RUNX1 regulates the CD34 gene in haematopoietic stem cells by mediating interactions with a distal regulatory element

Abstract

The transcription factor RUNX1 is essential to establish the haematopoietic gene expression programme; however, the mechanism of how it activates transcription of haematopoietic stem cell (HSC) genes is still elusive. Here, we obtained novel insights into RUNX1 function by studying regulation of the human CD34 gene, which is expressed in HSCs. Using transgenic mice carrying human CD34 PAC constructs, we identified a novel downstream regulatory element (DRE), which is bound by RUNX1 and is necessary for human CD34 expression in long-term (LT)-HSCs. Conditional deletion of Runx1 in mice harbouring human CD34 promoter-DRE constructs abrogates human CD34 expression. We demonstrate by chromosome conformation capture assays in LT-HSCs that the DRE physically interacts with the human CD34 promoter. Targeted mutagenesis of RUNX binding sites leads to perturbation of this interaction and decreased human CD34 expression in LT-HSCs. Overall, our in vivo data provide novel evidence about the role of RUNX1 in mediating interactions between distal and proximal elements of the HSC gene CD34.

Figure 1

A genomic region located between +17.4 and +19.6 kb of the human CD34 gene is necessary for its expression in SLAM⁺ LSKs. (A) Diagram of human CD34 genomic fragments used in transgenic mice. All fragments (A–C) were derived from PAC54A19 and digested as indicated. Thick vertical lines represent the eight hCD34 coding exons. (B) Flow cytometry was performed on bone marrow cells from transgenic mice carrying constructs A–C. Cells were stained with anti-mouse c-kit and sca-1 antibodies to identify the LSK (lineage⁻; Sca1⁺; and c-kit⁺ population), as indicated in the left panels; anti-murine CD48 and CD150/SLAM antibodies were used to identify the CD150 single positive population (SLAM⁺ cells) contained within the LSK compartment, as indicated in the middle panels; and anti-hCD34 antibody was used to quantify the amount of hCD34⁺ cells contained within the SLAM⁺ population (right panel). The percentage of the gated populations is noted. All plots display a representative example from each of the three different transgenic lines. T-test (two-tailed; type 3) showed that the data, obtained in three independent transgenic lines from construct A (n=3 per founder, a part from one founder in which we did not observe germ line transgene transmission), were not statistically different (P=0.55) from the percentages observed in eight independent transgenic mice for construct B (n=3 per founder, a part from 1 founder that did not display germ line transmission) and were instead statistically different (P=2.2, E−22) from three independent transgenic lines from construct C (n=3 per founder). (C) In silico sequence analysis was carried out to identify regions of interest contained in the 2.2-kb genomic region spanning from +17.4 and +19.6 kb. The most 3′ 0.8 kb, identified here as the downstream regulatory element (DRE), contains four RUNX sites and two E-Box/GATA motifs.

Figure 2

The DRE is necessary and sufficient for human CD34 expression in SLAM⁺ LSKs. (A) Construct A (described in Figure 1) was modified to specifically delete the DRE (construct D). (B) Bone marrow cells from mice carrying construct D have been analysed by flow cytometry, performed as described in Figure 1. Deletion of the DRE is sufficient to abolish hCD34 expression in SLAM⁺ LSKs (right panel). A representative FACS plot from one of the three founder lines is shown. T-test (two-tailed; type 3) indicated that the data obtained in three independent transgenic lines from construct D (n=3 per founder) were statistically different (P=2.8, E−21) from the percentages observed in nine independent transgenic mice for construct A (Figure 1). (C) Construct A was engineered to contain the DRE as the sole sequences 3′ of the human CD34 gene (construct E). (D) Flow cytometry of bone marrow cells from mice carrying construct E. The DRE is sufficient as a sole 3′ element to drive hCD34 expression in SLAM⁺ LSKs. A representative FACS plot from one of the nine founder lines is shown. T-test (two-tailed; type 3) showed that the percentages observed in nine independent mice transgenic for construct E (n=3 per founder, a part from two founders in which we did not observe germ line transgene transmission) were not statistically different from the data obtained in mice carrying construct A (P=0.15). See also Supplementary Table S1.

Figure 3

RUNX1 binds to the RUNX sites contained within the DRE. (A) Quantitative ChIP analysis was performed on primary human cord blood (CB) cells (>95% hCD34⁺) and HL60 cells (hCD34⁻). Primers and probes encompassing RUNX sites 1–3, and RUNX site 4, were used to amplify input genomic DNA, and DNA precipitated by antibodies against either normal IgG or Runx1 or histone 3 acetylated at lysines 9 and 14 (aH3-Ac). The Myf5 promoter was used as a negative control sequence. Values are normalized to input genomic DNA. The panel shows the values of fold enrichment obtained with anti-Runx1 antibodies (black columns for cord blood ▪ and dotted columns for HL60 ) compared with IgG (white columns for cord blood □ and diagonally barred columns for HL60 ), used as a negative control. Values obtained for the fold enrichment in cord blood cells are statistically different, as shown (bars indicate standard deviations). (B) The panel shows the values of fold enrichment obtained with an antibody against aH3-Ac (black columns for cord blood ▪ and dotted columns for HL60 ); IgG (white columns for cord blood □ and diagonally barred columns for HL60 ) served as control for the assay specificity. Values obtained for the fold enrichment in cord blood cells are statistically different, as indicated. All the data are representative of three independent experiments performed in duplicate. See also Supplementary Figure S1.

Figure 4

RUNX1 regulates human CD34 expression in SLAM⁺ LSKs. (A) Schematic of the breeding strategy used to obtain hCD34⁺ mice (hCD34^{18.3kb5′/DRE3′}) with conditional deletion of Runx1. hCD34 transgenic mice carrying construct E were bred to conditional Runx1 knockout (KO) mice (Runx1^F/F) and Mx1-Cre mice, to obtain interferon-inducible Runx1 gene excision. Mice have been treated with seven injections of PIPC and analysed 1 month later. (B) Flow cytometry analysis on bone marrow cells from PIPC-treated hCD34⁺ Runx1 WT or hCD34⁺ Runx1 KO mice. Representative FACS plots obtained from one WT and one KO mouse are shown. T-test (two-tailed; type 3) showed that the percentages observed in hCD34⁺ Runx1 WT (n=6) were statistically different (P=6.7, E−19) from the data obtained in hCD34⁺ Runx1 KO mice (n=6). See also Supplementary Figure S2.

Figure 5

The DRE interacts with the human CD34 promoter in SLAM⁺ LSKs. (A) Diagram showing the genomic position of the hCD34 promoter (Pr); the eight coding exons (thick vertical lines); the DRE; and location of the HindIII (H3) restriction enzyme sites (vertical arrows) used to perform the 3C assay. The block arrows represent the interaction of the Pr fragment (7.4 kb) with the DRE fragment (4.9 kb) and with six other fragments located in the 3′-flanking region and upstream of the DRE (block arrows 1–6). The distance of each H3 fragment from the DRE is indicated in kb (vertical numbers). (B) Relative crosslinking frequency of the Pr with the DRE and six other 3′ fragments located upstream of the DRE. Crosslinking frequency indicates how frequently distal genomic elements interact. Interaction of the 3′ fragments and the DRE with the Pr are rare in HL60 cells (hCD34⁻), whereas they are increased in frequency in KG1a cells (hCD34⁺), with the DRE showing a 6.2-fold enrichment in the relative crosslinking frequency over the hCD34⁻ HL60 cell line (n=4 per cell line; two independent 3C experiments). (C) On the left panel, the relative crosslinking frequency of the Pr and DRE in hCD34⁺ SLAM⁺ LSKs (n=4; black column; ▪) and hCD34⁻ lin⁺ cells (n=4; white column; □) from mice carrying construct A, as normalized to the levels in hCD34⁺ SLAM⁺ LSKs, is indicated. Interaction of the DRE with the Pr is rare in hCD34⁻ cells, whereas a 10-fold enrichment is observed in hCD34⁺ LT-HSCs. Only the relative crosslinking frequency between the Pr and the DRE has been analysed, given the small number of HSCs per mouse. On the right panel, Q-RT–PCR data verifying the expression levels of hCD34 in the SLAM⁺ LSKs and the lin⁺ cells utilized for the 3C assay are shown. Sorted SLAM⁺ LSKs have been pooled from a total of 25 mice transgenic for construct A. (D) On the left panel, the relative crosslinking frequency of the Pr and DRE in hCD34⁺ cord blood cells (n=4; black column; ▪) and hCD34⁻ cord blood cells (n=4; white column; □), as normalized to the levels in hCD34⁺ cord blood cells, is indicated. Interaction of the DRE with the Pr is weaker in hCD34⁻ cells, whereas an 8.7-fold enrichment is observed in hCD34⁺ cells. On the right panel, Q-RT–PCR data verifying the expression levels of hCD34 in the cord blood populations utilized for the 3C assay are shown. See also Supplementary Figure S3.

Figure 6

Intact RUNX sites within the DRE are necessary for human CD34 expression in SLAM⁺ LSKs and to maintain the promoter–DRE interaction. (A) Construct A (described in Figure 1) was modified to contain mutations in the Runx sites located in the DRE (construct F). The expanded section shows relative location of the mutated Runx sites in the DRE, and within the framed box the specific mutations are indicated relative to the wild-type sequence (Supplementary Figure S5). (B) Flow cytometry of bone marrow cells from mice carrying construct F. A representative example from one of the six founder lines is shown (three mice per each founder were analysed, a part from two founders in which we did not detect germ line transgene transmission). Mutation in the Runx sites strongly decreases hCD34 expression in SLAM⁺ LSKs in comparison with SLAM⁺ LSKs from mice carrying construct A, in terms of percentage (from 99.2±0.5% (s.d.) to 38.1±4.2% (s.d.), respectively; P=3.44, E−17) and mean fluorescence intensity (from 8617±125 (s.d.) to 2342±189 (s.d.), respectively; P=2.1, E−24). (C) LSK-HSCs from mice carrying construct F have been subdivided into a hCD34^low and a hCD34 intermediate (hCD34^int) population, in accordance to the hCD34 gating strategy adopted throughout this study, to perform 3C assays. In the upper panel, post-sorting analysis indicates the purity of the sorted population. The lower left panel shows Q-RT–PCR data, indicating the levels of hCD34 expression in the hCD34^low (black column; ▪) and the hCD34^int population (white column; □), normalized to the levels of hCD34 expression detected in LSK-HSCs from mice carrying construct A. The lower right panel indicates the relative crosslinking frequency of the promoter and DRE in the same hCD34^low (black column; ▪) and hCD34^int (white column; □) FACS-purified cells, as normalized to crosslinking frequency of LSK-HSCs from mice transgenic for construct A. Interaction of the DRE with the Pr fragment is less frequent in hCD34^low cells (n=4) than in hCD34^int LSKs (n=4), showing a 5.2-fold enrichment in the relative crosslinking frequency in cells with higher hCD34 expression. Sorted populations have been pooled from 18 mice transgenic for construct F. See also Supplementary Figure S4.