6372 Nucleic Acids Research, 2010, Vol. 38, No. 19 where we showed that only the promoter bound by PU.1 could be transactivated in its presence. The expression proﬁle of Elf-1 has been widely analysed during embryonic development. In chicken embryos, Elf-1 is expressed along the lining of the larger caliber and smaller caliber branching blood vessels, in the developing heart at Day 10 (3), in the developing dorsal aorta of the mouse embryos (17), in embryonic lymphoid organs (thymus and spleen) and a wide variety of epithelial cells and fetal liver (3). Elf-1 is speciﬁcally expressed in multiple haematopoietic cell types (Supplementary Figure S6) and we now show for the ﬁrst time that Elf-1 downregulation is important for erythroid diﬀerentiation. Scl and Lmo2 are known targets of Elf-1, and together with GATA-1, LDB1 and E2A constitute the major transcriptional acti- vator complex operating in erythroid cells (41). In light of our ﬁndings that Elf-1 downregulation is critical for eryth- roid diﬀerentiation, Elf-1 activation of Lmo2 and Scl therefore seems at ﬁrst glance contradictory. However, Elf-1 speciﬁcally activates only those regulatory elements within Scl and Lmo2 that are responsible for directing the expression of these genes in endothelial and blood stem/ progenitor cells. In contrast, the erythroid speciﬁc regula- tory elements of Scl and Lmo2 require autoregulation through Scl/Lmo2 containing complexes (6,7,28). Our results therefore highlight the need to understand the dy- namical nature of regulatory hierarchies and the need to perform functional assays in multiple lineages. Identiﬁcation of lineage-speciﬁc upstream regulators therefore will be essential to reconstruct diﬀerentiation state-speciﬁc regulatory networks as well as state transi- tions during diﬀerentiation. The control of myeloid versus erythroid diﬀerentiation of multipotent progenitors by PU.1 and GATA-1 has been the subject of extensive investigations (40,42–44). Current models suggest that PU.1 and GATA-1 are positive drivers of myeloid and erythroid diﬀerentiation, respectively, and direct cross-antagonistic interactions between the two determine myeloid versus erythroid cell fate choice. Our observations that (i) PU.1 is a direct upstream activator of Elf-1 in progenitor cells and (ii) Elf-1 itself inhibits terminal erythroid diﬀerentiation suggest that the model of PU.1/GATA-1 cross- antagonism as a simple molecular switch may need to be revised. Other known myeloid targets of PU.1, such as c-fms, promote myeloid diﬀerentiation but we now show that Elf-1 itself actually inhibits erythroid diﬀerentiation. Elf-1 has been shown to activate the PU.1 14 enhancer element (45) and is also known to be able to repress GATA-1 target genes (46,47). It is therefore possible that Elf-1 exerts its eﬀect largely through enhancing the antagonistic interaction of PU.1 with GATA-1. To this end, PU.1 and Elf-1 could also collaborate to directly repress GATA-1. Elf-1 is known to co-operate and interact with other members of the GATA family such as GATA-2 (48) and GATA-3 (49) to promote transcrip- tion but this does not exclude the possibility that Elf-1 interacts with GATA-1 to inhibit its action or repress its expression in a similar fashion to PU.1. Moreover, pRb, a binding partner of Elf-1 (21), is itself intrinsically required for terminal erythroid diﬀerentiation (50,51) suggesting that Elf-1 may have a wider role and thus link previously unsuspected regulators with the phenotype of PU.1 mediated suppression of erythroid diﬀerentiation. This study reports the ﬁrst description of the transcrip- tional regulation of the Ets factor Elf-1 and therefore rep- resents a critical advance in integrating this important regulator into the wider haematopoietic regulatory networks. Moreover, identiﬁcation of reciprocal activa- tion between Elf-1 and PU.1 together with identiﬁcation of a functional role for Elf-1 as a negative regulator of terminal erythroid diﬀerentiation have provided import- ant new insights into the transcriptional control of myelo-erythroid diﬀerentiation. Therefore, taken together, these results have expanded our understanding of haematopoietic regulatory networks and provided new insights into the molecular mechanisms that control erythropoiesis. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We are grateful to George Follows and Mary Janes for sharing results and to Richard Auburn from FlyChip for printing custom arrays. FUNDING Leukaemia & Lymphoma Research UK (07060); Cancer Research UK (C24474/A8043); The Wellcome Trust (WT074608MA); Leukaemia and Lymphoma Society (7165-07). Funding for open access charge: The Wellcome Trust (WT074608MA). Conﬂict of interest statement. None declared. REFERENCES 1. Seth,A. and Watson,D.K. (2005) ETS transcription factors and their emerging roles in human cancer. Eur. J. Cancer, 41, 2462–2478. 2. Leiden,J.M., Wang,C.Y., Petryniak,B., Markovitz,D.M., Nabel,G.J. and Thompson,C.B. (1992) A novel Ets-related transcription factor, Elf-1, binds to human immunodeﬁciency virus type 2 regulatory elements that are required for inducible trans activation in T cells. J. Virol., 66, 5890–5897. 3. Dube,A., Thai,S., Gaspar,J., Rudders,S., Libermann,T.A., Iruela-Arispe,L. and Oettgen,P. (2001) Elf-1 is a transcriptional regulator of the Tie2 gene during vascular development. Circ. Res., 88, 237–244. 4. Davis,J.N. and Roussel,M.F. 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