During erythropoiesis cells go through a progression of profound morphologic changes during which all but a limited subset of genes are silenced, presumably associated with incorporation into heterochromatin. Meanwhile other genes, such as beta-globin, become highly activated. The mechanisms by which higher order chromatin structure is regulated, and how it is linked to other aspects of gene activation such as subnuclear localization, epigenetic modifications and expression remains unknown. Generating cell lines and mice with targeted mutations of the endogenous beta-globin locus has proven to be a valuable approach to begin to link these processes. Unfortunately, standard gel based assays to assess generalized chromatin sensitivity are non-quantitative, time consuming, difficult to reproduce, require tremendous amounts of tissue, and only provide information on limited areas. Quantitative Chromatin PCR (QCP) has been recently developed and validated as a tool to sensitively and specifically screen large regions of the genome for DNase l hypersensitivity sites. The goal of this proposal is to extend this by validating QCP as a tool to quantitatively describe continuous """"""""profiles"""""""" of DNase l sensitivity over entire gene loci allowing us to correlate changes in this chromatin landscape with cellular morphology, sub-nuclear localization, histone modifications and expression during terminal differentiation, and to determine the effect of cis-acting mutations. QCP will be compared with standard DNase l assays in several model systems to simultaneously validate QCP and address questions regarding chromatin structure during erythropoiesis. Specifically we propose to 1) develop a continuous map (landscape) of chromatin sensitivity throughout the human beta globin locus and to correlate changes in patterns observed in QCP with those observed with traditional DNase l chromatin sensitivity assays, 2) demonstrate the utility of QCP by quantitatively defining changes in chromatin structure and determining how these correlate with changes in active expression in systems designed to address seminal questions of erythropoiesis. These include correlating active expression and chromatin structure of several genes during terminal differentiation, and determining how LCR mutations and additional targeted mutations of the endogenous locus affect chromatin structure, and 3) adapt QCP to study the endogenous mouse beta-globin locus in primary tissues of wildtype and mutant mice. ? ?
| Sawado, Tomoyuki; Halow, Jessica; Im, Hogune et al. (2008) H3 K79 dimethylation marks developmental activation of the beta-globin gene but is reduced upon LCR-mediated high-level transcription. Blood 112:406-14 |
