| Örjan
Wrange's Research Group
Örjan
Wrange, M.D., Ph.D., Professor Tel: +46 8 524 873 73
Sergey
Belikov, Ph.D., Docent, Reseacher Tel: +46 8 524 873 73
Carolina Åstrand, Graduate Student Tel: +46 8 524 873 81
Title:
Chromatin tructure and epigenic marks involved in gene regulation
by the glucocorticoid receptor
-
How is the
DNA of a silent promoter packaged in the living cell?
-
How is it unpacked when a gene is activated to be transcribed?
-
What is the structural effects of the gene activation process?
-
How do transcription factors and coactivators cooperate during gene activation?
-
How are histone proteins modified during gene regulation?
-
How
are histone modifications involved in recruitment and chromatin structure function?
By gaining more information on how genes are regulated in the normal case, we hope to understand how disturbed gene regulation may cause disease, such as Cancer, and how this might be treated. New and exciting potentials in this work is offered by the newly discovered enzymes that are involved in chemical modification of the histone proteins that organizes the DNA in the cell nucleus.
From yeast to man, the DNA in every living cell is packaged into a DNA-protein complex called chromatin, the smallest packaging unit of which is the nucleosome. Genes are switched ON or OFF by regulatory proteins such as the glucocorticoid receptor. This receptor is regulated by a hormone ligand i.e. glucocorticoid hormone. An early step in gene-activation is the glucocorticoid receptor mediated recruitment of co-activators. These are large multiprotein complexes that have enzymatic activities able to covalently modify the surrounding chromatin proteins or to remodel its structure by an ATP-dependent process. During gene activation these processes result in a more relaxed chromatin structure of the induced promoter and hence in a more accessible DNA.
Reconstitution of a massive chromatin remodeling event in a giant cell: We study chromatin remodeling in a cell of about 1 mm in diameter, the Xenopus oocyte. They are obtained from the African clawed toad (Xenopus laevis). By injection of in vitro transcribed mRNA coding for one or several proteins into the cytoplasm, the oocyte becomes programmed to produce the corresponding protein(s) (see figure). We then microinject reporter gene DNA into the oocyte nucleus. this DNA is efficiently assembled into chromatin by use of the stockpile of chromatin proteins in the oocyte.
The advantage with this model system is that we can inject up to a billion gene copies of a regulated gene in one single oocyte and then get all copies activated concomitantly e.g. by glucocorticoid hormone induction. The large copy number amplifies the structural information and can thus be analyzed at higher resolution than in a cell with a complex genome. The Xenopus oocyte is used as a live bioreactor for mechanistic studies of gene regulation in short term experiments.
These oocytes are not fertilized, cannot undergo any cell division and cease to function when energy stores run out.
Figure
legend: Above : Injection of an oocyte with synthetic mRNA into the cytoplasm
and single stranded DNA into the nucleus. Middle: The timeframe of the
experiment. Below: the intracellular second DNA strand synthesis that is
coupled to chromatin assembly.
Recent
publications
Holmqvist P-H, Belikov S, Zaret, K. S., Wrange Ö, (2005) FoxA1 binding to the MMTV LTR modulates chromatin structure and transcription. Exp. Cell Research vol 304, 593-603
Belikov S, Holmqvist PH, Åstrand C, Wrange Ö. (2004) Nuclear factor 1 and octamer transcription factor1 binding preset the chromatin structure of the mouse mammary tumor virus promoter for hormone induction. J Biol Chem. vol. 279 (48), 49857-67
Åstrand C, Klenka T, Wrange Ö, Belikov S, (2004) TSA reduces hormone-induced transcription of the MMTV promoter and has pleiotropic effects on its chromatin structure. Europ. J. Biochem. vol 271(6), 1153-1162
Belikov S, Åstrand C, Holmqvist P-H, Wrange Ö. (2004) Chromatin mediated restriction of NF1/CTF binding in a repressed and hormone activated promoter in vivo . Molec. Cellular Biol., vol. 24 (7), 3036-3047.
Contact
for information:
E-mail:
orjan.wrange@ki.se
Phone:
+46-(0)8-524 873 73
Fax: +46-(0)8-31 35 29
Mailing
address:
Örjan
Wrange
Dept.
of Cell and Molecular Biology
The
Medical Nobel Institute
Karolinska
Institutet
P.O.
Box 285
SE-171
77 Stockholm, Sweden
Funding:
The Swedish Cancer Foundation and the Swedish Research Council in Medicine are gratefully acknowledged for support.
| 
