Our long term objective is to determine the combination of transcriptiona]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 importantregulatorof hemopoiesis, whose early and restricted expression in ':his developmental system makes it an excellent candidate for this investigation. The proposed studies on Ikaros regulatory regions active in hemopoietic stem cells (HSC) provideus with an instructive entry point into the transcriptionalprograms that turn on the panoply of genes that specify HSC. For thefirst specific aim we will determine which of the previously identified genomic areas associated with tissue-specificDNAse I Hypersensitivity sites (DHSs) are minimally sufficient for driving expression in HSC and precursor populations.Distinctcombinations 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 regulatoryelements 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 evaluatethe 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 thefourth specific aim we will evaluate the requirementof these regulatoryunits for Ikaros expression in the HSCpopulation. In the course of this work, we will develop expression cassettes that target gene expression to 'he HSC and early lineage restrictedprecursors that are currentlyless accessible to genetic manipulation.We will also mark these rare cells in vivo with fluorescentreporter genes and evaluatethe utility of these markers for their purificationand study.This analysison Ikaros regulationwill identify the molecular codes for inductionof the HSC. This code, and the reagents generated in our attemptsto elucidate it, will increase our ability to manipulatehemopoietic development and may well be applied towards the in vitro generation of blood and immune cells and utilized in transplantationregimes.
