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Conditionally pathogenic genetic variants of a hematopoietic disease-suppressing enhancer

Soukup, Alexandra A., Matson, Daniel R., Liu, Peng, Johnson, Kirby D., Bresnick, Emery H.·2021·Science Advances
DOI 10.1126/sciadv.abk3521PMC PMC8664263
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Abstract

Noncoding genetic variants cause blood disease.

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Fig. 1. Gata2 Ets motif variant dysregulates hematopoiesis through a hematopoietic cell–autonomous mechanism. (A) Reciprocal transplantation of WT cells into WT and +9.5(Ets)−/− recipients. (B) CBCs after transplant. NS, not significant; WBCs, white blood cells; RBCs, red blood cells; PLTs, platelets. (C) Multilineage repopulating activity 16 weeks after competitive transplant (n = 4 to 10 per genotype). (D) Analysis of donor-derived LSK (Lin−CD48−Sca1+Kit+), HSC (Lin−CD48−Sca1+Kit+CD150+), MPP (Lin−CD48−Sca1+Kit+CD150−), LS−K (Lin−Sca1−Kit+), MEP (Lin−Sca1−Kit+FcγR−CD34−), CMP (Lin−Sca1−Kit+FcγR−CD34+), and GMP (Lin−Sca1−Kit+FcγR+CD34+) within bone marrow 16 weeks after transplant (n = 4 to 9 per genotype) (two-tailed unpaired Student’s t test).
Fig. 1. Gata2 Ets motif variant dysregulates hematopoiesis through a hematopoietic cell–autonomous mechanism. (A) Reciprocal transplantation of WT cells into WT and +9.5(Ets)−/− recipients. (B) CBCs after transplant. NS, not significant; WBCs, white blood cells; RBCs, red blood cells; PLTs, platelets. (C) Multilineage repopulating activity 16 weeks after competitive transplant (n = 4 to 10 per genotype). (D) Analysis of donor-derived LSK (Lin−CD48−Sca1+Kit+), HSC (Lin−CD48−Sca1+Kit+CD150+), MPP (Lin−CD48−Sca1+Kit+CD150−), LS−K (Lin−Sca1−Kit+), MEP (Lin−Sca1−Kit+FcγR−CD34−), CMP (Lin−Sca1−Kit+FcγR−CD34+), and GMP (Lin−Sca1−Kit+FcγR+CD34+) within bone marrow 16 weeks after transplant (n = 4 to 9 per genotype) (two-tailed unpaired Student’s t test).CC BY-NC · PubMed Central
Fig. 2. +9.5 Ets motif mitigates lethality induced by chronic stress. (A) Mice were given multiple doses of pI:C (10 mg/kg) or vehicle control as per Walter et al. (37). Treatment cycles were repeated every 56 days. (B) Survival [PBS: WT (n = 10), +9.5(Ets)−/− (n = 14); pI:C: WT (n = 14), +9.5(Ets)−/− (n = 20) from two experiments] (log-rank test). (C) Number of cells per single hindlimb. n = 5 to 10 per condition, Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Representative hematoxylin and eosin (H&E) staining of femurs. Each panel represents a single mouse. Scale bars, 50 μm.
Fig. 2. +9.5 Ets motif mitigates lethality induced by chronic stress. (A) Mice were given multiple doses of pI:C (10 mg/kg) or vehicle control as per Walter et al. (37). Treatment cycles were repeated every 56 days. (B) Survival [PBS: WT (n = 10), +9.5(Ets)−/− (n = 14); pI:C: WT (n = 14), +9.5(Ets)−/− (n = 20) from two experiments] (log-rank test). (C) Number of cells per single hindlimb. n = 5 to 10 per condition, Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001. (D) Representative hematoxylin and eosin (H&E) staining of femurs. Each panel represents a single mouse. Scale bars, 50 μm.CC BY-NC · PubMed Central
Fig. 3. +9.5 E-box accelerates regeneration kinetics. (A) Sequence of WT (+9.5+/+) and E-box variant [+9.5(E-box)−/−] mice. (B) Sequence of mutant mice. (C) Polymerase chain reaction (PCR) genotyping. (D) Genotyping results at weaning from +9.5(E-box)+/− × +9.5(E-box)+/− matings (chi-square test) (E) Steady-state blood parameters [+9.5+/+ (n = 9) and +9.5(E-box)−/− (n = 12)]. (F) Kaplan-Meier survival curve of mice following two 5-FU doses (250 mg/kg; days 0 and 11; n = 10 per genotype). P = 0.0167, log-rank test. (G) mRNA quantification from bone marrow after vehicle (PBS), 9-day 5-FU, or 11-day 5-FU (250 mg/kg) treatment (n = 5 to 11 per condition from five experiments). (H) Flow cytometry scheme for quantification of LSK (Lin−CD48−Sca1+Kit+), HSC (Lin−CD48−Sca1+Kit+CD150+), MPP (Lin−CD48−Sca1+Kit+CD150−), LS−K (Lin−Sca1−Kit+), MEP (Lin−Sca1−Kit+FcγR−CD34−), CMP (Lin−Sca1−Kit+FcγR−CD34+), and GMP (Lin−Sca1−Kit+FcγR+CD34+). (I) Quantification of HSC, MPP, and LSKs from total bone marrow after vehicle (PBS), 9-day 5-FU, or 11-day 5-FU (250 mg/kg) treatment (percentage of live bone marrow cells) [PBS: WT (n = 9), +9.5(E-box)−/− (n = 9); day 9: WT (n = 8), +9.5(E-box)−/− (n = 10); day 11: WT (n = 10), +9.5(E-box)−/− (n = 10)]. *P < 0.05, **P < 0.01, ***P < 0.001, Tukey’s multiple comparisons test.
Fig. 3. +9.5 E-box accelerates regeneration kinetics. (A) Sequence of WT (+9.5+/+) and E-box variant [+9.5(E-box)−/−] mice. (B) Sequence of mutant mice. (C) Polymerase chain reaction (PCR) genotyping. (D) Genotyping results at weaning from +9.5(E-box)+/− × +9.5(E-box)+/− matings (chi-square test) (E) Steady-state blood parameters [+9.5+/+ (n = 9) and +9.5(E-box)−/− (n = 12)]. (F) Kaplan-Meier survival curve of mice following two 5-FU doses (250 mg/kg; days 0 and 11; n = 10 per genotype). P = 0.0167, log-rank test. (G) mRNA quantification from bone marrow after vehicle (PBS), 9-day 5-FU, or 11-day 5-FU (250 mg/kg) treatment (n = 5 to 11 per condition from five experiments). (H) Flow cytometry scheme for quantification of LSK (Lin−CD48−Sca1+Kit+), HSC (Lin−CD48−Sca1+Kit+CD150+), MPP (Lin−CD48−Sca1+Kit+CD150−), LS−K (Lin−Sca1−Kit+), MEP (Lin−Sca1−Kit+FcγR−CD34−), CMP (Lin−Sca1−Kit+FcγR−CD34+), and GMP (Lin−Sca1−Kit+FcγR+CD34+). (I) Quantification of HSC, MPP, and LSKs from total bone marrow after vehicle (PBS), 9-day 5-FU, or 11-day 5-FU (250 mg/kg) treatment (percentage of live bone marrow cells) [PBS: WT (n = 9), +9.5(E-box)−/− (n = 9); day 9: WT (n = 8), +9.5(E-box)−/− (n = 10); day 11: WT (n = 10), +9.5(E-box)−/− (n = 10)]. *P < 0.05, **P < 0.01, ***P < 0.001, Tukey’s multiple comparisons test.CC BY-NC · PubMed Central
Fig. 4. Enhancer-engineered bone marrow failure mouse model. (A) Model depicting the motif configurations within the Gata2 +9.5 in WT, +9.5(Ets)+/−, +9.5+/−, and CH mice. (B) Genotypes from breeding schemes used to generate CH mice. Probability was calculated using chi-square tests. (C) Steady-state peripheral blood parameters. Data are represented as mean ± SEM; *P < 0.05 (Tukey’s multiple comparisons test). (D) Flow cytometric quantification of LT-HSC (Lin−Sca1+Kit+CD150+CD48−), LSKCD34+, LSKCD34−CD150−, and Lin−Kit+Sca1−. (E) qPCR of purified cells. (F) RNA-seq of LT-HSCs (Lin−Sca1+Kit+CD150+CD34−).
Fig. 4. Enhancer-engineered bone marrow failure mouse model. (A) Model depicting the motif configurations within the Gata2 +9.5 in WT, +9.5(Ets)+/−, +9.5+/−, and CH mice. (B) Genotypes from breeding schemes used to generate CH mice. Probability was calculated using chi-square tests. (C) Steady-state peripheral blood parameters. Data are represented as mean ± SEM; *P < 0.05 (Tukey’s multiple comparisons test). (D) Flow cytometric quantification of LT-HSC (Lin−Sca1+Kit+CD150+CD48−), LSKCD34+, LSKCD34−CD150−, and Lin−Kit+Sca1−. (E) qPCR of purified cells. (F) RNA-seq of LT-HSCs (Lin−Sca1+Kit+CD150+CD34−).CC BY-NC · PubMed Central
Fig. 5. Gata2 +9.5 E-box;GATA and Ets motif pro-regenerative functions. (A) Kaplan-Meier survival curve of mice following two 5-FU doses (250 mg/kg; days 0 and 11) [WT (n = 13), +9.5(Ets)+/− (n = 18), +9.5−/− (n = 7), and CH (n = 12) from two experiments]. Significance was determined using the log-rank test. (B) Flow cytometry scheme for quantification of LSK (Lin−CD48−Sca1+Kit+), HSC (Lin−CD48−Sca1+Kit+CD150+), and MPP (Lin−CD48−Sca1+Kit+CD150−). Bone marrow was harvested 9 or 10 days after vehicle (PBS) or 5-FU (250 mg/kg) treatment. (C) Quantification of HSC, MPP, and LSK from total bone marrow (percentage of live bone marrow cells) [PBS: WT (n = 11), +9.5(Ets)+/− (n = 6), +9.5−/− (n = 7), and CH (n = 6); day 9: WT (n = 12), +9.5(Ets)+/− (n = 8), +9.5−/− (n = 8), and CH (n = 4); day 10: WT (n = 12), +9.5(Ets)+/− (n = 9), +9.5−/− (n = 8), and CH (n = 5)]. False discovery rate–adjusted *P < 0.05, **P < 0.01, ***P < 0.001, Welch’s unequal variance t tests with Benjamini, Krieger, and Yekutieli multiple-test correction. (D) Representative H&E and anti-GATA2 staining of femurs. Each panel represents a single mouse. Scale bars, 50 μm. IHC, immunohistochemistry. (E) Quantification of GATA2+ mononuclear cells from (D) (n = 4 to 6 per condition from three experiments). (F) mRNA quantification from total bone marrow after vehicle (PBS), 9-day 5-FU, or 10-day 5-FU (250 mg/kg) treatment (n = 4 to 12 per condition from five experiments). (G) mRNA quantification from sorted HSC (Lin−Sca1+Kit+CD150+CD34−) or LSKCD34+ MPP (Lin−Sca1+Kit+CD34+) after vehicle (PBS), 7-day 5-FU, or 8-day 5-FU (250 mg/kg) treatment (n = 4 to 22 per condition from eight experiments). *P < 0.05, **P < 0.01, ***P < 0.001, Tukey’s multiple comparisons test.
Fig. 5. Gata2 +9.5 E-box;GATA and Ets motif pro-regenerative functions. (A) Kaplan-Meier survival curve of mice following two 5-FU doses (250 mg/kg; days 0 and 11) [WT (n = 13), +9.5(Ets)+/− (n = 18), +9.5−/− (n = 7), and CH (n = 12) from two experiments]. Significance was determined using the log-rank test. (B) Flow cytometry scheme for quantification of LSK (Lin−CD48−Sca1+Kit+), HSC (Lin−CD48−Sca1+Kit+CD150+), and MPP (Lin−CD48−Sca1+Kit+CD150−). Bone marrow was harvested 9 or 10 days after vehicle (PBS) or 5-FU (250 mg/kg) treatment. (C) Quantification of HSC, MPP, and LSK from total bone marrow (percentage of live bone marrow cells) [PBS: WT (n = 11), +9.5(Ets)+/− (n = 6), +9.5−/− (n = 7), and CH (n = 6); day 9: WT (n = 12), +9.5(Ets)+/− (n = 8), +9.5−/− (n = 8), and CH (n = 4); day 10: WT (n = 12), +9.5(Ets)+/− (n = 9), +9.5−/− (n = 8), and CH (n = 5)]. False discovery rate–adjusted *P < 0.05, **P < 0.01, ***P < 0.001, Welch’s unequal variance t tests with Benjamini, Krieger, and Yekutieli multiple-test correction. (D) Representative H&E and anti-GATA2 staining of femurs. Each panel represents a single mouse. Scale bars, 50 μm. IHC, immunohistochemistry. (E) Quantification of GATA2+ mononuclear cells from (D) (n = 4 to 6 per condition from three experiments). (F) mRNA quantification from total bone marrow after vehicle (PBS), 9-day 5-FU, or 10-day 5-FU (250 mg/kg) treatment (n = 4 to 12 per condition from five experiments). (G) mRNA quantification from sorted HSC (Lin−Sca1+Kit+CD150+CD34−) or LSKCD34+ MPP (Lin−Sca1+Kit+CD34+) after vehicle (PBS), 7-day 5-FU, or 8-day 5-FU (250 mg/kg) treatment (n = 4 to 22 per condition from eight experiments). *P < 0.05, **P < 0.01, ***P < 0.001, Tukey’s multiple comparisons test.CC BY-NC · PubMed Central
Fig. 6. Combinatorial E-box;GATA and Ets motif mutation abrogates LT-HSC repopulating activity. (A) Repopulating activity in peripheral blood 16 weeks after competitive transplant of total bone marrow (n = 9 to 10 per genotype) (two-tailed unpaired Student’s t test). (B) Percentage of donor-derived myeloid cells. (C) Percentage of donor-derived lymphoid cells. (D) Donor-derived hematopoiesis of LSK, HSC, and MPP within bone marrow 16 weeks after competitive transplant (n = 7 to 8 per genotype) (two-tailed unpaired Student’s t test). Donor-derived hematopoiesis of LS−K, CMP, GMP, and MEP within bone marrow 16 weeks after competitive transplant (n = 7 to 8 per genotype) (two-tailed unpaired Student’s t test). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t test.
Fig. 6. Combinatorial E-box;GATA and Ets motif mutation abrogates LT-HSC repopulating activity. (A) Repopulating activity in peripheral blood 16 weeks after competitive transplant of total bone marrow (n = 9 to 10 per genotype) (two-tailed unpaired Student’s t test). (B) Percentage of donor-derived myeloid cells. (C) Percentage of donor-derived lymphoid cells. (D) Donor-derived hematopoiesis of LSK, HSC, and MPP within bone marrow 16 weeks after competitive transplant (n = 7 to 8 per genotype) (two-tailed unpaired Student’s t test). Donor-derived hematopoiesis of LS−K, CMP, GMP, and MEP within bone marrow 16 weeks after competitive transplant (n = 7 to 8 per genotype) (two-tailed unpaired Student’s t test). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t test.CC BY-NC · PubMed Central
Fig. 7. E-box;GATA and Ets motif variants attenuate hematopoietic regenerative response to inflammation. (A) Flow cytometry scheme for quantification of LSK (Lin−Sca1+Kit+), HSC (Lin−Sca1+Kit+CD150+CD34−), CD150+CD34+ (Lin−Sca1+Kit+CD150+CD34+), CD150−CD34+ (Lin−Sca1+Kit+CD150−CD34+), and CD150−CD34− (Lin−Sca1+Kit+CD150−CD34−). Bone marrow was harvested 24 hours after a single dose of LPS (0.5 mg/kg) or vehicle control. (B) Quantification of HSPC populations identified in (A) (percentage of live bone marrow cells). N = 4 to 6 per condition from three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t test.
Fig. 7. E-box;GATA and Ets motif variants attenuate hematopoietic regenerative response to inflammation. (A) Flow cytometry scheme for quantification of LSK (Lin−Sca1+Kit+), HSC (Lin−Sca1+Kit+CD150+CD34−), CD150+CD34+ (Lin−Sca1+Kit+CD150+CD34+), CD150−CD34+ (Lin−Sca1+Kit+CD150−CD34+), and CD150−CD34− (Lin−Sca1+Kit+CD150−CD34−). Bone marrow was harvested 24 hours after a single dose of LPS (0.5 mg/kg) or vehicle control. (B) Quantification of HSPC populations identified in (A) (percentage of live bone marrow cells). N = 4 to 6 per condition from three experiments. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed unpaired Student’s t test.CC BY-NC · PubMed Central
Fig. 8. Enhancer variants disrupt clinically relevant stem cell mobilization. (A) Mice were given eight doses of rhG-CSF (125 μg/kg) over 5 days. (B) Quantification of peripheral blood neutrophils. N = 4 per condition from two experiments. (C) Flow cytometric quantification of HSC (Lin−Sca1+Kit+CD150+CD48−) from bone marrow (BM) or spleen. N = 3 to 9 per condition from six experiments. (D) CFUs from 0.1 ml of peripheral blood. N = 3 to 4 per condition from two experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Tukey’s multiple comparisons test. (E) Quantitation of CSF3R on LT-HSCs (Lin−CD48−Sca1+Kit+CD150+) by flow cytometry. (F) Experimental layout for ex vivo cytokine stimulation. (G) Representative phospho-flow cytometry quantitating pSTAT3 levels in all cells from WT (n = 3) and CH (n = 3) after a 5-min G-CSF stimulation. (H) Flow cytometric scheme for isolation of populations. (I) Representative phospho-flow plots of pSTAT3 levels in Gr1+ and Lin−Gr1−Kit+ populations. (J) Quantitation of (H). **P < 0.01, ***P < 0.001, Tukey’s multiple comparisons test.
Fig. 8. Enhancer variants disrupt clinically relevant stem cell mobilization. (A) Mice were given eight doses of rhG-CSF (125 μg/kg) over 5 days. (B) Quantification of peripheral blood neutrophils. N = 4 per condition from two experiments. (C) Flow cytometric quantification of HSC (Lin−Sca1+Kit+CD150+CD48−) from bone marrow (BM) or spleen. N = 3 to 9 per condition from six experiments. (D) CFUs from 0.1 ml of peripheral blood. N = 3 to 4 per condition from two experiments. *P < 0.05, **P < 0.01, ***P < 0.001, Tukey’s multiple comparisons test. (E) Quantitation of CSF3R on LT-HSCs (Lin−CD48−Sca1+Kit+CD150+) by flow cytometry. (F) Experimental layout for ex vivo cytokine stimulation. (G) Representative phospho-flow cytometry quantitating pSTAT3 levels in all cells from WT (n = 3) and CH (n = 3) after a 5-min G-CSF stimulation. (H) Flow cytometric scheme for isolation of populations. (I) Representative phospho-flow plots of pSTAT3 levels in Gr1+ and Lin−Gr1−Kit+ populations. (J) Quantitation of (H). **P < 0.01, ***P < 0.001, Tukey’s multiple comparisons test.CC BY-NC · PubMed Central
Fig. 9. Biological and pathological consequences of CP Gata2 enhancer variants. Top: The diagram illustrates the murine Gata2 +9.5 alleles analyzed here and their capacity to support hematopoiesis in the AGM, fetal liver, and bone marrow. The heat map depicts their relative activities as a percent of the WT activity. Bottom: The model illustrates the vital +9.5 enhancer activity to mediate adult hematopoiesis in response to stressors, including chemotherapy, inflammation, infection, and therapeutic HSC mobilization with G-CSF. As the motifs constituting the +9.5 enhancer are differentially important in distinct physiological and pathological contexts, genetic variants within these motifs differentially affect GATA2-dependent processes. Thus, we term these variants “conditionally pathogenic.”
Fig. 9. Biological and pathological consequences of CP Gata2 enhancer variants. Top: The diagram illustrates the murine Gata2 +9.5 alleles analyzed here and their capacity to support hematopoiesis in the AGM, fetal liver, and bone marrow. The heat map depicts their relative activities as a percent of the WT activity. Bottom: The model illustrates the vital +9.5 enhancer activity to mediate adult hematopoiesis in response to stressors, including chemotherapy, inflammation, infection, and therapeutic HSC mobilization with G-CSF. As the motifs constituting the +9.5 enhancer are differentially important in distinct physiological and pathological contexts, genetic variants within these motifs differentially affect GATA2-dependent processes. Thus, we term these variants “conditionally pathogenic.”CC BY-NC · PubMed Central

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Authors
Soukup, A. A., Matson, D. R., Liu, P., Johnson, K. D., & Bresnick, E. H.
Affiliations
  • Wisconsin Blood Cancer Research Institute, Department of Cell and Regenerative Biology, Wisconsin Institutes for Medical Research, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
  • University of Wisconsin Carbone Cancer Center, Department of Biostatistics and Medical Informatics, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2021-12-10
Volume
7
Issue
50
ISSN
2375-2548
DOI
10.1126/sciadv.abk3521
PMC
PMC8664263
Cited by
15
References
63
Language
en

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