Transcriptional regulation of the SCL locus: identification of an enhancer that targets the primitive erythroid lineage in vivo

Abstract

The stem cell leukemia (SCL) gene, also known as TAL-1, encodes a basic helix-loop-helix protein that is essential for the formation of all hematopoietic lineages, including primitive erythropoiesis. Appropriate transcriptional regulation is essential for the biological functions of SCL, and we have previously identified five distinct enhancers which target different subdomains of the normal SCL expression pattern. However, it is not known whether these SCL enhancers also regulate neighboring genes within the SCL locus, and the erythroid expression of SCL remains unexplained. Here, we have quantitated transcripts from SCL and neighboring genes in multiple hematopoietic cell types. Our results show striking coexpression of SCL and its immediate downstream neighbor, MAP17, suggesting that they share regulatory elements. A systematic survey of histone H3 and H4 acetylation throughout the SCL locus in different hematopoietic cell types identified several peaks of histone acetylation between SIL and MAP17, all of which corresponded to previously characterized SCL enhancers or to the MAP17 promoter. Downstream of MAP17 (and 40 kb downstream of SCL exon 1a), an additional peak of acetylation was identified in hematopoietic cells and was found to correlate with expression of SCL but not other neighboring genes. This +40 region is conserved in human-dog-mouse-rat sequence comparisons, functions as an erythroid cell-restricted enhancer in vitro, and directs beta-galactosidase expression to primitive, but not definitive, erythroblasts in transgenic mice. The SCL +40 enhancer provides a powerful tool for studying the molecular and cellular biology of the primitive erythroid lineage.

FIG. 1.

Coexpression of SCL and MAP17 in hematopoietic cell lines. (A) Diagram of the human SCL locus. (B) Relative levels of expression of the human SCL and neighboring genes as assessed by quantitative RT-PCR. Expression of β2M in the EM2 cell line is defined as 100% (black oval). Other transcript levels were normalized and are shown as various shades of grey, with white ovals representing 0%. Note the striking correlation between expression of SCL and MAP17. ND, not done. (C) Diagram of the murine SCL locus. (D) Relative levels of expression of murine SCL and neighboring genes. Expression of β2M in F4N is defined as 100% (black oval). Other transcript levels were normalized and are shown as various shades of grey, with white ovals representing 0%.

FIG. 2.

Chromatin immunoprecipitation of acetylated histone H3 throughout the murine SCL locus. A diagram of the murine SCL locus is shown beneath each set of four panels. Quantitative PCR was performed using primers located approximately every 1 kb throughout a 90-kb region. The increase over background was calculated by comparison with immunoprecipitates obtained using rabbit IgG. Peaks of H3 acetylation are labeled according to their positions relative to the first nucleotide of SCL exon la. (A) Adult mouse thymus; (B) BW5147 T-cell line; (C) embryonic day 14.5 fetal liver; (D) F4N erythroid cell line; (E) 416B myeloid progenitor cell line; (F) adult spleen mast cells; (G) RM26 myeloid cell line; (H) MDCT kidney cell line.

FIG. 3.

Chromatin immunoprecipitation of acetylated histone H3 throughout the human SCL locus. A diagram of the human SCL locus is shown at the bottom. Quantitative PCR was performed using primers located approximately every 1 kb throughout an 80-kb region. The increase was calculated by comparison with immunoprecipitates obtained using rabbit IgG. Peaks of H3 acetylation are labeled according to their positions relative to SCL exon 1a. (A) HPB-ALL T-cell line; (B) K562 myeloid-erythroid progenitor cell line; (C) HEL myeloid-erythroid cell line.

FIG. 4.

The +40 region is conserved and functions as an erythroid enhancer in vitro. (A) Schematic representation of a four-way sequence alignment of the MAP17 gene downstream region in human (Hs), dog (Cf), mouse (Mm), and rat (Rn) showing peaks of conserved sequence. Red boxes, coding exons; beige boxes, untranslated exons; blue boxes, repeat sequences. The 3.7-kb fragment incorporated into luciferase reporter constructs is shown at the bottom. (B) Stable transfection of hematopoietic cell lines with luciferase reporter constructs. Cell lines representing erythroid (F4N and J2E), myeloid progenitor (416B), or T-lymphoid (BW5147) cell types were transfected with reporter constructs containing various promoters (−0.9E3 SCL promoter, SV40 minimal promoter, or thymidine kinase promoter) with or without the +40 region. The histograms represent the mean (and standard deviation) luciferase activities (in relative light units [RLU]) of four independent pools normalized to the mean result obtained with a construct containing the corresponding promoter alone. The results shown were obtained in a single representative experiment. The same pattern of results was observed in at least three independent experiments for each cell line.

FIG. 5.

The +40 region targets expression to primitive erythroblasts in vivo. (A) Constructs used to generate transgenic mice. (B) Whole-mount (i to iii) and histological (iv to vi) sections of SV intron/lac/+40 transgenic mice (line 4092). (i) Lateral view of an E12.5 embryo. Note β-galactosidase expression in blood vessels and in the midbrain (arrow). (ii) High-power view of umbilical vessels from an E12.5 embryo. (iii) Yolk sacs from E13.5 nontransgenic (non-Tg) and transgenic (Tg) embryos. (iv) Sagittal section through E12.5 yolk sac blood island. (v) E12.5 umbilical vessel. (vi) E12.5 fetal liver. (C) Flow cytometric analysis (FACS) of β-galactosidase activity in circulating erythroid cells of SV intron/lac/+40 transgenic embryos (line 4092). Data from transgenic animals (solid lines) are shown superimposed on those from nontransgenic littermates (dashed lines). Similar results were obtained in at least three mice of each genotype for each time point. Similar data were also obtained at E12.5 and E14.5 in two additional independent transgenic lines (lines 4102 and 4274). (D) FACS analysis of β-galactosidase activity and Ter119 erythroid lineage marker expression in E14.5 fetal liver and adult bone marrow from SV intron/lac/+40 transgenic (Tg) animals (line 4092) and nontransgenic (non-Tg) littermates. The fetal liver results shown are representative of at least three embryos of each genotype, whereas the bone marrow data represent experiments in which at least four mice of each genotype (lines 4092, 4102, and 4274) were analyzed.