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• W2081372023 abstract "Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the megakaryocyte erythroid transcription factor GATA1. Hemopoietic-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, the 3′ 179 bp of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show mHS-3.5 binds key hemopoietic transcription factors in vivo and is required to maintain histone acetylation at the mGata1 locus in primary megakaryocytes. Analysis of GATA1-LacZ reporter gene expression in transgenic mice shows that a 25-bp element within the 3′-179 bp in mHS-3.5 is critical for megakaryocyte expression. In vitro three DNA binding activities A, B, and C bind to the core of the 25-bp element, and these binding sites are conserved through evolution. Activity A is the zinc finger transcription factor ZBP89 that also binds to other cis elements in the mGata1 locus. Activity B is of particular interest as it is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows sequence corresponding to the binding site for activity B is a previously unrecognized motif, present in the cis elements of the Fli1 gene, another important megakaryocyte-specific transcription factor. In summary, we have identified a motif and a DNA binding activity likely to be important in directing a megakaryocyte gene expression program that is distinct from that in red cells. Specification and differentiation of the megakaryocyte and erythroid lineages from a common bipotential progenitor provides a well studied model to dissect binary cell fate decisions. To understand how the distinct megakaryocyte- and erythroid-specific gene programs arise, we have examined the transcriptional regulation of the megakaryocyte erythroid transcription factor GATA1. Hemopoietic-specific mouse (m)GATA1 expression requires the mGata1 enhancer mHS-3.5. Within mHS-3.5, the 3′ 179 bp of mHS-3.5 are required for megakaryocyte but not red cell expression. Here, we show mHS-3.5 binds key hemopoietic transcription factors in vivo and is required to maintain histone acetylation at the mGata1 locus in primary megakaryocytes. Analysis of GATA1-LacZ reporter gene expression in transgenic mice shows that a 25-bp element within the 3′-179 bp in mHS-3.5 is critical for megakaryocyte expression. In vitro three DNA binding activities A, B, and C bind to the core of the 25-bp element, and these binding sites are conserved through evolution. Activity A is the zinc finger transcription factor ZBP89 that also binds to other cis elements in the mGata1 locus. Activity B is of particular interest as it is present in primary megakaryocytes but not red cells. Furthermore, mutation analysis in transgenic mice reveals activity B is required for megakaryocyte-specific enhancer function. Bioinformatic analysis shows sequence corresponding to the binding site for activity B is a previously unrecognized motif, present in the cis elements of the Fli1 gene, another important megakaryocyte-specific transcription factor. In summary, we have identified a motif and a DNA binding activity likely to be important in directing a megakaryocyte gene expression program that is distinct from that in red cells. Understanding the molecular basis of lineage specification from multipotential progenitors is a central question in biology. The question in its simplest and most tractable form is to understand how two different lineages arise from a common progenitor. Hemopoiesis has arguably been one of the most informative and well studied model systems in furthering our understanding of lineage determination. In hemopoiesis, the megakaryocytic and erythroid lineages have distinctive phenotypes and gene expression profiles, and yet arise from a common progenitor (1Akashi K. Traver D. Miyamoto T. Weissman I.L. Nature. 2000; 404: 193-197Crossref PubMed Scopus (1912) Google Scholar). The mechanism by which they are differentially specified has been extensively studied but is not well understood. Though lineage specification is regulated by external cues that are modulated by intracellular signaling pathways, it ultimately culminates in activation of uni-lineage programs of gene expression and repression of genes associated with alternative cell fate. Coordination of complex patterns of gene expression resulting in lineage specification is thought to be regulated, in part, by combinations of lineage-specific transcription factors. Therefore, in this model, in a common megakaryocyte-erythroid bipotential progenitor, lineage-specific (erythroid or megakaryocyte) combinations of transcription factors become expressed that direct differential specification of the erythroid and megakaryocyte lineages. The erythroid and megakaryocytic lineages share many critical hemopoietic transcription factors (e.g. GATA1, FOG-1, Gfi-1b, NF-E2p45, and SCL/TAL-1) but also express hemopoietic regulators unique to one or other lineage (such as RUNX-1, Meis1, and Fli1 in the megakaryocyte lineage and EKLF in erythroid cells). However, the detailed mechanisms by which these lineages utilize combinations of transcriptional regulators to direct lineage-specific programs of gene expression are unclear. One way to uncover the combination of regulators required for differential specification is to identify the DNA sequences (cis elements), and through them the DNA binding transcriptional regulators, required to specifically express genes in either red cells or megakaryocytes. In this study, we examine the cis elements required to direct expression of a key erythroid and megakaryocyte transcription factor GATA1, in megakaryocytes but not red cells. GATA1 is first expressed at low levels in the common myeloid progenitor (2Iwasaki H. Mizuno S. Wells R.A. Cantor A.B. Watanabe S. Akashi K. Immunity. 2003; 19: 451-462Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), and its expression is maintained in the megakaryocyte and erythroid lineages (reviewed in Ref. 3Valverde-Garduno V. Guyot B. Anguita E. Hamlett I. Porcher C. Vyas P. Blood. 2004; 104: 3106-3116Crossref PubMed Scopus (46) Google Scholar). In both lineages, sustained GATA1 expression is required for terminal maturation (4Fujiwara Y. Browne C.P. Cunniff K. Goff S.C. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12355-12358Crossref PubMed Scopus (619) Google Scholar, 5Shivdasani R.A. Fujiwara Y. McDevitt M.A. Orkin S.H. EMBO J. 1997; 16: 3965-3973Crossref PubMed Scopus (587) Google Scholar, 6Takahashi S. Komeno T. Suwabe N. Yoh K. Nakajima O. Nishimura S. Kuroha T. Yamamoto M. Blood. 1998; 92: 434-442Crossref PubMed Google Scholar, 7Vyas P. Ault K. Jackson C.W. Orkin S.H. Shivdasani R.A. Blood. 1999; 93: 2867-2875Crossref PubMed Google Scholar). GATA1 expression is regulated by a complex set of cis-acting regulatory elements. In mice, the hemopoietic promoter (mIE), an upstream enhancer 3.5 kilobases from the GATA1 hemopoietic transcription start site, HS1/G1HE/mHS-3.5 (hereafter referred to as mHS-3.5), and an element in the first mGata1 intron (HS 4/5 or mHS+3.5) (see Fig. 1A) are required to direct reporter gene expression to both erythroid cells and megakaryocytes in transgenic mice (8Ronchi A. Ciro M. Cairns L. Basilico L. Corbella P. Ricciardi-Castagnoli P. Cross M. Ghysdael J. Ottolenghi S. Genes Funct. 1997; 1: 245-258Crossref PubMed Scopus (20) Google Scholar, 9Onodera K. Takahashi S. Nishimura S. Ohta J. Motohashi H. Yomogida K. Hayashi N. Engel J.D. Yamamoto M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4487-4492Crossref PubMed Scopus (143) Google Scholar, 10McDevitt M.A. Fujiwara Y. Shivdasani R.A. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7976-7981Crossref PubMed Scopus (84) Google Scholar) Deletion of mHS-3.5 from the reporter construct extinguishes reporter gene expression in both red cells and megakaryocytes, highlighting a non-redundant enhancer function for mHS-3.5 in this assay (11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar, 12Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar). In contrast, germline deletion of a 7-kb region of genomic DNA including all of mHS-3.5 (ΔneoΔHS mice), virtually abrogates megakaryocyte GATA1 expression but GATA1 expression in red cells is unaffected (5Shivdasani R.A. Fujiwara Y. McDevitt M.A. Orkin S.H. EMBO J. 1997; 16: 3965-3973Crossref PubMed Scopus (587) Google Scholar, 10McDevitt M.A. Fujiwara Y. Shivdasani R.A. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7976-7981Crossref PubMed Scopus (84) Google Scholar), suggesting that mHS-3.5 plays a unique non-redundant role in megakaryocyte GATA1 expression, and that other elements in the mGata1 locus must compensate for loss of mHS-3.5 function in red cells. Furthermore, deletion analysis showed that within mHS-3.5, two distinct DNA sequences are important for enhancer activity in transgenic mice. First, a GATA site is absolutely required for mHS-3.5 enhancer activity in both red cells and megakaryocytes (11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar, 12Nishimura S. Takahashi S. Kuroha T. Suwabe N. Nagasawa T. Trainor C. Yamamoto M. Mol. Cell. Biol. 2000; 20: 713-723Crossref PubMed Scopus (100) Google Scholar). In vitro, this site and a neighboring E-box DNA element bind GATA factors and a pentameric hemopoietic transcription factor complex containing, at a minimum, GATA1-SCL/TAL-1-E2A-LMO2-LDB1 in erythroid and megakaryocytic cells (11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar). In vivo GATA1-SCL/TAL-1-E2A-LMO2-LDB1 binding is detected at mHS-3.5 in erythroid cells (3Valverde-Garduno V. Guyot B. Anguita E. Hamlett I. Porcher C. Vyas P. Blood. 2004; 104: 3106-3116Crossref PubMed Scopus (46) Google Scholar). Second, whereas the whole 312 bp of mHS-3.5 is required for megakaryocyte reporter gene expression, only the 5′ 133 bp is necessary for red cell reporter gene expression (11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar). These data suggest that the 3′ 179 bp of mHS-3.5 binds trans-acting factors that cooperate with proteins that bind the GATA site, to direct megakaryocyte-specific enhancer activity. Therefore, as a prelude to identifying trans-acting factors required for megakaryocyte-specific GATA1 expression, and megakaryocyte-specific gene activation in general, we set out to pinpoint the cis-acting sequences within mHS-3.5 mediating megakaryocyte-specific enhancer activity. Primary Cells and Cell Lines—All cell culture reagents were purchased from Invitrogen (Paisley, UK), unless indicated otherwise. ΔneoΔHS mice have been previously described (5Shivdasani R.A. Fujiwara Y. McDevitt M.A. Orkin S.H. EMBO J. 1997; 16: 3965-3973Crossref PubMed Scopus (587) Google Scholar). Briefly, a genomic region between 12,000 and 2600 bp upstream of the hemopoietic Gata1 transcription start site, and encompassing the mHS-3.5 element (Fig. 1B), was deleted by homologous recombination in ΔneoΔHS mice. To isolate mouse primary wild-type (WT) 3The abbreviations used are: WT, wild type; EMSA, electrophoretic mobility shift assay; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; ChIP, chromatin immunoprecipitation assay; FDG, fluorescein di-(β-d-galactopyranoside; FACS, fluorescent-activated cell sorting. C57Bl/6 or ΔneoΔHS megakaryocytes, mice were administered intraperitoneal 5-fluorouracil (5-FU, 0.15 g/kg, Sigma). After 8 days, bone marrow was flushed from the femurs and tibias. Red cells were lysed using ACK buffer (Cambrex Bioscience, Verviers, Belgium). Bone marrow cells were resuspended at 5 × 106 cells/ml in serum-free StemPro 34 SFM medium (Stem Cell Technology, London, UK) supplemented with glutamine, antibiotics, and Tpo (1% conditioned medium, Ref. 13Villeval J.L. Cohen-Solal K. Tulliez M. Giraudier S. Guichard J. Burstein S.A. Cramer E.M. Vainchenker W. Wendling F. Blood. 1997; 90: 4369-4383Crossref PubMed Google Scholar). After 3 days in culture, cells labeled with biotin-conjugated anti-Ter119 (cat. 553672), anti-B220 (cat. 553085), anti-Gr1 (cat. 553124) and anti-Mac1 (cat. 557395) antibodies (all from BD Pharmingen, Oxford, UK) were removed using streptavidin-coated magnetic beads and a large cell depletion column (both from Miltenyi Biotech, Bergisch Galdbach, Germany). The remaining cell population was more than 95% megakaryocytes as assessed by May-Grunewald Giemsa staining and expression of CD61 (BD Pharmingen), which was assessed by flow cytometry. Primary red cells were purified from the spleen of phenylhydrazine-treated mice. Briefly, spleen cells labeled with unconjugated anti-B220 (cat. 553084), anti-Gr1 (cat. 553123), anti-Mac1 (cat. 553308), and anti-CD2 (cat. 553109) antibodies (all from BD Pharmingen) were removed using goat anti-rat IgG coated magnetic beads and a LD depletion column (both from Miltenyi Biotech). More than 90% of the unlabeled cells were Ter119+ve red cells as assessed by flow cytometry and May-Grunewald Giemsa staining. Fetal livers were obtained from E13.5/14.5 C57Bl/6 embryos. Culture and derivation of the murine megakaryoblastic leukemia cell line L8057 (14Ishida Y. Levin J. Baker G. Stenberg P.E. Yamada Y. Sasaki H. Inoue T. Exp. Hematol. 1993; 21: 289-298PubMed Google Scholar), C2C12 myoblast cell line (15Yaffe D. Saxel O. Nature. 1977; 270: 725-727Crossref PubMed Scopus (1562) Google Scholar), and 3T3-L1 pre-adipocyte cell line (16Green H. Meuth M. Cell. 1974; 3: 127-133Abstract Full Text PDF PubMed Scopus (820) Google Scholar) have been previously described. Constructs—For constructs WT, GK3, GK8, and GS12 shown in Fig. 5, a PCR fragment extending from A to I (see Fig. 2B) was obtained using a common upstream primer 5′-TTGTTCGGTACCGGATTCGTCAGGCCTGCAATGGGCTCCC-3′ and a specific downstream primer, which was either WT sequence or sequences corresponding to mutants GK3, GK8 or GS12 (see Fig. 3B). PCR products were then cloned into the Asp718 site of the 5′-3′-LacZ vector (10McDevitt M.A. Fujiwara Y. Shivdasani R.A. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7976-7981Crossref PubMed Scopus (84) Google Scholar) using Asp718 sites that were introduced into the PCR primers. All constructs were sequenced prior to use.FIGURE 2A conserved 25-bp sequence is required for the megakaryocyte-specific enhancer activity of mHS-3.5 in transgenic mice. A, mouse Gata1 locus with the hemopoietic-specific IE promoter and five coding exons (depicted as black boxes) is shown on top.An arrow shows the location of mHS-3.5. Below, a map of the vector 5′-3′ LacZ is shown with the LacZ coding sequence fused in-frame with the initiator ATG of the Gata1 gene. Below left, four genomic DNA fragments (A-D to A-J) and their location in mHS-3.5 are shown: closed circle, GATA site, open circle, E-box, black box, conserved GC-rich sequence, gray boxes, mouse/human conserved sequences. These fragments were attached to the 5′-end of 5′-3′-LacZ to produce constructs A-D to A-J. To the right is shown the size of each attached fragment and a summary of β-galactosidase expression in different cell types in transient transgenic embryos injected with these constructs. Column 1 shows the number of β-galactosidase expressing embryos/total number transgenic embryos. An embryo was defined as expressing β-galactosidase if >0.5% of cells stained with X-gal substrate. Column 2 shows the range of β-galactosidase-expressing cells (%). 200 cells were counted. Results for A-D and A-E constructs are taken from Ref. 11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar and is shown for clarity. Below, photographs of representative X-gal staining of fetal liver megakaryocytes (red arrowheads) and definitive erythroblasts (black arrows) from E14.5 embryos transgenic for A-I or A-J constructs. Original magnification is ×40. The black bar represents 10 μm. B, sequence alignment of mouse, human, and dog HS-3.5 elements. Positions of GATA1 and SCL/TAL-1 binding sites and borders of constructs A-D, A-E, A-I, and A-J are shown. Asterisks show conserved nucleotides in all three species.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 3Sequence-specific DNA activities that bind the 25-bp region (E-I) in megakaryoblast nuclear extracts. A, protein-DNA complexes detected by EMSA bind the E-I region of mHS-3.5. In the first panel (lanes 1-3), nuclear extracts from L8057 cell were incubated with the WT probe (for the sequence see B), either alone (-)(lane 1) or with antibodies directed against Sp1 (lane 2) or Sp3 (lane 3). In the second panel, nuclear extracts of E13.5/14.5 mouse fetal liver (FL)(lane 4), mouse primary megakaryocytes (1° Meg.)(lane 5), C2C12 mouse myoblasts (lane 6), or 3T3-L1 mouse adipocytes (lane 7) were used. DNA-protein complexes A, B, and C are indicated on the side of the autoradiograph along with Sp1 (*) and Sp3 (**)-containing complexes. B, nucleotide sequence of DNA fragments used to map the binding sites for complexes binding to E-I region. The wild-type sequence is shown on top and mutant sequences below. Boundaries of the E-I region are shown, and mutated bases are in bold. Right, summary of the EMSA analysis shown in C indicates the ability of each mutated sequence to bind complexes A, B, and C. C, DNA-protein complexes binding to the E-I region detected by EMSA. Nuclear extracts from L8057 cells were incubated with WT probe shown in B, either alone (-)(lanes 1, 12, and 23) or with increasing amount of unlabeled competitors (open triangles, 20- or 50-fold molar excess) (lanes 2-11, 13-22, and 23-35). DNA binding activities are indicated as in A. D, summary of DNA binding sequences required for binding of Sp1, Sp3, and complexes A, B, and C to the E-I region. Black lines show bases required for DNA binding and dashed lines bases that affect but are not required for binding. E, WT or indicated mutated labeled probes were incubated with L8057 nuclear extracts, either alone (-)(lanes 1, 4, 7, 10, and 13) or with antibodies directed against Sp1 (lanes 2, 5, 8, 11, and 14) or Sp3 (lanes 3, 6, 9, 12, and 15). DNA binding activities are indicated as in A.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Chromatin Immunoprecipitation (ChIP)—ChIP experiments on mouse primary megakaryocytes and erythroid cells were performed as previously described (3Valverde-Garduno V. Guyot B. Anguita E. Hamlett I. Porcher C. Vyas P. Blood. 2004; 104: 3106-3116Crossref PubMed Scopus (46) Google Scholar, 17Guyot B. Valverde-Garduno V. Porcher C. Vyas P. Blood. 2004; 104: 89-91Crossref PubMed Scopus (23) Google Scholar), except that 5 × 105 megakaryocytes were used for each immunoprecipitation. For the ZBP-89 ChIP, a polyclonal rabbit antiserum from Rockland (ref. 100-401-685, Gilbertsville, PA) was used. Transgenics Procedures—Standard techniques were used to isolate transgene sequences for DNA purification and for pronuclear injection of CD-1 (Charles River Laboratories, MA and MRC Harwell, UK) and B6CBAF1/J (Jackson Laboratories, Bar Harbor, ME) fertilized eggs. Chimeric fetuses (thereafter called transient transgenics) were sacrificed at E13.5-14.5, genotyped by PCR using LacZ and RapSyn primers as previously described (10McDevitt M.A. Fujiwara Y. Shivdasani R.A. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7976-7981Crossref PubMed Scopus (84) Google Scholar) and analyzed for β-galactosidase expression as detailed below. β-Galactosidase Assays—β-galactosidase expressing fetal liver cells (E13.5-14.5) were analyzed either visually by X-gal staining (Sigma) (10McDevitt M.A. Fujiwara Y. Shivdasani R.A. Orkin S.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7976-7981Crossref PubMed Scopus (84) Google Scholar), or by flow-cytometry using fluorescein di-(β-d-galactopyranoside) (FDG, Sigma) and lineage-specific antibody staining. For FDG staining, fetal liver cells were loaded with FDG as previously described (18Fiering S.N. Roederer M. Nolan G.P. Micklem D.R. Parks D.R. Herzenberg L.A. Cytometry. 1991; 12: 291-301Crossref PubMed Scopus (165) Google Scholar) and stained with biotin- or PE-conjugated antibodies directed against Ter119, Mac1, or CD61 surface markers or their respective isotype controls (all from BD Pharmingen). APC-conjugated streptavidin was used as secondary antibody for biotin-conjugated primary antibodies. FACS analysis was performed using a CyAn machine and Summit software (Dako Cytomation, Cambridge, UK). Hoechst 33258 Molecular Probes, Eugene, OR) was used to exclude dead cells. Micrograph images were taken with a BX60 microscope and a 40/0.75 objective (Olympus, London, UK), using a Qicam camera (Qimaging, Burnaby, Canada) and Openlab software (Improvision, Coventry, UK). Statistical Analysis of Reporter Gene Expression—Analysis to determine the statistical significance of differences in the frequencies of LacZ-expressing transgenic embryos when different mGata1-LacZ transgenes were tested was performed with a binomial test (Graphpad). Electromobility Shift Assay (EMSA)—Nuclear extract preparations and DNA binding assays were performed as previously reported (11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar). Antibodies used were: anti-Sp1 (sc-59), anti-Sp3 (sc-644) (Santa Cruz Biotechnology (Calne, UK) and the anti-ZBP-89 antiserum used in for ChIP. Sequences of oligonucleotides used in EMSA are shown in Fig. 3B. Bioinformatics—Multispecies mHS-3.5 alignments were performed with ClustalW (MacVector Accerlys, UK). The GenBank™ accession numbers for mouse, human, and dog Gata1 genes are AF 136574, AF 136573, and NW139919, respectively. Sequences of Fli1 genes were obtained from Ensembl data base. Accession numbers for the Fli1 genes are: ENSMUSG00000016087 (Mus musculus), ENSRNOG00000008904 (Rattus norvegicus), ENSG00000151702 (Homo sapiens), and ENSP-TRG00000004467 (Pan troglodytes). 1500-bp upstream of the transcription start site(s) were analyzed using MacVector to look for sequences similar to the E-I region. Dog (Canis familiaris) and opossum (Monodelphis domestica) Fli1 genes are not annotated in the current releases and were located using BLAST software with the mouse Fli1 cDNA sequence and a highly conserved regulatory sequence in the Fli1 gene promoter (19Starck J. Doubeikovski A. Sarrazin S. Gonnet C. Rao G. Skoultchi A. Godet J. Dusanter-Fourt I. Morle F. Mol. Cell. Biol. 1999; 19: 121-135Crossref PubMed Scopus (62) Google Scholar). mHS-3.5 Is Required for Hyperacetylation of Histone H3 within the mGata1 Locus in Primary Megakaryocytes but Not Red Cells—To establish when during megakaryocytic differentiation mHS-3.5 is required for GATA1 expression, we isolated fetal primary common myeloid progenitors (CMP), megakaryocyte erythroid progenitors (MEP), megakaryocyte progenitors (MkP), and primary megakaryocytes from wild-type mice and mice with deletion of mHS-3.5 (ΔneoΔHS mice) and examined GATA1 mRNA levels by quantitative real-time Taqman PCR. GATA1 levels were similar in wild-type and ΔneoΔHS CMP and MEP but specifically decreased in ΔneoΔHS MkP and megakaryocytes to 5% of wild-type levels (data not shown and Ref. 20Kuhl C. Atzberger A. Iborra Nieswandt B. Porcher C. Vyas P. Mol. Cell. Biol. 2005; 25: 8592-8606Crossref PubMed Scopus (63) Google Scholar). This suggests that mHS-3.5 plays a critical non-redundant role at the level of a megakaryocyte progenitor and megakaryocyte precursors. Next, we examined in vivo acetylation of histone H3 in WT and ΔneoΔHS primary megakaryocytes and red cells to determine how loss of mHS-3.5 affected chromatin structure in the two lineages (contrast Fig. 1, A with B). In both WT megakaryocytes and red cells there is a domain enriched for hyperacetylated histone H3 between the mIE promoter and mHS+3.5, the mGata1 intron cis element. In addition, a smaller peak of acetylation is seen at mHS-3.5. Lastly, a peak of enrichment of acetylated H3 is also detected at the ubiquitous DNase I hypersensitive site mHS+20, that probably marks the promoter of the mHdac6 gene (3Valverde-Garduno V. Guyot B. Anguita E. Hamlett I. Porcher C. Vyas P. Blood. 2004; 104: 3106-3116Crossref PubMed Scopus (46) Google Scholar). Importantly, only in megakaryocytes, but not red cells, is deletion of mHS-3.5 associated with a striking loss of the domain enriched in hyperacetylated histone H3 between mIE and mHS+3.5. This loss was specific to this region of the mGata1 locus as enrichment of hyperacetylation of H3 was maintained at mHS+20. These data highlight the critical, non-redundant role of mHS-3.5 in maintaining a domain of hyperacetylated chromatin in the mGata1 locus in and around the Gata1 gene in primary megakaryocytes but not red cells. In part this domain of acetylation may be maintained by binding of GATA1 and SCL (supplementary Fig. S1, A and B, respectively) to mHS-3.5 in primary megakaryocytes. A 25-bp Element within mHS-3.5 Is Required for Megakaryocyte-specific Enhancer Activity—We then proceeded to more precisely map sequences within mHS-3.5 required for megakaryocyte enhancer activity. We previously showed that whereas a 317-bp region within mHS-3.5 (11Vyas P. McDevitt M.A. Cantor A.B. Katz S.G. Fujiwara Y. Orkin S.H. Development. 1999; 126: 2799-2811Crossref PubMed Google Scholar) (Fig. 2A, construct A-D) was required for enhancer activity in megakaryocytes, the 5′ 133 bp (construct A-E) was active only in red cells and not megakaryocytes (Fig. 2A, construct A-E). In the region between E and D within mHS-3.5, there are a number of blocks of sequence conserved between human, dog, and mouse (Fig. 2B). Next, two additional 3′ mHS-3.5 deletional constructs were tested for enhancer activity in fetal liver cells from E13.5 transient transgenics embryos (Fig. 2A, constructs A-I and A-J). Both constructs were able to direct LacZ expression in fetal liver megakaryocytes, as well as red cells. These data suggest that a 25-bp fragment within mHS-3.5 (hereafter called E-I), present in construct A-I but not A-E, is required to direct reporter gene expression to fetal liver megakaryocytes in a transgenic mouse assay. Within E-I there are 17 bp, within a 24-bp block, that are conserved through evolution (Fig. 2B). The E-I Region Binds a Sequence-specific DNA Binding Activity Present in Mouse Primary Megakaryocytes but Not Primary Red Cells—We then examined in vitro DNA binding by nuclear proteins using an EMSA with a probe encompassing sequences E-I (Fig. 3B), to identify potential DNA binding transcriptional regulators responsible for megakaryocyte-specific enhancer activity. Five retarded bands were observed using nuclear extracts from the megakaryoblast cell line, L8057 (Fig. 3A, lane 1). Given the high GC content of the E-I region we hypothesized that some of the retarded bands may reflect binding by the Sp family of transcription factors. This was confirmed by supershift assays, and two DNA-protein complexes contained Sp1 and Sp3 (Fig. 3A, lanes 2 and 3). However, as we could not detect in vivo binding, by chromatin immunoprecipitation, of either Sp1 or Sp3 to mHS-3.5 in either primary red cells or primary megakaryocytes, (though binding of both factors was detected at mHS+20) (data not shown), we did not further investigate the role of Sp1 and Sp3. The three remaining DNA-protein complexes were called A, B, and C. We then investigated the tissue-specific expression of these complexes by EMSA using nuclear extracts from E13.5 fetal liver (which is mainly composed of erythroid cells), primary fetal liver derived megakaryocytes, C2C12 myoblasts and 3T3-L1 adipocytes (Fig. 3A, lanes 4-7). A number of other cell types were also studied (data not shown). As expected we detected binding of transcription factors Sp1 and Sp3 in all nuclear extracts, given the ubiquitous expression of Sp1 and Sp3 (21Suske G. Gene (Amst.). 1999; 238: 291-300Crossref PubMed Scopus (985) Google Scholar). The other three DNA binding activities (A, B, and C) were expressed in both hemopoietic and non-hemopoietic cells with cell-type specific differences in the abundance of the binding activity. Note-worthy for this study was that complex B was barely detected in fetal liver (mainly erythroid cells) but was easily detectable in primary megakaryocytes. To map where Sp1, Sp3, A, B, and C were binding in vitro within the DNA fragment E-I, a series of 2-bp scanning mutations were made in wild-type sequence to generate a series of mutant oligonucleotides (Fig. 3B) that were used as competitor in EMSA assays with wild-type sequence as probe. By recording which probes failed to abrogate binding of the 5 activities (Sp1, Sp3, A, B, and C), we could delineate the in vitro DNA binding sites of Sp1/3, A, B, and C in E-I (Fig. 3C; the data are summarized in" @default.
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