Early B cell factor (EBF) is a transcription factor essential for specification and commitment to the B cell fate. into Hoxa9 function and regulation during lymphoid and B cell development. Furthermore they suggest that failure to upregulate Flt3 provides a molecular basis for the lymphoid/early B cell deficiencies in (7-9). EBF and E2A synergize to induce the early program of B lineage gene expression including the B lineage Lappaconite HBr commitment factor Pax5 (10). Together these factors coordinate critical early B lineage differentiation events and restrict alternative developmental programs. The importance of E2A EBF and Pax5 in regulation of B lymphopoiesis is underscored by the retention of developmental plasticity in cell Lappaconite HBr lines derived from mice deficient in any of these B lineage regulators (11-13). Currently a comprehensive understanding of the genetic networks these factors regulate that facilitate B cell fate specification and commitment is far from complete. Cell lines derived Lappaconite HBr from gene-targeted mice are valuable tools for the identification and characterization of genetic circuits that regulate cellular differentiation pathways (11-13). Importantly they circumvent limitations imposed by molecular manipulation of rare populations and the Lappaconite HBr developmental heterogeneity inherent to ex vivo isolated immunophenotypically defined subsets. Long-term expanded is direct. Chromatin immunoprecipitation (ChIP) analysis revealed binding of Hoxa9 to the promoter in vivo and data obtained from knockdown and ectopic expression studies revealed that modulation of Hoxa9 levels altered Flt3 transcription and expression. Although EBF and Hoxa9 inversely correlate during B cell differentiation EBF does not directly regulate transcription. These data provide new information regarding the role of Hoxa9 in regulation of lymphopoiesis and B cell development and address the role of EBF in silencing a Hoxa9-driven progenitor program. Materials and Methods Icam2 Mice C57Bl6 mice were purchased from The Jackson Laboratory (Bar Harbor ME). (F) 5??CCAGTCTTTTGCATAACCAT-3′ and (R) 5′-AGGTCCTTCTTGTCCTCTTC-3′; (F) 5′-TCCTACCCTATTGTCACAGG-3′ and (R) 5′-GAATACTGAGGGTGGCTGTA-3′; (F) 5′-CTGCAGACATTCTAGCACTC-3′ and (R) 5′-AACTGAAGCTCAGGGTAGAC-3′; (F) 5′-AAGGAGTTCTCTGGGGATAG-3′ and (R) 5′-AACCATGGTCCTCCTAGACT-3′; (F) 5′-TATTAAAACGCTCCTGTGGT-3′ and (R) 5′-AGGCCCTCATAGAGATTTTC-3′; (F) 5′-GGCCTATCTCACAGGTTGT-3′ and (R) 5′-GGAAGAAGATGCTAATGGTG-3′; (F) 5′-CATCCAAGACAACATCTCCT-3′ and (R) 5′-CCCTGAAGTCAACGTAGAAG-3′; (F) 5′-TCTCTCCAGACTACCACACC-3′ and (R) 5′-CACACTCTGTACATTCCTGGT-3′; and (F) 5′-AAGGGAGAAATCAAATGCTCT-3′ and (R) 5′-CCTCCTCCTTTTCACACAGTA-3′. Sequences of the remaining primers used in this study have been published (20 22 Relative mRNA expression levels were normalized to using the 2 2?ΔΔCT method. RNA purification and microarray analysis RNA was prepared from three independent flasks of test value cutoff 0.05). The microarray data were deposited in the Gene Expression Omnibus database under accession number “type”:”entrez-geo” attrs :”text”:”GSE16002″ term_id :”16002″GSE16002 (http://www.ncbi.nlm.nih.gov/geo) and the analysis used in the study provided in Supplemental Tables I and II. ChIP assays ChIP analysis was performed using the EZ-ChIP kit (Millipore Bedford MA) according to the manufacturer’s instructions. Briefly 2 × 107 test. Lappaconite HBr Results Comparative analysis of transcripts are expressed in gene and our previous findings transcripts were very low to undetectable in (transcripts were high in transcripts correlated well with surface expression of Flt3 in the cell lines (Fig. 1in Fig. 1represent the staining pattern of CD34 and CD27 within the Pre-Pro-B/Pro-B-enriched fraction and the within the Pre-B/sIg+ subset. Expression of CD34 and CD27 is absent from the vast majority of BCPs. This in vivo analysis is consistent with our cell line data and shows that B cell differentiation is accompanied by downregulation of a cellular program preferentially expressed by primitive hematopoietic progenitors. Downregulation of HoxA transcription accompanies B cell fate specification To identify novel genetic events that accompany B cell fate specification we took a functional genomics approach. The and cells express a novel transcriptome Next we focused on transcripts differentially expressed in transcripts in transcripts transcripts in transcripts was confirmed by real-time RT-PCR (Fig. 2gene cluster was unique to the clonal line.