A highly orchestrated gene expression pattern endows stem cells with the capacity to differentiate into diverse cell types. Assembly of cell type-specific chromatin domains and transcriptional networks in stem cells and multipotent progenitors is poorly understood, despite powerful experimental systems, such as mouse bone marrow hematopoietic stem cells (HSCs) and embryonic stem (ES) cell-derived hematopoietic precursors. HoxB4, Bmi 1, beta-catenin, TAL-1 and GATA-2 transcription factors control HSC self-renewal and/or function. We hypothesize that comparing transcriptional mechanisms regulating production of these factors will yield important principles vis-a-vis stem cell biology and hematopoiesis. Studies to test this hypothesis will be instituted with the following Specific Aims. As no examples exist in which the native nucleoprotein structure of an endogenous chromatin domain has been defined in HSCs, this work falls under the rubric of """"""""pilot and feasibility"""""""" studies.
Aim 1. To determine if the histone modification pattern of the GATA-2 domain is developmentally dynamic. GATA-2 is upregulated in erythroid precursors upon targeted disruption of GATA-1, and GATA-1 represses GATA-2 transcription. Using GATA-1-null G 1E cells, we showed that repression involves GATA-l-dependent displacement of GATA-2 from an upstream region (-2.8 kb) and reduced histone acetylation domain-wide. We hypothesize that GATA-2 maintains acetylation and GATA-1 abrogates autoregulation by displacing GATA-2, reconfiguring nucleoprotein complexes, and reducing acetylation. We will determine if this mechanism is operational in HSCs and progenitors and ES cell-derived hematopoietic precursors.
Aim 2. To determine the importance of an upstream GATA factor binding region of the GATA-2 locus. Our studies provided correlative evidence that GATA-2 and GATA-1 binding to the -2.8 kb region confer activation and repression, respectively. We will test this model by generating ES cells with a deletion of GATA sites within the -2.8 kb region and will construct a polydactyl zinc finger protein that prevents GATA factor binding to this region. The impact of the deletion and the inhibitor on GATA-2 expression and hematopoiesis in vitro will be assessed. The studies will define the regulation and function of GATA-2 as a prelude to developing principles for how chromatin domains and transcriptional networks assemble in and control HSCs.
| Martowicz, Melissa L; Grass, Jeffrey A; Boyer, Meghan E et al. (2005) Dynamic GATA factor interplay at a multicomponent regulatory region of the GATA-2 locus. J Biol Chem 280:1724-32 |
