
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

39229096
10.1101/2024.08.16.607812
preprint
1
Article
The Clonal Hematopoiesis-associated Gene Srcap Plays an Essential Role in Hematopoiesis
http://orcid.org/0000-0002-4509-208X
Wong Terrence N. 1*
Mychalowych Anna 1
Feldpausch Ellie R. 1
Carson Alexander 1
Karpova Darja 2
Link Daniel C. 2
1 Division of Hematology-Oncology, University of Michigan, Ann Arbor, Michigan.
2 Division of Oncology, Washington University School of Medicine, St. Louis, Missouri.
Authorship

Contributions: T.N.W. and D.C.L. initiated the project, designed the research, and wrote the paper with input from other authors. T.N.W., A.M., E.R.F., D.K., and A.C. performed the research and analyzed the data.

* Corresponding author: Terrence N. Wong, University of Michigan, 109 Zina Pitcher Place, BSRB 2053, Ann Arbor, MI, 48109;tnwong@med.umich.edu
19 8 2024
2024.08.16.607812https://creativecommons.org/licenses/by-nd/4.0/ This work is licensed under a Creative Commons Attribution-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, and only so long as attribution is given to the creator. The license allows for commercial use.
nihpp-2024.08.16.607812.pdf
Somatic mutations arising in hematopoietic stem cells (HSCs) may provide the latter with a fitness advantage, allowing the mutant HSC to clonally expand. Such mutations have been recurrently identified in the chromatin modifier, SRCAP, in both non-malignant and leukemic clones, suggesting that this gene plays a significant role in hematopoiesis. We generated a conditional Srcap loss of function murine model and determined the consequences of hematopoietic-specific loss of this gene. We show that Srcap is essential for normal fetal liver erythropoiesis and monocytopoiesis. In Srcap deficient fetal livers, the number of phenotypic HSCs is similar to that of controls, but these HSCs exhibit a profound repopulating defect. Likewise, conditional deletion of Srcap during adult hematopoiesis results in a rapid loss of HSCs. Loss of Srcap is associated with evidence of increased DNA damage in HSCs and lineage-restricted progenitors as assessed by y-H2AX expression. Consistent with this finding, we observed strong transcriptional upregulation of the p53 pathway in Srcap deficient erythroid precursors. Collectively our data highlight the importance of Srcap in maintaining HSC function and supporting hematopoietic differentiation and suggests that it plays an essential role in maintaining genomic integrity.
==== Body
pmcIntroduction

Hematopoietic stem cells (HSCs) acquire somatic mutations with age.1 A subset of these mutations, which arise in specific genes, may provide HSCs with a fitness advantage, allowing them to clonally expand in a process termed clonal hematopoiesis (CH).2,3 Such mutations can significantly alter the behavior of both HSCs and the maturing hematopoietic populations that are derived from them. These impacts may involve their competitive fitness,4 response to cellular stress,5–7 leukemogenic potential,8,9 inflammatory status,10 and interactions with non-hematopoietic tissues11 with potential implications in the development of both leukemia8,9 and non-hematologic disorders.12 As such, understanding the role that genes that are recurrently mutated in CH and malignant leukemias have in hematopoietic populations is of significant biological and clinical importance.

SRCAP is a member of the Snf2 family of ATPases.13 Along with its ortholog EP400, SRCAP is responsible for the incorporation of the variant histone H2A.Z into the nucleosome. H2A.Z incorporation has been associated with multiple intracellular processes including heterochromatin regulation, the DNA damage response, and transcriptional regulation including both activation and repression.13 On a cellular level, H2A.Z is essential for the proper functioning of both embryonic14 and tissue-specific15,16 stem cells, including both their self-renewal and differentiation. Not surprisingly, the dysregulation of H2A.Z has been linked to the development of multiple malignancies.17,18 However, the mechanisms underlying how this dysregulation promotes carcinogenesis remain poorly understood. The Srcap complex has also been shown to play an integral role in non-malignant hematopoiesis with hematopoietic-specific loss of Znhit1, a component of this complex, impacting both HSC function (quiescence and repopulating ability) and hematopoietic differentiation.15,19

We previously assessed for CH in 81 patients who had received prior treatment with cytotoxic therapy.7 In addition to mutations in well-characterized CH genes (e.g. DNMT3A, TET2, TP53, PPM1D), we identified mutations in SRCAP with a variant allele frequency (VAF) > 0.1% in ~16% of these patients. SRCAP mutations have also been observed to arise in CH emerging after allogeneic transplantation20 and after treatment for myelodysplastic syndrome (MDS).21 In terms of malignant hematologic disease, mutations in SRCAP have been identified in 0.9% of MDS cases22 and in 5% of core binding factor AML (CBF-AML) cases as a member of the founding clone.23 Germline mutations in SRCAP have previously been associated with the genetic disorder Floating-Harbor syndrome (FHS), which is characterized by abnormal facial features, skeletal malformations, and delayed speech development.24 However, unlike FHS-associated mutations, which typically occur in exons 33–34 of SRCAP, CH and AML-associated mutations (which include truncating frameshift and nonsense mutations) have been identified throughout the entire coding region of SRCAP. Interestingly, the majority of CBF-AML associated SRCAP mutations are truncating in nature.23 These findings suggest that SRCAP plays a significant role in the regulation of hematopoiesis and that a disruption to SRCAP function may perturb non-malignant hematopoiesis with potential leukemogenic consequences. For these reasons, we assessed how loss of Srcap impacts hematopoiesis in mice.

Materials and methods

Mouse models

Mice were maintained under standard pathogen-free conditions with animal studies conducted in accordance with IACUC-approved University of Michigan protocols. Using CRISPR/Cas9, the Transgenic, Knockout and Micro-Injection Core at Washington University School of Medicine modified the germline of mice on a C57Bl/6 background, inserting a loxP site before exon 11 and after exon 16 of the Srcap gene. Proper insertion of the loxP sites was verified with targeted next-generation sequencing. Vav-iCre (strain 008610) and Mx1-Cre (strain 003556) mice were purchased from The Jackson Laboratory.

For experiments involving adult mice, experimental and control mice were age (8–12 weeks) and sex-matched. For timed mating experiments, 1–3 females were housed with a male for 2 days, separated, and monitored with serial weight checks to assess for pregnancy. Floxing of Srcap with the Mx1-Cre strain was induced by the administration of 1.0 μg/g of poly(I:C) (Cytiva) every other day for five total doses as previously described.25

Antibodies

Flow cytometry was performed with the following antibodies (Biolegend unless otherwise noted): Ly-6C/G (RB6–8C5, Gr-1), Ly6G (1A8), Ly6C (HK1.4), CD115 (AFS98), F4/80 (BM8), CD11b (M1/70), Ly-6A/E (D7, Sca1), CD3e (145–2C11), CD16/32 (93, Fc 2R/ Fc 3R), CD34 (RAM34), CD45R (RA3–6B2, B220), CD45.1 (A20, Ly5.1), CD45.2 (104, Ly5.2), CD48 (HM48–1), CD71 (BD Biosciences, C2F2, TfR1), CD117 (2B8, ckit), CD150 (TC15–12F12.2, SLAM), CD201 (ebioscience, 1560, Epcr), CD86 (GL-1), TER-119 (Ter-119), CD44 (IM7), Ki-67(Invitrogen, SolA15), and γ-H2AX (Ser139) (EMD Millipore, JBW301). Cell fixation and permeabilization were performed using the FoxP3 Transcription Factor Fixation/Permeabilization Kit (ebioscience) as per manufacturer’s instructions.

Flow cytometry

Cells were stained using standard protocols. Flow cytometry data were collected on a 14-color, 3-laser LSRFortessa (BD Biosciences) or a 10-color, 4-laser Gallios (Beckman Coulter) flow cytometer. Cell sorting was performed with a Sony MA900 cell sorter.

RNA expression

RNA was isolated from cell populations using Trizol LS (Thermo Fisher) as per manufacturer’s instructions. RNA-Seq was performed on ribo-depleted RNA with the library preparation and sequencing performed by the University of Michigan Advanced Genomics Core and analysis performed by the University of Michigan Bioinformatics Core.

Bone marrow transplantation

For transplantation experiments, Ly5.1/5.2 recipient mice were conditioned with 1,000–1,100 cGy in split dosing from either a 137cesium or x-ray source prior to the retro-orbital transplantation of bone marrow or fetal liver cells. Prophylactic antibiotics (trimethoprim-sulfamethoxazole (Alpharma) or neomycin/polymyxin B (Sigma)) were given during the initial two weeks following transplantation.

Quantification and statistical analysis

Analysis and quantification of flow cytometry data were performed using FlowJo software (Tree Star). Gene Set Enrichment Analysis (GSEA) of RNA-Seq data was performed with GSEA software,26 using the Hallmark gene set annotation pathways. Statistical analysis was done using GraphPad Prism 9 with the specific analytic tests performed detailed in the appropriate figure legends. Unless otherwise noted, data is represented as the mean ± the standard error of the mean.

Results

Generation of a mouse model with conditional loss of Srcap.

In mice, germline homozygous loss of Srcap results in early embryonic lethality.27 Therefore, to investigate the impact of Srcap loss on hematopoiesis, we used CRISPR/Cas9 to generate a conditional Srcap loss of function murine model with loxP sites flanking exons 11–16 of the gene (Figure 1A). Excision of these exons results in the loss of Srcap’s catalytic ATPase domain and a frameshift mutation. The breeding of Vav1-iCre+/−Srcapfl/+ and Srcapfl/fl mice together produced no pups with the Vav1-iCre+/−Srcapfl/fl genotype. However, 50 of the first 211 embryos (26 litters) assessed from E13.5 to E16.5 possessed this genotype, consistent with Mendelian genetics. PCR genotyping of sorted fetal liver erythroid progenitor cells from Vav1-iCre+/−Srcapfl/fl embryos showed efficient floxing (Figure 1B), while RNA-Seq data from the corresponding fetal liver stage 1 erythroid progenitor cells showed that exons 11–16 were efficiently excised from the Srcap mRNA transcript (Figure 1C). Thus, Srcap is efficiently deleted in hematopoietic cells in Vav1-iCre+/−Srcapfl/fl mice with loss of this gene resulting in embryonic lethality. Consistent with this, Srcap deficient E13.5/E14.5 embryos displayed a significantly paler phenotype than their corresponding control littermates with either full expression or haploinsufficiency of Srcap (Figure 1D).

Loss of Srcap causes aberrant fetal hematopoiesis.

We first analyzed how Srcap loss impacts mature hematopoietic populations in the fetal liver. Mutations in SRCAP have been recurrently identified in myelodysplastic syndrome, suggesting that this gene may play a role in hematopoietic maturation. Consistent with their appearance after H&E staining, Vav1-iCre+/−Srcapfl/fl fetal livers possessed a significantly decreased number of fetal liver cells, with a marked reduction in erythroid Ter119+ cells (Figure 2A–B, Supplemental Figure 1A–B). Interestingly, the number of stage 1 erythroid progenitors (as assessed with CD44 and Ter119 staining along with cell size) was similar between Vav1-iCre+/−Srcapfl/fl embryos and other genotypes. In contrast, the number of later stage erythroid progenitors was significantly reduced (Figure 2C–D, Supplemental Figure 1C). Thus, fetal liver erythroid progenitor populations may display differing tolerances to Srcap loss, with more mature erythroid populations being particularly sensitive to loss of this gene.

We also assessed how loss of Srcap impacts fetal liver myeloid cell maturation. Although the number of CD11b+CD115− phenotypic fetal liver granulocytes was similar in embryos with normal expression, haploinsufficiency, and complete loss of Srcap, embryos with complete loss of this gene had significantly fewer phenotypic CD115+ monocytes (Figures 3A–3B; Supplemental Figure 2A). To ensure that this phenotype was not due to downregulation of CD115, we also used multiple CD115-independent strategies to differentiate between granulocytes and monocytes These included assessing the surface expression of Ly6C (Figures 3A–3B; Supplemental Figure 2B), Ly6G, and F4/80 (Supplemental Figures 2C–E). Results from these different approaches all demonstrated a marked decline in phenotypic monocytes in concordance with the results obtained with CD115, suggesting that monocytic cells are particularly sensitive to the loss of Srcap.

To determine if Srcap loss also impacts earlier stages of fetal liver myeloid development, we assessed the number of myeloid progenitors using CD34 and CD16/32 staining. Embryos with loss of Srcap had decreased numbers of common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte-erythrocyte progenitors (MEPs) (Figure 3D; Supplemental Figure 2F) with a particularly marked decline in CMPs. GMPs have previously been demonstrated to be heterogeneous populations and, using cell surface markers, divisible into granulocyte-biased and monocyte-biased cells.28 Consistent with the decrease in mature monocytic populations, the GMP populations of embryos with Srcap loss were characterized by a marked loss of monocyte-committed progenitors (MPs) in contrast to more immature Ly6C− cells or granulocytic-committed progenitors (GPs) (Figure 3E–F). In total, these data suggest that monocytic populations are particularly sensitive to Srcap loss with almost no monocytic-biased progenitors or mature monocytic populations present upon loss of this gene. Collectively, these data show that Srcap is required for normal monocytic and erythroid development during fetal hematopoiesis in mice.

Loss of Srcap abrogates fetal HSC function.

In addition to MDS, mutations in SRCAP have also been recurrently identified in AML. Mutations associated with the development of myeloid malignancies have often been found to provide HSCs with a fitness advantage (e.g. mutations in DNMT3A and TET2).4,29 However, several classes of myeloid malignancy-associated mutations (e.g. impacting the spliceosome or cohesin complexes) have paradoxically been observed to induce defects in HSC function.30,31 We therefore analyzed how Srcap loss impacts immature hematopoietic populations. In contrast to more differentiated populations, phenotypic CD150+CD48− KLS HSCs were present at similar numbers in Vav1-iCre+/−Srcapfl/fl fetal livers when compared to other genotypes (Figures 4A–B; Supplemental Figure 3A). Interestingly, surface Sca-1 expression was increased in Srcap knockout HSCs (Figure 4A). To avoid potential confounding issues secondary to Sca-1 marker dysregulation, we also classified phenotypic HSCs with Sca-1-independent methods. Using the previously characterized EPCR32 (Figure 4C; Supplemental Figures 3B–C) and CD8633 (Supplemental Figures 3D–F) markers, we similarly identified a similar number of phenotypic fetal liver HSCs in embryos with and without Srcap. When assessed at this time point, hematopoietic progenitor populations lacking in Srcap exhibited increased cellular proliferation as assessed by Ki-67 and 7-AAD staining when compared to corresponding cells with normal expression of Srcap or Srcap haploinsufficiency (Supplemental Figure 3G). This was particularly apparent in the immature CD150+CD48− KLS compartment and may be secondary to the need to compensate for the previously demonstrated decline in more mature hematopoietic populations associated with the loss of Srcap.

We next analyzed the functional properties of fetal liver HSPCs lacking Srcap by assessing their ability to repopulate following transplantation. HSPCs with normal Srcap expression and HSPCs with Srcap haploinsufficiency showed a similar ability to compete against wild-type adult bone marrow HSCs in transplantation assays. In contrast, HSPCs lacking Srcap failed to repopulate lethally irradiated recipients both at early and later time points (Figure 4D) with a failure to contribute to either the myeloid or lymphoid lineages (Figure 4E). Following transplantation, Srcap deficient HSPCs also did not reconstitute any of the recipient hematopoietic progenitor populations (Figure 4F). Thus, although phenotypic HSCs are present in Srcap deficient fetal livers, they are functionally impaired.

Loss of Srcap abrogates adult HSC function.

Next, we assessed whether adult HSCs recapitulated the phenotypes of their fetal liver counterparts. Mx1-Cre+/−Srcapfl/fl mice treated with poly(I:C) began exhibiting signs of illness and died within two-three weeks of drug administration (Figure 5A). Mx1-Cre+/−Srcapfl/fl mice sacrificed at the first sign of illness displayed a decline in peripheral blood counts (Figure 5B) and a progressive loss of bone marrow c-kit+ cells (Figure 5C), suggesting that Srcap plays an essential role in maintaining adult hematopoiesis. To determine if this role is cell-intrinsic in nature, we non-competitively transplanted 5×106 bone marrow cells with inducible loss of Srcap (Mx1-Cre+/−Srcapfl/fl ; Cre+) or control (Srcapfl/fl) bone marrow cells into lethally irradiated recipients followed by poly(I:C) treatment 5–6 weeks post-transplantation. As expected, control bone marrow cells efficiently repopulated the recipient hematopoietic compartment. However, Cre+ bone marrow cells failed to reconstitute hematopoietic populations in recipient mice (Figure 5D). This included both the myeloid and lymphoid compartments (Supplemental Figure 4A) and the bone marrow hematopoietic progenitor populations (Figure 5E). Of note, we observed an initial decline in Cre+ repopulation even before the administration of poly(I:C), potentially due to leakiness of the Mx1-Cre transgene caused by post-transplantation inflammation. These data show that hematopoietic-intrinsic Srcap is essential for the proper function of adult HSCs.

In clonal hematopoiesis and in myeloid malignancies, SRCAP mutations are heterozygous in nature. However, fetal liver hematopoiesis is normal in Vav1-iCre+/−Srcapfl/+ mice, with no significant defects either at steady state or after transplantation (see Figures 2–4). We next determined whether the same was true for adult HSCs by competing bone marrow cells from mice with constitutive germline Srcap haploinsufficiency (Srcap+/− genotype) against an equal number of age and sex-matched wild-type competitors in transplantation assays. Like fetal liver hematopoietic cells, adult bone marrow cells with heterozygous loss of Srcap maintained an overall normal repopulating capacity both in primary (Figure 5F) and secondary (Figure 5G) transplantation experiments. Interestingly, Srcap+/− bone marrow cells showed a similar ability to repopulate the myeloid compartment as wild-type control cells after both primary and secondary transplantation. In contrast, a modest advantage in repopulating the lymphoid compartment after primary transplantation (Supplementary Figure 4B) was observed, which does not persist after secondary transplantation (Supplementary Figure 4C). This is similar to the phenotype observed upon transplanting HSCs with heterozygous expression of an Srcap truncation mutant allele.34

Collectively, these data show that adult and fetal liver hematopoiesis is maintained in Srcap haploinsufficient mice, with no evidence that Srcap haploinsufficiency confers a fitness advantage to HSCs.

Cellular impacts of Srcap loss

Finally, we investigated the transcriptomic consequences of Srcap loss. We focused on fetal liver stage 1 erythroid progenitors, which is the stage prior to where we observed a significant loss in the erythroid population. We used RNA-Seq to compare cells with normal Srcap expression, heterozygous loss of Srcap, and complete loss of Srcap. Consistent with the phenotypic consequences of Srcap deficiency, cells with normal Srcap expression or heterozygous loss of Srcap were similar transcriptionally, while both were distinct from cells having complete Srcap loss. (Figure 6A). The top upregulated gene expression pathway in Srcap deficient erythroid cells was the p53 pathway (Figures 6B–C; Supplemental Table 1). Other top hits included inflammatory signaling and apoptosis pathways. Interestingly, Srcap deficient fetal liver hematopoiesis was associated with increased CD44 erythroid progenitor (Figure 2C) and Sca1 HSPC (Figure 4A) surface expression, consistent with the reported increase in these two surface markers following exposure to inflammatory signaling.35,36

SRCAP has previously been proposed to play a direct role in DNA double-strand break repair,37 and the presence of a commonly identified Srcap truncation mutation was recently demonstrated to alter the expression of multiple genes involved in the repair of DNA damage.34 Thus, we assessed if the transcriptional upregulation of p53 target genes associated with Srcap loss was accompanied by evidence of increased DNA damage, focusing on the fetal liver HSPC compartment due to the robust DNA damage machinery maintained in fetal liver HSCs.38 In control embryos, γ-H2AX median fluorescent intensity (MFI) increased as hematopoietic progenitors matured from HSCs to more differentiated progenitors and was higher in cells in the G2M phase of the cell cycle versus cells with 2N DNA content (Figure 6D–E), consistent with increased γ-H2AX levels in actively replicating cells.39 Compared to controls, Vav1-iCre+/−Srcapfl/fl hematopoietic progenitors had an increased γ-H2AX MFI in all assessed stages of hematopoietic progenitor maturation and in cells with either 2N or 4N DNA content, with the increase particularly prominent in the immature HSC compartment. Collectively, these data suggest that the loss of Srcap is associated with evidence of increased DNA damage and concurrent activation of the p53 pathway.

Discussion

In this study, we generated a conditional Srcap loss of function murine model to determine the functional consequences of hematopoietic-specific loss of this gene. We discovered that hematopoietic-specific Srcap loss is embryonic lethal. Certain hematopoietic populations were particularly sensitive to Srcap loss with its ablation resulting in the early decline of fetal liver erythroid progenitor and monocytic populations while phenotypic granulocytic and HSPC populations were initially preserved. However, fetal liver HSPCs lacking Srcap failed to repopulate in bone marrow transplantations assays, and adult HSPCs with induced loss of Srcap failed to maintain hematopoiesis, indicating that this gene is essential for both hematopoietic maturation and HSPC function.

Indeed, AML-associated mutations in SRCAP appear to be exclusively heterozygous in nature,23 suggesting that, in hematopoietic populations harboring such mutations, the function of SRCAP is perturbed, but not completely eliminated. Supporting this, previous studies have demonstrated that alterations in Srcap activity, whether through loss of the Znhit1 subunit of the Srcap complex15,19 or via expression of a truncated Srcap mutant,34 can impact the normal functioning of HSPCs. In our murine model of Srcap loss, heterozygous loss of this gene does not adversely impact HSPC repopulating ability, indicating that hematopoietic progenitors that maintain at least one wild-type SRCAP allele remain capable of sustaining hematopoiesis. Interestingly, similar to a recently published murine model with expression of a commonly observed Srcap truncation mutant,34 hematopoietic bone marrow cells with heterozygous loss of Srcap displayed a lymphoid bias after transplantation. How the different SRCAP frameshift and nonsense mutations observed in human hematopoiesis impact SRCAP activity (e.g. the degree to which they abrogate wild-type function through loss of the wild-type allele or partial dominant negative activity or provide a novel gain of function) remains to be determined.

To investigate the cellular consequences of SRCAP deficiency in hematopoietic populations, we performed RNA-Sequencing on Vav1-iCre+/−Srcapfl/fl fetal liver stage 1 erythroid progenitors. With complete loss of Srcap, this population was associated with the significant upregulation of p53, apoptotic, and inflammatory pathways. Interestingly, shRNA-induced knockdown of SRCAP in a non-hematopoietic cell line has been similarly associated with a more modest upregulation of inflammatory pathways and the p53 pathway.40 Upregulation of such pathways may be common downstream results of SRCAP loss in hematopoietic populations, providing a potential explanation as to why some degree of SRCAP expression is required to maintain hematopoiesis. It is also possible that the significant upregulation of p53 and inflammatory pathways associated with complete Srcap loss is masking milder impacts loss of this gene has on other cellular pathways. Indeed, previous publications have also identified both SRCAP and H2AZ as playing a critical role in the cellular response to DNA damage.34,37,41 The degree to which milder perturbations of SRCAP impact such pathways in human clonally expanded and/or leukemic populations harboring SRCAP mutations, the dependence of these impacts on mutation-induced alterations to SRCAP’s function in the chromatin deposition of H2AZ, and the mechanisms through which this may promote clonal expansion and leukemic transformation all remain open areas of investigation.

Supplementary Material

Supplement 1 Supplemental Figure 1. Loss of Srcap causes aberrant hematopoietic maturation. (A) Representative H&E stains of fetal livers with the indicated genotypes. (B) Percentage of fetal liver cells that were Ter119+. (C) Percentage of fetal liver erythroid progenitors at the indicated stage of maturation based on forward scatter, Ter119 staining, and CD44 staining.

Supplemental Figure 2. Loss of Srcap causes aberrant fetal myeloid maturation. (A) Percentage of fetal liver cells classified as phenotypic CD11b+CD115− granulocytes or CD11b+CD115+ monocytes as described in Fig. 3A. (B) Percentage of fetal liver cells classified as Ly6CIntCD11b+ granulocytes or Ly6CHighCD11bInt monocytes as described in Fig. 3A. (C) Representative flow plots identifying fetal liver myeloid populations using the markers CD11b, Ly6G, F4/80, and CD115. (D) Total number of fetal liver cells classified as phenotypic CD11b+Ly6G+ granulocytes or F4/80+CD115Int macrophages as described in C. (E) Percentage of fetal liver cells classified as phenotypic CD11b+Ly6G+ granulocytes or F4/80+CD115Int macrophages as described in C. (F) Percentage of phenotypic CMPs, GMPs, and MEPs among fetal liver cells. ** P ≤ 0.01. *** P ≤ 0.001. **** P ≤ 0.0001. Significance was determined with an ordinary one-way ANOVA with multiple comparisons.

Supplemental Figure 3. HSCs with loss of Srcap are initially maintained in the fetal liver. (A) Percentage of fetal liver cells classified as phenotypic HSPCs using c-kit, Sca1, CD150 and CD48. (B) Representative flow plots of fetal liver HSPCs using c-kit and EPCR as markers. Cells were initially gated as CD45+ and lineage negative. (C) Percentage of fetal liver cells classified as phenotypic HSPCs using c-kit, EPCR, CD150 and CD48. (D) Representative flow plots of fetal liver HSPCs using c-kit and CD86 as markers. Cells were initially gated as CD45+ and lineage negative. (E) Total number of phenotypic fetal liver HSPCs using c-kit, CD86, CD150 and CD48. (F) Percentage of fetal liver cells classified as HSPCs using c-kit, CD86, CD150 and CD48. (G) Cell-cycle distribution of fetal liver cells. 2N, Ki-67 low cells were classified as in G0, 2N, Ki-67 high cells were classified as in G1, and 4N cells were classified as in G2-M/S. * P ≤ 0.05. ** P ≤ 0.01. *** P ≤ 0.001. **** P ≤ 0.0001. Significance was determined with an ordinary one-way ANOVA with multiple comparisons.

Supplemental Figure 4. Loss of Srcap abrogates HSC function. (A) Percentage peripheral blood donor (Ly5.2) chimerism in the myeloid (Gr1+ and CD115+) and lymphoid (B220+) lineages following non-competitive transplantation of 5×106 Mx1 Cre+/−Srcapfl/fl or Srcapfl/fl bone marrow cells. (B) Percentage peripheral blood donor (Ly5.2) chimerism in the myeloid (Gr1+ and CD115+) and lymphoid (B220+) lineages following competitive transplantation of 2.5×106 Srcap+/− or Srcap+/+ bone marrow cells against an equal number of competitor cells. (C) Percentage peripheral blood donor (Ly5.2) chimerism in the myeloid (Gr1+ and CD115+) and lymphoid (B220+) lineages after re-transplantation of Srcap+/− or Srcap+/+ bone marrow cells. Significance was assessed with a two-way ANOVA. * P ≤ 0.05. *** P ≤ 0.001. **** P ≤ 0.0001.

Supplement 2 Supplemental Table 1. Upregulated gene pathways in stage 1 fetal liver erythroid progenitors with loss of Srcap.

Acknowledgements

This work was supported by National Institutes of Health, National Cancer Institute grant K08 CA197369 (to T.N. Wong) and by the Leukemia Research Foundation. At Washington University School of Medicine, technical support was provided by the Transgenic, Knockout and Micro-Injection and High-Speed Cell Sorting Cores. At the University of Michigan, technical support was provided by the Flow Cytometry, Advanced Genomics, and Bioinformatics Cores.

Figure 1. Generation and initial characterization of a mouse model with conditional loss of Srcap.

(A) Genetic model for conditional loss of Srcap in cells expressing Cre recombinase. (B) Representative PCR genotyping reaction showing efficient Cre-induced excision in sorted fetal liver erythroid progenitor cells. (C) Representative RNA-Seq reads of excised Srcap exons as displayed by IGV Viewer from sorted fetal liver stage 1 erythroid progenitor cells. (D) Representative pictures of murine embryos with the indicated genotypes.

Figure 2. Loss of Srcap causes aberrant fetal erythroid maturation.

(A) Total number of fetal liver cells. (B) Total number of Ter119+ fetal liver cells. (C) Representative flow plots of fetal erythroid maturation. (D) Total number of fetal liver erythroid progenitor cells grouped by their stage of maturation based on forward scatter, Ter119 staining, and CD44 staining. ** P ≤ 0.01. **** P ≤ 0.0001. Significance was determined with an ordinary one-way ANOVA with multiple comparisons.

Figure 3. Loss of Srcap causes impaired monocytic differentiation.

(A) Representative flow plots identifying fetal liver myeloid populations using the markers CD115, Ly6C, and CD11b. (B) Total number of fetal liver cells classified as phenotypic CD11b+CD115− granulocytes or CD11b+CD115+ monocytes as indicated in A. (C) Total number of fetal liver cells classified as Ly6CIntCD11b+ granulocytes or Ly6CHighCD11bInt monocytes as indicated in A. (D) Total number of phenotypic fetal liver common myeloid progenitors (CMPs), granulocyte-monocyte progenitors (GMPs), and megakaryocyte–erythrocyte progenitors (MEPs). (E) Representative flow plots identifying monocytic and granulocytic progenitors within the GMP compartment using Ly6C and CD115 as markers. (F) Percentage of Ly6C− cells, monocyte-committed progenitors (MP), and granulocyte-committed progenitors (GP) within the GMP compartment. ** P ≤ 0.01. *** P ≤ 0.001. **** P ≤ 0.0001. Significance was determined with an ordinary one-way ANOVA with multiple comparisons.

Figure 4. Loss of Srcap abrogates fetal HSC function.

(A) Representative flow plots of fetal liver HSPCs using c-kit and Sca1 as markers. Cells were initially gated as CD45+ and lineage negative. (B) Total number of phenotypic fetal liver HSPCs using c-kit, Sca1, CD150 and CD48 as markers. (C) Total number of phenotypic fetal liver HSPCs using c-kit, EPCR, CD150 and CD48 as markers. (D) Percentage donor (Ly5.2) chimerism in the peripheral blood following competitive transplantation of 1×106 Ly5.2 fetal liver cells of the indicated genotypes against 2×106 wild-type Ly5.1 bone marrow cells. Vav Cre+/−Srcapfl/fl cells were pooled from multiple embryos prior to transplantation (8 recipients in two individual experiments). Cells from embryos with full Srcap expression (6 donors) or haploinsufficient loss of Srcap (3 donors) were transplanted individually. (E) Percentage donor (Ly5.2) chimerism in the peripheral blood myeloid (Gr1+ and CD115+) and lymphoid (B220+ and CD3+) compartments 18 weeks following transplantation of fetal liver cells. (F) Percentage bone marrow donor chimerism 18 weeks following transplantation of fetal liver cells. * P ≤ 0.05. **** P ≤ 0.0001. Significance for D was determined with a two-way ANOVA. Significance was otherwise determined with an ordinary one-way ANOVA with multiple comparisons.

Figure 5. Loss of Srcap abrogates adult HSC function.

(A) Survival of mice following poly(I:C) administration to induce loss of Srcap. Survival was measured from the date of the last dose of poly(I:C), and significance assessed with a Mantel-Cox test. (B) Hematopoietic parameters following poly(I:C) administration. Mx1 Cre+/− Scrapfl/- mice were analyzed upon exhibiting signs of distress with control mice analyzed at similar time points. Significance was assessed with an unpaired t-test. (C) Representative bone marrow flow plots following poly(I:C) administration. Cells were initially gated as lineage negative. (D) Percentage donor (Ly5.2) chimerism in the indicated tissues following non-competitive transplantation of 5×106 Mx1 Cre+/−Srcapfl/fl or Srcapfl/fl bone marrow cells (8 recipients per group in two independent experiments) and poly(I:C) administration. Significance was assessed with a two-way ANOVA. (E) Percentage bone marrow donor chimerism 11–12 weeks following poly(I:C) administration with significance assessed using an unpaired t-test. (F) Percentage peripheral blood donor (Ly5.2) chimerism following competitive transplantation of 2.5×106 Srcap+/− or Srcap+/+ bone marrow cells against an equal number of competitor cells (14–18 recipients per group in 2 independent experiments). Significance was assessed with a two-way ANOVA. (G) Percentage peripheral blood donor (Ly5.2) chimerism after re-transplantation of Srcap+/− or Srcap+/+ bone marrow cells (5 recipients per group). The input chimerism is the chimerism of the bone marrow KLS cells at the time of re-transplantation. Significance was assessed with a two-way ANOVA. ** P ≤ 0.01. *** P ≤ 0.001. **** P ≤ 0.0001.

Figure 6. Cellular impacts of Srcap loss.

(A) Principal component analysis of RNA-Seq data from stage 1 fetal liver erythroid progenitors from Vav Cre+/−Srcapfl/fl embryos (n=4), Vav Cre+/−Srcapfl/+ embryos (n=2) and Srcapfl/fl embryos (n=2). (B) Volcano plot of differentially expressed genes in RNA-Seq data of stage 1 fetal liver erythroid progenitor cells with complete loss of Srcap (n=4) versus cells with either full or heterozygous loss of Srcap expression (n=4). Genes with mean DeSeq2 values > 50 are shown. Genes are highlighted if they are in the P53, apoptosis, TNFα via NFκβ, or IL2/STAT5 GSEA Hallmark pathways and among the top 250 most significantly dysregulated genes. Genes highlighted in the latter two pathways were not also present in the first two. (C) P53 pathway and TNFα via NFκβ GSEA enrichment plots from the analysis in B. These had the two highest normalized enrichment scores among the GSEA Hallmark pathways. (D) Representative flow plots showing γ-H2AX levels in CD150+CD48− KSL cells. DNA content was assessed with 7-AAD staining. (E) γ-H2AX MFI in the fetal liver hematopoietic progenitor compartment. The MFIs were normalized to the average MFI of control CD150+CD48− KLS cells having 2N DNA content with either full or heterozygous loss of Srcap expression. Significance was determined with an ordinary one-way ANOVA with multiple comparisons. *** P ≤ 0.001. **** P ≤ 0.0001.

Key Points:

(1) Srcap plays an essential role in supporting normal hematopoietic differentiation. and in maintaining HSC function.

(2) Loss of Srcap is associated with evidence of increased DNA damage and transcriptional upregulation of the p53 pathway.

Conflict-of-interest disclosure

The authors declare no competing financial interests.
==== Refs
References

1. Welch JS , Ley TJ , Link DC , The origin and evolution of mutations in acute myeloid leukemia. Cell. 2012;150 (2 ):264–278.22817890
2. Genovese G , Kahler AK , Handsaker RE , Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N Engl J Med. 2014;371 (26 ):2477–2487.25426838
3. Jaiswal S , Fontanillas P , Flannick J , Age-related clonal hematopoiesis associated with adverse outcomes. N Engl J Med. 2014;371 (26 ):2488–2498.25426837
4. Challen GA , Sun D , Jeong M , Dnmt3a is essential for hematopoietic stem cell differentiation. Nat Genet. 2011;44 (1 ):23–31.22138693
5. Hsu JI , Dayaram T , Tovy A , PPM1D Mutations Drive Clonal Hematopoiesis in Response to Cytotoxic Chemotherapy. Cell Stem Cell. 2018;23 (5 ):700–713 e706.30388424
6. Kahn JD , Miller PG , Silver AJ , PPM1D-truncating mutations confer resistance to chemotherapy and sensitivity to PPM1D inhibition in hematopoietic cells. Blood. 2018;132 (11 ):1095–1105.29954749
7. Wong TN , Miller CA , Jotte MRM , Cellular stressors contribute to the expansion of hematopoietic clones of varying leukemic potential. Nat Commun. 2018;9 (1 ):455.29386642
8. Abelson S , Collord G , Ng SWK , Prediction of acute myeloid leukaemia risk in healthy individuals. Nature. 2018;559 (7714 ):400–404.29988082
9. Weeks LD , Niroula A , Neuberg D , Prediction of risk for myeloid malignancy in clonal hematopoiesis. NEJM Evid. 2023;2 (5 ).
10. Cai Z , Kotzin JJ , Ramdas B , Inhibition of Inflammatory Signaling in Tet2 Mutant Preleukemic Cells Mitigates Stress-Induced Abnormalities and Clonal Hematopoiesis. Cell Stem Cell. 2018;23 (6 ):833–849 e835.30526882
11. Sano S , Oshima K , Wang Y , Tet2-Mediated Clonal Hematopoiesis Accelerates Heart Failure Through a Mechanism Involving the IL-1beta/NLRP3 Inflammasome. J Am Coll Cardiol. 2018;71 (8 ):875–886.29471939
12. Jaiswal S , Natarajan P , Silver AJ , Clonal Hematopoiesis and Risk of Atherosclerotic Cardiovascular Disease. N Engl J Med. 2017;377 (2 ):111–121.28636844
13. Giaimo BD , Ferrante F , Herchenrother A , Hake SB , Borggrefe T . The histone variant H2A.Z in gene regulation. Epigenetics Chromatin. 2019;12 (1 ):37.31200754
14. Hu G , Cui K , Northrup D , H2A.Z facilitates access of active and repressive complexes to chromatin in embryonic stem cell self-renewal and differentiation. Cell Stem Cell. 2013;12 (2 ):180–192.23260488
15. Sun S , Jiang N , Jiang Y , Chromatin remodeler Znhit1 preserves hematopoietic stem cell quiescence by determining the accessibility of distal enhancers. Leukemia. 2020;34 (12 ):3348–3358.32694618
16. Zhao B , Chen Y , Jiang N , Znhit1 controls intestinal stem cell maintenance by regulating H2A.Z incorporation. Nat Commun. 2019;10 (1 ):1071.30842416
17. Hua S , Kallen CB , Dhar R , Genomic analysis of estrogen cascade reveals histone variant H2A.Z associated with breast cancer progression. Mol Syst Biol. 2008;4 :188.18414489
18. Kim K , Punj V , Choi J , Gene dysregulation by histone variant H2A.Z in bladder cancer. Epigenetics Chromatin. 2013;6 (1 ):34.24279307
19. Ye B , Liu B , Yang L , Suppression of SRCAP chromatin remodelling complex and restriction of lymphoid lineage commitment by Pcid2. Nat Commun. 2017;8 (1 ):1518.29138493
20. Wong WH , Bhatt S , Trinkaus K , Engraftment of rare, pathogenic donor hematopoietic mutations in unrelated hematopoietic stem cell transplantation. Sci Transl Med. 2020;12 (526 ).
21. da Silva-Coelho P , Kroeze LI , Yoshida K , Clonal evolution in myelodysplastic syndromes. Nat Commun. 2017;8 :15099.28429724
22. Beauchamp EM , Leventhal M , Bernard E , ZBTB33 is mutated in clonal hematopoiesis and myelodysplastic syndromes and impacts RNA splicing. Blood Cancer Discov. 2021;2 (5 ):500–517.
23. Jahn N , Terzer T , Strang E , Genomic heterogeneity in core-binding factor acute myeloid leukemia and its clinical implication. Blood Adv. 2020;4 (24 ):6342–6352.33351131
24. Seifert W , Meinecke P , Kruger G , Expanded spectrum of exon 33 and 34 mutations in SRCAP and follow-up in patients with Floating-Harbor syndrome. BMC Med Genet. 2014;15 :127.25433523
25. Nakada D , Saunders TL , Morrison SJ . Lkb1 regulates cell cycle and energy metabolism in haematopoietic stem cells. Nature. 2010;468 (7324 ):653–658.21124450
26. Subramanian A , Tamayo P , Mootha VK , Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A. 2005;102 (43 ):15545–15550.16199517
27. Ye B , Yang L , Qian G , The chromatin remodeler SRCAP promotes self-renewal of intestinal stem cells. EMBO J. 2020;39 (13 ):e103786.32449550
28. Yanez A , Coetzee SG , Olsson A , Granulocyte-Monocyte Progenitors and Monocyte-Dendritic Cell Progenitors Independently Produce Functionally Distinct Monocytes. Immunity. 2017;47 (5 ):890–902 e894.29166589
29. Fuster JJ , MacLauchlan S , Zuriaga MA , Clonal hematopoiesis associated with TET2 deficiency accelerates atherosclerosis development in mice. Science. 2017;355 (6327 ):842–847.28104796
30. Shirai CL , Ley JN , White BS , Mutant U2AF1 Expression Alters Hematopoiesis and Pre-mRNA Splicing In Vivo. Cancer Cell. 2015;27 (5 ):631–643.25965570
31. Wang T , Glover B , Hadwiger G , Miller CA , di Martino O , Welch JS . Smc3 is required for mouse embryonic and adult hematopoiesis. Exp Hematol. 2019;70 :70–84 e76.30553776
32. Subramaniam A , Talkhoncheh MS , Magnusson M , Larsson J . Endothelial protein C receptor (EPCR) expression marks human fetal liver hematopoietic stem cells. Haematologica. 2019;104 (2 ):e47–e50.30026339
33. Kanayama M , Izumi Y , Yamauchi Y , CD86-based analysis enables observation of bona fide hematopoietic responses. Blood. 2020;136 (10 ):1144–1154.32438398
34. Chen CW , Zhang L , Dutta R , SRCAP mutations drive clonal hematopoiesis through epigenetic and DNA repair dysregulation. Cell Stem Cell. 2023.
35. Johnson P , Ruffell B . CD44 and its role in inflammation and inflammatory diseases. Inflamm Allergy Drug Targets. 2009;8 (3 ):208–220.19601881
36. Malek TR , Danis KM , Codias EK . Tumor necrosis factor synergistically acts with IFN-gamma to regulate Ly-6A/E expression in T lymphocytes, thymocytes and bone marrow cells. J Immunol. 1989;142 (6 ):1929–1936.2493502
37. Dong S , Han J , Chen H , The human SRCAP chromatin remodeling complex promotes DNA-end resection. Curr Biol. 2014;24 (18 ):2097–2110.25176633
38. Biswas A , Roy IM , Babu PC , The Periostin/Integrin-alphav Axis Regulates the Size of Hematopoietic Stem Cell Pool in the Fetal Liver. Stem Cell Reports. 2020;15 (2 ):340–357.32735820
39. Vallabhaneni H , Lynch PJ , Chen G , High Basal Levels of gammaH2AX in Human Induced Pluripotent Stem Cells Are Linked to Replication-Associated DNA Damage and Repair. Stem Cells. 2018;36 (10 ):1501–1513.29873142
40. Jostes S , Vardabasso C , Dong J , H2A.Z chaperones converge on E2F target genes for melanoma cell proliferation. Genes Dev. 2024.
41. Xu Y , Ayrapetov MK , Xu C , Gursoy-Yuzugullu O , Hu Y , Price BD . Histone H2A.Z controls a critical chromatin remodeling step required for DNA double-strand break repair. Mol Cell. 2012;48 (5 ):723–733.23122415
