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Files in this Data Supplement:
Fig S1. The Ainv18 tet operon does not interfere with the transcriptional control imparted by Peklf. Analysis of GFP expression in response to 24 hours of doxycycline treatment at day 2 of EB differentiation by qRT-PCR (one representative experiment). An untargeted (Ainv18) and a GFP cDNA-targeted Ainv18 ES cell clone (ploxGFP) were included as negative and positive controls, respectively.
Fig S2. Peklf-controlled GFP transgene expression is specific to committed erythroid cells at EB day 8. (A-F) FACS analysis of GFP expression in EBs at day 8 of differentiation. About 20% of all cells express GFP. All GFP+ cells fall either into the highly proliferative compartment as indicated by the expression of the transferrin receptor (CD71, B) (Carotta et al., 2004; Zambidis et al., 2005) and integrin gpIIb (CD41, C) (Ferkowicz et al., 2003; Mikkola et al., 2003), or are already restricted to the erythroid fate (Ter119+, D). However, GFP expression is not found in mature cells of the granulocyte-macrophage (Gr1+/Mac1+, E) or megakaryocytic (CD42d+, F) lineages.
Carotta, S., Pilat, S., Mairhofer, A., Schmidt, U., Dolznig, H., Steinlein, P. and Beug, H. (2004). Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells. Blood 104, 1873-1880.
Ferkowicz, M. J., Starr, M., Xie, X., Li, W., Johnson, S. A., Shelley, W. C., Morrison, P. R. and Yoder, M. C. (2003). CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo. Development 130, 4393-4403.
Mikkola, H. K., Fujiwara, Y., Schlaeger, T. M., Traver, D. and Orkin, S. H. (2003). Expression of CD41 marks the initiation of definitive hematopoiesis in the mouse embryo. Blood 101, 508-516.
Zambidis, E. T., Peault, B., Park, T. S., Bunz, F. and Civin, C. I. (2005). Hematopoietic differentiation of human embryonic stem cells progresses through sequential hematoendothelial, primitive, and definitive stages resembling human yolk sac development. Blood 106, 860-870.
Fig. S3. Hematopoietic regulatory regions of the Gata1 locus contain conserved putative Smad binding sites required for megakaryocytic Gata1 expression. (A) A multi-species phylogenetic alignment of the Gata1 locus upstream of exon 2 identifies four peaks of highly conserved non-coding sequence (shaded in red) that are located within previously reported hematopoietic cis-regulatory regions: erythroid promoter exon 1 (ex1E) (Ito et al., 1993); Gata1 hematopoietic enhancer (G1HE) (McDevitt et al., 1997; Onodera, 1997; Ronchi et al., 1997); intronic enhancer (Onodera et al., 1997). Ex1E itself is not sufficient to confer high-level transcription in transgenic mice (McDevitt et al., 1997; Onodera et al., 1997). However, G1HE plus ex1E are sufficient to give expression in primitive erythroid cells in the yolk sac (E8.5), whereas G1HE plus ex1E and the intronic enhancer are required to give expression in definitive erythroid cells in the fetal liver (E12.5) (Onodera et al., 1997) (reviewed by Ferreira et al., 2005). Also depicted are the testis promoter exon 1 (ex1T) (Ito et al., 1993) and DNaseI hypersensitive sites (HS) (McDevitt et al., 1997). Alignment color code: repeat or low complexity DNA (blue); UTR (tan); exon (brown); DNaseI hypersensitive site (orange); restriction site (black) according to McDevitt et al. and Onodera et al. (McDevitt et al., 1997; Onodera et al., 1997). (B) Three of the four Gata1 alignment peaks (Peaks 1, 2 and 4) display clusters of conserved Smad binding motifs (blue) and GC-rich blocks of perfect homology (purple) in the vicinity of conserved WGATAR motifs (red) in a layout similar to the cis-regulatory regions of the Eklf locus (see Fig. 3). By contrast, the testis promoter region of the Gata1 gene (G1TAR) does not contain any conserved Smad binding motifs (data not shown), arguing for their specific role in the hematopoietic regulation of Gata1 expression. Of particular interest is the peak region at hypersensitive site 1 (HS1) that falls into the hematopoietic enhancer (HE) region of the Gata1 locus (G1HE/HS1), which contains a WGATAR motif surrounded by Smad binding motifs and a GC-rich block of conservation. The WGATAR motif is necessary for expression of a transgene in erythroid as well as megakaryocytic cells, as are the sequence elements directly downstream of the WGATAR motif (Nishimura et al., 2000; Vyas et al., 1999). Previous reports had suggested these sequence elements contain an E-box, but failed to show binding by a bHLH factor complex (Nishimura et al., 2000; Vyas et al., 1999). Similarly, a more recent study demonstrated that the conserved GC-rich block is required for megakaryocytic Gata1 expression, but neither ETS factors nor Runx1 could be shown to bind (Guyot et al., 2006). Instead, we identify conserved putative Smad binding sites based on known consensus motifs (see Materials and methods) and GC-rich sequences. Therefore, we propose that the G1HE upstream enhancer is cooperatively activated by Gata2 and Smad5 in a progenitor population of erythroid-megakaryocytic potential, which would explain the transgene expression pattern mentioned above. Upon erythroid lineage commitment and differentiation, Gata1 expression no longer requires any of the sequence elements located downstream of the WGATAR in G1HE (Nishimura et al., 2000; Vyas et al., 1999), arguing that Gata1 autoregulation occurs independent of Smad binding. In support of this model, we show Gata2 occupancy at G1HE/HS1 in vivo in differentiating EBs at the progenitor stage, followed by a switch to Gata1 occupancy at EB day 6 upon erythroid commitment (Fig. 5). Interestingly, the HS1 region is not required for Gata1 expression in eosinophils (Guyot et al., 2004). This strongly suggests that the activation of Gata1 expression via the G1HE/HS1 upstream enhancer occurs within a progenitor population of erythroid-megakaryocytic potential during hematopoietic development, analogous to our model of Eklf activation via its upstream enhancer. In addition, known CP2 sites (pink) (Bose et al., 2006), as well as putative Klf transcription factor binding sites (light blue) and unassigned blocks of perfect homology (green) are depicted. Peak 1 and 2 as well as peak 3 (not shown) constitute the three cis-regulatory elements of the previously described Gata1 minigene (Ohneda et al., 2002). Peak 4 falls within a region of histone hyperacetylation (Valverde-Garduno et al., 2004).
Bose, F., Fugazza, C., Casalgrandi, M., Capelli, A., Cunningham, J. M., Zhao, Q., Jane, S. M., Ottolenghi, S. and Ronchi, A. (2006). Functional interaction of CP2 with GATA-1 in the regulation of erythroid promoters. Mol. Cell. Biol. 26, 3942-3954.
Ferreira, R., Ohneda, K., Yamamoto, M. and Philipsen, S. (2005). GATA1 function, a paradigm for transcription factors in hematopoiesis. Mol. Cell. Biol. 25, 1215-1227.
Guyot, B., Valverde-Garduno, V., Porcher, C. and Vyas, P. (2004). Deletion of the major GATA1 enhancer HS 1 does not affect eosinophil GATA1 expression and eosinophil differentiation. Blood 104, 89-91.
Guyot, B., Murai, K., Fujiwara, Y., Valverde-Garduno, V., Hammett, M., Wells, S., Dear, N., Orkin, S. H., Porcher, C. and Vyas, P. (2006). Characterization of a megakaryocyte-specific enhancer of the key hemopoietic transcription factor GATA1. J. Biol. Chem. 281, 13733-13742.
Ito, E., Toki, T., Ishihara, H., Ohtani, H., Gu, L., Yokoyama, M., Engel, J. D. and Yamamoto, M. (1993). Erythroid transcription factor GATA-1 is abundantly transcribed in mouse testis. Nature 362, 466-468.
McDevitt, M. A., Fujiwara, Y., Shivdasani, R. A. and Orkin, S. H. (1997). An upstream, DNase I hypersensitive region of the hematopoietic-expressed transcription factor GATA-1 gene confers developmental specificity in transgenic mice. Proc. Natl. Acad. Sci. USA 94, 7976-7981.
Nishimura, S., Takahashi, S., Kuroha, T., Suwabe, N., Nagasawa, T., Trainor, C. and Yamamoto, M. (2000). A GATA box in the GATA-1 gene hematopoietic enhancer is a critical element in the network of GATA factors and sites that regulate this gene. Mol. Cell. Biol. 20, 713-723.
Ohneda, K., Shimizu, R., Nishimura, S., Muraosa, Y., Takahashi, S., Engel, J. D. and Yamamoto, M. (2002). A minigene containing four discrete cis elements recapitulates GATA-1 gene expression in vivo. Genes Cells 7, 1243-1254.
Onodera, K., Takahashi, S., Nishimura, S., Ohta, J., Motohashi, H., Yomogida, K., Hayashi, N., Engel, J. D. and Yamamoto, M. (1997). GATA-1 transcription is controlled by distinct regulatory mechanisms during primitive and definitive erythropoiesis. Proc. Natl. Acad. Sci. USA 94, 4487-4492.
Ronchi, A., Ciro, M., Cairns, L., Basilico, L., Corbella, P., Ricciardi-Castagnoli, P., Cross, M., Ghysdael, J. and Ottolenghi, S. (1997). Molecular heterogeneity of regulatory elements of the mouse GATA-1 gene. Genes Funct. 1, 245-258.
Valverde-Garduno, V., Guyot, B., Anguita, E., Hamlett, I., Porcher, C. and Vyas, P. (2004). Differences in the chromatin structure and cis-element organization of the human and mouse GATA1 loci: implications for cis-element identification. Blood 104, 3106-3116.
Vyas, P., McDevitt, M. A., Cantor, A. B., Katz, S. G., Fujiwara, Y. and Orkin, S. H. (1999). Different sequence requirements for expression in erythroid and megakaryocytic cells within a regulatory element upstream of the GATA-1 gene. Development 126, 2799-2811.
Fig S4. Eklf expression is upregulated upon G1E-ER-Gata1 differentiation. Western blot analysis of Eklf expression in whole-cell extracts from untreated cells (−) or cells treated with estradiol for 14 hours (+). Hsp90 levels were used as a loading control.
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