Örjan Wrange MD, PhD, Professor
Chromatin, epigenetics and gene regulation
- The organization and function of chromatin in the living cell.
- The DNA access for transcription factors in chromatin.
- Chromatin presetting by various transcription factors.
- Transcription regulation by steroid receptors.
- The role of linker histone H1 in chromatin structure and function.
- The mechanism of action of antiandrogens used for treatment of prostate cancer.
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). These steroid receptors are activated by binding of their corresponding hormone, such as glucocorticoid hormone Cortisol or androgenic hormone Testosterone. Hormone binding induces nuclear translocation and sequence specific DNA binding of the receptor to its response elements on DNA. Here the receptor recruits coactivators such as chromatin remodelers leading to chromatin opening near the receptor binding site and, finally, to transcription activation. Several other DNA binding transcription factors interact with the steroid receptors and have their binding sites close to the receptor response elements such as FoxA1, Nuclear factor 1 (NF1) and Oct1 and others.
We use a hormone-responsive gene regulatory element of the mouse mammary tumor virus (MMTV), 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 toad (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 concentration of expressed protein is estimated e.g. by immunoblotting and can be controlled by the amount of injected mRNA. This is exploited to address quantitative effects of transcription factors in vivo. We also microinject the MMTV driven reporter DNA construct into the oocyte nucleus in circular single stranded form. This DNA rapidly 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 footprinting with the methylation pattern developed by primer extension. Chromatin related methods, such as digestion with DNase I or micrococcal nuclease as well as ChIP assay can be applied as well. The nuclear uptake of receptor(s) and other proteins may be analyzed by Western blot of manually dissected nuclei.
Top: Injection of an oocyte with synthetic mRNA into the cytoplasm and single stranded DNA into the nucleus. Middle: The timeframe of the experiment. Bottom: the intracellular second DNA strand synthesis that is coupled to chromatin assembly.
This is a curiosity-driven basic research project focusing on how genes are regulated in the normal cell. This knowledge is exploited in translational research on the mechanism of action and efficiency of different drugs currently used in treatment of prostate cancer.
- Örjan Wrange, M. D. Ph.D. professor Tel: 5248 7373
- Sergey Belikov, Ph.D. Docent, Senior Researcher Tel: 5248 7903
FoxA1 corrupts the antiandrogenic effect of Bicalutamide but only weakly attenuates the effect of MDV3100 (Enzalutamide).
Molec. Cell. Endocrinol. 2013, vol. 365, 95-107
Linker histone subtypes differ in their effect on nucleosomal spacing in vivo.
J. Mol. Biol., 2012, Mar 21.
FoxA1 and glucocorticoid receptor crosstalk via histone H4K16 acetylation at a hormone regulated enhancer.
Exp Cell Res. 2012, vol. 318 (1), 61-74.
FoxA1 binding directs chromatin structure and functional response of a glucocorticoid receptor-regulated promoter.
Mol Cell. Biol., 2009, vol. 29 (20) 5413-5425
Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter.
Exp Cell Res. 2009 vol. 315(15), 2604-2615
The Swedish Cancerfoundation and the Swedish Research Council in Medicine are gratefully acknowledged for their support.