Hematopoiesis, the differentiation of distinct lineages of blood cells from a common progenitor, is intricately controlled at the transcriptional level. Detailed knowledge of the transcription factors and their molecular interactions is essential to a complete understanding of normal hematopoiesis as well as an array of disorders that arise from de-regulated transcriptional control. In addition, transcriptional regulation in hematopoiesis is a paradigm for reprogramming pluripotent stem cells and is therefore a key area of inquiry in bioengineering as well. Currently, hematopoietic transcription factors are modulated indirectly through their expression levels by genetic approaches, such as transgenes, engineered knock-out/down animal models and RNA interference. These techniques, which are slow in onset, persistent in duration, and global in effect, are ill-suited to dissect transcriptional pathways an probe transient interactions that typify transcriptional control. Direct chemical control of transcriptional activity i.e. at the protein/DNA level, is currently unavailable for the vast majorty of hematopoietic regulators. Additionally the strong structural conservation among members of transcription factor families confounds simple screening of chemical libraries against these transcriptional factors. Here, we explore a physicochemical strategy to isolate activators of structurally homologous members of the ETS-family transcription factors PU.1 and Ets-1, which play distinct roles in hematopoietic cell-fate determination. Specifically, we will screen a phage display library against cognate PU.1/DNA and Ets-1/DNA complexes under osmotically variable conditions (alternating conditions of normal and elevated osmolality) to isolate peptides that selectively stabilize the binding of PU.1 or Ets-1 to their cognate binding sites. Our proposal is based on our recent studies showing that cognate site binding by PU.1 is exquisitely sensitive to osmotic stress, but Ets-1 is insensitive, even though the DNA binding domains of the two proteins are structurally superimposable. After further optimizing sequence candidates using combinatorial libraries, we will determine the ability of lead candidates to localize in the nucleiof hematopoietic progenitor cells and specifically activate PU.1 or Ets-1 target genes. Given the emerging interest in PU.1 and Ets-1 in a broad range of experimental models of hematopoiesis, the peptides generated from this research are expected to become useful reagents for interrogating PU.1 and Ets-1 target genes as well as templates for targeted diagnostics and therapeutics in hematologic, rheumatologic, and infectious diseases.
This proposal is aimed at developing new compounds that act directly on and activate cellular regulators involved in the development of blood cells from their precursors. These compounds are designed to overcome limitations of existing genetic methods and enable previously inaccessible experimental studies into the function and interactions of these essential regulators. In addition, we anticipate these novel compounds to serve as templates for new diagnostics and therapeutics for hematologic, rheumatologic, and infectious diseases in which these regulators have been recently implicated.
