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发表于 2009-3-5 10:48 |只看该作者 |倒序浏览 |打印

a Department of Hematology, Cambridge Institute for Medical Research, University of Cambridge, Cambridge, United Kingdom;
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b Centro Andaluz de Biologia del Desarrollo, Universidad Pablo de Olavide, Seville, Spain;# u1 g8 N3 C0 N
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c University of Massachusetts Medical School, Department of Pediatrics (Hematology/Oncology Division) and Department of Cancer Biology, Worcester, Massachusetts, USA;

d The Laboratory of Lymphocyte Signaling and Development, The Babraham Institute, Babraham, Cambridge, United Kingdom$ v' S4 K# Q1 z
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Key Words. Hematopoietic stem cell ? Transcription factor SCL/tal-1 ? Transcriptional regulation ? Experimental models

Correspondence: Berthold G?ttgens, D.Phil., Department of Hematology, Cambridge Institute for Medical Research, Cambridge University, Hills Road, Cambridge CB2 2XY, U.K. Telephone: 44-1223-336829; Fax: 44-1223-762670; e-mail: bg200@cam.ac.uk8 q5 ~+ y6 d  Y

ABSTRACT
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Appropriate control of cell type–specific gene expression lies at the heart of development. Hematopoiesis provides a prime example of this process, in which key transcription factors play central roles in cell fate specification and subsequent differentiation . Moreover, aberrant expression of the same key transcription factors is often associated with the development of leukemia . Accurate transcriptional regulation is therefore clearly critical, but in most cases the underlying molecular mechanisms remain poorly understood.

The stem cell leukemia gene (SCL), also known as TAL-1, encodes a basic helix-loop-helix transcription factor, which was first identified through its ectopic expression in T-cell acute lymphoblastic leukemia (T-ALL). SCL is normally expressed in hemangioblasts, hematopoietic stem cells (HSCs), erythroid cells, megakaryocytes, and mast cells as well as angioblasts, mature endothelial cells, smooth muscle myocytes, and specific areas of the central nervous system . Targeted deletion has shown that SCL is essential for the formation of HSCs during mouse embryonic development and for the remodeling of primary yolk sac vasculature . Moreover, ectopic expression of SCL during Zebrafish development resulted in excessive formation of hemangioblasts and blood cells at the expense of other mesodermal fates . In the adult, studies of conditional SCL knockout mice demonstrated that SCL is not required for self-renewal or long-term repopulation activity of HSCs but that short-term repopulating capacity of SCL-deleted HSCs is severely impaired . The same studies also revealed that SCL is vital for adult megakaryopoiesis and erythropoiesis, which was consistent with previous reports demonstrating that enforced expression of SCL in purified normal human hematopoietic CD34  cells increased the number of erythroid and megakaryocyte colonies .

We have systematically dissected the mechanisms regulating transcription of the SCL locus. So far, these studies have resulted in the identification of five independent enhancers, each of which targets expression to a specific subdomain of the normal SCL expression pattern . Of particular note, a 3' element (the SCL  18/19 enhancer) was found to direct expression to embryonic endothelium and to most adult and embryonic hematopoietic progenitors and long-term repopulating stem cells . Moreover, this enhancer was able to rescue early hematopoietic progenitors and yolk sac angiogenesis in SCL–/– mouse embryos when it was used to drive expression of an SCL cDNA .! K% r9 P, K, E
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The  18/19 enhancer was originally characterized as a 5,245-bp fragment. This fragment has been used to direct HSC expression of several proteins, including SCL itself , Cre recombinase , tetracycline transactivator , the TVA avian retroviral receptor protein , and oncogenic fusion proteins . Interpretation of these and future studies using this experimental approach will require detailed knowledge of the precise cell types targeted. However, apart from hematopoietic progenitors and stem cells, there has been no detailed analysis of the activity of this fragment in adult mice. A 644-bp core  19 enhancer has been shown to target hematopoietic cells and endothelium at a single time point during embryonic development. However, no phenotypic characterization of the hematopoietic cells was performed, and activity of the core enhancer has not been studied at other developmental stages or in adult mice. In this article, we report a detailed analysis of the activity of the 5,245-bp  18/19 enhancer and the 644-bp  19 core enhancer in transgenic mice.4 ^, \$ d$ w1 ^8 t

MATERIALS AND METHODS) Q7 S+ n! q* D
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5,245-bp SCL  18/19 Enhancer Targets ?-Galactosidase Expression to Adult Endothelium, Megakaryocytes, and Mast Cells
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Activity of the 5,245-bp SCL  18/19 enhancer fragment in adult tissues was assessed in transgenic mice carrying the  6E5/Lac/ 18/19 reporter construct (Fig. 1A). As shown in Figure 1B, histochemical analysis of tissue sections demonstrated ?-galactosidase activity in endothelial cells in multiple tissues, including kidney, heart, liver, lung, spleen, and skin, in three out of six lines analyzed. The widespread endothelial activity of the SCL  18/19 enhancer during embryonic development  is therefore maintained in adult mice.% a+ F3 t) Q4 ^3 q1 R! L' u

Figure 1. The 5,245-bp SCL  18/19 enhancer fragment is active in adult endothelium. (A): Diagram of the murine SCL locus indicating the position of the 5,245-bp BglII fragment containing the SCL  18/19 enhancer. Shown underneath is the transgenic reporter construct, which contained the SCL exon 4 promoter ( 6E5 fragment) and a lacZ reporter gene in addition to the  18/19 3' enhancer fragment. (B): Three transgenic lines carrying the  6E5/lac/ 18/19 construct were analyzed for lacZ expression in adult tissues by histochemical analysis of tissue sections. Blue staining indicates ?-galactosidase activity. No staining was observed in nontransgenic controls. Two lines showed widespread endothelial lacZ expression, whereas no expression was observed in the third line. The results shown are for line 2262 . Kidney, arrowheads indicate ?-galactosidase–positive glomerular endothelial cells. Heart, arrow indicates ?-galactosidase staining in the endocardium. Liver, widespread endothelial ?-galactosidase activity was observed including staining in sublobular veins (sv). Lung, endothelial cells lining vessels and alveolar ducts were positive (a, artery; b, bronchiole; d, alveolar duct). Spleen, a network of endothelial cells was positive (see arrowheads). Skin, endothelial cells stained positive for ?-galactosidase in the dermis (arrow points to lining endothelium of a small vessel). (C): High-power view of section of the spleen showing a megakaryocyte staining positive for ?-galactosidase activity (line 2262 ). (D): Primary bone marrow mast cells derived as described  were analyzed by flow cytometry demonstrating that approximately 50% of the resultant FcRcIgE-positive mast cells expressed the ?-galactosidase transgene (detected using FDG). Abbreviations: FDG, fluorescent ?-galactosidase substrate fluorescein di–?-D-galactopyranoside; SCL, stem cell leukemia.
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Within the hematopoietic system, SCL is expressed in the erythroid, megakaryocyte, and mast cell lineages . It has previously been reported that the SCL  18/19 enhancer does not display significant activity in Ter119  erythroid cells , but activity of the enhancer in megakaryocytes and mast cells was not investigated. Examination of X-gal–stained bone marrow cytospin preparations and histological sections of the spleen from two independent lines revealed that approximately 50% of megakaryocytes expressed the transgene (Fig. 1C and data not shown). To study enhancer activity in mast cells, primary bone marrow mast cells were derived as described . FACS analysis demonstrated that approximately half of the resultant FcRcIgE-positive mast cells expressed the ?-galactosidase transgene (Fig. 1D). These data suggest that the SCL  18/19 enhancer is capable of maintaining transgene expression when stem and progenitor cells differentiate into mature mast cells and megakaryocytes. Because all HSCs express ?-galactosidase in the 6E5/lac/18/19 transgenic lines used here, expression of ?-galactosidase in only half of the differentiated mast cells and megakaryocytes may represent position-dependent variegation.

644-bp  19 Core Enhancer Targets ?-Galactosidase Expression to Endothelium and Blood Throughout Early Embryogenesis but Not in Adult Mice1 W) n3 d5 i' q6 o9 t8 A- {

We have previously used F0 transgenic analysis to identify a 644-bp core  19 enhancer located within the 5,245-bp  18/19 fragment. This core enhancer was sufficient to target expression at E11.5 to endothelial cells as well as to rare round cells in the fetal liver and to clusters of cells attached to the ventral wall of the dorsal aorta . No phenotypic analysis of the presumed hematopoietic cells was performed, and activity of the enhancer was not studied at any other time points. To carry out a detailed analysis of the biological activity of the  19 core enhancer, five lines of transgenic mice were generated in which the  19 core enhancer was linked to a minimal promoter and lacZ reporter cassette (SV/lac/19; Fig. 2A). We had shown previously that the  18/19 enhancer did not have locus control region activity . Transgene activity was dependent on integration sites, and consequently transgene copy number did not correlate with expression levels and some transgenic lines did not express at all . In the current study, two out of five SV/lac/19 lines showed detectable ?-galactosidase expression and a similar pattern of enhancer activity at E11.5. Given our previous observation that the  18/19 enhancer did not have locus control region activity, it was not unexpected that three SV/lac/19 transgenic lines showed no expression.

Figure 2. The  19 core enhancer targets ?-galactosidase expression to endothelium and blood throughout early embryogenesis but not in adult mice. (A): Diagram of the murine SCL locus indicating the position of the 5,245-bp BglII fragment containing the SCL  18/19 enhancer. Shown underneath are the  6E5/lac/ 18/19 and SV/lac/19 transgenic reporter constructs indicating the position of the 644-bp  19 core enhancer within the larger 5,245-bp  18/19 enhancer fragment. (B): Analysis of SV/lac/19 embryos (line 588) by whole-mount staining for ?-galactosidase activity. E7.5, staining in the extraembryonic region destined to form the yolk sac blood islands; E8.5, prominent staining in the developing vasculature; E9.5, endothelial staining in developing intersomitic vessels; E12.5, widespread endothelial staining. Blue staining indicates ?-galactosidase activity. No staining was observed in nontransgenic controls. No ?-galactosidase activity was detectable in adult tissues (data not shown). (C, D): Flow cytometric analysis of fetal liver and adult bone marrow demonstrated that approximately 4% of E11.5 fetal liver cells expressed the transgene in line 588, most of which also expressed the hematopoietic progenitor marker c-kit. However, no transgene expression was observed in adult bone marrow (compare plots for nontransgenic,  6E5/lac/ 18/19 , and SV/lac/19 ). Abbreviations: FDG, fluorescent ?-galactosidase substrate fluorescein di–?-D-galactopyranoside; PI, propidium iodide; SCL, stem cell leukemia.

Enhancer activity was then studied in the two expressing transgenic lines at multiple time points during embryonic development (Fig. 2B). At E7.5, lacZ staining was observed in the extra-embryonic region destined to form the yolk sac blood islands. From E8.5, prominent staining was observed in the developing vasculature, with prominent staining in intersomitic vessels at E9.5. Endothelial staining persisted at E12.5 but progressively diminished at later developmental stages and was absent in adult mice (data not shown). Loss of activity of the  19 core enhancer in the adult was also observed in the hematopoietic system. As shown in Figure 2C, FACS analysis of E11.5 fetal liver demonstrated that approximately 4% of cells expressed the transgene, most of which also expressed c-kit, a marker of hematopoietic progenitors and stem cells. However, similar analysis of adult bone marrow (Fig. 2D) revealed no detectable activity of the  19 core enhancer in any of the SV/lac/19 transgenic lines, in contrast to transgenic lines carrying lacZ driven by the 5,245-bp SCL  18/19 enhancer. These data therefore demonstrate that activity of the 644-bp  19 core enhancer during development is similar to that of the 5,245-bp  18/19 enhancer. However, the  19 core enhancer was not sufficient to produce ?-galactosidase expression in adult mice.
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SCL  19 Core Enhancer Targets PLAP Expression to Endothelium and Hematopoietic Cells During Embryogenesis and in Adult Mice

The 6E5/lac/18/19 and SV/lac/19 constructs contain the SCL 6E5 and simian virus SV40 minimal promoters, respectively. The absence of ?-galactosidase expression in adult SV/lac/19 transgenic mice might therefore have been a consequence of using these two different promoters. However, we have recently shown that the SV40 minimal promoter in connection with the SCL 18/19 enhancer can reproducibly drive expression of the tetracycline transactivator gene in adult bone marrow . Failure of expression in adult SV/lac/19 transgenic mice would also be consistent with the suggestion that the  19 core enhancer may require additional sequences outside the core enhancer to maintain expression throughout ontogeny. Alternatively, the ?-galactosidase reporter gene may not faithfully report enhancer activity in adult mice, a scenario that has been suggested previously . To distinguish these possibilities, ?-galactosidase was replaced by the mammalian reporter gene human PLAP. Previous studies have demonstrated that PLAP functions as a robust reporter in transgenic mice and can be detected in tissue sections as well as by FACS .2 A0 @% K  G( g1 X. h) F' X
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Unlike the SV/lac/19 construct, the SV/PLAP/19 construct reproducibly directed expression in adult tissues, with five out of six transgenic lines analyzed showing strong expression in adult hematopoietic and endothelial cells. Embryonic expression in hematopoietic and endothelial cells was present in three out of three lines tested. At E11.5, transgene expression was observed in endothelial cells, including those in the yolk sac and fetal liver, as well as in clusters of round cells attached to the ventral wall of the dorsal aorta (Fig. 3B). FACS analysis demonstrated that fetal liver cells expressing the PLAP transgene also expressed the progenitor marker c-kit (Fig. 3C). In the bone marrow, approximately half of the transgene-positive cells were c-kit positive (Fig. 3C). Expression in adult endothelium was observed in all tissues examined, including the intestine, lung, and pancreas (Fig. 3D). Taken together, these results suggested that the  19 core enhancer contains all sequences necessary for cell type–specific activity in postnatal mice but only in the context of some reporter genes.

Figure 3. The SCL  19 core enhancer targets PLAP expression to endothelium and hematopoietic cells during embryogenesis and in adult mice. (A): Diagram of the murine SCL locus indicating the position of the 5,245-bp BglII fragment containing the SCL  18/19 enhancer. Shown underneath are the  6E5/lac/ 18/19, SV/lac/19, and SV/PLAP/19 transgenic reporter constructs. (B): Histochemical detection of PLAP activity (purple stain) in sections of SV/PLAP/19 transgenic E11.5 mouse embryos showed expression in endothelium (see section of yolk sac), endothelial cells, and round (haematopoietic) cells in the fetal liver and clusters of round cells attached to the ventral wall of the dorsal aorta. Similar data were obtained in three lines. The data shown are for line 1772. (C): The SV/PLAP/19 reporter construct is active in fetal liver and adult bone marrow hematopoietic cells. Flow cytometric analysis demonstrated that fetal liver cells expressing PLAP also expressed the progenitor marker c-kit. In bone marrow, less than half of the PLAP  cells also expressed c-kit (data shown are for line 1791). (D): The SV/PLAP/19 reporter construct is active in adult endothelium. Shown are histological sections demonstrating endothelial PLAP activity (blue staining) in the intestine, lung, and pancreas. Similar results were obtained for five out of six lines analyzed. The data shown are for line 1772. Abbreviations: PLAP, placental alkaline phosphatase; SCL, stem cell leukemia.. ~% |* i+ Z; @' q( D3 T
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SCL  19 Core Enhancer Targets Long-Term Repopulating HSCs3 g. Y, E# P( g9 _3 b2 t3 n
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The ability to target long-term repopulating HSCs is a key feature of the 5,245-bp SCL  18/19 enhancer fragment . To assess whether the  19 core enhancer was also sufficient to drive expression in HSCs, PLAP  and PLAP– cells from SV/PLAP/19 transgenic adult bone marrow or E12.5 fetal liver were used in long-term repopulation studies. PCR for the donor PLAP transgene was used to assess engraftment and confirm multilineage contribution 4 to 6 months after transplantation.( J. B' g/ `# c& v$ @" ~+ _2 Z1 I

As shown in Table 1, 4 out of 13 animals transplanted with 103 to 104 PLAP  fetal liver cells showed long-term hematopoietic engraftment. Similarly, 4 out of 14 animals transplanted with 103 to 5 x 103 bone marrow cells showed long-term hematopoietic contribution of PLAP  donor cells. Subsequent multilineage reconstitution analysis in three positive animals from each group demonstrated contribution of PLAP  cells to all hematopoietic lineages analyzed in all recipients tested (Fig. 4 and data not shown). Long-term engraftment was occasionally observed in recipients of 105 PLAP– fetal liver or bone marrow cells, indicating that a minority of HSCs were not targeted in SV/PLAP/19 lines. Taken together, these results suggest that the  19 core enhancer is active in most long-term repopulating HSCs in fetal liver and bone marrow." d* y6 i" }( C3 H

Table 1. The  19 core enhancer targets long-term repopulating hematopoietic stem cells in SV/PLAP/19 transgenic mice

Figure 4. The  19 core enhancer targets long-term repopulating hematopoietic stem cells in SV/PLAP/19 transgenic mice. Multilineage reconstitution analysis demonstrated that PLAP-positive hematopoietic stem cells contribute to all lineages. Hematopoietic cell populations were isolated by fluorescence-activated cell sorting where necessary, and genomic DNA was analyzed by polymerase chain reaction for presence of the PLAP transgene (Bl, peripheral blood; BM, bone marrow; Th, thymus; S, spleen; LN, lineage-negative cell; T, T cells; B, B cells; E, erythroid cells; M, monocytes/macrophages; –, nontransgenic control; w, water control;  , transgenic control). Three animals transplanted with fetal liver and three animals transplanted with bone marrow PLAP  cells were analyzed for multilineage contribution. All six animals showed multilineage contribution (representative data shown). Data shown are for line 1791.

SCL  19 Core Enhancer Targets Mature Megakaryocytes and Bone Marrow Mast Cells but Not Definitive Erythroid Cells
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As shown above, the 5,245-bp fragment of the SCL 18/19 enhancer is active in mast cells and megakaryocytes. Examination of bone marrow sections from SV/PLAP/19 transgenic mice showed that in two out of three lines analyzed, the  19 core enhancer was also strongly active in a large proportion of megakaryocytes (Fig. 5A). Staining in the third line was weaker yet clearly positive compared with nontransgenic controls. In addition, the  19 core enhancer was active in 95% of bone marrow mast cells, derived by culturing in the presence cytokines, as previously described . The phenotype of the resultant cells was confirmed by expression of c-kit and positive staining with toluidine blue (Fig. 5B). These observations suggested that the  19 core enhancer is capable of maintaining transgene expression when immature progenitors differentiate into mature mast cells and megakaryocytes.

Figure 5. Survey of hematopoietic PLAP expression in SV/PLAP/19 transgenic mice. (A): The  19 core enhancer drives expression in bone marrow megakaryocytes in SV/PLAP/19 transgenic mice. Shown are low- and high-magnification views of bone marrow sections stained for PLAP activity (purple). (B): The  19 core enhancer is active in mast cells in SV/PLAP/19 transgenic mice. Bone marrow mast cells were derived as previously described , and their phenotype was confirmed by expression of c-kit and positive staining with toluidine blue. Flow cytometric analysis demonstrated that 95% of mast cells expressed the PLAP transgene. (C): Immature erythroid cells express PLAP in SV/PLAP/19 transgenic mice. Five stages (S1–S5) of increasing erythroid maturation were distinguished by flow cytometric analysis using the CD71 and Ter119 markers. S1 and S2 correspond to the CFU-E/proerythoblast stages; S3 and S4, respectively, to early and late basophilic erythroblasts; and S5 to orthochromatophilic erythroblasts. Shown is the distribution of PLAP– (blue) compared with PLAP  (red) cells within these five stages in E15.5 fetal liver from SV/PLAP/19 transgenic embryos. (D): The percentage of PLAP  cells sharply decreases with increasing erythroid maturation. The graph shows the proportion of PLAP  (red) and PLAP– (blue) cells for all stages (S1–S5) of erythroid differentiation analyzed. (E): Expression of endogenous SCL is not downregulated during erythroid differentiation. SCL transcripts were quantified using real-time reverse transcription–polymerase chain reaction in fetal liver cells sorted from the regions S1–S5. SCL expression levels were normalized against three control RNAs (GAPDH, ?-actin, and 18S rRNA). (F): Splenic Mac-1  myeloid and B220  B-lymphoid cells express PLAP in SV/PLAP/19 transgenic mice. Splenocytes were analyzed by flow cytometry using the Mac1 and B220 markers in combination with an antibody against PLAP. (G): Thymic CD4- and CD8-positive T lymphocytes express PLAP in SV/PLAP/19 transgenic mice. Thymocytes were analyzed by flow cytometry using the CD4 and CD8 markers in combination with an antibody against PLAP. Data shown are for line 1791. Abbreviations: GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PLAP, placental alkaline phosphatase; SCL, stem cell leukemia.. Z1 }! d- O* T0 C+ }

Previous studies showed that the 5,245-bp SCL  18/19 enhancer directed ?-galactosidase expression to only a small minority of Ter119  erythroid cells . By contrast, analysis of E15.5 fetal liver and adult bone marrow showed PLAP activity in up to 49% of Ter119  erythroid cells (data not shown). FACS analysis using the CD71 and Ter119 markers to distinguish five stages (S1 to S5; Fig. 5C) of increasing erythroblast maturation demonstrated that the proportion of transgene-positive cells at each stage decreased from 96% (S1) to 3% (S5) (Figs. 5C, 5D). Moreover, the level of PLAP expression progressively decreased with increasing erythroid differentiation. Mean PLAP expression levels of PLAP  cells declined approximately 10-fold between regions S1 and S3 (data not shown). Analogous results were obtained from analysis of spleen and bone marrow cells (data not shown). These results indicate that activity of the  19 core enhancer rapidly diminished during early erythroblast maturation. The different patterns of reporter gene expression observed in the 6E5/lacZ/ 18/19 mice (few transgene-positive erythroid cells ) and the SV/PLAP/19 mice (significant levels of transgene expression but only in the most immature erythroid cells) is likely to reflect differences in mRNA and/or protein stability between the two different reporter genes. However, our data do not exclude the possibility that sequences outside the  19 core enhancer (but within the 6E5/lacZ/ 18/19 cassette) are able to repress enhancer activity in erythroid cells.& W6 ]/ ]% e: _8 t/ k6 O6 S+ g" s$ U
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SCL is generally thought to be upregulated during erythroid maturation , yet one study also reported potential down-regulation . To compare endogenous SCL transcription with activity of the  19 core enhancer, SCL transcripts were quantified using real-time RT-PCR in fetal liver cells sorted from the regions S1 to S5, thus allowing us to assess SCL expression levels in prospectively isolated primary cells of increasing erythroid maturation. Expression levels were normalized against three control RNAs, namely GAPDH, ?-actin, and 18S rRNA (Fig. 5E). This analysis demonstrated that SCL was expressed at high levels throughout erythroid maturation (cycle threshold numbers in similar range as GAPDH). Moreover, when normalized against GAPDH and 18S rRNA, SCL expression levels increased during erythroid maturation, whereas they remained constant when normalized against ?-actin (Fig. 5E). Importantly, regardless of the normalization control used and in contrast to the SV/PLAP/19 reporter gene, levels of endogenous SCL expression did not decrease during erythroid differentiation. This marked discrepancy is unlikely to be due to the presence of long-lived SCL mRNA species because we have shown previously that increased stability cannot account for increased SCL mRNA levels during erythroid differentiation of mouse erythroleukemia cells . Taken together, our results therefore indicate that the  19 core enhancer is insufficient to maintain SCL expression during definitive erythroid differentiation and suggest that an additional regulatory element is required to express SCL during the later stages of erythropoiesis.+ r3 k' G" v5 _! c$ ]2 q' O- [
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Activity of the  19 Core Enhancer in Other Hematopoietic Lineages+ c. s! K: w& _9 u

FACS analysis demonstrated PLAP expression in spleen and bone marrow of five out of six SV/PLAP/19 transgenic lines analyzed. In the highly expressing line 1791, PLAP  cells included approximately half of Mac1  myeloid and B220  B-lymphoid cells (Fig. 5E). FACS analysis of adult thymus showed that a significant proportion of CD4  and CD8  thymocytes expressed the PLAP transgene in two out of four lines analyzed (Fig. 5G and data not shown). Reporter gene expression in lymphoid cells had previously been seen in some transgenic lines carrying the larger 5,245-bp  18/19 enhancer transgene . Endogenous SCL is not expressed in most thymocytes, B cells, or Mac  bone marrow cells. Our observation that the  19 core enhancer and the 5,245-bp  18/19 enhancer are active in these cell types suggests the existence of a silencer present elsewhere in the endogenous SCL locus. Failure to downregulate SCL is associated with T-ALL, and characterizing the mechanisms responsible for SCL repression therefore represents an important future goal.0 o! t  b6 A6 \2 [# x/ B6 g
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DISCUSSION
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The SCL  18/19 stem cell enhancer is one of the most popular tools to engineer transgene expression in HSCs in vivo. Therefore, comprehensive analysis of the in vivo activity of the SCL 3' enhancer within and outside the hematopoietic system not only provides further insight into transcriptional regulation of SCL but will also be vital to interpret current and future mouse models generated using this powerful regulatory element.+ O: i  W: S" K

ACKNOWLEDGMENTS

Orkin SH. Diversification of haematopoietic stem cells to specific lineages. Nat Rev Genet 2000;1:57–64.7 y3 K" M* S9 P/ @* h6 F5 i# A

Tenen DG. Disruption of differentiation in human cancer: AML shows the way. Nat Rev Cancer 2003;3:89–101.
% K' I/ O# \- [
Begley CG, Green AR. The SCL gene: from case report to critical hematopoietic regulator. Blood 1999;93:2760–2770.

Shivdasani RA, Mayer EL, Orkin SH. Absence of blood formation in mice lacking the T-cell leukaemia oncoprotein tal-1/SCL. Nature 1995;373:432–434.! A6 H) J2 o. [+ R, c6 w

Robb L, Lyons I, Li R et al. Absence of yolk sac hematopoiesis from mice with a targeted disruption of the scl gene. Proc Natl Acad Sci U S A 1995;92:7075–7079.- ^8 O% i2 L- o5 Q* N' T
) g. C( f' h7 w0 s" t+ L: x
Porcher C, Swat W, Rockwell K et al. The T cell leukemia oncoprotein SCL/tal-1 is essential for development of all hematopoietic lineages. Cell 1996;86:47–57.

Robb L, Elwood NJ, Elefanty AG et al. The scl gene product is required for the generation of all hematopoietic lineages in the adult mouse. EMBO J 1996;15:4123–4129.
- f) v2 Q: v5 t9 M4 Y3 ?8 V. ^, R3 F
Visvader JE, Fujiwara Y, Orkin SH. Unsuspected role for the T-cell leukemia protein SCL/tal-1 in vascular development. Genes Dev 1998;12:473–479.; b( \$ ]1 j- U' w+ q* v
: ?& d. z4 m& W& L! k9 ?$ J/ B
Gering M, Rodaway AR, G?ttgens B et al. The SCL gene specifies haemangioblast development from early mesoderm. EMBO J 1998;17:4029–4045.

Mikkola HK, Klintman J, Yang H et al. Haematopoietic stem cells retain long-term repopulating activity and multipotency in the absence of stem-cell leukaemia SCL/tal-1 gene. Nature 2003;421:547–551.

Curtis DJ, Hall MA, Van Stekelenburg LJ et al. SCL is required for normal function of short-term repopulating hematopoietic stem cells. Blood 2004;103:3342–3348.4 `, f$ M+ ?% ^  T* w% Z- D

Hall MA, Curtis DJ, Metcalf D et al. The critical regulator of embryonic hematopoiesis, SCL, is vital in the adult for megakaryopoiesis, erythropoiesis, and lineage choice in CFU-S12. Proc Natl Acad Sci U S A 2003;100:992–997." @" {. v  K  z% Y& e
$ F- @( k. ^: x! n& C3 c
Elwood NJ, Zogos H, Pereira DS et al. Enhanced megakaryocyte and erythroid development from normal human CD34( ) cells: consequence of enforced expression of SCL. Blood 1998;91:3756–3765.$ y  |4 E3 o: U) F) k& y9 r
3 [. c: d7 U! n; T2 v
G?ttgens B, Barton LM, Gilbert JG et al. Analysis of vertebrate SCL loci identifies conserved enhancers. Nat Biotechnol 2000;18:181–186.5 d6 W0 T" O, K) c: |- x! Y  l

G?ttgens B, McLaughlin F, Bockamp EO et al. Transcription of the SCL gene in erythroid and CD34 positive primitive myeloid cells is controlled by a complex network of lineage-restricted chromatin-dependent and chromatin-independent regulatory elements. Oncogene 1997;15:2419–2428.

G?ttgens B, Nastos A, Kinston S et al. Establishing the transcriptional programme for blood: the SCL stem cell enhancer is regulated by a multi protein complex containing Ets and GATA factors. EMBO J 2002;21:3039–3050.% N3 d+ C( G8 X

Sinclair AM, G?ttgens B, Barton LM et al. Distinct 5μ SCL enhancers direct transcription to developing brain, spinal cord, and endothelium: neural expression is mediated by GATA factor binding sites. Dev Biol 1999;209:128–142.
/ I+ v$ N1 F7 X
S芍nchez M, G?ttgens B, Sinclair AM et al. An SCL 3' enhancer targets developing endothelium together with embryonic and adult haematopoietic progenitors. Development 1999;126:3891–3904.
: p8 |, y( o" `* k' \% U* W
S芍nchez MJ, Bockamp EO, Miller J et al. Selective rescue of early haematopoietic progenitors in Scl(–/–) mice by expressing Scl under the control of a stem cell enhancer. Development 2001;128:4815–4827.
: Q3 W3 C! q; k" D# b) q) n2 J+ x
Gothert JR, Gustin SE, Hall MA et al. In vivo fate-tracing studies using the SCL stem cell enhancer: embryonic hematopoietic stem cells significantly contribute to adult hematopoiesis. Blood 2004;105:2724–2732.
8 {, a" m8 H. M1 C9 K, m# O1 g
Koschmieder S, G?ttgens B, Zhang P et al. Inducible chronic phase of myeloid leukemia with expansion of hematopoietic stem cells in a transgenic model of BCR-ABL leukemogenesis. Blood 2005;105:324–334.1 \& }! b3 \$ X8 M; d

Murphy GJ, G?ttgens B, Vegiopoulos A et al. Manipulation of mouse hematopoietic progenitors by specific retroviral infection. J Biol Chem 2003;278:43556–43563." \+ E" k: E  x2 K( P3 X( m! Z  q
  ^8 g3 e# V( V4 O: `: w
Eguchi M, Eguchi-Ishimae M, Green A et al. Directing oncogenic fusion genes into stem cells via an SCL enhancer. Proc Natl Acad Sci U S A 2005;102:1133–1138.
5 j& X, K/ e2 m" N7 q
Melcher R, Grosch HW, Hasilik A. Plasmid vectors with a 5μ-hybrid intron facilitate high-level glycoprotein expression in CHO-cells. Biochim Bio-phys Acta 2002;1575:49–53.. z& v$ ~0 F+ ]! {& O( b$ k
, J3 M( G3 R' x
Buchman AR, Berg P. Comparison of intron-dependent and intron-independent gene expression. Mol Cell Biol 1988;8:4395–4405.

Palmiter RD, Sandgren EP, Avarbock MR et al. Heterologous introns can enhance expression of transgenes in mice. Proc Natl Acad Sci U S A 1991;88:478–482.
& Y0 _. z3 V, I! U' I
Brinster RL, Allen JM, Behringer RR et al. Introns increase transcriptional efficiency in transgenic mice. Proc Natl Acad Sci U S A 1988;85:836–840.

Choi T, Huang M, Gorman C et al. A generic intron increases gene expression in transgenic mice. Mol Cell Biol 1991;11:3070–3074.
- i: n5 G2 _& d& z
Costello PS, Turner M, Walters AE et al. Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells. Oncogene 1996;13:2595–2605.: J& T( u, l; `$ s

Miles C, S芍nchez MJ, Sinclair A et al. Expression of the Ly-6E.1 (Sca-1) transgene in adult hematopoietic stem cells and the developing mouse embryo. Development 1997;124:537–547.
- H3 C8 [  c0 c: c
Socolovsky M, Nam H, Fleming MD et al. Ineffective erythropoiesis in Stat5a(–/–)5b(–/–) mice due to decreased survival of early erythroblasts. Blood 2001;98:3261–3273.

DePrimo SE, Stambrook PJ, Stringer JR. Human placental alkaline phosphatase as a histochemical marker of gene expression in transgenic mice. Transgenic Res 1996;5:459–466.5 F* M* d1 {, ~/ s. y" U; J8 n+ K

Bockamp EO, Fordham JL, G?ttgens B et al. Transcriptional regulation of the stem cell leukemia gene by PU.1 and Elf-1. J Biol Chem 1998;273:29032–29042.7 w' I! W. P! x5 t# E: Z

Cohen-Tannoudji M, Morello D, Babinet C. Unexpected position-dependent expression of H-2 and beta 2-microglobulin/lacZ transgenes. Mol Reprod Dev 1992;33:149–159.

Cohen-Tannoudji M, Vandormael-Pournin S, Drezen J et al. lacZ sequences prevent regulated expression of housekeeping genes. Mech Dev 2000;90:29–39.

Mehtali M, LeMeur M, Lathe R. The methylation-free status of a housekeeping transgene is lost at high copy number. Gene 1990;91:179–184.
( T+ D, i( G! S
Ogilvy S, Elefanty AG, Visvader J et al. Transcriptional regulation of vav, a gene expressed throughout the hematopoietic compartment. Blood 1998;91:419–430.5 D3 g, |4 P& t7 X7 H8 |- {
, M. O; v+ H' T& \; R! A  y+ x9 e6 @
Garrick D, Sutherland H, Robertson G et al. Variegated expression of a globin transgene correlates with chromatin accessibility but not methylation status. Nucleic Acids Res 1996;24:4902–4909.
& e8 y  K  d0 ^
Guy LG, Kothary R, Wall L. Position effects in mice carrying a lacZ trans-gene in cis with the beta-globin LCR can be explained by a graded model. Nucleic Acids Res 1997;25:4400–4407.

Knoll BJ, Rothblum KN, Longley M. Two gene duplication events in the evolution of the human heat-stable alkaline phosphatases. Gene 1987;60:267–276.# H# w* v! z6 X4 h) p! W) c

Knoll BJ, Rothblum KN, Longley M. Nucleotide sequence of the human placental alkaline phosphatase gene: evolution of the 5' flanking region by deletion/substitution. J Biol Chem 1988;263:12020–12027.

Henthorn PS, Knoll BJ, Raducha M et al. Products of two common alleles at the locus for human placental alkaline phosphatase differ by seven amino acids. Proc Natl Acad Sci U S A 1986;83:5597–5601., D/ d. T( x7 _1 T) n) |
4 W* r& B5 e7 [( k
DePrimo SE, Cao J, Hersh MN et al. Use of human placental alkaline phosphatase transgenes to detect somatic mutation in mice in situ. Methods 1998;16:49–61.9 k& M" v1 w0 M
7 f4 T+ v- B: h& b. T, z/ p  l
Carvajal JJ, Cox D, Summerbell D et al. A BAC transgenic analysis of the Mrf4/Myf5 locus reveals interdigitated elements that control activation and maintenance of gene expression during muscle development. Development 2001;128:1857–1868.4 K- u9 g6 L( y+ i4 Q
/ n2 d& C* u% ^4 m3 l/ C
Chen B, Gandhi R, Baltimore D. CD4 down-modulation during infection of human T cells with human immunodeficiency virus type 1 involves independent activities of vpu, env, and nef. J Virol 1996;70:6044–6053.
+ P1 k6 [  h) q5 \: i7 [7 F
Gandhi RT, Chen BK, Straus SE et al. HIV-1 directly kills CD4  T cells by a Fas-independent mechanism. J Exp Med 1998;187:1113–1122." Z/ M) D; t8 w' Y; E$ j
3 c$ u4 _. O: \
Jacob J, Baltimore D. Modelling T-cell memory by genetic marking of memory T cells in vivo. Nature 1999;399:593–597.6 r5 q3 G0 ?* K# Q" }7 z
6 z- d1 q# P5 w7 n7 k0 V
Cepko C, Ryder EF, Austin CP et al. Lineage analysis using retrovirus vectors. Methods Enzymol 1995;254:387–419.0 p7 a  c  _2 ?2 E- X1 K

Lobe CG, Koop KE, Kreppner W et al. Z/AP, a double reporter for cre-mediated recombination. Dev Biol 1999;208:281–292.# q% S( U1 \/ n% o! ?' q- m

Ziegler S, Burki K, Skoda RC. A 2-kb c-mpl promoter fragment is sufficient to direct expression to the megakaryocytic lineage and sites of embryonic hematopoiesis in transgenic mice. Blood 2002;100:1072–1074.
- {3 T# m- z) L. T5 O) f$ {
Shelburne CP, McCoy ME, Piekorz R et al. Stat5 expression is critical for mast cell development and survival. Blood 2003;102:1290–1297.
; ^4 k5 T3 [% N) b" h9 c7 `
Aplan PD, Nakahara K, Orkin SH et al. The SCL gene product: a positive regulator of erythroid differentiation. EMBO J 1992;11:4073–4081.' N) M1 G5 T- q5 P) B- o3 _
3 }0 A7 k% Q- A# u
Visvader J, Begley CG, Adams JM. Differential expression of the LYL, SCL and E2A helix-loop-helix genes within the hemopoietic system. Oncogene 1991;6:187–194., y$ ?( T4 U, b: c, l4 B
; c2 e  @, b# I7 m
Mouthon MA, Bernard O, Mitjavila MT et al. Expression of tal-1 and GATA-binding proteins during human hematopoiesis. Blood 1993;81:647–655.
) [8 F; h$ @) `4 X$ V5 ^3 C
Murrell AM, Bockamp EO, G?ttgens B et al. Discordant regulation of SCL/TAL-1 mRNA and protein during erythroid differentiation. Oncogene 1995;11:131–139.; q0 ?# k3 y& H& U
+ X" @, N; e0 K  N" A) X% n% F
Ma X, Robin C, Ottersbach K et al. The Ly-6A (Sca-1) GFP transgene is expressed in all adult mouse hematopoietic stem cells. STEM CELLS 2002;20:514–521.
  Y+ H- W# X1 A4 }( }& D5 a0 S1 c
Spangrude GJ, Brooks DM. Mouse strain variability in the expression of the hematopoietic stem cell antigen Ly-6A/E by bone marrow cells. Blood 1993;82:3327–3332.5 q9 u2 l  S( F; J- I, m& H8 v
$ P( Y8 x& c2 s' a* \
Okuno Y, Huettner CS, Radomska HS et al. Distal elements are critical for human CD34 expression in vivo. Blood 2002;100:4420–4426.& Y9 R* w, M& O7 C) P1 l' r4 \
, m6 G, F; f8 V5 @
Chen CZ, Li M, de Graaf D et al. Identification of endoglin as a functional marker that defines long-term repopulating hematopoietic stem cells. Proc Natl Acad Sci U S A 2002;99:15468–15473.: C* m, I% q0 A7 n

Ferrando AA, Herblot S, Palomero T et al. Biallelic transcriptional activation of oncogenic transcription factors in T-cell acute lymphoblastic leukemia. Blood 2004;103:1909–1911.(Lev Silbersteina, Mar赤a-J)

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