The SCL 40 Enhancer Targets the Midbrain Together with Primitive

MOLECULAR AND CELLULAR BIOLOGY, Oct. 2007, p. 7206–7219 Vol. 27, No. 200270-7306/07/$08.00�0 doi:10.1128/MCB.00931-07Copyright © 2007, American Society for Microbiology. All Rights Reserved.

The SCL �40 Enhancer Targets the Midbrain Together with Primitiveand Definitive Hematopoiesis and Is Regulated by SCL and

GATA Proteins�

S. Ogilvy, R. Ferreira, S. G. Piltz, J. M. Bowen, B. Gottgens, and A. R. Green*Department of Haematology, Cambridge Institute for Medical Research, University of Cambridge, Hills Road,

Cambridge CB2 0XY, United Kingdom

Received 25 May 2007/Returned for modification 29 June 2007/Accepted 9 August 2007

The SCL/Tal-1 gene encodes a basic helix-loop-helix transcription factor with key roles in hematopoietic andneural development. SCL is expressed in, and required for, both primitive and definitive erythropoiesis. Thus far,we have identified only one erythroid SCL enhancer. Located 40 kb downstream of exon 1a, the �40 enhancerdisplays activity in primitive erythroblasts. We demonstrate here that a 3.7-kb fragment containing this element alsotargets expression to the midbrain, a known site of endogenous SCL expression. Although the 3.7-kb construct wasactive in primitive, but not definitive, erythroblasts, a larger 5.0-kb fragment, encompassing the 3.7-kb region, wasactive in both fetal and adult definitive hematopoietic cells. This included Ter119� erythroid cells along with fetalliver erythroid and myeloid progenitors. Unlike two other SCL hematopoietic enhancers (�18/19 and �4), �40enhancer transgenes were inactive in the endothelium. A conserved 400-bp core region, essential for both hemato-poietic and midbrain �40 enhancer activity in embryos, relied on two GATA/E-box motifs and was bound in vivoby GATA-1 and SCL in erythroid cells. These results suggest a model in which the SCL �18/19 and/or �4 enhancersinitiate SCL expression in early mesodermal derivatives capable of generating blood and endothelium, withsubsequent activation of the �40 enhancer via an autoregulatory loop.

The vertebrate hematopoietic system is highly conservedacross evolution, as are the expression patterns and functionsof its key regulatory genes. The stem cell leukemia gene SCL(or Tal-1) encodes a basic helix-loop-helix transcription factorwhich is essential for the development of primitive and defin-itive hematopoiesis (29, 31, 36). Although SCL is not requiredfor the self-renewal or long-term reconstituting activity of adulthematopoietic stem cells (HSCs), those lacking SCL have im-paired short-term repopulating capacity (6, 17, 21, 35). Con-tinued SCL expression is also important for normal erythroidand megakaryocytic differentiation (17, 21).

Appropriate transcriptional regulation is essential for thebiological functions of SCL, and we have therefore systemati-cally investigated the mechanisms regulating murine SCL tran-scription. Six independent enhancers have been identified todate, each directing expression to a specific subdomain of thenormal SCL expression pattern (7, 11, 12, 33, 38). The SCL�18/19 stem cell enhancer (numbering reflects the distance inkilobases from the start of SCL exon 1a) directs transgeneexpression to HSCs and progenitors in both adult and fetalliver, as well as to embryonic endothelium (33, 34, 37). The �4enhancer is active in embryonic endothelial (and hematopoi-etic) cells (12), but not adult hematopoietic cells. Ets andGATA family transcription factors have been implicated incontrolling SCL expression in HSCs and progenitors throughthese two enhancers (12, 13).

SCL is also expressed in both the primitive and definitive

erythroid lineages (9, 14). Since none of the known SCL en-hancers directed transgene expression to erythroid cells be-yond early progenitor stages (11, 13, 33, 34, 38), we postulatedthe existence of a distinct erythroid enhancer, responsible formaintaining SCL expression following erythroid commitment(34). A systematic survey of histone acetylation across the SCLlocus resulted in identification of the �40 enhancer, a novelelement with erythroid specificity in vitro and in transgenicmice (7). A 3.7-kb fragment containing the �40 region, di-rected transgene expression to primitive, but not definitive,erythroblasts in vivo.

In addition to its pivotal role in hematopoiesis, SCL exhibitsa conserved pattern of expression in the central nervous system(CNS), and particularly in specific regions of the diencephalon,mesencephalon, metencephalon, and spinal cord (9, 38, 39). Inthe spinal cord, SCL promotes glial and neuronal progenitorsto adopt astrocyte and V2b interneuron fates, respectively(23). SCL is also essential for the development of neurons inCNS regions known to express SCL (5). Conditional deletionof SCL in neurons resulted in growth retardation and prema-ture death, along with behavioral and visual reflex abnormal-ities (5). Three different SCL enhancers that target expressionto distinct SCL-expressing regions of the CNS have been iden-tified (11, 38).

In this paper, we describe a detailed analysis of the �40enhancer. In addition to its hematopoietic activity, the 3.7-kb�40 enhancer targets specific midbrain neurons in a patternconsistent with endogenous SCL expression and indistinguish-able from that of SCL promoter 1a. Investigation of a 5-kb �40enhancer fragment revealed activity in adult definitive ery-throid and progenitor cells, as well as in the primitive erythroidcells targeted by the 3.7-kb enhancer. We have also identifieda conserved 400-bp �40 enhancer core region, and our results

* Corresponding author. Mailing address: Department of Haema-tology, CIMR, University of Cambridge, Hills Road, Cambridge CB20XY, United Kingdom. Phone: 44 1223 336820. Fax: 44 1223 762670.E-mail: [email protected].

� Published ahead of print on 20 August 2007.

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demonstrate that its activity requires two conserved GATA/E-box motifs found therein. Moreover, both GATA-1 and SCLproteins bind the core in vivo in erythroid cells, suggesting thatSCL transcription is subject to positive autoregulation via the�40 enhancer.

MATERIALS AND METHODS

Generation of �40 constructs. The 3.7-kb enhancer fragment (�40/3.7), SV/lac/�40 and SV intron/lac/�40 transgenes [referred to here as SV (intron)lac/�40/3.7] were as described previously (7). Shorter �40 fragments were PCRamplified (Extensor hi-fidelity mix; ABgene, Epsom, United Kingdom) fromgenomic 129 embryonic stem cell DNA and cloned into the SalI and KpnI sitesof SV intron/lac/�40/3.7 after removal of the 3.7-kb fragment. To generate thedeletion constructs, short fragments located immediately 5� or 3� of the 0.4-kbcore were PCR amplified using 3� or 5� primers (respectively) containing anRsrII site and subcloned into SV intron/lac/�40/3.7 or SV/luc/�40/3.7 usingRsrII and a unique upstream or downstream site. Point mutants were generatedby site-directed mutagenesis (QuikChange; Stratagene, La Jolla, CA). Fragmentscontaining the desired mutations were subcloned into the original 3.7-kb en-hancer. A short fragment, running from nucleotide 97342 of GenBank sequenceAJ297131 (the 5� end of the 5.0-kb enhancer �40/5.0) to the RsrII site internalto �40/3.7, was PCR amplified and inserted into SV intron/lac/�40/3.7 andSV/luc/�40/3.7, resulting in SV intron/lac/�40/5.0 and SV/luc/�40/5.0. All PCRproducts used were sequenced prior to their use in cloning. Luciferase reporterconstructs were made in pBluescript II (Stratagene, La Jolla, CA) and containedthe simian virus 40 (SV40) minimal promoter, firefly luciferase coding region,and SV40 poly(A) (all from pGL2-Promoter; Promega, Southampton, UnitedKingdom) with or without �40 fragments. Further details are available from theauthors on request.

Stable transfection assays. F4N (8) and BW5147 (30) cell lines were grown inRPMI 1640 with L-glutamine (Invitrogen, Carlsbad, CA) as described previously(7). Transfection assays of enhancer activity using luciferase reporter constructswere done as described previously (7). Experiments were repeated at least threetimes per cell line or experimental series. Results are expressed as mean lucif-erase activity (in relative light units) � standard deviation, where the mean of thefour pools of the SV/luc “promoter alone”-transfected cells was normalized to 1and the activity in each of the four pools transfected with the various �40construct-containing plasmids compared.

Generation and analysis of transgenic mice. Transgene fragment preparation,generation of transgenic mice, PCR-based genotyping, and 5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside (X-Gal) staining were all performed as describedpreviously (7). Animals bearing the �40/3.7 fragment and its derivatives wereadditionally genotyped using transgene-specific primers SV40-forward (5�-GGTCTG GTG TCA AAA ATA ATA A-3�) and �40-reverse (5�-ACT ACT TGTCAT TTG TTG CTA A-3�). Details of PCR protocols are available on request.X-Gal-stained embryos were embedded in paraffin, sectioned (5 �m), and coun-terstained with neutral red (Sigma, St. Louis, MO).

Flow cytometric analysis of �-Gal activity. Single-cell suspensions from he-matopoietic tissues were analyzed for �-galactosidase (�-Gal) activity as de-scribed previously (26), except that no red cell lysis was done. A FACScaliburrunning CellQuest software (BD Biosciences Immunocytometry Systems, SanJose, CA) was used for data acquisition and analysis. Phycoerythrin-conjugatedrat anti-mouse Ter119, anti-Mac-1, anti-c-Kit, and rat immunoglobulin G2b(IgG2b) isotype control antibodies, along with biotinylated anti-CD34 andstreptavidin-allophycocyanin were purchased from BD Biosciences Pharmingen(San Diego, CA). A forward scatter gate excluded debris, whereas dead cellswere excluded by propidium iodide (Sigma) uptake. Fetal blood samples wereobtained as described previously (27). Briefly, pregnant females were sacrificed,and the closed uterus was washed. The uterine wall and yolk sac were removedwithout disrupting the umbilical cord. Umbilical vessels were then clamped andsevered, and the embryo was washed in phosphate-buffered saline containing 2%fetal calf serum (PBS-2% FCS). After transfer to fresh PBS-2% FCS, the jugularveins and cervical arteries were severed, and the embryo was allowed to bleedout. For fetal blood analyses, between 0.5 � 104 and 2 � 104 viable cells wereexamined. Similarly, between 2 � 104 and 10 � 104 viable fetal liver cellswere analyzed. For adult tissues, between 0.5� 104 and 2 � 104 viable cells wereexamined.

In vitro assays of colony-forming activity in sorted populations. Bone marrowand fetal liver suspensions were stained as described above, except that sterilitywas maintained throughout. Approximately 2 � 104 cells each was then analyzed.For fetal liver, �-Gal-positive (�-Gal�) samples were pooled before sorting, and

the genotypes of individual embryos were confirmed retrospectively by PCR onyolk sac DNA. �-Gal� and �-Gal-negative (�-Gal�) populations were sortedinto PBS-5% FCS using a MOFLO cell sorter (Dako, Ely, United Kingdom). InSV intron/lac/�40/5.0 transgenic bone marrow, the average percentages of gatedcells in the �-Gal� and �-Gal� fractions were 1.3% and 97%, respectively. Usingfetal liver, the corresponding values were 1.6% and 87% for SV intron/lac/�40/3.7 and 16% and 69% for SV intron/lac/�40/5.0, respectively. A sample of eachsorted fraction was taken for reanalysis. Although purities varied between indi-vidual experiments, regardless of the tissue sorted, the �-Gal� fraction was over97% pure, such that contamination of the �-Gal� sort population with �-Gal�

cells cannot account for the vast majority of colonies being formed from the�-Gal� sorted population. Viable cell counts were done on the remainder, and0.5 � 104 to 1.25 � 104 viable cells were plated in Methocult (M3434; Stem CellTechnologies, Vancouver, Canada) according to the manufacturer’s protocol. Astained, unsorted sample was treated similarly. A total of 12.5 � 103 unfraction-ated, 5 � 103 �-Gal� or 12.5 � 103 �-Gal� viable bone marrow cells were plated,respectively. Similarly, 104 unfractionated, 103 �-Gal� or 104 �-Gal� fetal livercells were plated. Erythroid and myeloid (granulocyte-macrophage CFU) colo-nies were scored after 8 to 10 days, and the distributions of colony-forming cellsin the sorted �-Gal� and �-Gal� fractions were calculated as described previ-ously (33).

Chromatin immunoprecipitation assays. Chromatin immunoprecipitation as-says were performed as described previously (7) in the F4N erythroleukemia cellline (8), using rabbit anti-SCL (a kind gift from C. Porcher, Oxford, UnitedKingdom), rabbit anti-GATA-1 (AB11963; Abcam, Cambridge, United King-dom), and nonspecific rabbit IgG (Sigma-Aldrich). Enrichment was determinedusing SYBR green real-time quantitative PCR (Stratagene) as described previ-ously (7). The following primer pairs were used to amplify the SCL �16 tran-scriptionally inactive negative control, the �-globin HS-8 positive control, and the�40 enhancer regions, respectively: NegF (5�-GCA ATG AAC CTC CGA ACTGG-3�) and NegR (5�-CGT CTA AGA AGG TGC CCA CAG-3�), PosF (5�-GTC TCC CTT AGG TAG AGT AG-3�) and PosR (5�-GTG GCT CTT TCTTGG AGA GG-3�), and �40F (5�-TCC TAA AGC CTT GGT GCC TG-3�) and�40R (5�-GAG CTG GCG ATA AGG AAG AGG-3�). The levels of enrich-ment with the specific antibodies were normalized and calculated as the changein enrichment compared to that for nonspecific IgG. Results shown are a rep-resentative experiment of three independent experiments.

RESULTS

Characterization of hematopoietic and midbrain expressiondriven by the 3.7-kb �40 enhancer element. We recently de-scribed a novel regulatory element for SCL, the �40 enhancer,which targets in vivo lacZ transgene activity to distinct sites ofendogenous SCL expression, namely, primitive erythroid cellsof the peripheral blood, along with the midbrain (7). To inves-tigate the onset of transgene expression, we analyzed �-Galactivity in transgenic embryos carrying the SV intron/lac/�40/3.7 transgene (Fig. 1A) during early mouse embryonic devel-opment. As shown in Fig. 1B, panel i, by embryonic day 7.5(E7.5), the SV intron/lac/�40/3.7 transgene is expressed in aring of cells within the extraembryonic mesoderm of whole-mount embryos. Sagittal sections revealed that staining wasconfined to small groups of cells lying at the junction betweenextraembryonic tissue and the embryo proper (data notshown). At E8.5, staining was limited to the allantois, yolk sacblood islands, and to cells within the developing blood vesselsof the embryo proper (Fig. 1B, panels ii and iii), including theheart rudiment (Fig. 1B, panel iv) and paired dorsal aortae(Fig. 1B, panel v). By contrast to previously characterized SCLregulatory elements, there was no apparent �-Gal activity inthe endothelium lining the dorsal aorta or other major bloodvessels (Fig. 1B, panels v and vi, respectively) (see also refer-ences 12, 13, 33, and 38). Analysis of whole-mount embryosfrom E9.5 onwards indicated that hematopoietic transgeneexpression was localized to blood vessels (Fig. 1B, panels vii to

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FIG. 1. �40/3.7 transgene expression during mouse development. Enhancer activity in blood coincides with the initiation of hematopoi-esis. (A) Diagram of the SV (intron) lac/�40/3.7 transgene(s). (B) Whole mounts and sections from X-Gal-stained SV intron/lac/�40/3.7transgenic embryos (line 4092) at various stages of development. (i) A whole-mount E7.5 transgenic embryo showing staining in theextraembryonic mesoderm. (ii) An E8.5 embryo. (iii and vi) Cross sections of the same embryo with high-power views (iv and vi, respectively)showing staining in cells located in the yolk sac blood vessel (Bv) lumen (iii), the heart (Ht) rudiment (iii and iv), and the dorsal aortae (DA)(iii and v). Arrowheads in panels v and vi indicate the endothelium surrounding the dorsal aortae (v) and other major blood vessels (vi). (vi)Cells within the lumen of a large blood vessel (Bv), but not the surrounding endothelial lining (arrowhead), were �-galactosidase positive.(vii) An E9.5 embryo with staining localized to the blood vessels, but not midbrain. (viii) An E10.5 embryo showing staining in both bloodand midbrain. (ix) An E11.5 transgenic embryo. An arrowhead indicates the diencephalic extension. (x to xii) Sagittal sections through anE11.5 embryo showing a major blood vessel (the arrowhead indicates the endothelial lining) (x), yolk sac (xi), and the developing heart (xii).High-power lateral (xiii) and dorsal (xiv) views of the E11.5 embryo shown in panel ix. Lines marked a and b indicate the plane of thecorresponding coronal sections shown in panels xv and xvi, respectively. (xiv to xvi) Stained intertectal axonal projections crossing at themidline (arrows), diencephalic extensions (arrowheads), and cells within blood vessels (Bv) are indicated. (xvii) An X-Gal-stained whole-mount E18.5 brain with staining in the mesencephalon. (xviii to xx) Whole-mount X-Gal-stained brains from adult SV intron/lac/�40/3.7transgenic (line 4092) (xviii and xix) and SCL-lacZ knock-in mice (line 252) (xx), both displaying �-galactosidase activity in the superior (SC)and inferior (IC) colliculi. Note the staining in the blood vessels (Bv) of the SCL-lacZ knock-in mouse, but not in the SV intron/lac/�40/3.7transgenic mouse.

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ix). At E11.5, blood vessel and yolk sac endothelium (Fig. 1B,panels x and xi, respectively), as well as endocardium (Fig. 1B,panel xii), appeared �-Gal�. Generalized staining of circulat-ing blood cells made it difficult to identify clusters of hemato-poietic cells on the ventral wall of the dorsal aorta thought tocontain HSCs or to unequivocally detect staining in them (datanot shown). As noted previously, expression in circulating ery-throid cells declined sharply with the shift from primitive todefinitive erythropoiesis after E12.5 (data not shown) (7).

In the midbrain, lacZ activity was first detected at E10.5 (Fig.1B, panel viii). At E11.5, expression was evident in the anterolat-eral midbrain (Fig. 1B, panels ix, xiii, and xvi) with diencephalicextensions (Fig. 1B, panel ix) and axonal projections to the roof ofthe midbrain (Fig. 1B, panels xiv and xvi). By E18.5, the �-Gal�

axonal projections had spread caudally to cover the tectum (Fig.1B, panel xvi). Midbrain expression was maintained in adult miceand was particularly prominent in the superior (SC) and inferior(IC) colliculi (Fig. 1B, panels xviii and xix). The same pattern wasseen in three SV (intron) lac/�40/3.7 lines (line 4092, 4102, and4274) and it appeared identical to that previously observed foranother SCL neural element which lies immediately upstream ofexon 1a (termed promoter 1a; see reference 38). With one im-portant exception, the pattern also matched that observed inbrain from fetal and adult mice with lacZ knocked into the SCLlocus (Fig. 1B, panel xx) (9, 39). Whereas the knock-in constructgave rise to lacZ expression in blood vessels (Fig. 1B, panel xx),consistent with SCL expression in adult endothelium and ourprevious identification of two SCL endothelial enhancers (12, 33,38), the �40/3.7 construct did not target adult blood vessels (Fig.1B, panels xviii and xix) or the cells therein (data not shown; alsosee Fig. 6C, panel vii).

The 3.7-kb �40 enhancer is active in fetal liver hematopoi-etic progenitors. We have previously noted that the 3.7-kb �40enhancer was inactive in the vast majority of definitive hema-topoietic cells in the fetal liver at E14.5, a time point at whichmost cells are definitive erythroblasts (7). This was surprisingsince most erythroid regulatory elements do not discriminate

between the primitive and definitive erythroid lineages. Herewe confirm our previous observation (Table 1) but also dem-onstrate the existence of a small, but significant, �-Gal� pop-ulation in both transgenic E12.5 (Fig. 2A and Table 1) andE14.5 (Table 1) fetal liver. At E12.5, the greater part of this�-Gal� population did not express the erythroid lineage sur-face marker Ter119, but a significant proportion expressedMac-1, CD34, and/or c-Kit, all markers found on multipotentprogenitors and HSCs from fetal liver (Fig. 2A and Table 1).LacZ expression in adult bone marrow and spleen was notsignificantly different from LacZ expression in nontransgeniccontrols (Table 1).

To confirm progenitor function, �-Gal� cells were sortedfrom E12.5 fetal liver and assayed for their ability to formdefinitive hematopoietic colonies in vitro. As shown in Table 2,compared to the �-Gal� population, the �-Gal� populationwas enriched (20-fold) for both erythroid and myeloid colo-nies. Our results demonstrate that the 3.7-kb �40 element wasactive in approximately 27% of sorted erythroid progenitorsand 26% of sorted myeloid progenitors.

The minimal region required for �40 activity centers on anarea of high sequence homology containing multiple conservedtranscription factor binding motifs. To refine a core regionresponsible for enhancer activity, we generated reporter con-structs containing subfragments of the 3.7-kb region. Thesecentered on a region of high homology in human/dog/mouse/rat sequence alignments (Fig. 2B) (7). The whole 3.7-kb frag-ment directed expression to E12.5 embryonic midbrain andblood, as did both 0.9-kb and 0.7-kb subfragments (Fig. 2C,panels i and ii, and Table 3). However, no activity was detectedin either brain or blood at E12.5 using a smaller 0.4-kb sub-fragment (Fig. 2C, panel iii, and Table 3). In addition, the levelof activity (measured by rapidity and intensity of X-Gal stain-ing) observed with SV intron/lac/�40/0.9 and SV intron/lac/�40/0.7 appeared generally lower than with SV (intron) lac/�40/3.7, suggesting that while specificity was maintained withthe shorter �40 regions, maximal enhancer function was lost.

TABLE 1. Analysis of in vivo �40 transgene activity in hematopoietic populations

Genotype Tissue Total �-Gal� (%)a Ter119� �-Gal� (%)b Mac-1� �-Gal� (%)b CD34� �-Gal� (%)b c-Kit� �-Gal� (%)b

�40/3.7 E12.5 FLc 1.47 � 0.46* 16 � 5.5 45 � 5.1 65 � 7.8 91 � 5.2E14.5 FLc 0.36 � 0.09* 36 � 11 ND ND 86 � 3.8BMe 0.09 � 0.03Spleene 0.08 � 0.01

�40/5.0 E12.5 FLd 9.73 � 2.05* 51 � 11 16 � 5.8 33 � 6.4 60 � 11E14.5 FLd 4.59 � 1.37* 53 � 10 26 � 8.6 23 � 7.5 68 � 11BMe 1.66 � 0.32* 57 � 31 ND ND NDSpleene 1.75 � 0.85*** 37 � 17 ND ND ND

SCL 3�E BMe 1.93 � 0.56** 9.1 � 3.6 ND ND NDSpleene 2.12 � 0.52** 5.4 � 2.0 ND ND ND

a Total �-galactosidase-positive (�-Gal�) cells (mean � standard deviation SD�) as a percentage of total viable cells analyzed. In analogous samples fromnontransgenic controls analyzed concomitantly, �0.22% of the total viable cells were �-Gal�. Significant differences between the nontransgenic and transgenicpopulations, where P � 0.001, P � 0.005, or P � 0.02, are marked with one, two, or three asterisks, respectively. Otherwise, there was no significant difference in at test (P 0.05). ND, not done.

b Total Ter119, Mac-1, CD34, or c-Kit marker-positive, �-Gal� cells (mean � SD) as a percentage of total �-Gal� cells analyzed.c Fetal liver (FL) results from line 4092 embryos.d Fetal liver (FL) results from line 5405 embryos.e Bone marrow (BM) and spleen were from adult ( 12 weeks of age) SV intron/lac/�40/3.7 (line 4092; also representative of lines 4102 and 4274), SV

intron/lac/�40/5.0 (line 5402; also representative of lines 5400 and 5405), and SCL 3�E (�6E5/lacZ/3�E, line 2269) transgenic mice. For all results shown, at least threeembryos or mice of each genotype were analyzed.



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FIG. 2. The �40/3.7 enhancer is active in E12.5 fetal liver, and a 700-bp subfragment is sufficient for �40 activity in vivo. (A) Flow cytometric analysis(FACS) of �-galactosidase activity and cell surface marker expression in E12.5 fetal liver cells from a line 4092 SV intron/lac/�40/3.7 transgenic (Tg)embryo (top row) and nontransgenic (non-Tg) littermate (bottom row). Cells were stained simultaneously for �-Gal activity and the erythroid lineagemarker Ter119, Mac-1, CD34, or c-Kit as indicated. Comparable data were obtained in two independent experiments in which 10 embryos of eachgenotype were analyzed. (B) Schematic representation of a four-way sequence alignment of the MAP17 gene downstream region in humans (Homosapiens [Hs]), dogs (Canis familiaris [Cf]), mice (Mus musculus [Mm]), and rats (Rattus norvegicus [Rn], showing peaks of sequence homology (modifiedfrom Delabesse et al. [7]). Red boxes, coding exons; beige boxes, untranslated exons; blue boxes, repeat sequences. The relative positions of the various�40 fragments incorporated into lacZ reporter constructs are shown below the graph of sequence homology. (C) Whole-mount X-Gal-stained E12.5transgenic embryos bearing the SV intron/lac/�40/3.7 (line 4092) (i), SV intron/lac/�40/0.7 (line 6320) (ii), and SV intron/lac/�40/0.4 (iii) (F0 transgenic)constructs. (D) FACS analysis of �-Gal activity in adult bone marrow (BM) from a nontransgenic littermate, an SV intron/lac/�40/3.7 transgenic (line4092), and two SV intron/lac/�40/0.7 transgenic mice representing independent lines (6281 and 6320). Cells were stained simultaneously for �-Gal activityand the erythroid lineage marker Ter119. Similar results were obtained in an additional two SV intron/lac/�40/0.7 transgenic lines (lines 6278 and 6559).

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As with SV (intron) lac/�40/3.7 transgenic mice, there was nodetectable �-Gal activity in adult bone marrow from micerepresenting four independent SV intron/lac/�40/0.7 trans-genic lines (Fig. 2D and data not shown). These data demon-strate that sequences within the 0.7-kb fragment are sufficientto direct �-Gal expression to both midbrain and primitiveerythroblasts.

A four-way human/dog/mouse/rat sequence alignment ofthis region is shown in Fig. 3. The 0.7-kb fragment and the 3�boundary of the 0.4-kb fragment are indicated. Several con-served consensus binding sites for hematopoietic transcriptionfactors were identified, including two GATA sites, two E-boxes

(CANNTG), and two Ets (GGAW) sites. Both GATA sites liein close proximity to an E-box. In all four species, the 5� GATAsite and E-box were separated by 9 bp, and the 3� GATA siteand E-box were separated by 6 bp. This spacing indicates that,for each GATA/E-box motif, DNA-bound GATA and basichelix-loop-helix proteins would be on the same face of thedouble helix and capable of interacting.

A 400-bp core region is required for 3.7-kb enhancer activityin vivo and contains GATA/E-box motifs with both essentialand redundant functions. Although apparently not sufficientfor �40 transgene activity in vivo, the high level of sequenceconservation within the �40/0.4-kb core indicated that this

TABLE 2. Hematopoietic progenitors in transgenic fetal liver and adult bone marrow

Tissue, genotype, and cellpopulationa

No. of erythroidcoloniesb

% Total sorted erythroidprogenitorsc

No. of myeloidcoloniesb

% Total sorted myeloidprogenitorsc

E12.5 FLSV intron/lac/�40/3.7

�-Gal� sorted 21 � 11 27 127 � 37 26�-Gal� sorted 1.1 � 0.7 73 6.9 � 3.8 74Stained, unsorted 1.3 � 0.3 11 � 1.2

SV intron/lac/�40/5.0�-Gal� sorted 11 � 4.1 83 70 � 16 72�-Gal� sorted 0.5 � 0.5 17 6.3 � 1.7 28Stained, unsorted 1.7 � 0.7 11 � 1.8

BMSV intron/lac/�40/5.0

�-Gal� sorted 26 � 17 12 65 � 34 8�-Gal� sorted 2.5 � 2.1 88 9.9 � 7.3 92Stained, unsorted 3.1 � 2.0 10 � 4.7

a FL, fetal liver; BM, bone marrow.b The mean number (� standard deviation) of erythroid colonies (erythroid burst-forming unit and granulocyte-sythrocyte-monocyte-megakaryocyte CFU) or

myeloid colonies (granulocyte-macrophage colony-forming cells) formed per 1,000 plated E12.5 fetal liver or 5,000 bone marrow cells, respectively.c The distribution of erythroid (erythroid burst-forming unit and granulocyte-sythrocyte-monocyte-megakaryocyte CFU) or myeloid (granulocyte-macrophage CFU)

progenitors in each fraction, given as a percentage of total sorted (erythroid or myeloid) progenitors. Data shown are pooled from two independent fetal liver experiments each(lines 4092 and 5405) and three bone marrow (line 5402) experiments. The difference between the results obtained with the �-Gal� and �-Gal� sorted populations wassignificant in all cases (P � 0.000001).

TABLE 3. Analysis of �40 regions required for in vivo activity in E12.5 embryos

ConstructNo. of embryos with expression of the construct/total no. of embryosa

Blood Midbrain No expression Ectopic expression

SV (intron) lac/�40/3.7 4/6 5/6 0/6 3/6SV intron/lac/�40/0.9 1b/3 3/3 0/3 3/3SV intron/lac/�40/0.7 2b/8 5c/8 3/8 1/8SV intron/lac/�40/0.4 0/4 0/4 3/4 1/4

SV intron/lac/�40/3.7 �GE1 � 2 0/10 0/10 4/10 6d/10SV intron/lac/�40/3.7 �GE1 2/6 3c/6 2/6 3/6SV intron/lac/�40/3.7 �GE2 4/13 1/13 7/13 3d/13

SV intron/lac/�40/3.7 mGE1 � 2 0/11 0/11 4/11 7d/11SV intron/lac/�40/3.7 mGE1 4/8 4/8 2/8 2/8SV intron/lac/�40/3.7 mGE2 2/4 0/4 1/4 1/4

SV intron/lac/�40/5.0 9/10 9/10 1/10 3/10

a Results represent four SV (intron) lac/�40/3.7 transgenic lines and two F0 transgenic E12.5 embryos, five SV intron/lac/�40/0.7 transgenic lines and three F0transgenic E12.5 embryos, and three SV intron/lac/�40/5.0 transgenic lines and seven F0 transgenic E12.5 embryos, respectively. All other results denote F0 transgenicembryos analyzed at E12.5.

b Weak staining and/or staining in only a proportion of circulating blood cells.c Weak staining in midbrain.d Ectopic staining in the brains of some (1 of 10 SV intron/lac/�40/3.7 �GE1 � 2, 1 of 13 SV intron/lac/�40/3.7 �GE2, and 3 of 11 SV intron/lac/�40/3.7 mGE1

� 2) embryos, which was apparently not related to the endogenous SCL midbrain expression pattern.



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FIG. 3. The �40 core region contains multiple conserved transcription factor recognition sequences. A four-way sequence alignment of the�40/0.7 region (boxed) in human SCL (Homo sapiens [hsscl]), dog SCL (Canis familiaris [cfscl]), mouse SCL (Mus musculus [mmscl]), and rat SCL



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region might be necessary for �40 function. We thereforedeleted from the original �40/3.7-kb fragment, either the prox-imal 200 bp (from the beginning of the �40/0.4 region to aninternal RsrII site) containing the 5� GATA/E-box motif, thedistal 200 bp (from the internal RsrII site to the end ofthe �40/0.4 region) containing the 3� GATA/E-box motif, orthe entire 400-bp �40/0.4 region (Fig. 3 and 4). Luciferasereporter constructs containing each of these deletion frag-ments (SV/luc/�40/3.7 �GE1, SV/luc/�40/3.7 �GE2 and SV/luc/�40/3.7 �GE1 � 2) were tested for enhancer activity bystable transfection using the erythroid F4N cell line (Fig. 4A).Deletion of the entire 400-bp core region or its 5� half resultedin almost complete loss of enhancer function, whereas deletionof the 3� 200 bp had little or no effect (Fig. 4A).

The same �40 deletion fragments were used to generatecorresponding lacZ reporter constructs (SV intron/lac/�40/3.7�GE1, SV intron/lac/�40/3.7 �GE2, and SV intron/lac/�40/3.7 �GE1 � 2). F0 transgenic embryos were generated andanalyzed for �-Gal activity at E12.5. Consistent with our trans-fection data, transgene activity was not detected in either themidbrain or blood of any of 10 embryos bearing the SV intron/lac/�40/3.7 �GE1 � 2 transgene (Fig. 4B, panel i, and Table3). These data demonstrate that the 400-bp core region isnecessary for appropriate transgene action in vivo. One-third

of the SV intron/lac/�40/3.7 �GE1 transgenic embryos ana-lyzed displayed �-Gal activity in both blood and midbrain (Fig.4B, panel ii, and Table 3), indicating that the 5� half of the400-bp core region is not required for enhancer activity ineither tissue. Similarly, 4 of the 13 SV intron/lac/�40/3.7 �GE2transgenic embryos had �-Gal activity in peripheral blood,whereas 1 of 13 had appropriate expression in the midbrain(Fig. 4B, panel iii, and Table 3). Taken together, these datademonstrate that the 5� and 3� halves of the 400-bp core regionhave redundant functions in determining the cell type speci-ficity of enhancer action in blood (primitive erythropoiesis)and midbrain.

To investigate the importance of individual binding siteswithin the 0.4-kb core region, we mutated individually, or incombination, each of the two conserved GATA and E-boxrecognition sites (Fig. 5A) and tested their enhancer activity invitro in F4N cells (Fig. 5B). Compared to the original 3.7-kbfragment, there was an almost complete loss of activity whenall four sites were mutated, suggesting that one or more ofthese sites are essential for enhancer activity in this cell line.Mutation of either the 5� GATA (data not shown), 5� E-box(data not shown), or both sites (Fig. 5B), also resulted inalmost complete loss of enhancer activity, whereas the effect ofmutating the 3� GATA and/or E-box sites was minimal (Fig. 5B

(Rattus norvegicus [rnscl]) genes, showing potential Ets, GATA, and E-box (helix-loop-helix factor) binding sites. An RsrII restriction enzymerecognition site located between the two GATA/E-box motifs and present only in the mouse sequence is indicated. Completely conserved residuesare shown by white letters on a black background, and residues conserved in three of the four species are shown by white letters on a graybackground. The �40/0.4 core region fragment shares the same 5� boundary as the �40/0.7 fragment, and its 3� border is indicated by a blackarrowhead. Gaps introduced to maximize alignment are indicated by the dashes.

FIG. 4. The �40/0.4 core region, containing GATA/E-box motifs, is required for in vivo activity. (A) Schematic representation of the SVintron/luc/�40/3.7 deletion constructs and in vitro luciferase assays of their enhancer activity in stably transfected F4N cells compared to theoriginal �40/3.7 wild-type construct. Histograms represent the mean (plus standard deviation [error bar]) luciferase activity (in relative light units[RLU]) of four independent pools, normalized to the mean result obtained with SV/luc. The results shown are representative of those observedin at least three independent experiments. (B) Whole-mount X-Gal-stained E12.5 F0 transgenic embryos bearing the SV intron/lac/�40/3.7�GE1 � 2 (i), SV intron/lac/�40/3.7 �GE1 (ii), or SV intron/lac/�40/3.7 �GE2 (iii) deletion constructs in which the entire �40/0.4-kb region orthe 5� or the 3� 0.2 kb, respectively, had been deleted. Midbrain staining in the SV intron/lac/�40/3.7 �GE1 embryo shown in panel ii is indicatedby an arrowhead.



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and data not shown). These data are consistent with the resultsobserved using deletions within the 3.7-kb fragment (Fig. 4A).

Transgenic analysis was then performed using mutant SVintron/lac/�40/3.7 constructs in which either the 5� GATA/E-box motif, the 3� GATA/E-box motif, or all four sites weremutated (Fig. 5C and Table 3). All 11 F0 SV intron/lac/�40/3.7mGE1 � 2 transgenic E12.5 embryos, in which both 5� and 3�GATA/E-box motifs were mutated, lacked appropriate expres-sion in both blood and midbrain (Fig. 5C, panel i, and Table 3).

Mutation of the 5� GATA/E-box motif resulted in expressionin the midbrain and blood in four out of eight transgenic E12.5embryos (Fig. 5C, panel ii, and Table 3). In contrast, mutationof the 3� GATA/E-box motif resulted in expression in blood (intwo of four transgenic E12.5 embryos), but not in the midbrain(Fig. 5C, panel iii, and Table 3). Again, these observations arein line with those obtained using deletion constructs (Fig. 4Band Table 3).

To identify proteins bound to the conserved GATA/E-box

FIG. 5. GATA/E-box motifs, present in the �40 enhancer core, are necessary for its in vivo activity. (A) Sequences of the two wild-typeGATA/E-box motifs found within the �40/0.4 core with the corresponding mutated sequences (mGE1 and mGE2) shown below. The individualresidues that differ between the wild-type �40/3.7 and mutated fragments are shown in white letters on a black background. (B) Schematicrepresentations of the SV intron/luc/�40/3.7 mutation constructs and in vitro luciferase assays of their enhancer activity in stably transfected F4Ncells compared to the original �40/3.7 wild-type construct. Histograms represent the mean (plus standard deviation [SD] [error bar]) luciferaseactivity (in relative light units [RLU]) of four independent pools, normalized to the mean result obtained with SV/luc. The results shown arerepresentative of those observed in at least three independent experiments. (C) Whole-mount X-Gal-stained E12.5 F0 transgenic embryos bearingthe SV intron/lac/�40/3.7 mGE1 � 2 (i), SV intron/lac/�40/3.7 mGE1 (ii), or SV intron/lac/�40/3.7 mGE2 (iii) mutation constructs in which bothmotifs, the 5� GATA/E-box motif or the 3� GATA/E-box motif, respectively, were mutated. (D and E) Chromatin immunoprecipitation of GATA-1(D) and SCL (E) proteins in the F4N erythroid cell line, analyzed by real-time PCR. The results are presented as mean change in enrichment (plusSD [error bar]) obtained using the GATA-1 and SCL antibodies compared to rabbit IgG. The transcriptionally inactive SCL �16 region and the�-globin HS-8 were used as negative (Neg) and positive (Pos) controls, respectively.



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motifs in vivo, we performed chromatin immunoprecipitationin the erythroid cell line F4N. Antibodies against GATA-1 andSCL were used. In erythroid cells, these proteins are part oftranscription activation complexes that specifically targetGATA/E-box motifs (32, 42). A 3.5-fold enrichment for the�40 enhancer genomic region was observed when using anti-GATA-1 antibody, compared to nonspecific rabbit IgG (Fig.5D). By contrast, there was no enrichment for a transcription-ally inactive negative-control region, located in the 16-kb re-gion upstream of SCL exon 1 (7). An �-globin HS-8 positive-control region, containing a GATA/E-box motif known to bebound by SCL and GATA-1 (3), showed a 3.6-fold enrichment.Using an anti-SCL antibody, a 20-fold enrichment for the �40enhancer was seen (Fig. 5E). Again, no enrichment was ob-served for the �16 negative control, whereas the �-globinHS-8 region showed a 14-fold enrichment. These results con-firm that, in F4N erythroid cells, GATA/E-box motifs presentin the �40 enhancer are physically bound by GATA-1 andSCL.

Taken together, our results demonstrate that at least one�40 GATA/E-box motif is required for blood activity of the�40 enhancer. Furthermore, out of a total of 38 independenttransgenic embryos, in which the 3� GATA/E-box motif waseither mutated or absent, only one embryo displayed �-Galactivity in the midbrain. This observation suggests that the 3�GATA/E-box motif, although not absolutely essential, is par-ticularly important for midbrain enhancer function and thatthe 5� GATA/E-box motif can only partially compensate for itsloss.

A larger �40/5.0-kb enhancer fragment directs expressionto adult blood and definitive bone marrow progenitors. Wehave previously reported that the 3.7-kb �40 enhancer is activein primitive, but not definitive, erythroblasts (7), and yet, SCLis expressed in both the primitive and definitive erythroid lin-eages. A region of relatively high sequence conservation liesimmediately upstream of the 3.7-kb fragment (Fig. 6A), indi-cating that additional upstream sequences may be required forfull �40 erythroid enhancer activity. To address this possibility,luciferase and lacZ reporter constructs were generated inwhich 1.3 kb of upstream sequence was added to the original3.7-kb fragment (Fig. 6A). In stable transfection assays usingthe erythroid cell line F4N, the 5.0-kb enhancer was 10-foldmore active than the 3.7-kb enhancer (Fig. 6B). By contrast,neither the 5.0-kb nor the 3.7-kb enhancer fragments wereactive in the T-cell line BW5147 that does not express SCL(Fig. 6B). The 5.0-kb fragment therefore maintains the lineagespecificity previously noted for the 3.7-kb fragment (7).

In transgenic midgestation embryos, the 5.0-kb fragmentgave rise to the same pattern of expression as that of the 3.7-kbfragment. However, SV intron/lac/�40/5.0 embryos displayedan apparently higher level of �-Gal activity (indicated byquicker, more intense X-Gal staining) and less inappropriateectopic expression than 3.7-kb enhancer counterparts (Table3). In particular, activity was noted in the midbrain and bloodin 9 of 10 independent E12.5 F0 transgenic embryos or lines(Fig. 6C, panel i, and Table 3). Transgene activity was alreadyevident in the extraembryonic mesoderm at E7.5 (Fig. 6C,panels ii and iii). At E12.5, midbrain staining was evident in thepretectum, tegmentum, and tectum (Fig. 6C, panel iv). Mid-brain expression continued postnatally, particularly in the su-

perior and inferior colliculi of the adult brain (Fig. 6C, panelv). Further, in adult mice representing three independenttransgenic lines, and in stark contrast to the �40/3.7 transgenicmice, there was abundant staining in blood vessels (compareFig. 6C, panel v, with 1B, panel xviii), specifically in circulatingblood cells (compare panels vi and vii in Fig. 6C).

Flow cytometric analysis (fluorescence-activated cell sorting[FACS]) analysis of �-Gal activity in peripheral blood estab-lished that the 5.0-kb fragment directed expression to circulat-ing erythroid cells at both E12.5 and E14.5 (Fig. 6D), timepoints when primitive and definitive erythroid cells predomi-nate, respectively. By contrast, the 3.7-kb enhancer was notactive in peripheral blood at E14.5 (7; also data not shown). InE12.5 fetal liver, the 5.0-kb enhancer gave rise to �-Gal activityin a greater proportion of cells than the 3.7-kb enhancer(10% versus 1.5%, respectively) (Table 1; compare Fig. 6Eto 2A). With the 5.0-kb enhancer, approximately half of the�-Gal� cells were also Ter119� (Table 1) and significant pro-portions were positive for Mac-1, CD34, and c-Kit (Table 1). InE14.5 fetal liver, a similar pattern was observed, with the 5.0-kbfragment again far more active than the 3.7-kb fragment (4.6%versus 0.36%; see also Table 1). Moreover, in marked contrastto the 3.7-kb fragment, the 5.0-kb fragment gave rise to clearactivity (particularly in Ter119� erythroid cells) in adult bonemarrow (Fig. 6E and Table 1) and spleen (Table 1), but notthymus (data not shown).

To confirm progenitor function, �-Gal� cells were sortedfrom fetal liver or adult bone marrow and subjected to in vitrocolony-forming assays. The 5.0-kb enhancer was active in83% and 72% of fractionated erythroid and myeloid E12.5fetal liver progenitors, respectively, substantially higher per-centages than were obtained using the 3.7-kb enhancer (Table2). There was significant enrichment for erythroid (20-fold)and myeloid (10-fold) progenitors in the �-Gal� fractioncompared to the sorted �-Gal� population. Similarly, in adultbone marrow, erythroid (10-fold) and myeloid (7-fold)progenitors were also enriched in the �-Gal� fraction, with12% and 8% of sorted erythroid and myeloid progenitorsfound in the �-Gal� fraction (Table 2).

DISCUSSION

SCL �40 enhancer activity centers on a 400-bp core and ismediated by GATA/E-box motifs. In this paper, we demon-strate that a 0.7-kb SCL �40 enhancer fragment is sufficientfor activity in embryonic blood and midbrain. A smaller 0.4-kbcore region and the two conserved GATA/E-box motifs itcontains were shown to be essential for enhancer function inboth transfection studies and transgenic mice. Moreover, bothGATA-1 and SCL bind this region in definitive erythroid cells.In the context of the 3.7-kb enhancer, mutation of both GATA/E-box motifs resulted in loss of enhancer function, whereaseither motif was able to independently support activity inblood. By contrast, the 3� GATA/E-box motif was particularlyimportant for midbrain enhancer function.

GATA/E-box motifs are not present in the SCL promoter orin five other SCL enhancers characterized so far (4, 11–13).However, GATA/E-box motifs have been described in a num-ber of hematopoietically expressed genes, including EKLF (1,2), GATA-1 (25, 41), �-globin (3), glycophorin A (20), EPCR



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FIG. 6. The �40/5.0-kb enhancer fragment targets additional definitive hematopoietic sites in vivo. (A) Schematic representation of a four-waysequence alignment of the MAP17 gene downstream region in humans (Homo sapiens [Hs]), dogs (Canis familiaris [Cf]), mice (Mus musculus[Mm]), and rats (Rattus norvegicus [Rn]), showing peaks of sequence homology (modified from Delabesse et al. [7]) and the position of the �40/5.0fragment compared to the �40/3.7 region. Red boxes, coding exons; beige boxes, untranslated exons; blue boxes, repeat sequences. (B) In vitroluciferase assays of �40 fragment enhancer activity in murine F4N erythroid and BW5147 T-lymphoid cells stably transfected with luciferasereporter constructs containing either the SV40 minimal promoter alone (SV/luc) or the promoter with the �40/3.7-kb (SV/luc/�40/3.7) or the�40/5.0-kb (SV/luc/�40/5.0) fragment, respectively. Histograms represent the mean (� SD) luciferase activity (in relative light units [RLU]) offour independent pools, normalized to the mean result obtained with SV/luc. The results shown are representative of those observed in at leastthree independent experiments per cell line. (C) �-Galactosidase activity in SV intron/lac/�40/5.0 transgenic mice. Whole-mount E12.5 (line 5400)(i) and E7.5 (line 5405) (ii) transgenic embryos stained with X-Gal. In panel ii, maternally derived tissue (Mat) and the embryo proper (Em) areindicated. (iii) A sagittal section through another X-Gal-stained E7.5 embryo showing the allantois (Al) and defined clusters of expressing cellsin the region of extraembryonic mesoderm which gives rise to yolk sac blood islands (Bi). (iv) A sagittal section through an X-Gal-stained E12.5transgenic embryo (line 5405) showing staining in the pretectum (PT), tegmentum (Tg), and tectum (T). (v) A whole-mount X-Gal-stainedtransgenic adult brain (line 5400). (vi and vii) X-Gal-stained peripheral blood samples from an adult SV intron/lac/�40/5.0 (line 5402) transgenicmouse (vi) and SV/lac/�40/3.7 (line 4274) transgenic mouse (vii). At E12.5 (i and iv) and in the adult brain (v), staining was localized to bloodvessels and midbrain regions (particularly the superior and inferior colliculi of adult brain). Examples shown in panels i and v and vi are



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(22), Cdc6 (40), and protein 4.2 (43). Many of the motifspresent in these genes are in agreement with, but do notnecessarily exactly match, the consensus sequence described byWadman and colleagues, of an E-box (CAGGTG) and aGATA binding site [(A/T)GATA(A/G)] separated by 8 to 10bases (42). In erythroid cells, GATA/E-box motifs mediate therecruitment of a complex containing SCL, E2A, LMO2, LDB1,and GATA-1 (32, 42). Within the SCL �40 enhancer, thestructure of the 5� GATA/E-box motif is generally in accordwith that of the consensus sequence, with the GATA andE-box binding sites separated by 9 bases. By contrast, the 3�GATA/E-box motif exhibits 6-bp spacing. Such spacing waspreviously shown to preclude recruitment of the SCL complexin erythroid cell lines (42). This is consistent with our obser-vation that the 3� GATA/E-box motif was particularly impor-tant for �40 enhancer activity in the midbrain but contributedlittle to luciferase activity in erythroid cells in vitro. However,some flexibility in the nature and arrangement of sites withinGATA/E-box motifs exists. Indeed, partially overlappingGATA and E-box binding sites are present in motifs found inboth the erythroid lineage marker gene glycophorin A (20) andin the human Cdc6 gene (40), which is expressed inmegakaryocytic cells. Interestingly, in primary hematopoieticcells, the glycophorin A promoter is bound in vivo by a com-plex containing SCL and GATA-1, and the assembly of thiscomplex is necessary for promoter activation (20). This impliesthat the spacing described by Wadman and colleagues (42) isnot an absolute requirement for function. With reference tothe �40 enhancer, complexes containing different componentspresumably exhibit differential affinity for the 5� and 3� GATA/E-box motifs. It may be particularly relevant that erythroidcells contain high levels of GATA-1 (10), whereas the midbrainregions in which the �40 enhancer is active express GATA-2and GATA-3 (18, 24), but not GATA-1 (38). However, dele-tion of the 5� motif did not completely abolish hematopoieticactivity in transgenic mice, suggesting that, despite its distinctstructure, the 3� motif can compensate to some extent for lossof the 5� motif in vivo. Similarly, the reverse is also true.

The �40 enhancer is active in both primitive and definitivehematopoietic cells in vivo. Compared to the 3.7-kb construct,the 5.0-kb �40 construct gave rise to increased activity inerythroid cells in transfection assays and more robust reporterexpression in transgenic embryos. Both were active in E7.5extraembryonic mesoderm, primitive erythroblasts, and defin-itive fetal liver progenitors. Importantly and in contrast to the3.7-kb construct, the 5.0-kb construct also directed expressionto erythroid cells in adult blood, bone marrow, and spleen, aswell as to erythroid and myeloid bone marrow progenitors.Several mechanisms might account for this more extensive

expression pattern. The 1.3-kb region, which distinguishes thetwo constructs, lies directly 5� of the 3.7-kb fragment and mightconceivably contain a distinct, novel enhancer with activity indefinitive erythroid cells. However, analysis of the aligned up-stream region uncovered no further conserved GATA or E-box sites and few other conserved factor binding sites. Theconcept of a separate enhancer responsible for activity in de-finitive hematopoietic cells is rendered less likely by the factthat the 3.7-kb construct does target a minority of definitivefetal liver progenitors. We therefore favor the possibility thatour observations reflect quantitative differences in the activityof the 3.7-kb and 5.0-kb constructs, with better maintenance of5.0-kb construct function in adult hematopoiesis. Rather thanrepresenting an independent enhancer, sequences present inthe 5.0-kb, but not 3.7-kb, fragment, may facilitate increased orcontinued expression by maintaining an open chromatin do-main and/or counteracting nearby negative influences. Thelatter may include integration site effects (19) or a repressiveeffect of the �-galactosidase reporter itself (15, 16, 26, 37).

In addition to the �40 enhancer, two other SCL enhancers(�4 and �18/19) have been identified that are active in hema-topoietic cells. These direct expression to HSCs, hematopoieticprogenitors, and endothelium, but not to erythroid cells be-yond early progenitor stages (12, 33). A reporter constructdriven by the �18/19 enhancer is active in frog hemangioblasts,and it has been proposed that enhancer activity in both endo-thelial and hematopoietic progenitors is characteristic ofgenes, such as SCL, which exhibit hemangioblast expression(13, 28). The absence of �40 enhancer activity in endothelialcells indicates that it is unlikely to be active in hemangioblasts.Our results suggest a model in which the �18/19 and/or the �4enhancers are responsible for initiation of SCL transcription inearly mesodermal derivatives with the potential to generateboth blood and endothelium. By contrast, the �40 enhancermay be recruited at a later stage of development, perhaps viaan autoregulatory loop, thereafter playing a key role in direct-ing SCL expression in the erythroid lineage.

Midbrain �40 enhancer activity closely mimics that of theendogenous SCL promoter. Expression of SCL in the midbrainand spinal cord (9, 38, 39) is maintained throughout vertebrateevolution. In spinal cord, SCL directs neuronal progenitors toadopt particular cell fates (23). During midbrain development,SCL is expressed mainly in the posterior and inferior commis-sure, and in the adult, in the superior and inferior colliculi (9,39). These CNS regions, where SCL plays a nonredundantrole, are associated with the processing of visual and auditoryreflexes, and of pain-related information (5). We have nowshown that the �40 enhancer directs lacZ transgene expres-sion to the midbrain in a pattern indistinguishable from that

representative of at least three transgenic embryos or adult mice from each of three independent lines (lines 5400, 5402, and 5405), respectively.Panel ii is representative of seven transgenic embryos from line 5405. (D) Flow cytometric analysis (FACS) of �-galactosidase activity in peripheralblood from a line 5405 SV intron/lac/�40/5.0 transgenic embryo (solid lines) and nontransgenic littermate (dashed lines) at E12.5 and E14.5 asindicated. Similar results were obtained from at least three embryos of each genotype for each time point. Analogous data were also obtained atE12.5 in an additional independent transgenic line (line 5400). (E) FACS analysis of �-galactosidase activity and Ter119 expression in E12.5 fetalliver (FL) and adult bone marrow (BM) from an SV intron/lac/�40/5.0 transgenic embryo (line 5405) or mouse (line 5402), respectively. Cells werestained simultaneously for �-galactosidase activity and the erythroid lineage marker Ter119. Similar fetal liver results were obtained with at leastfour transgenic embryos. Comparable bone marrow results were obtained with at least three transgenic mice from each of three independent SVintron/lac/�40/5.0 transgenic lines (lines 5400, 5402, and 5405).



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generated by SCL promoter 1a (38). Both target the sameregions of embryonic midbrain, including the ventrolateral nu-clei, the posterior and inferior commissure, and the dience-phalic extensions. Similarly, in the adult, both direct expressionto the superior and inferior colliculi. Their apparent redundantfunctions may relate to the biological importance of SCL forthe development of these midbrain regions. Further, both el-ements appear to be regulated by GATA proteins (this studyand reference 38). However, this likely reflects distinct mech-anisms, since promoter 1a lacks a recognizable GATA/E-boxmotif. As suggested above for hematopoietic cells, binding ofGATA factors to SCL promoter 1a in neuronal cells mightinitiate expression, thereby generating SCL protein subse-quently involved in �40 midbrain enhancer activity.

Although the promoter 1a and the �40 enhancer elementshave very similar activities in midbrain, their activity in blooddiffers. Unlike the �40 enhancer, promoter 1a does not directlacZ expression to hematopoietic cells in transgenic mice (38).The GATA/E-box sites found within the �40 enhancer arenecessary for both hematopoietic and midbrain activity; how-ever, they are not sufficient, since the 0.4-kb �40 core fragmentwas inactive in both transfection studies and transgenic mice.Further analysis of the �40 enhancer, its constituent sites, andmode of action is likely to provide additional valuable insightsinto SCL transcriptional regulation.

ACKNOWLEDGMENTS

We acknowledge and thank the following: Paula Braker, Sarah Ped-ley, James Harrison, and Denise Weekly for expert generation andcare of the transgenic mice; Eric Delabesse for important intellectualand practical contributions to this work, particularly at its inception;Scott Oldham and Beverley Haynes for valuable help with histology;Mike Chapman and Ian Donaldson for help with bioinformatics; LindaScott for help with hematopoietic colony assays; and CatherinePorcher for the anti-SCL antibody.

This work was supported by The Wellcome Trust.This work is dedicated to the memory of Isabelle Anne Bouhon

(1969 to 2005), a valued friend and colleague.

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