Örjan Wrange emeritus
Örjan Wrange, M. D., Ph.D. professor emeritus
(Örjan retired Aug 2015 and the laboratory was closed in December 2015)
- DNA binding affinity of steroid receptors in vivo.
- The mechanism of action of antiandrogens for treatment of prostate cancer.
- The organization and function of chromatin in the living cell.
- The DNA access for transcription factors in chromatin.
- Chromatin presetting and cooperative binding by transcription factors.
DNA in every living cell is folded 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 (GR) and the androgen receptor (AR). The gene regulatory activity of each steroid receptor is triggered by the binding of its corresponding hormone, such as Cortisol for GR and Testosterone for AR. Hormone binding induces a cytosol-to-nuclear translocation and sequence specific DNA binding of the receptor to its recognition sequence on DNA , i.e. the enhancer. Once DNA-bound the receptor recruits other protein complexes referred to as co-activators or chromatin remodelers that are able to open the chromatin structure and make the DNA more accessible for transcription, i.e. gene expression. The genes to become activated are selected by the protein-DNA binding reaction of the steroid receptor together with several other DNA binding transcription factors that bind cooperatively to the DNA at adjacent sites thus forming composite DNA response elements. The factors that bind cooperatively with steroid receptors may also interact with DNA before the steroid receptor has been activated by hormone and is then referred to as pioneer factors since they have the capacity to preset the steroid response element. Factors that are involved in these events are e.g. FoxA1, Nuclear factor 1 (NF1) and Oct1 and others. The combination of these different cooperatively bound factors control the selectivity of the DNA binding event for each type of steroid receptor and for each DNA. This selection of regulatory DNA sites to be accessible for each steroid receptor determines the genetic program of each cell type. Different genes will thus be activated in different cell types by one and the same steroid receptor-hormone complex. The more than 200 different cell types of the human body thus are able to express a selective combination of genes to provide their different specialized characters. The DNA binding transcription factors thus transforms the genetic code into biological activity. My focus has been to understand how this is possible?
We used a hormone-responsive gene regulatory element of the mouse mammary tumor virus (MMTV) as well as other DNA elements to study the interactions between different DNA binding factors during the gene induction event. We reconstitute this genetic switch in Xenopus oocyte, a giant cell of about 1.2 mm diameter. These oocytes are obtained from the ovarium of the African clawed frog (Xenopus Laevis), they are not fertilized, cannot undergo any cell division and cease to function when energy stores run out. The oocyte may be programmed to produce any protein(s) at will by injection of the corresponding mRNA(s). The intranuclear concentration of expressed hormone receptor protein may be quantified using tritium-labeled steroid hormone treated Xenopus oocytes from which nuclei
may be manually isolated. This information was exploited to address quantitative effects of transcription factors in vivo. The various DNA constructs were injected into the oocyte nucleus in circular single stranded form. This DNA then undergoes second strand synthesis and is efficiently assembled into chromatin by use of the stockpile of chromatin proteins stored in the oocyte. Hence the Xenopus oocyte is used as a live test-tube for mechanistic studies of gene regulation in short term experiments. Another advantage with this system is its capacity to reconstitute more than a billion gene copies into chromatin organized minichromosomes which can be activated concomitantly, e.g. by hormone induction (see above). The large copy number amplifies the structural information that can thus be analyzed at higher resolution than in a cell with a complex genome. The high copy number also allows the specific protein-DNA binding to be quantitatively monitored in vivo by dimethyl sulphate (DMS) methylation protection, i.e. footprinting. Chromatin structure was monitored by DNase I or micrococcal nuclease.
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.
This is a curiosity-driven basic research project focused on how genes are regulated in the normal cell. This knowledge was exploited in translational research on the mechanism of action and efficiency of different drugs currently used in treatment of prostate cancer.
Quantification of transcription factor-DNA binding affinity in a living cell.
Belikov S, Berg O, Wrange Ö
Nucleic Acids Res. 2016 Apr;44(7):3045-58
The polyglutamine-expanded androgen receptor has increased DNA binding and reduced transcriptional activity.
Belikov S, Bott LC, Fischbeck KH and Wrange Ö.
Biochem. Biophys. Reports 2015 vol. 3, 134-139. doi:10.1016/j.bbrep.2015.07.014
FoxA1 corrupts the antiandrogenic effect of bicalutamide but only weakly attenuates the effect of MDV3100 (Enzalutamide™).
Belikov S, Öberg C, Jääskeläinen T, Rahkama V, Palvimo J, Wrange Ö
Mol. Cell. Endocrinol. 2013 Jan;365(1):95-107
Linker histone subtypes differ in their effect on nucleosomal spacing in vivo.
Öberg C, Izzo A, Schneider R, Wrange Ö, Belikov S
J. Mol. Biol. 2012 Jun;419(3-4):183-97
FoxA1 and glucocorticoid receptor crosstalk via histone H4K16 acetylation at a hormone regulated enhancer.
Belikov S, Holmqvist P, Astrand C, Wrange Ö
Exp. Cell Res. 2012 Jan;318(1):61-74