at different
times after overexpression of vHnf1 (15min to 2h); [NOT INCLUDED IN
THE PROJECT]
2. To do an in silico search of vHNF1-binding sites within the regulatory
regions of the Fgf3. For that purpose the student will have to use the
vHNF1-binding motifs published in mice (Cereghini, 1996, Kim et al.,
2005), and search for those in the regulatory regions of the mFgf3 and
cFgf3 gene. A detail study of the putative functional-binding sites is
required.
Lab people
This will be done in collaboration with Ferran Aragón and Cristina Pujades (Dev.
Biol. Group, DCEXS-UPF).
References
Aragón, F., Vázquez-Echeverría, C., Ulloa, E., Reber, M., Cereghini, S., Alsina,
B., Giraldez, F., Pujades, C. (2005). Dev Dyn 234, 567-576
Barbacci, E., Reber, M., Ott, MO., Breillat, C., Huetz, F., Cereghini, S. (1999).
Development 126: 4795-4805
Cereghini, S. (1996) FASEB J. Feb;10(2):267-82
Kim FA, Sing A, Kaneko T, Bieman M, Stallwood N, Sadl VS, Cordes SP.
(2005) Mech Dev. Nov 3;
HNF1=vHNF1=vAPF=LFB3 encoded by TCF2 gene
2. Project
This project mainly consists of computationally detecting vHNF1 sites in the
promoter of FGF3 in chicken and mouse. In addition, a preliminar
characterization of the whole regulatory region is also a secondary objective.
For fulfiling the project requirements, several computational tools will be used in
order to access and to process genomic data from chicken, mouse and human.
No programming skills are demanded, although Perl and Gawk languages can
be very useful to speed the research. Internet efficient use is essential.
This project is oriented to people aiming to use the computers for extracting
useful biological knowledge. Some rudimentary ideas about eukaryotic gene
regulation are necessary. If the results are interesting enough, additional
experimental tests could be used to validate them, but do not expect a priori to
get the proper answer easily.
The final documentation must contain each step of the following protocol clearly
explained. It is recommended to write a log or a README file with all of the
operations that were performed during the project. All of the input files must be
uploaded in a web page to permit other researchers to repeat the tests. The
documentation format is HTML, and abundant use of snapshots explaining the
concepts and the results is a must. The working language in the documentation
must be English. The writing style must be scientific style.
3. Working protocol
The following steps are a preliminar proposal to achieve the best results:
1) Search and read the two references that contain the list of characteristic
binding motifs of vHNF1.
2) Find the FGF3 gene in mouse, chicken and mouse: characterization and
promoter extraction (UCSC genome browser).
3) General analysis: global and local alignments of mouse/chicken promoters,
CpG islands test.
4) Jaspar/Promo/Transfac research study about vHNF1 and FGF3.
5) Motifs: exact pattern matching or with substitutions
Construction of matrices using the published motifs.
(RSA
tools).
6) MatScan: searching the vHNF1 sites with position weight matrices
7) TF-map alignment between chicken and mouse.
8) Repeat the test with other members of the FGF family.
9) Documentation
Personal sessions with the advisor:
1) An introductory session to explain the main lines of the project.
2) In the middle, a session to evaluate the progress of the project.
3) The final interview with the experimentalist and the bioinformatician.
Project sessions:
The project sessions during the laboratory hours may be used for having short
interviews in which small doubts can be solved. The assistance of the teacher
to these ordinary sessions must be previously solicited by e-mail by students
24h before.