Mini simpozij infrastrukturnih centrov Medicinske fakultete Univerze v Ljubljani, bo potekal 21. junija 2018 med 14:00 in 17:00 v srednji predavalnici na Medicinski fakulteti Univerze v Ljubljani na Korytkovi 2.
Taste of Genomics: the 70th anniversary of Prof. Radovan Komel and 13th CFGBC Scientific Symposium will take place from June 20th-21st 2018 at the University of Ljubljana, Faculty of Medicine, Slovenia.
1Centre for functional Genomics and Bio-Chips, Faculty of Medicine, University of Ljubljana, Slovenia,2Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Slovenia
Icer is a gene that has a role in circadian events. It is important in regulation of melatonin synthesis and it shows high expression in different neurological tissues, especially in the ones that form the HPA axis . We already showed that it affects the circadian expression of different clock-controlled genes in adrenal glands. We mechanistically explained the effect on Per1 expression, which is one of the most important genes that form molecular circadian clock . ICER arises from CREM gene as a product of alternative splicing and is expressed in different isoforms. One of differentiations of ICER isoforms is whether they contain exon g or not. For the purposes of qPCR we could not design a primer pair in such a way that we could amplify all Icer isoforms in a single run. We can observe separately isoforms that contain exon g (Icer) and the ones that do not (Icer g). In an experiment with forskolin treatment (initiator of cAMP signalling), we observed that two isoforms react differently. They also exhibit different circadian profile in mice adrenal glands. With the use of molecular modelling and 3-D structures of proteins from the same family of transcription factors (e.g. CREB) we constructed a model of both ICER isoforms and predict how they bind to DNA. As a template, we took a sequence of Per1 promoter that was already shown that ICER binds to. The difference is in a small exon g that allows isoform ICER to bind DNA more tightly than ICER g. We expressed both ICER isoforms with His-tag in bacteria and purified both proteins on Ni-exchanged sepharose columns. With EMSA we validated the 3-D molecular model structure. With the titration of fluorescent-labelled probes, we showed that ICER affinity to DNA is stronger than ICERg. The question what is the biological role of both isoforms still needs to be answered. One of the approaches are siRNA transfections, which are still in progress (see poster of T. Bensa).
References: 1. Kell CA, Dehghani F, Wicht H, Molina CA, Korf H-W, Stehle JH (2004) Distribution of transcription factor inducible cyclicAMP early repressor (ICER) in rodent brain and pituitary. J. Comp. Neurol. 478, 379–394. 2. Zmrzljak UP, Korenčič A, Košir R, Goličnik M, Sassone-Corsi P, Rozman D (2013) Inducible cAMP Early Repressor Regulates the Period 1 Gene of the Hepatic and Adrenal Clocks. J. Biol. Chem. 288, 10318–10327.
2006 - University of Ljubljana, Faculty of Medicine, Center for Functional Genomics and Bio-chips.