PU.1 levels are critical for normal hematopoiesis and, even modest alterations can lead to leukemia and lymphoma. To achieve optimal expression of the PU.1 gene at different stages of hematopoiesis, multiple interactions are required between its regulatory elements to form fine chromatin architecture. While significant advances have been made in understanding which PU.1 locus chromatin states are active or silent, nothing is known how these states are propagated and maintained. The overall goal of the proposed project is to understand the role of antisense RNAs in the regulation of the PU.1 gene. Therefore I will (1) Characterize the timing, order, cellular localization, and levels of the natural noncoding antisense and coding PU.1 transcripts and formation of the active chromatin architecture within the PU.1 gene locus during cell development; (2) Investigate the functional effect of nuclear ablation of antisense concoding RNAs ablation on the formation of the active chromatin configuration and the expression of the PU.1 gene and, ultimately, on hematopoietic stem cell (HSC) fate; (3) Study the functional role of antisense noncoding RNAs in tissue culture and transgenic systems; and (4) Investigate the functional connection between the expression of the antisense noncoding RNAs and the occurrence of the other epigenetic marks, such as chromatin compaction, covalent histone modification, and DNA methylation. Accomplishment of these aims will enable us to understand the roles of antisense noncoding RNAs corresponding to PU.1 gene locus in the regulation of hematopoietic stem cell differentiation and to identify naturally occurring RNAmediated regulatory mechanisms, the manipulation of which might lead to critical changes in cell fate and provide knowledge of how changes in antisense RNAs can lead to disorders of hematopoietic stem cell function and leukemia.