etin and stem cell factor modestly enhance globin gene expression; a cocktail of Epo, granulocyte monoctye-colony stimulating factor and interleukin-3 does not. BMCs derived from -117 Greek HPFH b-YAC transgenic mice recapitulated the HPFH phenotype. A c-globin-specific synthetic Zn finger activator protein , induced c-globin synthesis in wildtype PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19674025 b-YAC BMCs, as does enforced expression of other fetal- specific transcriptional activators including FKLF and FGIF. Differentiation into terminal erythrocytes is not required, since globin gene expression patterns are similar in both our BMC and erythroid cell populations. CID-dependent bone marrow cells were established from Ac-luc b-luc b-YAC transgenic mice. These cells were utilized to develop and benchmark a robust HTS assay to identify compounds that 1) induced c-globin gene expression, 2) did not induce b-globin gene 10 High-Throughput Lysine vasopressin site screen for Fetal Hemoglobin Inducers expression, 3) were not cytotoxic and 4) did not directly affect luciferase enzymatic activity. The HTS surveyed 121,035 compounds from various libraries and yielded 232 hits that induced cluc 2- to 11-fold. Of these, 211 were reconfirmed for c-luc induction, and 124 met the constraints of the four listed criteria following a PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19673983 4-assay, 10 point dose-response secondary screen. Six of the top 124 actives were cherry-picked for further analysis and conformation in other cell-based systems. Our first assessment was in wild-type b-YAC BMCs, where normal c-globin transcript and protein levels could be measured in the multi-potential mouse BMC background, rather than luciferase activity. Using the optimum dose for each compound, four of the six compounds induced c-globin transcription 2- to 26-fold and had increased F cells ranging from 9.9 to 16% in wild-type bYAC BMCs. ELISA concurred with these data; an approximately 2- to 4-fold increase in HbF was observed; the same pattern of change seen in mRNA levels was reflected in protein levels. Although different cell lines have been used extensively to screen chemical compound libraries for HbF inducers, these lines do not undergo hemoglobin switching. Thus, the output of these screens may yield false-positive hits that do not correlate with their ability to induce HbF in primary human erythroid progenitors. The dual luciferase reporter system established in primary mouse BMCs immortalized by the CID and used for the HTS described in this report more closely mimics human erythroid progenitors. Our studies in erythroid progenitors generated from adult human primary CD34+ stem cells confirmed the validity of using the CIDdependent b-YAC BMCs for the HTS. In addition, the data demonstrated that this system recapitulated erythropoiesis and the c to b-globin switch in a manner similar to that previously published using another liquid culture system,. The advantage of the human progenitor terminal differentiation protocol is that we produce mature erythrocytes and mimic the c- to b-globin switch similar to normal erythropoiesis in vivo, thereby adding further credibility to our hit identification. The fact that HbF was induced by lead compounds 7, 42, 87 and 208 to levels greater than sodium butyrate suggests that these agents are candidates for further drug development using medicinal chemistry approaches to treat 
b-hemoglobinopathies. 11 High-Throughput Screen for Fetal Hemoglobin Inducers Lipopolysaccharide, a major component of gram-negative bacteria cell membrane, is a well-char