Mechanism governing a stem cell-generating cis-regulatory element

Abstract

The unremitting demand to replenish differentiated cells in tissues requires efficient mechanisms to generate and regulate stem and progenitor cells. Although master regulatory transcription factors, including GATA binding protein-2 (GATA-2), have crucial roles in these mechanisms, how such factors are controlled in developmentally dynamic systems is poorly understood. Previously, we described five dispersed Gata2 locus sequences, termed the -77, -3.9, -2.8, -1.8, and +9.5 GATA switch sites, which contain evolutionarily conserved GATA motifs occupied by GATA-2 and GATA-1 in hematopoietic precursors and erythroid cells, respectively. Despite common attributes of transcriptional enhancers, targeted deletions of the -2.8, -1.8, and +9.5 sites revealed distinct and unpredictable contributions to Gata2 expression and hematopoiesis. Herein, we describe the targeted deletion of the -3.9 site and mechanistically compare the -3.9 site with other GATA switch sites. The -3.9(-/-) mice were viable and exhibited normal Gata2 expression and steady-state hematopoiesis in the embryo and adult. We established a Gata2 repression/reactivation assay, which revealed unique +9.5 site activity to mediate GATA factor-dependent chromatin structural transitions. Loss-of-function analyses provided evidence for a mechanism in which a mediator of long-range transcriptional control [LIM domain binding 1 (LDB1)] and a chromatin remodeler [Brahma related gene 1 (BRG1)] synergize through the +9.5 site, conferring expression of GATA-2, which is known to promote the genesis and survival of hematopoietic stem cells.

Keywords: HSCs; cis element.

Fig. 1.

The −3.9 GATA switch site bears hallmarks of an important cis-regulatory element. (A) Sequence alignment of the −3.9 site demonstrates conservation among vertebrates. The WGATAR motifs and intervening sequence that were removed by homologous recombination are indicated. (B) ChIP-sequencing profiles for factor occupancy and histone modifications at the Gata2 locus mined from existing datasets (, –72). BM, bone marrow; H3K27a, acetylation of H3 at lysine 27; H3K4m1, monomethylation of histone H3 at lysine 4; MEL, murine erythroleukemia cells. (C) Strategy for targeted deletion of the −3.9 site. Following NeoR excision, the targeted allele has a 126-bp Xba I-to-Not I fragment containing a single LoxP site substituted for the GATA motifs and intervening sequence. Arrowheads indicate positions of primers used for genotype determination. (D) Representative gel shows PCR-based strategy to distinguish WT and targeted alleles following NeoR excision. (E) Genotypes of viable pups from mating −3.9^+/− males and females determined at the time of weaning. Expected numbers of pups based on Mendelian ratios are shown in parenthesis. (F) Representative −3.9^+/+ and −3.9^−/− embryos at E12.5.

Fig. 2.

The −3.9 site is dispensable for Gata2 expression during hematopoiesis. (A) Whole-mount immunostaining of CD31⁺ cells (magenta) and c-Kit⁺ cells (green) within the aorta region of E10.5 embryos. The −3.9^−/− embryos were compared with WT littermates and with +9.5^−/− embryos that almost entirely lack c-Kit⁺ HSCs (25). (Scale bars, 100 μM.) Quantitation of the number of c-Kit⁺ cells per dorsal aorta (DA) (four embryos each for −3.9^+/+ and −3.9^−/−; two embryos for +9.5^−/−) (mean ± SEM). (B) Quantitative analysis of Gata2 mRNA in E13.5 livers [six litters: −3.9^+/+ (n = 12), −3.9^+/− (n = 20), −3.9^−/− (n = 18)] and brains [four litters: −3.9^+/+ (n = 9), −3.9^+/− (n = 14), −3.9^−/− (n = 11)] (mean ± SEM). −RT, no reverse transcriptase. (C) Ter119⁺ and Lin⁻ populations were sequentially isolated from bone marrow via magnetic bead separation. Enrichment of the distinct populations was confirmed in −3.9^+/+ bone marrow samples by measuring the expression of the lineage-restricted genes Mpl (Lin⁻) and Hbb-b1 (Ter119⁺). (D) Comparison of Gata2 mRNA expression in Lin⁻ and Ter119⁺ cells from three independent isolations (mean ± SEM). Fluorescence-activated cell sorting of Sca-1⁺ and c-Kit⁺ double-positive cells from Lin⁻ cells of −3.9^+/+ and −3.9^−/− mice (E) and comparison of Gata2 expression in Lin⁻Sca⁺Kit⁺ cells from two independent biological replicates (mean ± SD) (F) are shown. ***P < 0.001 (two-tailed unpaired Student t test).

Fig. 3.

GATA switch site mutations abrogate chromatin accessibility at −3.9 and +9.5 sites. (A) DNaseI hypersensitivity at the Gata2 locus in fetal liver mined from mouse Encyclopedia of DNA Elements data (48). (B) Quantitative FAIRE analysis of GATA switch sites in WT fetal liver (n = 4, mean ± SEM). The promoters of the actively transcribed RNA Polymerase II (RPII215) and inactive Keratin 5 (Krt5) genes were used as positive and negative controls, respectively. (C) Quantitative FAIRE analysis of chromatin accessibility at and surrounding the −3.9 and +9.5 sites (n = 4, mean ± SEM). The dashed line illustrates the average FAIRE signal at the Krt5 promoter. (D) Allele-specific FAIRE analysis of WT and mutated (Mt) alleles in fetal liver cells from −3.9^+/− (n = 4) and +9.5^+/− (n = 5) E13.5 embryos (mean ± SEM). Primers used for the allele-specific FAIRE analysis are indicated in Table S3. (E) Allele-specific analysis of Gata2 primary transcripts from WT and Mt alleles in +9.5^+/− E13.5 fetal liver and brain (n = 8) and adult bone marrow (n = 3) samples (mean ± SEM). *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed unpaired Student t test).

Fig. 4.

Unique propensity of TAL1 and LDB1 to occupy the +9.5 site. (A) ChIP-sequencing profiles of factor occupancy at the Gata2 locus in lineage-negative bone marrow cells mined from existing datasets (37). (B) Quantitative ChIP analysis of TAL1 and LDB1 occupancy at GATA switch sites of the Gata2 locus in E13.5 fetal liver (n = 3, mean ± SEM). ***P < 0.001. The Necdin promoter was used as a negative control. (C) Allele-specific ChIP analysis of TAL1 and LDB1 occupancy at WT and Mt alleles in fetal liver from E13.5 +9.5^+/− embryos (n = 4, mean ± SEM). ***P < 0.001 (two-tailed unpaired Student t test). PI, preimmune.

Fig. 5.

Gata2 repression/reactivation assay. Evidence for distinct functional properties of the GATA switch sites is illustrated. (A) Schematic representation of the experimental strategy for Gata2 repression and reactivation in G1E–ER–GATA-1 cells. Treatment of G1E–ER–GATA-1 cells with β-estradiol activates ER–GATA-1, leading to loss of Gata2 transcripts and protein by 24 h (10). Washout of β-estradiol reverses Gata2 repression, leading to restoration of GATA-2 by 48-h treatment. (B) Quantitative real-time PCR was used to measure Gata2 primary transcripts during locus reactivation. After 24 h, β-estradiol treatment (+estradiol) cells were washed in PBS and cultured in media without β-estradiol (washout) for an additional 24 h. RNA was isolated and analyzed before β-estradiol treatment (0 h); after 24 h of β-estradiol treatment; and 2, 6, 12, and 24 h following washout (n = 4, mean ± SEM). (C) Representative Western blots of GATA-2 and ER–GATA-1 from samples isolated at the same times as the corresponding RNA samples. (D) Relative chromatin occupancy of ER-GATA-1 (○) and GATA-2 (●) during Gata2 reactivation using quantitative ChIP (n = 3, mean ± SEM).

Fig. 6.

Molecular attributes of the +9.5 site revealed by the repression/reactivation assay. (A) Quantitative FAIRE analysis of chromatin accessibility at GATA switch sites in untreated G1E-ER-GATA-1 cells (0 h). The inactive Necdin locus was used as a negative control (n = 4, mean ± SEM). (B) Quantitative FAIRE analysis of chromatin accessibility at GATA switch sites upon Gata2 repression and reactivation. FAIRE signals for the 0-h times were normalized to 1.0 (n = 4, mean ± SEM). (C) Representative Western blots of GATA-2, TAL1, and LDB1 protein levels at the same times analyzed by ChIP. The asterisk represents a nonspecific band. (D) Quantitative ChIP analysis of TAL1 and LDB1 chromatin occupancy at the active (0 h), repressed (+24 h), and reactivated (WO) Gata2 locus. The βmajor promoter was used as a positive control (n = 4, mean ± SEM). **P < 0.01; ***P < 0.001. (E) Quantitative ChIP analysis of BRG1 chromatin occupancy at the active (0 h) and repressed (+24 h) Gata2 locus and control sites (βmajor and Necdin) (n = 3, mean ± SEM). ***P < 0.001. WO, washout.

Fig. 7.

Mechanism underlying +9.5 site function. (A) Knockdown strategy. Cells were transfected with siRNA twice with a 24-h interval and harvested 48 h after the first transfection. (B) Representative Western blots of GATA-2, TAL1, LDB1, and BRG1 following knockdown of Ldb1 and Brg1 mRNAs individually or in combination. The asterisk represents a nonspecific band. Schematic representation (C) and Western blots (D) of siRNA-mediated factor knockdown strategy during Gata2 repression/reactivation. G1E–ER–GATA-1 cells were induced with β-estradiol (0 h) and transfected twice with factor-specific or control siRNAs at 6 and 24 h postinduction. β-estradiol was washed out at the time of the second transfection. Protein and RNA samples were collected at 24 and 48 h. −, β-estradiol uninduced at 0 h; +, β-estradiol induced at 24 h; WO, β-estradiol washout. All conditions received specific siRNA or nontargeting control siRNA at same molar concentration. (E) ChIP analysis of TAL1 occupancy at the +9.5 and −1.8 sites quantitated under individual knockdown or LDB1 and BRG1 combined knockdown conditions (n = 4, mean ± SEM). **P < 0.01; ***P < 0.001. (F) FAIRE analysis of chromatin accessibility of +9.5 and −1.8 sites following LDB1 and BRG1 individual or LDB1/BRG1 combined knockdown (n = 3, mean ± SEM). ***P < 0.001. (G) ChIP analysis of GATA-1 occupancy at GATA switch sites following LDB1/BRG1 combined knockdown during GATA-2 reactivation (n = 4, mean ± SEM). **P < 0.01; ***P < 0.001. (H) Type I coherent feed-forward loop network motif that controls Gata2 expression. (I) Model depicts GATA switch-site chromatin architecture and its relationship to Gata2 expression. The red X indicates that the motif has been deleted.