Our long-term objective is to determine the combination of transcriptional inputs that are necessary and sufficient for inducing the hemopoietic cell fate. To do so we propose to study the transcriptional regulation of Ikaros, an important regulator of hemopoiesis, whose early and restricted expression in this developmental system makes it an excellent candidate for this investigation. The proposed studies on Ikaros regulatory regions active in hemopoietic stem cells (HSC) provide us with an instructive entry point into the transcriptional programs that turn on the panoply of genes that specify HSC. For the first specific aim we will determine which of the previously identified genomic areas associated with tissue-specific DNAse I Hypersensitivity sites (DHSs) are minimally sufficient for driving expression in HSC and precursor populations. Distinct combinations of DHS driving expression of fluorescent reporters will be tested for activity at the early steps of the hemo-lymphoid hierarchy in transgenic mice. For the second specific aim we will delineate the regulatory elements within these regions responsible for activity. Mutational analysis will be performed on sequences that are conserved across species. For the third specific aim we will identify the nuclear factors responsible for activity of the Ikaros regulatory elements in HSC using three different approaches (biochemical, bio-informatics and genetics). In the second part of this aim we will functionally evaluate the role of the identified factors for activity of the regulatory region upon their ectopic expression in a variety of cell types derived from the transgenic lines. For the fourth specific aim we will evaluate the requirement of these regulatory units for Ikaros expression in the HSC population. In the course of this work, we will develop expression cassettes that target gene expression to the HSC and early lineage restricted precursors that are currently less accessible to genetic manipulation. We will also mark these rare cells in vivo with fluorescent reporter genes and evaluate the utility of these markers for their purification and study. This analysis on Ikaros regulation will identify the molecular codes for induction of the HSC. This code, and the reagents generated in our attempts to elucidate it, will increase our ability to manipulate hemopoietic development and may well be applied towards them in vitro generation of blood and immune cells and utilized in transplantation regimes.
