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Human corrupt but do not abrogate GATA-2 function

Koichi R. Katsumuraa,b, Charu Mehtaa,b, Kyle J. Hewitta,b, Alexandra A. Soukupa,b, Isabela Fraga de Andradea,b, Erik A. Ranheimc, Kirby D. Johnsona,b, and Emery H. Bresnicka,b,1

aUniversity of Wisconsin–Madison Research Program, Department of Cell and Regenerative Biology, Wisconsin Institutes for Medical Research, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705; bUniversity of Wisconsin Carbone Cancer Center, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705; and cDepartment of Pathology and Laboratory Medicine, University of Wisconsin School of Medicine and Public Health, Madison, WI 53705

Edited by Stuart H. Orkin, Children’s Hospital and the Dana Farber Cancer Institute, Howard Hughes Medical Institute and Harvard Medical School, Boston, MA, and approved September 4, 2018 (received for review July 31, 2018) By inducing the generation and function of hematopoietic stem 2 function include the embryonic-lethal Gata2 intronic (+9.5) and progenitor cells, the master regulator of hematopoiesis GATA- mutant with defective HSC genesis (18) and erythroid 2 controls the production of all blood cell types. Heterozygous precursor function (19) and distal (−77) (20) enhancer mutant GATA2 mutations cause immunodeficiency, myelodysplastic syn- with defective myelo-erythroid progenitor differentiation. The drome, and . GATA2 disease mutations results with these models, and the finding that GATA-2 overexpression commonly disrupt residues that mediate DNA binding in suppresses hematopoiesis (22), indicate that or cis-elements within a vital GATA2 intronic enhancer, suggesting GATA-2 levels/activity must be constrained within a physiological a haploinsufficiency mechanism of pathogenesis. Mutations also window. occur in GATA2 coding regions distinct from the DNA-binding In accord with critical GATA-2 functions discovered in mice, carboxyl-terminal (C-finger), including the amino- heterozygous human GATA2 mutations are pathogenic and terminal zinc finger (N-finger), and N-finger function is not estab- cause immunodeficiency that often progresses to MDS and AML lished. Whether distinct mutations differentially impact GATA-2 (23, 24). GATA2 mutations also cause other AML-linked fa- mechanisms is unknown. Here, we demonstrate that N-finger mu- milial diseases, and GATA2 is mutated frequently in high-risk GENETICS tations decreased GATA-2 chromatin occupancy and attenuated tar- MDS (25). GATA2 mutations often occur in the DNA binding C- get regulation. We developed a genetic complementation finger and inhibit DNA binding (26). GATA2 +9.5 enhancer assay to quantify GATA-2 function in myeloid progenitor cells from mutations decrease GATA-2 expression (18, 27). In 3q21q26 Gata2 −77 enhancer-mutant mice. GATA-2 complementation in- AML, the −77 enhancer is repositioned next to MECOM encoding creased erythroid and myeloid differentiation. While GATA-2 the EVI1 (28, 29). Decreased GATA2, concomitant with disease mutants were not competent to induce erythroid differ- elevated EVI1, underlies this malignancy. In addition, GATA- − + entiation of Lin Kit myeloid progenitors, unexpectedly, they 2 overexpression in AML can predict poor prognosis (30). In promoted myeloid differentiation and proliferation. As the aggregate, mouse and human data emphasize the need to avert myelopoiesis-promoting activity of GATA-2 mutants exceeded declines and increases in GATA-2, both being pathogenic. that of GATA-2, GATA2 disease mutations are not strictly inhib- GATA-2 establishes and maintains cell-type–specific genetic itory. Thus, we propose that the haploinsufficiency paradigm networks, and heterozygous GATA2 mutations that reduce GATA- does not fully explain GATA-2–linked pathogenesis, and an amal- 2 levels/activity may differentially affect network integrity in distinct gamation of qualitative and quantitative defects instigated by GATA2 mutations underlies the complex of GATA- Significance 2–dependent pathologies. GATA-2 functions in stem and progenitor cells to control blood GATA-2 | hematopoiesis | MDS | AML | leukemia cell development, and its mutations cause blood diseases (im- munodeficiency, myelodysplasia, and myeloid leukemia). How echanisms underlying the heterogeneous malignancy acute GATA-2 mutations cause these diseases is unclear. We innovated Mmyeloid leukemia (AML) are incompletely understood, a genetic complementation assay to analyze functional ramifi- and there is a vital need to develop efficacious therapies (1). cations of GATA-2 disease mutations. The activities of GATA- Although major progress has been made in developing molecu- 2 and mutants were quantified in blood progenitor cells from larly targeted and transplant therapies, the 5-y survival of geri- mice engineered to express a low level of GATA-2 due to de- atric and pediatric AML patients remains at 10–20% and 60– letion of an essential Gata2 enhancer. Unexpectedly, the mu- 70%, respectively (2). Elucidating how myeloid cell genetic tants were not only competent to induce myeloid cells, but their networks are corrupted may unveil opportunities for AML bio- activities exceeded that of GATA-2. These results transform the marker and therapeutics development. Rigorous studies have current paradigm that disease mutations are solely inhibitory, defined AML genetic and epigenetic landscapes and the vexing and ectopically low GATA-2 levels/activity constitute the disease clonal evolution during disease progression (3–9). Germline mechanism. mutations that predispose to (MDS) and AML, such as those disrupting GATA-2 expression and Author contributions: K.R.K. and E.H.B. designed research; K.R.K., C.M., K.J.H., A.A.S., I.F.d.A., function (10–12), have the potential to reveal clues regarding and K.D.J. performed research; K.D.J. contributed new reagents/analytic tools; K.R.K., E.A.R., mechanisms governing disease initiation and progression. and E.H.B. analyzed data; and K.R.K. and E.H.B. wrote the paper. GATA-2 is essential for multilineage hematopoiesis (13), The authors declare no conflict of interest. triggers hemogenic to produce hematopoietic stem This article is a PNAS Direct Submission. cells (HSCs) (14, 15), regulates HSC activity (16–18), and stim- Published under the PNAS license. ulates myelo-erythroid differentiation, pro- 1To whom correspondence should be addressed. Email: [email protected]. liferation, and survival (19–21). Gata2-null mice exhibit impaired This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. multilineage hematopoiesis and die at ∼embryonic day (E) 10.5 1073/pnas.1813015115/-/DCSupplemental. (13). Additional instructive mouse models for analyzing GATA- Published online October 9, 2018.

www.pnas.org/cgi/doi/10.1073/pnas.1813015115 PNAS | vol. 115 | no. 43 | E10109–E10118 Downloaded by guest on October 1, 2021 contexts (26). Inadequate or excessive target gene activity would These results indicate that GATA-2 N-finger integrity promotes both corrupt networks. Oncogenic Ras-dependent, multisite GATA- chromatin occupancy at target without grossly changing 2 phosphorylation, coupled with GATA-2–dependent positive auto- nuclear localization. regulation of GATA2 transcription, can elevate GATA-2 levels/ activity and therefore disrupt physiological GATA-2 function (31, Structure-Based Design of GATA-2 Mutants to Dissect GATA-2 32). Because GATA-2 stimulates AML cell proliferation and sur- Leukemia Mutant Mechanisms. GATA factor N-fingers are highly vival in vitro (32), elevating or reducing GATA-2 may instigate or conserved (Fig. 2A), implying important functions. To assess contribute to leukemogenesis. whether the inhibitory consequences of N-finger leukemia mu- GATA factor C-fingers mediate DNA binding (33, 34), and C- tants are recapitulated by a that disrupts zinc finger finger mutations impair GATA-2 function (23, 24). Although N- structure, C295A was engineered into GATA-2 (Fig. 2A). Under finger function remains enigmatic, N-finger mutations occur in conditions in which GATA-2, C295A, and R307W were expressed patients with erythroleukemia (35) and AML with biallelic muta- at comparable levels (Fig. 2B), both C295A and R307W signifi- tion of CEBPA (36, 37). The N-finger was reported to bind DNA cantly decreased induction of GATA-2 target in with sequence-specificity in vitro (38, 39). The GATA-1 N-finger MAE cells (Fig. 2C). Similar to the N-finger disease mutants, binds the critical coregulator FOG-1 (40). This interaction is me- C295A was exclusively nuclear-localized (Fig. 2D). diated by GATA-1 V205 (40), and V205 mutation disrupts ery- Previously, we demonstrated that Ras signaling induces multi- throid maturation in mice and generates familial dyserythropoietic site GATA-2 phosphorylation and increases GATA-2–dependent in humans (41). Although the GATA-1 and GATA-2 N- transcriptional activation (31, 32). We tested whether N-finger fingers are well conserved, GATA-2–expressing hematopoietic leukemia mutants are competent to respond to Ras signaling. stem and progenitor cells (HSPCs) do not express FOG-1. Expression of Ras(G12V) shifted GATA-2 migration to a slow- R216 mutations in patients with X-linked gray syndrome mobility species (2.2-fold increase), which we demonstrated pre- (42) attenuate GATA-1 function without influencing FOG- viously to be a hyperphosphorylated isoform (Fig. 2E). Ras 1 binding (43). This mutation reduces binding to sites contain- (G12V) also induced a mobility shift with R307W (2.4-fold in- ing single or palindromic GATA motifs. Analogous GATA-2 crease) and increased its capacity to activate Hdc expression (R307W) and GATA-3 (R276) residues can be mutated in leu- sevenfold, comparable to the fold-induction achieved with Ras kemia patients. Because the GATA-2 N-finger can be mutated in (G12V)/GATA-2 (Fig. 2 E and F). Thus, although R307W- leukemia, and unlike GATA-1, FOG-1 is not expressed in the mediated transcriptional activation was strongly compromised, it GATA-2–expressing cells, dissecting molecular consequences of remained competent to respond to Ras(G12V). these mutations has the potential to inform GATA factor mech- Because the C-finger mediates DNA binding (33), and N- anisms and pathologies. Herein, we analyzed the mechanistic finger function in vivo is unresolved, it was instructive to com- ramifications of N-finger disease mutations in diverse systems, pare the consequences of cysteine mutations in the N- and C- including a genetic complementation assay to quantify GATA-2 fingers. We engineered C349A to disrupt C-finger structure (Fig. function in Gata2 −77 enhancer mutant primary myelo-erythroid 2A) and analyzed its activity in MAE cells. The predominant progenitor cells. Our discovery that GATA-2 disease mutations C349A isoform exhibited a slow mobility, even without Ras unexpectedly enhance select GATA-2 functions in primary cells (G12V) expression, and the intensity of this isoform was 2.3-fold demands a reconsideration of the paradigm that inhibitory disease greater than that of GATA-2 (Fig. 2G). This is consistent with mutations strictly decrease GATA-2 levels/activity. These results our report that the T354M disease mutant linked to MDS/AML provide a perspective into the haploinsufficiency model of GATA- (11, 12) exhibits reduced chromatin binding and is predomi- 2-linked pathologies. nantly hyperphosphorylated without Ras(G12V) (31); however, multisite phosphorylated T354M does not increase target gene Results expression, based on impaired chromatin binding. Ras(G12V) GATA-2 N-Finger Increases GATA-2 Endogenous Target Gene Chromatin also induced the slow mobility C295A isoform 1.7-fold (Fig. 2G). Occupancy and Activation. Because human disease mutations can Whereas Ras(G12V) increased GATA-2– and C295A-dependent unveil unique mechanistic insights, we analyzed the functional Hdc induction fivefold, C349A was inactive in the absence or consequences of GATA-2 N-finger mutations (R293Q, P304H, presence of Ras(G12V) (Fig. 2H), consistent with the disrupted C- R307W, A318T, and R330Q) (Fig. 1A) from AML patients in a finger that would not be competent for chromatin binding. mouse aortic endothelial (MAE) cell assay (31, 32) in which ex- GATA-1 V205 binds FOG-1, which mediates transcriptional ogenous GATA-2 expression activates endogenous target genes. activation and repression of many GATA-1 target genes (40, 46, Increasing levels of murine GATA-2 (98% sequence identity to 47). FOG-1 is not expressed in GATA-2–expressing cells (e.g., human) or mutants were expressed in MAE cells, ensuring that MAE cells) and does not mediate GATA-2 biological activity in mutants were analyzed at levels resembling that of GATA-2 (Fig. HSPCs. In principle, the GATA-2 residue equivalent to GATA-1 1B). While GATA-2 activated the target genes Hdc, Gfi1,and V205 might mediate transcriptional regulation through a related Grb10 (14, 19, 20, 31), N-finger leukemia mutations attenuated or novel mechanism. We tested this model by engineering mu- the transcriptional response (Fig. 1C). tations at GATA-2 V296, equivalent to GATA-1 V205, and To elucidate the mechanism underlying the compromised ac- analyzing its activity in MAE cells at expression levels resembling tivity of N-finger mutants, we tested whether the subcellular lo- GATA-2 (Fig. 2I). V296G and V296M retained the capacity to calization of the mutants resembles or differs from that of induce Hdc (Fig. 2J). Thus, GATA-2 N-finger–dependent tran- GATA-2. Immunofluorescence analysis indicated that GATA- scriptional activation is impaired by leukemia mutations, requires 2 and the N-finger mutants were exclusively nuclear-localized in N-finger structure, and has distinct molecular determinants from MAE cells (Fig. 1D). Using a quantitative chromatin immuno- the GATA-1 N-finger. precipitation (ChIP) assay with anti-HA antibody, we compared GATA-2 and R307W N-finger mutant activities to occupy chromatin. GATA-2 Genetic Complementation Assay with Gata2 −77 Enhancer R307W was analyzed, because this mutation strongly inhibited target Mutant Progenitor Cells: GATA-2 Leukemia Mutants Promote Myelopoiesis. gene induction. The R307W mutation decreased, but did not abro- To elucidate the functional consequences of GATA-2 mutations, gate, GATA-2 occupancy at the Gata2 +9.5 enhancer (18, 44), Hdc we compared the activity of expressed wild-type and mutant +3.7 (31), and Lyl1 (45) loci (Fig. 1E). GATA-2 did not GATA-2 in primary mouse bone marrow and fetal liver HSPCs. + + occupy Gata2 −18.3, Hdc +11.2, Lyl1 −1.3, and Necdin promoter GATA-2 or R307W expression in −77 / bone marrow cells sites, providing evidence for specificity of chromatin occupancy. decreased CFU-GM (SI Appendix, Fig. S1), consistent with the

E10110 | www.pnas.org/cgi/doi/10.1073/pnas.1813015115 Katsumura et al. Downloaded by guest on October 1, 2021 − − report that GATA-2 overexpression suppresses bone marrow of −77 / cells. The −77 enhancer decreased Gata2 ex- HSPCs (22). We also expressed wild-type or mutant GATA-2 in pression by 69% (Fig. 3C). − − − − mouse fetal liver lineage-negative (Lin ) hematopoietic precur- As described previously (20), −77 / Lin myelo-erythroid sors (Fig. 3 A and B). Although we predicted that R307W and progenitor cells have little to no capacity to generate erythroid the T354M C-finger leukemia mutant would be inactive or have colonies (CFU-E and BFU-E) (Fig. 3D). Wild-type progenitors attenuated activity, unexpectedly, they increased CFU-GM formed very few myeloid colonies (CFU-GM) (Fig. 3D), which + + − in −77 / Lin fetal liver cells (Fig. 3B, Right). reflected the 1-d culture with erythroid factors postinfection and Because human GATA-2 disease mutations are heterozygous differed from our report in which wild-type progenitors were + + (23, 24), expressing GATA-2 mutants in wild-type (−77 / ) he- analyzed without culturing generate large numbers of CFU-GM matopoietic progenitors is not optimal for elucidating physio- (20). HA–GATA-2 expression rescued CFU-E and BFU-E, and logical or pathological mechanisms. We therefore devised a increased CFU-GM (Fig. 3D). Comparison of GATA-2, R307W, − − genetic complementation assay using −77 / fetal liver myelo- and T354M activities revealed that R307W, but not T354M, erythroid progenitor cells (20), in which endogenous GATA- resembled GATA-2 in rescuing BFU-E at similar expression − − 2 expression is reduced. It was not possible to use −77 / bone levels (Fig. 3D). Unexpectedly, this analysis revealed that marrow, because this homozygous mutation is embryonic-lethal R307W and T354M increased CFU-GM 7- and 2.5-fold greater (20). Genetic complementation analysis in mutant cells is a pow- than GATA-2 (Fig. 3D). To test whether rescue involved GATA- erful strategy to dissect mechanisms, as exemplified by studies with 2 expression at levels resembling endogenous GATA-2 or over- GATA-1 (48–51). While the MAE system enables quantification of expression, we titrated the GATA-2–expressing and GATA-2 activity to regulate endogenous target genes (31), no analyzed HA–GATA-2 and endogenous GATA-2 expression si- GATA-2 genetic complementation systems have been described. multaneously with anti–GATA-2 antibody. HA–GATA-2 levels − Using Lin myelo-erythroid progenitor cells, we compared GATA-2 that rescued CFU-E and BFU-E and elevated CFU-GM were and mutant activities to induce myeloid and erythroid differentiation comparable to that of endogenous GATA-2 (Fig. 3E). Furthermore,

N-ZnF C-ZnF A 1 480 C 150

Hdc GENETICS 100 V296 R330 ** ** ** 50 R307 ** *** ** *** ** ** *** ** ****** ** R293 A318 0 ** 9 Gfi1 P304 6 GATA-2 R293Q P304H * * * B * Empty 3 ** -3 GATA-2 0 50 15 x 10

r Grb10 50 Tubulin M 10 GATA-2 R307W A318T R330Q mRNA Levels (Relative Units) * * Empty 5 * ** * ** ** * * -3 *** GATA-2 0 50

x 10 GATA-2 R293Q P304H R307W A318T R330Q r 50 Tubulin Empty M D Empty GATA-2 R293Q P304H R307W A318T R330Q

HA

DAPI

Merge

0.045 E A-2 * * PI anti-HA Empty GAT R307W -3 0.030 * * 50 GATA-2 x 10 * r 50 Tubulin 0.015 * M

(Relative Units) 0

HA-GATA-2 Occupancy A-2 A-2 A-2 Empty Empty Empty Empty AT Empty Empty Empty AT GATA-2R307W GATA-2 R307W GATA-2R307W G R307W GATA-2R307W GAT R307W G R307W Gata2 (+9.5) Gata2 (-18.3) Hdc (+3.7) Hdc (+11.2) Lyl1 Pro Lyl1 (-1.3) Necdin Pro

Fig. 1. GATA-2 N-finger leukemia mutations attenuate chromatin occupancy and target gene activation. (A) GATA-2 structural model based on human GATA-3 crystal structure. (B) Representative Western blot analysis with anti-HA antibody of wild-type and mutant transiently expressed in MAE cells. (C) qRT-PCR analysis of mRNA levels of GATA-2 target genes in MAE cells transiently expressing HA–GATA-2 and mutant proteins (n = 6). (D) Immunoflu- orescence analysis with anti-HA antibody in MAE cells expressing HA–GATA-2 and mutant proteins. (Scale bars, 10 μm.) (E) Quantitative ChIP analysis of HA– GATA-2 in MAE cells transiently expressing HA–GATA-2 or HA-R307W (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

Katsumura et al. PNAS | vol. 115 | no. 43 | E10111 Downloaded by guest on October 1, 2021 B GATA-2 C295A R307W

C295A C349A Empty

A -3 R R W R W R GATA-2 L D L N 50

P G T A x 10

r Tubulin T T T N 50 A G T G M T H T D 36 C Hdc A Y T P G L Q V 24 C C C C 12 ** *** *** N ++ N N ++ N ** *** Zn Zn *** V A A A 0 A C C A C C 15 G R E G L Y G T C G L Y Gfi1 10 V205 N-Finger mRNA Levels (Relative Units) 5 GATA-1 LPLPPCEARECVNCGATATPLWRRDRTGHYLCNACGLYHKMNG * * * * GATA-2 KARSCSEGRECVNCGATATPLWRRDGTGHYLCNACGLYHKMNG 0 V296 GATA-2 C295A R307W Empty GATA-2 Hyper

HA DAPI Merge -3 D E 50 Phos Hypo Ras(G12V) x 10 15 Empty r 50 M Tubulin GATA-2 R307W Ras(G12V) 1.5 GATA-2 F **

1.0 **

C295A 0.5 * *

(Relative Units) 0 Hdc mRNA Levels GATA-2 R307W Ras(G12V) GATA-2 Hyper G Phos H 300 50 Hypo ** **

-3 GATA-2 (dark) Hyper 200 50 Phos Hypo x 10 100 ** r Ras(G12V) * M 15 (Relative Units)

50 Tubulin Hdc mRNA Levels 0 GATA-2 GATA-2 C295A C295A C349A C349A Ras(G12V) Ras(G12V) 210 I GATA-2 V296G V296M C295A J Empty -3 140 50 GATA-2

x 10 70 r 50 Tubulin

M * *

(Relative Units) 0 Hdc mRNA Levels GATA-2 V296G V296M C295A Empty

Fig. 2. Structural determinants of GATA-2 function. (A, Upper) Schematic representation of C295A and C349A mutants. (Lower) Murine GATA-1 and GATA-2 N-finger amino acid sequences. (B) Representative Western blot analysis with anti-HA antibody of MAE cells transiently expressing HA–GATA-2 or mutant proteins. (C) qRT-PCR analysis of mRNA levels of GATA-2 target genes in MAE cells transiently expressing HA–GATA-2 or mutant proteins (n = 4). (D) Immunofluorescence analysis with anti-HA antibody in MAE cells expressing HA–GATA-2 or the C295A mutant. (Scale bars, 10 μm.) (E) Representative Western blot analysis with anti-HA antibody of HA–GATA-2 and HA–R307W transiently expressed in MAE cells with or without Ras(G12V). (F)qRT-PCR analysis of mRNA levels of Hdc genes in MAE cells transiently expressing HA–GATA-2 and R307W with or without Ras(G12V) (n = 5). (G) Representative Western blot analysis with anti-HA antibody of HA–GATA-2 or mutant proteins transiently expressed in MAE cells with or without Ras(G12V). This Western blot utilized a large SDS/PAGE gel to achieve maximal isoform separation. (H) qRT-PCR analysis of Hdc mRNA levels in MAE cells transiently expressing HA–GATA-2 or mutant proteins with or without Ras(G12V) (n = 3). (I) Representative Western blot analysis with anti-HA antibody of MAE cells expressing HA–GATA- 2 or mutant proteins. (J) qRT-PCR analysis of Hdc mRNA levels in MAE cells transiently expressing HA–GATA-2 or mutant proteins (n = 5). *P < 0.05, **P < 0.01, ***P < 0.001.

the level of R307W that strongly induced CFU-GM was comparable (Fig. 3 G and H). Flow cytometric analysis revealed that R307W + + to that of endogenous GATA-2 (Fig. 3F). These results highlight or T354M increased Mac1 Gr1 myeloid cells (Fig. 3I). the unique utility of the genetic complementation assay for quali- By eliminating the culture step with Epo and factor, tatively and quantitatively analyzing GATA-2–dependent pro- which favors erythroid precursors at the expense of myelo- genitor activity. Importantly, the assay was conducted under erythroid progenitors, we improved the genetic complementa- conditions in which ectopically introduced wild-type or mutant tion assay for analyzing both erythroid and myeloid differentia- GATA-2 was expressed at physiological GATA-2 levels. tion. We also utilized a more refined myelo-erythroid progenitor − + Because R307W and T354M increased the percentage of population, FACS-purified Lin Kit myelo-erythroid progeni- − CFU-GM among the colonies from 37% to 86% (R307W, P = tors, rather than the bead-sorted Lin population. CFU activity − + 0.0014) or 92% (T354M, P = 0.0009), we analyzed colony cel- was assayed immediately after Lin Kit cell isolation to mini- lularity. were more abundant in colonies derived mize the probability of cellularity transitions ex vivo (Fig. 4A). − − from R307W- or T354M-expressing progenitors, in comparison Gata2 expression was threefold lower in −77 / progenitors in with colonies derived from GATA-2–expressing progenitors comparison with wild-type progenitors (Fig. 4B). Resembling

E10112 | www.pnas.org/cgi/doi/10.1073/pnas.1813015115 Katsumura et al. Downloaded by guest on October 1, 2021 -77+/+ -77-/- Fetal liver Lin- cell Infection Culture with Colony A (E14.5) isolation Epo and SCF assay

BFU-E CFU-GM Lin- Cells B +/+ C 1.5 -77 18 21 ** ** EmptyGATA-2R307WT354M Cells 12 14 1.0 -3 - GATA-2 50 0.5 6 7 ** x 10 50 Tubulin r Colonies per 1000 Lin M 0 0 (Relative Units) 0 -77+/+ -77-/-

A-2 A-2 Gata2 mRNA Levels Empty R307WT354M Empty R307WT354M GAT GAT -/- -77-/- -77 D E GATA-2 -77+/+ -3 -3 EmptyGATA-2R307WT354M HA-GATA-2 50 HA-GATA-2 -77+/+ 50 GATA-2 -77+/+ x 10 x 10

-/- r -/- r Tubulin 50 -77 50 Tubulin -77 M M CFU-E BFU-E CFU-GM CFU-E BFU-E CFU-GM 900 16 100 800 20 10 ** ** *** ** *** * ** 7.5 * ** 12 75 600 15 *

600 Cells Cells

- ** - ***** *** 8 50 400 * 10 5.0 * *** * 300 4 25 200 * 5 2.5 Colonies per Colonies per 1000 Lin 1000 Lin 0 0 0 0 0 0 GATA-2 GATA-2 GATA-2 Empty Empty Empty EmptyEmpty T354M EmptyEmpty T354M EmptyEmpty T354M GATA-2R307W GATA-2R307W GATA-2R307W BFU-E CFU-GM +/+ -/- 10 60 -77 -77 ****** F 8 ** GATA-2 R307W ** 40

EmptyEmpty Cells 6 *** -3 - * ** HA-GATA-2 * *** GATA-2 4 50 20 +/+ x 10 ** -77

r 2 50 Tubulin ** ** -/- Colonies per -77 GENETICS M 1000 Lin 0 0 GATA-2 R307W GATA-2 R307W +/+ EmptyEmpty -/- EmptyEmpty G -77 -77 Empty Empty GATA-2 R307W T354M Pro Neu Ery Neu Neu Neu Neu Ery Ery Neu Ery Neu H 75 / Basophilic Orthochromatic Enucleated

Promyeloblast erythroblast erythroblast ** ** 50 -77+/+ -77-/- ** 25 ** *** ** Percent of Cells 0 A-2 ATA-2 EmptyEmptyGATA-2R307WT354MEmptyEmptyGATA-2R307WT354MEmptyEmptyGATA-2R307WT354MEmptyEmptyG R307WT354MEmptyEmptyGATA-2R307WT354M EmptyEmptyGAT R307WT354M Empty GATA-2 R307W T354M I 45 105 8.97 105 25.8 105 38.0 105 48.0 ** ** ** 4 4 4 4 10 2.61 10 4.81 10 20.3 10 26.1 30 ** -/- Cells (%)

-77 + 103 103 103 103 15 0 0 0 0 Gr1 + 87.5 65.1 35.6 15.3 0 Mac1 0103 104 105 0103 104 105 0103 104 105 0103 104 105 Gr1 Mac1 EmptyGATA-2R307WT354M

Fig. 3. GATA-2 leukemia mutants increase myeloid progenitor cell activity in a primary cell genetic complementation assay. (A) Schematic representation of + + genetic complementation assay. (B, Left) Representative Western blot analysis of −77 / fetal liver cells expressing HA–GATA-2 with anti-HA antibody. (Right) Quantitative analysis of CFU activity of −77+/+ fetal liver cells (n = 3). (C) qRT-PCR analysis of Gata2 mRNA levels in Lin− cells from wild-type or −77−/− fetal livers (n = 5). (D, Upper) Representative Western blot analysis with anti-HA antibody of −77−/− fetal liver cells expressing HA–GATA-2 or mutants. (Lower) − − + + Quantitative analysis of CFU activity of −77 / fetal liver cells (n = 7). −77 / fetal liver cells infected with control vector were used as control. (E, Upper) − − Representative Western blot analysis with anti–GATA-2 antibody (recognizes both HA–GATA-2 and endogenous GATA-2) of −77 / fetal liver cells expressing − − + + HA–GATA-2. (Lower) Quantitative analysis of CFU activity of −77 / fetal liver cells (n = 3). −77 / fetal liver cells infected with control vector were used as − − control. (F, Left) Representative Western blot analysis with anti-GATA-2 antibody of −77 / fetal liver cells expressing HA–GATA-2 or R307W. (Right) Quantitative analysis of CFU activity of −77−/− fetal liver cells (n = 4). −77+/+ fetal liver cells infected with control vector were used as control. Significance relative to −77−/− cells infected with empty vector was evaluated. (G) Representative image of Giemsa-stained cells from colonies. (Scale bars, 20 μm.) Ery, erythroblasts; Mac, macrophage; Neu, ; Pro, proerythroblast/promyeloblast. (H) Quantification of Giemsa stain (n = 3). (I) Flow cytometric analysis of cells isolated from colonies and stained with Gr1 and Mac1 (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001.

− − − − + − − Lin cells, −77 / Lin Kit cells produced almost no colonies erythroid cells in colonies from R307W-expressing −77 / mye- (Fig. 4C). GATA-2 expression elevated BFU-E and CFU-GM loid progenitors in comparison with GATA-2–expressing pro- − − 6.4- and 2.6-fold, respectively (Fig. 4D). While R307W did not genitors (Fig. 4 F and G). were abundant in −77 / rescue BFU-E, it increased CFU-GM 3.9-fold relative to the cells infected with empty vector, as described (20). Thus, R307W GATA-2 control (Fig. 4D). T354M also increased CFU-GM but preferentially induces granulocytes. The distinct morphology of − − − + not BFU-E (Fig. 4E). Morphological analysis revealed only rare the cells derived from these −77 / Lin Kit progenitors,

Katsumura et al. PNAS | vol. 115 | no. 43 | E10113 Downloaded by guest on October 1, 2021 A Fetal liver B - + C -/- - + (E14.5) Lin Kit Cells -77 Lin Kit Cells 1.2 - + Lin Kit cell 0.8 isolation ** 0.4 Infection (Relative Units)

mRNA Levels Gata2 mRNA 0 +/+ -/- Empty GATA-2 R307W Colony assay -77 -77 -77-/- D E BFU-E CFU-GM BFU-E CFU-GM 6 75 12 90 ** *** ***

Cells *** *** Cells + + 8 60 4 50 ** Kit Kit - - ** *** ** 30 +/+ 2 25 4 * -77 -77-/- Colonies per Colonies per 0 0 0 0 1000 Lin 1000 Lin EmptyEmpty EmptyEmpty GATA-2R307W GATA-2R307W EmptyGATA-2T354MR307W EmptyGATA-2T354MR307W +/+ F -77 -77-/- Empty Empty GATA-2 R307W

Mac Neu Ery Mac Neu Ery Neu Mac Ery Pro Neu Neu Neu

90 Macrophage G Proerythroblast/ Basophilic Orthochromatic Enucleated Granulocyte Promyeloblast erythroblast erythroblast * 60 -77+/+ -77-/- * 30 ** ** ** Percent of Cells 0

EmptyEmptyGATA-2R307WEmptyEmptyGATA-2R307WEmptyEmptyGATA-2R307WEmptyEmptyGATA-2R307WEmptyEmptyGATA-2R307WEmptyEmptyGATA-2R307W 1.5 1.8 4.5 2.4 1.5 1.5 H Gata2 Gata2 Mpo Ctsg Elane Myb * (HA) *** ** ** 1.0 1.2 3.0 1.0 1.6 ** 1.0 ** * 0.5 0.6 1.5 0.8 0.5 0.5

0 0 0 0 0 0 7.5 4.5 3 2.4 2.1 3 Kit Tal1 Lyl1 Slc4a1 Alas2 Epb4.9 ** ** ** ** 5.0 3.0 2 1.6 1.4 2 ** * 2.5 ** * 1.5 1 0.8 0.7 1 mRNA Levels (Relative Units) mRNA 0 0 0 0 0 0 A-2 EmptyEmpty EmptyEmpty EmptyEmptyGATA-2R307W EmptyEmptyGATA-2R307W GATA-2R307W EmptyEmptyGAT R307W EmptyEmptyGATA-2R307W GATA-2R307W +/+ -77 -77-/-

− + Fig. 4. GATA-2 leukemia mutants stimulate myelopoiesis ex vivo. (A) Schematic representation of rescue assay with Lin Kit cells. (B) qRT-PCR analysis of − + − − Gata2 mRNA levels in Lin Kit cells from wild-type or −77 / fetal livers (n = 3). (C) Representative image of the dishes subjected to CFU assay. (D) Quantitative − − + + analysis of CFU activity of −77 / myeloid progenitor cells. −77 / fetal liver cells infected with control vector were used as control (n = 8). (E) Quantitative analysis of CFU activity of −77−/− Lin−Kit+ cells (n = 3). (F) Representative image of Giemsa-stained cells from colonies derived from Lin−Kit+ cells. (Scale bars, 20 μm.) Ery, erythroblasts; Mac, macrophage; Neu, neutrophil; Pro, proerythroblast/promyeloblast. (G) Quantification of Giemsa stain (n = 4). (H) qRT-PCR analysis of mRNA levels in cells isolated from colonies (n = 8). *P < 0.05, **P < 0.01, ***P < 0.001.

− − − in comparison with the −77 / Lin cells (Fig. 3) reflected the inconsequential for increasing CFU-GM. We tested C349A, in assay modification in which the progenitors analyzed in Fig. 4 which the cysteine mutation destroys the structural integrity of were immediately subjected to colony assay, rather than cul- the DNA-binding C-finger. C349A had little to no activity in the tured for 1-d postinfection. Under both conditions, however, genetic complementation assay, using CFU as a read-out (SI R307W induced granulocytes. Appendix, Fig. S2 A and B), suggesting that C-finger structure Because R307W and T354M chromatin binding is reduced, and DNA binding competence is required to induce CFU-GM. based on ChIP analysis at select target genes, and they increased To assess whether additional GATA-2 leukemia mutants in- CFU-GM, we asked whether GATA-2 DNA binding activity is duce CFU-GM, we analyzed GATA-2 A318T, which has been

E10114 | www.pnas.org/cgi/doi/10.1073/pnas.1813015115 Katsumura et al. Downloaded by guest on October 1, 2021 detected more frequently than R307W. A318T induced CFU- CFU analysis that the leukemia mutants are more effective than − − + GM in Lin cells and Lin Kit cells (SI Appendix,Fig.S2 GATA-2 in inducing CFU-GM. C–E), and granulocytes were more abundant in colonies from − − + −77 Lin cells and −77 Lin Kit cells (SI Appendix, Fig. S2 F and GATA-2 Leukemia Mutant Stimulates Myelo-Erythroid Progenitor Cell G). Thus, all GATA-2 leukemia mutants tested gain a function Cycle Progression. Consistent with the increased colony number, to increase CFU-GM, differing from GATA-2 and the inactive GATA-2 or R307W expression significantly increased the num- C349A mutant. ber of cells within colonies. R307W increased cell numbers The gain-of-function myelopoiesis-stimulating activity of twofold greater than GATA-2 (Fig. 5A). Because GATA-2 (21, GATA-2 leukemia mutants was surprising, given the paradigm 52) and select target genes regulate progression, and that mutations create insufficient GATA-2 activities/levels to R307W has strong activity to induce granulocytes, we compared control stem and progenitor cell transitions and function. In GATA-2 and R307W activities to impact cell cycle progression − − − + certain cases, cis-element mutations in MDS/AML decrease in the −77 / Lin Kit cell genetic complementation assay. GATA-2 expression, consistent with haploinsufficiency. How- GATA-2 or R307W expression significantly increased S and G2/M + + ever, because elevated GATA-2 would deregulate target genes, phase cells in the Mac1 Gr1 myeloid cell population (Fig. 5 which include multiple disease-linked genes (14, 19, 20, 32), and B and C). A greater percentage of R307W-expressing cells resided can correlate with poor prognosis of AML (30), ectopically low in S phase in comparison with GATA-2–expressing cells. R307W or high GATA-2 levels/activity will disrupt the integrity of ge- was not more effective than GATA-2 in stimulating cell cycle + − − − netic networks that ensure normal hematopoiesis. progression of Mac1 Gr1 cells or Mac1 Gr1 cells (Fig. 5C). + + Gene-expression analysis in R307W-expressing cells isolated The Mac1 Gr1 myeloid cell population in S phase decreased − − + + from colonies revealed elevated myeloid gene (Mpo, Ctsg, and significantly in −77 / vs. −77 / cells (Fig. 5 D and E). Cell − − − + Elane) and reduced erythroid gene (Slc4a1, Epb4.9, and Alas2) survival was analyzed in the −77 / Lin Kit cell genetic com- expression (Fig. 4H). Genes expressed in HSPCs and erythroid plementation assay. Although altered survival was not detected in − − + + − − cells (e.g., Myb, Kit, and Tal1) were reduced in −77 / cells, and Mac1 Gr1 cells and Mac1 Gr1 cells, GATA-2 or R307W ex- + − GATA-2 rescued expression. While R307W did not affect Tal1 pression significantly increased live cells in Mac1 Gr1 cells (Fig. expression, it was more effective than GATA-2 in elevating Myb 5F). Analysis of the erythroid cell population revealed the loss of − − expression (Fig. 4H). These results reinforce the conclusion from this population in −77 / cells. However, there was no obvious GENETICS )

5 9 Empty GATA-2 R307W A ** B 400 Cells

6 * + 200 400 Gr1

3 + 200 ** 100 200

0 Mac1 0 -/- Cell Number (x10 0 0 EmptyGATA-2R307W 0 50 100 150 200 0 50 100 150 200 0 50 100 150 200

-77 DAPI -/- + + -/- + - -/- - - -77 Mac1 Gr1 -77 Mac1 Gr1 -77 Mac1 Gr1 C 100 100 100 *** ** *** 75 ** 75 ** 75 50 ** 50 50 *** ** ** *** 25 25 25 ** ** **

Percent of Cells 0 0 0

ATA-2 ATA-2 ATA-2 EmptyGATA-2R307WEmptyGATA-2R307WEmptyGATA-2R307W EmptyG R307WEmptyGATA-2R307WEmptyGATA-2R307W EmptyGATA-2R307WEmptyG R307WEmptyG R307W G1 S G2/M G1 S G2/M G1 S G2/M -/- + + -77 Mac1 Gr1 DE+/+ -/- -77 -77 G1 S G2/M 400 300 100 16 10.0

Cells * 75 12 7.5 + 200 200

Gr1 50 8 5.0

+ 100 25 4 2.5 Percent of Cells Mac1 00 0 0 0 0040 80 120 40 80 120 DAPI -77+/+ -77-/- -/- + + -/- + - -/- - - -77 Mac1 Gr1 -77 Mac1 Gr1 -77 Mac1 Gr1 F 100 100 100 Live Early Late Dead Live Early Late Dead Live Early Late Dead 80 80 *** 80 60 60 60 *** * 40 40 * 40 * ** 20 20 ** 20

Percent of Cells 0 0 0 Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty Empty R307W R307W R307W R307W R307W R307W R307W R307W R307W R307W R307W R307W GATA-2 GATA-2 GATA-2 GATA-2 GATA-2 GATA-2 GATA-2 GATA-2 GATA-2 GATA-2 GATA-2 GATA-2

-77+/+ -77-/-

Fig. 5. GATA-2 leukemia mutant increases cell cycle progression in a genetic complementation assay. (A) Quantification of cell number in colonies derived from −77−/− Lin−Kit+ cells infected with empty vector, GATA-2 expression vector, or R307W expression vector. (B) Flow cytometric analysis of cell cycle in − − + + + + + + −77 / Mac-1 Gr-1 cells isolated from colonies. (C) Quantification of cell cycle status (n = 5). (D) Flow cytometric analysis of cell cycle in −77 / Mac-1 Gr-1 cells − − + + + − and −77 / Mac-1 Gr-1 cells. (E) Quantification of cell cycle status (n = 4). (F) Quantification of early apoptotic (Annexin V DRAQ7 ), late apoptotic (Annexin + + − + V DRAQ7 ), and dead cells (Annexin V DRAQ7 ) by flow cytometry (n = 4). *P < 0.05, **P < 0.01, ***P < 0.001.

Katsumura et al. PNAS | vol. 115 | no. 43 | E10115 Downloaded by guest on October 1, 2021 change in survival of R1 population erythroid precursor cells (SI A Appendix,Fig.S3). This analysis indicates that GATA-2 stimulates GATA-2 R307W T354M A318T C295A C349A V296M −/− cell cycle progression in −77 myelo-erythroid progenitor cells, Gene and while the leukemia mutant not only retains this activity, its Regulation Chromatin activity can exceed that of GATA-2. Occupancy ND ND ND ND Colony Discussion Formation ND ND Erythroid The paradigm for how GATA2 mutations instigate MDS/AML Proliferation ND ND involves haploinsufficiency (27): inadequate GATA-2 production Myeloid to fulfill its requirement to establish/maintain genetic networks Proliferation ND ND that govern HSPC transitions. Heterozygous GATA2 coding re- gion mutations inhibit DNA binding or corrupt the ORF, while Low High +9.5 enhancer mutations reduce GATA2 expression. Both classes of mutations yield subphysiological GATA-2 levels. High-level B Myeloid Progenitors GATA-2 expression in humans has been correlated with poor (-77-/- Lin-Kit+) prognosis of AML (30), and GATA-2 overexpression can suppress bone marrow hematopoiesis in mice (22). Mutant We devised a genetic complementation assay that enables – Control quantification of GATA-2 dependent myelo-erythroid pro- GATA-2 genitor differentiation, endogenous target gene regulation, and cellular functions. We discovered that GATA-2 mutants pre- dicted to be inactive or to have attenuated activity retain activity in primary cells. Of particular interest were their activities to Macrophage-biased Myelo-erythroid Excessive granulocyte differentiation differentiation differentiation/proliferation induce granulocytes and stimulate cell cycle progression, which −/− exceeded that of GATA-2. Whereas GATA-2 induced −77 erythroid granulocyte macrophage myeloid progenitor cells to produce both erythroid and myeloid cells, R307W selectively increased granulocytes (Fig. 6). Thus, Fig. 6. GATA-2 leukemia mutations: gain-of-function and loss-of-function leukemia mutations corrupt, without abrogating, GATA-2 function. consequences. (A) Molecular activities of wild-type and mutant GATA-2. It is therefore instructive to consider the relationship between DNA binding capacity of T354M was described previously (31). ND, not de- our findings and AML pathogenesis involving GATA2 muta- termined. (B) The −77−/− myeloid progenitor cells differentiate preferentially tions. We propose that insufficient or elevated GATA-2 levels/ into macrophages ex vivo. While GATA-2 expression induces erythroid cells activity corrupt GATA-2–dependent genetic network integrity, and granulocytes, GATA-2 leukemia mutants stimulate granulocyte differ- and both GATA-2 loss-of-function and gain-of-function may entiation and proliferation, and this activity can exceed that of GATA-2. constitute pathogenic mechanisms. Thus, GATA-2 leukemia mutants exhibit a gain-of-function activity to stimulate myelopoiesis, with a concomitant loss of activity to stimulate Although the functional consequences of GATA-2 N-finger . mutations detected in a subset of AML patients were unclear, prior studies implicated the GATA-1 N-finger in DNA binding in certain contexts (38). However, whether the N-finger is an GATA-2–mediated Hdc induction. GATA-1 R216 is important essential determinant of chromatin occupancy in physiological for GATA-1 recognition of palindromic GATA-motifs (59), contexts is uncertain. Herein, we demonstrated that N-finger and in vivo analysis demonstrated that R216 mutation de- leukemia mutations resembled C-finger mutations in attenuat- creased occupancy at sites containing single GATA-motifs ing GATA-2 chromatin binding and target gene activation. (60). Our studies revealed that GATA-2 R307W strongly in- However, analysis of C295A and C349A mutations that disrupt creased CFU-GM. Mutations of this conserved of N- and C-finger structure, respectively, indicated that N-finger GATA-1, GATA-2, and GATA-3 were described in hemato- mutants were more effective than C-finger mutants in activating logic malignancy and anemia patients. target genes. N-finger, but not C-finger, mutants were responsive How do GATA-2 leukemia mutants induce myeloid cell pro- to Ras(G12V)-mediated GATA-2 activation. These results liferation despite their crippled transcriptional activity? It is in- highlight functional differences in N- and C-finger mutants. structive to consider the consequences of mutating GATA-1 C-finger mutants have been described as germline mutations V205, which strongly reduces GATA-1–mediated activation and in familial MDS/AML (10–12). Somatic N-finger mutations were repression of target genes that require FOG-1, the majority of reported in patients with acute erythroid leukemia (35) and GATA-1 target genes. V205 mutations inhibit chromatin occu- AML with biallelic mutation of CEBPA (53). Analyses of familial MDS/AML have identified the co-occurrence of T354M with pancy at select target genes and induce ectopic chromatin oc- acquired ASXL1 mutations (54, 55). Other mutations reported cupancy at sites not normally occupied by GATA-1 (47). This to co-occur with GATA2 mutations include NRAS, WT1, and chromatin redistribution mechanism may have broader appli- DDX41, among others (56, 57). In addition, CDC25C mutations cability to mutations that influence DNA – in familial platelet disorder, which predisposes to AML, can sequence-specificity or coregulator transcription factor interac- involve subsequent GATA2 somatic mutations (58). Further tions and therefore indirectly impact chromatin occupancy. Be- studies with large patient cohorts are required to rigorously an- cause various factors are implicated in binding GATA factors, in alyze genotype– relationships. principle, mutations that impact chromatin occupancy might The GATA-1 N-finger is an essential determinant of GATA- inhibit or enhance such interactions, thereby altering networks 1function(40).V205mediates FOG-1 binding and FOG-1– established/maintained by interactors. In addition, DNA binding- dependent transcriptional activation and repression (40). impaired GATA factor mutants might retain the capacity to be While R216 does not impact FOG-1 binding, it contributes to recruited into chromatin complexes via –protein interac- the regulation of select target genes (43). Our study revealed tions. Regardless of these potential mechanisms, it will be in- that GATA-2 R307, the structural equivalent of GATA-1 structive to consider the relationship between the unexpected R216, is critical for GATA-2 function, while GATA-2 V296, myelopoiesis-inducing activity resulting from human GATA-2 which is equivalent to GATA-1 V205, is dispensable for disease mutations and leukemogenesis.

E10116 | www.pnas.org/cgi/doi/10.1073/pnas.1813015115 Katsumura et al. Downloaded by guest on October 1, 2021 Materials and Methods temperature. After washing, slides were blocked with 10% BlokHen (Aves Cell Culture. MAE cells (31) were maintained in medium 200 supplemented Labs) in 0.1% Tween 20 in PBS for 1 h at 37 °C and then incubated with low-serum growth supplement (Thermo Fisher Scientific) and 1% penicillin/ primary antibody (anti-HA, Covance HA11) in 2% BlokHen at 4 °C overnight. streptomycin (Thermo Fisher Scientific). Cells were transfected with After washing, slides were incubated with secondary antibody for 1 h at Nucleofector II (Lonza). Fetal liver hematopoietic precursor cells were cul- 37 °C. Slides were washed and mounted using Vectashield mounting me- tured in StemPro-34 (Thermo Fisher Scientific) with 1× nutrient supplement dium with DAPI (Vector Laboratories). with 2 mM glutamax (Thermo Fisher Scientific), 1% penicillin/streptomycin − − + (Thermo Fisher Scientific), 100 μM monothioglycerol (Sigma), 1 μMdexa- Colony Forming Unit Assay. For CFU assays, dissociated Lin cells or Lin Kit methasone (Sigma), 0.5 U/mL of , and 1% conditioned medium cells from E14.5 fetal livers were plated in duplicate in Methocult M3434 from a Kit ligand-producing Chinese hamster ovary (CHO) cells. Cells were complete media (StemCell Technologies) at 1 × 103 cells per 35-mm plate. cultured in a humidified incubator at 37 °C and 5% carbon dioxide (61). Plates were incubated for 8 d, and colonies were identified and enumerated. For subsequent analysis, cells were isolated from the plates using PBS con- Plasmids. Murine GATA-2 cDNA was cloned into pcDNA4TOFHA vector (kindly taining 50% calf bovine serum and centrifuged for 10 min to remove provided by Danny Reinberg, New York University, New York) and pMSCV methylcellulose. vector (kindly provided by Mitchell Weiss, St. Jude Children’s Research Hospital, − + Memphis, TN). XZ-201Ras(G12V) was kindly provided by Jing Zhang, Univer- Flow Cytometry. For Lin Kit cell sorting, E14.5 fetal liver cells were dissoci- – sity of Wisconsin Madison, Madison, WI. ated and resuspended in PBS with 10% FBS and passed through 25-μm cell strainers to obtain single-cell suspensions before antibody staining. After Quantitative Real-Time RT-PCR. Total RNA was purified with TRIzol (Thermo cells were stained with FITC-conjugated CD5 (11-0051-85), CD8 (11-0081-85), μ Fisher Scientific). cDNA was prepared by annealing 1 g of RNA with 250 ng of CD19 (11-0193), IgM (11-5890), Il7Ra (11-1271), AA4.1 (11-5892; Thermo a 1:5 mixture of random hexamer and oligo (dT) primers heated at 68 °C for Fisher Scientific), B220 (103206), CD3 (100306), CD4 (100406), TER-119 10 min. This was followed by incubation with Murine Moloney Leukemia (116206), and PE Cy7-conjugated c-Kit (105814; Biolegend) antibodies, Virus Reverse Transcriptase (Thermo Fisher Scientific) with 10 mM DTT, RNAsin − + Lin Kit cells were collected on a FACSAria II cell sorter (BD Biosciences). For (Promega), and 0.5 mM dNTPs at 42 °C for 1 h. This mixture was heat- analysis of myeloid cells, cells isolated from colonies were fixed in 2% inactivatedat95°Cfor5minand diluted to a final volume of 100 μL. paraformaldehyde for 10 min at 37 °C. After permeabilization overnight at −20 °C in 95% methanol, cells were incubated for 1 h in HBSS/4% FBS at Quantitative ChIP. ChIP analysis in MAE cells was conducted as described 4 °C. After incubation with Fc block on ice for 15 min, cells were stained with previously (62). Samples containing 3 × 106 cells were cross-linked with 1% anti-mouse Mac1-APCe780 (47-0112-82; Thermo Fisher Scientific) and anti- formaldehyde for 10 min. Lysates were immunoprecipitated with rabbit polyclonal anti-HA antibody using rabbit preimmune serum (Covance) as a mouse Gr1-PE-Cy7 (108416; Biolegend) at room temperature for 30 min. GENETICS control. DNA was quantitated by real-time PCR (Applied Biosystems Viia DAPI was added at this stage for cell cycle analysis. Cells were washed twice 7 instrument) with SYBR green fluorescence, and the amount of product was in PBS before analysis and analyzed on a LSR II flow cytometer (BD Biosci- determined relative to a standard curve created from serial dilution of ences). The data were analyzed using FlowJo v10.1 software (TreeStar) and input chromatin. ModFit LT software (Verity Software House).

Protein Analysis. Protein samples were isolated by centrifugation of 1 × 106 Apoptosis Assay. To quantify apoptosis after Mac1/Gr1 staining, cells were cells from each condition, washing with cold PBS, and lysing in SDS sample washed in Annexin V buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl2,pH7.4) buffer (25 mM Tris, pH 6.8, 2% β-mercaptoethanol, 3% SDS, 0.005% bro- and stained with Annexin V-Pacific blue (A35122; TermoFisher) and DRAQ7 mophenol blue, 5% glycerol). Samples were boiled for 10 min and stored (ab109292; Abcam) for 15 min in the dark at room temperature. at −80 °C. Samples were resolved by SDS/PAGE, and proteins were detected by semiquantitative Western blotting with ECL Plus (Pierce). For primary Statistical Analysis. Statistical significance was determined by Student’s t-test fetal liver cells, FEMTO supersignal (Pierce) was used. using web-based GraphPad (https://www.graphpad.com).

Immunofluorescence. Cells were cytospun and fixed with 3.7% paraformal- ACKNOWLEDGMENTS. This work was supported by National Institutes of dehyde in PBS for 10 min at room temperature. Slides were washed with PBS, Health Grants DK68634 and DK50107 (to E.H.B.) and K01DK113117 (to and cells were permeabilized with 0.2% Triton X-100 for 10 min at room K.J.H.), and by the Carbone Cancer Center P30CA014520.

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