HIRA, a DiGeorge Syndrome Candidate Gene, Confers Proper Chromatin Accessibility on HSCs and Supports All Stages of Hematopoiesis

Article

HIRA, a DiGeorge Syndrome Candidate Gene, Confers Proper Chromatin Accessibility on HSCs and Supports All Stages of Hematopoiesis Graphical Abstract

Authors Chao Chen, Ming-an Sun, Claude Warzecha, ..., Todd Macfarlan, Paul Love, Keiko Ozato

Correspondence [email protected]

In Brief Chen et al. show that the histone H3.3 chaperone HIRA directs development and survival of adult hematopoietic stem cells (HSCs). Hira deletion impairs development of all lineages of hematopoiesis.

Highlights d

HIRA confers chromatin accessibility that defines the HSC transcriptome

d

Long-term HSCs in adult Hira KO incur increased DNA damage and apoptosis

d

Hira KO has some similarity to DiGeorge syndromes, which lack the HIRA locus

d

Fetal hematopoiesis is spared in Hira-KO mice

Chen et al., 2020, Cell Reports 30, 2136–2149 February 18, 2020 https://doi.org/10.1016/j.celrep.2020.01.062

Cell Reports

Article HIRA, a DiGeorge Syndrome Candidate Gene, Confers Proper Chromatin Accessibility on HSCs and Supports All Stages of Hematopoiesis Chao Chen,1,5 Ming-an Sun,2 Claude Warzecha,3 Mahesh Bachu,1 Anup Dey,1 Tiyun Wu,1 Peter D Adams,4 Todd Macfarlan,2 Paul Love,3 and Keiko Ozato1,6,* 1Molecular Genetics of Immunity Section, Division of Developmental Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA 2Mammalian Epigenome Reprogramming Section, Division of Developmental Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA 3Hematopoiesis and Lymphocyte Biology Section, Division of Developmental Biology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA 4Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037, USA 5Present address: Division of Hematology/Oncology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA 6Lead Contact *Correspondence: [email protected] https://doi.org/10.1016/j.celrep.2020.01.062

SUMMARY

HIRA is a histone chaperone that deposits the histone variant H3.3 in transcriptionally active genes. In DiGeorge syndromes, a DNA stretch encompassing HIRA is deleted. The syndromes manifest varied abnormalities, including immunodeficiency and thrombocytopenia. HIRA is essential in mice, as total knockout (KO) results in early embryonic death. However, the role of HIRA in hematopoiesis is poorly understood. We investigate hematopoietic cell-specific Hira deletion in mice and show that it dramatically reduces bone marrow hematopoietic stem cells (HSCs), resulting in anemia, thrombocytopenia, and lymphocytopenia. In contrast, fetal hematopoiesis is normal in Hira-KO mice, although fetal HSCs lack the reconstitution capacity. Transcriptome analysis reveals that HIRA is required for expression of many transcription factors and signaling molecules critical for HSCs. ATAC-seq analysis demonstrates that HIRA establishes HSC-specific DNA accessibility, including the SPIB/PU.1 sites. Together, HIRA provides a chromatin environment essential for HSCs, thereby steering their development and survival. INTRODUCTION Hematopoiesis is a dynamic process that originates from hematopoietic stem cells (HSCs). HSCs possess a dual function, that is, self-renewal and lineage specification (Ng and Alexander, 2017; Pouzolles et al., 2016). HSC functions are dictated by a number of specific transcription factors, chromatin regulators, and histone modifications leading to epigenetic regulation, without causing genetic alterations (Cullen et al., 2014; Li et al., 2011; Olsson et al., 2016; Wilson et al., 2010). Aberrant epige-

netic modifications can derail HSC development, causing defective erythropoiesis, impaired immunity, and even blood cell malignancy. HIRA is a conserved histone chaperone, which deposits the histone variant H3.3 onto chromatin in a transcription-coupled manner (Allis and Jenuwein, 2016; Hammond et al., 2017; Talbert and Henikoff, 2017). This process is distinct from replication-coupled deposition of core histones. HIRA forms a foursubunit complex and localizes to numerous sites in the genome (Pchelintsev et al., 2013; Ricketts et al., 2015). Recent studies show that HIRA directs H3.3 deposition not only on transcriptionally active genes but bivalent genes in embryonic stem cells and DNA damage sites (Adam et al., 2013; Banaszynski et al., 2013; Goldberg et al., 2010). It has been shown that HIRA is important for the development and function of many cell types, ranging from germ cells, muscle cells, and endothelial cells to neurons, affecting diverse biological processes (Dilg et al., 2016; Dutta et al., 2010; Li and Jiao, 2017; Loppin et al., 2005; Nashun et al., 2015; Yang et al., 2016). It also regulates senescence and tumor growth (Rai et al., 2014). HIRA is essential in the mouse, as standard Hira knockout (KO) causes early embryonic lethality (Roberts et al., 2002). Consistent with this, HIRA is reported to have important roles in embryonic stem cells from both mouse and human, by regulating their metabolism and differentiation (Meshorer et al., 2006; Zhu et al., 2017). The HIRA gene maps to Chr22q11.21 in humans. In patients with DiGeorge syndromes, also referred to as Chr22q11 deletion syndromes, the genomic region encompassing HIRA, a 5-3 Mb DNA stretch, is deleted (Akar and Adekile, 2007; D’Antoni et al., 2004; Lindsay, 2001; Sullivan, 2019). The deleted regions contain not only HIRA but additional genes, including TBX1, involved in embryonic development. Patients with hemizygous deletion manifest diverse clinical features, ranging from cardiac malformation and craniofacial and limb anomaly to psychiatric disorders (McDonald-McGinn and Sullivan, 2011). DiGeorge syndromes are also associated with

2136 Cell Reports 30, 2136–2149, February 18, 2020 This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Figure 1. Generation and Characterization of Hira Conditional KO Mice (A) qRT-PCR showing relative expression of Hira mRNA (normalized to Hprt) from indicated cells. FL, fetal liver; BM, bone marrow; SP, spleen; BMDMs, BMderived macrophages. (B–D) qRT-PCR and immunoblot analysis of Hira mRNA and protein in BM (B), spleen (C), and thymus (D). Hira mRNA values are the average values from three independent experiment in duplicate ± SD. (E–I) Photographs of mice (E), spleen (F), thymus (G), bone marrow (H), and lymph node (I) from WT (Hiraf/f) and Hira-KO (Hiraf/f Vav-Cre) littermates. (J) Survival rates of WT and Hira-KO mice (n = 50 mice per group). (K) The weight of mice (n = 29 mice per group), spleen (n = 15 mice per group), and thymus (n = 13 mice per group). (L) Numbers of nucleated cells in BM (n = 12 mice per group), spleen (n = 10 mice per group), thymus (n = 9 mice per group), and lymph nodes (n = 6 mice per group). Data represent average value ± SD. *p < 0.05 and **p < 0.01; ns, not significant.

immunodeficiency, including thymic defects and thrombocytopenia (Davies, 2013; Klocperk et al., 2014; Patel et al., 2012). Despite its clear link to DiGeorge syndromes and reported importance in development, HIRA’s role in hematopoietic development has been poorly understood. Here we conditionally knocked out the Hira gene in early hematopoietic cells in the mouse. Hira KO caused a massive loss of bone marrow (BM) HSCs, resulting in a marked failure in generating all cells in the hematopoietic lineage. Hira-KO mice suffered from a severe depletion of T and B lymphocytes, monocytes, and platelets, along with reduced red blood cells (RBCs). However, Hira KO did not prevent fetal hematopoiesis, allowing embryonic and neonatal survival. Gene profiling analysis of BM HSCs identified a number of transcription factors and signaling

molecules as HIRA target genes. Assay for transposase-accessible chromatin followed by sequencing (ATAC-seq) analysis revealed that the absence of HIRA globally reduces chromatin accessibility on regions important for transcription of HSC-specific genes. Our results demonstrate that HIRA establishes a proper epigenetic state in chromatin and helps their development and survival. RESULTS Expression of Hira in Hematopoietic Cells and Vav-CreBased Conditional Hira KO We first examined Hira mRNA expression in various hematopoietic cells. qRT-PCR data in Figure 1A showed that Hira

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transcripts were broadly expressed in most, if not all, cells of the hematopoietic lineage, from fetal liver (FL) and BM LSK (Lin
Sca1+cKit+) cells, hematopoietic progenitors, including common lymphoid progenitors (CLPs), common myeloid progenitors (CMPs), granulocyte-macrophage progenitors (GMPs), and megakaryocyte-erythroid progenitors (MEPs), and mature immune cells. The highest Hira expression was found in BM B cells and thymic double-positive cells. LSK cells had intermediate levels of Hira, while BM-derived macrophages (BMDMs) had the lowest amounts of Hira transcripts. To construct Hiraf/f mice, Lox-p sites were introduced into the sequence between exon 6 and exon 8, which would remove exon 7 and the flanking introns. This would result in a frameshift with a premature stop codon in exon 8 (Figure S1A). The strategy to delete exon 7 was previously used to construct total Hira KO (Roberts et al., 2002). To delete Hira in hematopoietic cells, Hiraf/f mice were crossed with mice with Vav-Cre, which disrupts floxed genes in early hematopoietic cells, including HSCs (Aggarwal et al., 2012; Dey et al., 2019; Stadtfeld and Graf, 2005). qRT-PCR data from Hira-KO BM, spleen, and thymus confirmed the loss of exon 7 transcripts, with some residual exon 9 transcripts, as expected (Figures 1B–1D). Furthermore, RNA sequencing (RNA-seq) analysis in Figure S1B verified the absence of exon 7 RNA peaks in Hira-KO LSK cells. These data were further substantiated by qRT-PCR (Figure S1C). Immunoblot analysis corroborated the absence of HIRA protein in BM, spleen, and thymus (Figures 1B–1D). Hira-KO mice were born with the expected Mendelian ratios (Figure S1D) and lived normally up to 9 weeks but appeared to die earlier than wildtype (WT) mice (Figure 1J). Also, Hira-KO mice were generally smaller than WT (Figure 1E). Their spleen and lymph nodes (LNs), but not thymus, were smaller in size, weighing less than WT counterparts (Figures 1F, 1G, and 1I; quantification in Figure 1K). Accordingly, the total cell recovery from BM, spleen, and LNs was significantly lower in Hira-KO mice than that in WT mice (Figure 1L). In addition, Hira-KO BM looked paler than that of WT littermates (Figure 1H). Hira-KO Mice Suffer from Anemia, Thrombocytopenia, and Severe Depletion of Myeloid and Lymphoid Cells To determine the effect of Hira KO on steady-state hematopoiesis, the composition of hematopoietic cells was examined in peripheral blood, collected from 8- to 16-week-old Hira-KO mice. The number of RBCs were reduced by about 30% in Hira-KO mice, accompanied by reduced hemoglobin levels, although the mean red cell volume (MCV) was similar to that of WT RBCs (Figures 2A–2C). Interestingly, even heterozygous Hira-KO mice exhibited slightly decreased RBC counts and hemoglobin levels relative to WT mice, indicating haploinsufficiency. Total white blood cells (WBCs) and platelet counts were markedly lower in Hira-KO mice than WT mice (reduced by 83% and 64%, respectively; Figures 2D and 2E). Again, the number of these cells in heterozygous mice were lower than those in WT mice, confirming haploinsufficiency, a feature of DiGeorge syndromes. Furthermore, the total number of lymphocytes was lower in Hira-KO mice than WT mice (Figure 2F); in line with these data, fluorescence-activated cell sorting (FACS) analysis (Figures 2K and 2L) revealed a profound reduction in B220+

2138 Cell Reports 30, 2136–2149, February 18, 2020

cells, indicating a serious B cell deficiency. The total numbers of CD3+ cells were also reduced in Hira-KO peripheral blood, although not obvious in percentage in FACS profiles (Figures 2K and 2L). Also, the number of monocytes decreased significantly (Figure 2J). FACS analysis of Cd11b+Gr1
cells confirmed a reduction in the number of monocytes (Figures 2K and 2M). Although the number was not affected (Figures 2G, 2H, 2K, and 2M), granulocytes (Cd11b+, Gr1+) displayed conspicuous morphological abnormality, showing hypersegmented nuclei, resembling neutrophils of myelodysplasia (Figure 2N) (Zhou et al., 2015). Furthermore, the number of basophils but not eosinophils was lower in Hira-KO mice (Figure 2I). Together, these results show that development of all blood cell types was grossly dysregulated in Hira-KO mice. Hira Deletion Causes a Drastic Reduction in Long-Term HSCs in BM and Inhibits Their Progression to Early Progenitors The above results suggested that Hira KO causes a serious developmental defect that broadly alters hematopoiesis. We thus examined BM cells for the presence of HSCs and early progenitors. FACS analysis in Figure 3A (left panel) showed that the percentage of LSK cells was substantially lower in Hira-KO than WT BM. Accordingly, the total number of LSK cells was markedly lower in Hira-KO BM than WT BM (Figure 3B). We then tested CD48 and CD150 markers in the LSK population and found that CD150+, C48
cells were also drastically reduced in HiraKO cells relative to WT cells (Figures 3A and 3B). CD150+, C48
cells are designated as long-term (LT) HSCs capable of self-renewal and differentiation into short-term HSCs or lineage-directed progenitors (Challen et al., 2009; Mitroulis et al., 2018). These cells are also called SLAM HSCs (Oguro et al., 2013). Although LSK cells contain HSCs, LK (Lin
Sca1
cKit+) cells are composed largely of progenitors. We found that the LK cell population was also reduced in Hira-KO BM (Figures 3A and 3B). Furthermore, cells carrying markers for CLPs and CMPs were lower in Hira-KO BM than WT BM, whereas GMPs were comparable between HIRA-KO and WT BM (Figures 3C– 3F; see markers in the figure legend). In addition, MEPs were lower in Hira-KO BM. MEPs generate megakaryocytes (Mkps), which then develop into platelets (Psaila et al., 2016; Tong and Lodish, 2004). We examined Mkps (CD150high, CD41high) in the LK population and found a significant reduction in Hira-KO cells compared with WT cells (Figures 3G and 3H). Consistent with the meager presence of Mkp, the Mkp colony-forming capacity was drastically lower in Hira-KO BM compared with WT BM (Figures 3I and 3J). These finding demonstrate that the loss of Hira prevents development and survival of HSCs and their progression into early progenitors. Lymphoid and Myeloid Lineage Differentiation Is Derailed in Hira-KO Mice Having found a large reduction in HSCs and progenitors in HiraKO mice, it was important to study cells in peripheral blood and organs. Data in Figure 3K and Figure S2A showed that RBCs are slightly lower in Hira-KO BM but higher in Hira-KO spleen. The higher RBC counts in spleen may be due to a peripheral compensatory mechanism covering stem cell defects (Gordon

Figure 2. Hira-KO Mice Suffer from Anemia, Lymphocytopenia, and Thrombopenia (A–J) Complete blood counts (CBCs) in peripheral blood from WT and Hira-KO littermates are shown (n = 13 mice per group). RBCs, red blood cells (A); Hemoglobin (B); MCV, mean red cell volume (C); WBCs, white blood cells (D); Platelets (E); Lymphocytes (F); Granulocytes (G); Eosinophils (H); Basophils (I); Monocytes (J). (K–M) One of the representative FACS profiles (K) and the number of cells (L and M) for B220+, CD3+, CD11b+/Gr1+, and CD11b+/Gr1
cells in peripheral blood (n = 15 mice per group). Values represent the average of 15 separate experiments ± SD. (N) Representative images of peripheral blood smear stained by Wright-Giemsa from three pairs of WT and Hira-KO littermates. Note that neutrophils from HiraKO peripheral blood are hypersegmented. *p < 0.05 and **p < 0.01; ns, not significant.

et al., 2008). In addition, the numbers of cells carrying markers for neutrophils (CD11b+Ly6GhighLy6Clow), monocytes (CD11b+ Ly6GlowLy6Chigh), and dendritic cell (CD11b+CD11c+) were lower in Hira-KO BM than WT BM, although the numbers of CD11b+F4/80+ cells were similar in Hira-KO and WT BM (Figure 3L; Figure S2B). Next, we tested the number of T and B lymphocytes in spleen, thymus, and LNs. As shown in Figure 3M and Figures S2C–S2E, B220+CD19+ B cells were dramatically lower in BM, spleen, and LNs from Hira-KO mice, less than 15% of those in WT organs. Further tests for differentiating B cells showed that pre-B cells (B220+CD43
IgM
IgD
) were markedly reduced in Hira-KO BM, although pro-B cells (B220+CD43+) were not significantly different (Hardy and Hayakawa, 2001). Consequently, cells generated from pre-B cells, namely, immature (B220+CD43
IgM+IgD
) and mature (B220+CD43
IgM+

IgD+) B cells were almost absent in Hira-KO BM (Figure 3N; Figure S2C). These results suggest that B cell development was arrested at the pro-B-to-pre-B transition in Hira-KO mice. Tests for the T cell lineage found that the number of thymic cells in Hira-KO mice were similar in number and composition as WT mice. In addition, there was no difference in double-positive and single CD4+ or CD8+ cells in WT and Hira KO thymi (Figure 3O; Figure S2F). These results indicate that Hira deletion does not grossly affects thymic development. However, CD4+ and CD8+ T cells in spleen and LNs were much lower in Hira-KO mice relative to WT mice (Figures 3P, 3Q; Figure S2F). The reduction of T cells in peripheral organs indicates a post-thymic defect in T cell development in Hira-KO mice. Together, these results show that HIRA deficiency not only affects HSC development in BM but exerts adverse effects

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Figure 3. Hira Deletion Results in HSC Defects and Impaired Hematopoiesis (A–H) Representative FACS profiles of BM cells harvested from two femurs and two tibias from WT and Hira-KO littermates. (A) One of the FACS profiles of LT-HSC (Lin
Sca1+cKit+CD150+D48
), LSK (Lin
Sca1+cKit+), and LK (Lin
Sca1
cKit+) populations. (B) Total cell counts for LT-HSC (n = 3 mice per group), LSK (n = 13 mice per group), and LK (n = 13 mice per group) cells in WT and Hira-KO BM cells. (C and D) One of 11 FACS profiles (C) and total cell counts (D) for CLPs (Lin
Sca1lowcKitlowIL7R+) in WT and Hira-KO BM cells (n = 11 mice per group). (E and F) One of 10 FACS profiles (E) and total cell counts (F) for CMPs (Lin
Sca1
cKit+CD16/32medCD34+), GMPs (Lin
Sca1
cKit+CD16/32highCD34+), and MEPs (Lin
Sca1
cKit+CD16/32lowCD34
) (n = 10 mice per group). (G and H) One of 4 FACS profiles and total cell counts for megakaryocytes (Mkp, Lin
Sca1
cKit+CD150+CD41+) (n = 4 mice per group). Data represent the average value ± SD. (I) Megakaryocyte colony-forming assay. BM cells from WT or Hira-KO mice were cultured in indicated medium for eight days. Colonies were stained for acetylcholinesterase activity.

(legend continued on next page)

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on discreet steps of subsequent lymphoid and myeloid cell differentiation. HIRA Is Required for the Acquisition of BM HSC Stem Cell Property: BM Reconstitution Experiments To address the root cause of reduced LT-HSCs in Hira-KO BM, we asked whether LT-HSCs are defective in survival. To this end, we tested possible DNA damage. HSCs are susceptible to various stress, including pathogen stimulation and oxidative stress, leading to DNA damage, the process controlled in part by the p53 pathway (Liu et al., 2009; Takizawa et al., 2017; Walter et al., 2015). Phosphorylated (g)-H2AX has been used to detect DNA damage in various cells, including HSCs (Kataoka et al., 2006; Rossi et al., 2007). g-H2AX staining was visualized for the LT-HSC fraction using FACS analysis. Data in Figures 4A and 4B showed noticeably higher staining in Hira-KO cells than WT cells, with a significant increase in mean fluorescence intensity (MFI) values. Presumably as a result, Hira-KO LT-HSCs displayed increased apoptosis, as evidenced by increased annexin V staining (Figures 4C and 4D). These results indicate that HIRA is required for HSC genome integrity and that its loss leads to apoptotic cell death. Cell cycle profiles presented in Figures S3A and S3B, nevertheless, did not show a discernible difference between Hira-KO and WT HSCs. To determine whether Hira-KO HSCs possess the key HSC property (i.e., self-renewal and progenitor differentiation capacity), competitive BM transplantation experiments were performed. Sorted LSK cells from WT or Hira KO (both CD45.2+) were co-transplanted into lethally irradiated recipient mice (WT, CD45.1+) at a 1:1 ratio along with WT competitor cells (CD45.1+) (Figure 4E). Mice were allowed to recover for 16 weeks, and then FACS analyses were performed to determine whether CD45.2+ donor cells could reconstitute peripheral blood, spleen, and BM (Figures 4F–4H; Figure S3 for FACS data). Hira-KO donor cells failed to give rise to B, T and myeloid cells in peripheral blood, while WT cells generated these cells to represent 50% of reconstituted cells (Figure 4F). Furthermore, Hira-KO donor cells did not produce LSK, nor LK cells in BM, illustrating defective self-renewal capacity. Consequently, Hira KO donors did not give rise to either B220+ or CD11b+ cells in BM, consistent with defective progenitor development (Figure 4G). As a result, Hira-KO donor cells did not contribute to repopulation of B cells, T cells, and myeloid cells in spleen (Figure 4H). The failure of Hira-KO cells to reconstitute the recipient mice was not due to defective homing, as CFSE-labeled LSK cells from WT and Hira-KO mice, when injected into irradiated CD45.1 WT mice, migrated to BM in a comparable manner (Figure 4I). These

results show that Hira-KO HSCs are prone to DNA damage and apoptosis and lack the LT self-renewal capacity as well as the ability to produce progenitor cells. HIRA Is Dispensable for Fetal Hematopoiesis During embryogenesis, HSCs emerge in the aorta-gonad-mesonephros region on embryonic day 10.5 (E10.5) and subsequently migrate into the FL on E11.5. FL hematopoiesis reaches its peak on E14.5, and then it shifts to the BM (Aggarwal et al., 2012). Prior to the emergence of HSCs, FL is populated by yolk sac-derived erythroid-myeloid progenitors capable of rapid differentiation (McGrath et al., 2015). Given the normal birthrate of Hira-KO mice, we sought to clarify whether Hira disruption alters embryonic hematopoiesis in FL. Hira-KO embryos and FL appeared normal on E14.5 (Figure 5A), and the number of nucleated cells in FL was similar in WT and Hira-KO embryos (Figure 5B). qRTPCR analysis verified the absence of Hira exon 7 transcripts, and immunoblot analysis confirmed the lack of HIRA protein in KO FL cells (Figures S4A and S4B). FACS analysis found little to no difference in the frequencies and total numbers of LK, LSK, and LT-HSC cells in Hira-KO and WT FL (Figure 5C; Figure S4C). We also examined FL erythroid cells at varying stages of differentiation and found virtually no difference between HiraKO and WT embryos (Figure 5D; Figure S4D). Likewise, cells carrying B220+CD19+, CD5+CD3+, and CD11b+Gr1+ markers were similar in numbers in FL of WT and Hira-KO embryos (Figure 5E; Figure S4E). These data show that Hira is dispensable for hematopoiesis in FL up to E14.5, pointing to a differential role of HIRA in fetal and adult hematopoiesis. Hira-KO Fetal Liver HSCs Lack Long-Term Self-Renewal Capacity Transplantation experiments were performed to determine the potential of FL HSCs to repopulate BM. FL cells from WT or Hira-KO embryos (1 3 106, CD45.2+) were injected into lethally irradiated WT recipients together with the same number of BM cells from WT CD45.1 mice. Cells reconstituted in peripheral blood of the recipient mice were tested 4, 8, and 16 weeks later using FACS analyses (Figures 5F–5I; Figure S5 for FACS data). Hira-KO FL cells did not give rise to mononuclear cells in peripheral blood to a detectable degree. Similarly, Hira-KO FL cells did not generate T and B lymphocytes and myeloid cells in both BM and spleen (Figures 5J and 5K). In contrast, WT FL cells successfully reconstituted lymphocytes and myeloid cells in peripheral blood. Likewise, only WT FL cells, but not Hira-KO cells, gave rise to LK and LSK cells in the recipient BM (Figure 5J). CSFE-stained WT and Hira-KO

(J) Quantification of megakaryocyte colonies generated from WT and Hira-KO BM cells. Data are the average colony numbers from five independent experiment in duplicate ± SD. (K) Numbers of erythrocytes (Ter119+) in WT and Hira-KO BM (n = 6 mice per group) and spleen (SP; n = 4 mice per group). (L) Numbers of neutrophils (CD11b+Ly6GhighLy6Clow cells), monocytes (CD11b+Ly6GlowLy6Chigh cells), macrophages (MF; CD11b+F4/80+), and dendritic cells (DC; CD11b+CD11c+) in WT and Hira-KO BM (n = 9 mice per group). (M) Numbers of total B cells (B220+CD19+) in BM (n = 6 mice per group), spleen (n = 9 mice per group), and lymph nodes (n = 4 mice per group). (N) Numbers of B cells at different differentiation stages in BM (n = 6 mice per group): pro-B cells (B220+CD43+), pre-B cells (B220+CD43
IgM
IgD
), immature B cells (B220+CD43
IgM+IgD
), and mature cells (B220+CD43
IgM+IgD+). (O–Q) Numbers of CD4+, CD8+, and CD4
CD8
double-negative T cells in thymus (O) (n = 11 mice per group), spleen (P) (n = 8 mice per group), and lymph nodes (Q) (n = 4 mice per group). Data represent the average cell count ± SD. *p < 0.05 and **p < 0.01; ns, not significant.

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Figure 4. Hira-KO HSCs Are Prone to DNA Damage and Apoptosis and Lack Self-Renewal Capacity (A) Live BM LT-HSCs were first stained for LT-HSC cell surface makers, and cells were then fixed, permeabilized, and stained with g-H2AX antibody, and g-H2AX signals in LT-HSC (Lin
Sca1+cKit+CD150+D48
) population were analyzed using FACS. (B) The average MFI of g-H2AX signals calculated from three independent samples prepared from independently bred littermates. (C) BM LT-HSCs were stained with annexin V for 30 min and analyzed using FACS. (D) The percentage of cell death in Hira-KO and WT cells (n = 3). (E) LSK cells sorted from WT (CD45.2) or Hira-KO BM (CD45.2) were mixed in a 1:1 ratio with WT competitor LSK cells from CD45.1 mice and injected into lethally irradiated CD45.1 WT mice. (F–H) Sixteen weeks after cell transfer, recipient mice were analyzed for the relative contribution of donor engraftment (percentage CD45.2 cells) in (F) peripheral blood (n = 6 mice per group), (G) BM (n = 5 mice per group), and (H) spleen (n = 3 mice per group). Values are the average percentages of donor cells ± SD. (I) Homing capacity of WT and Hira-KO HSCs was assessed as follows. CFSE-labeled LSK cells were injected into lethally irradiated recipients. 24 h later, CFSEstained cells in BM were detected using FACS. Data represent the average percentage of CFSE+ cells from three independent experiments. **p < 0.01; ns, not significant.

FL LSK cells migrated to the recipient tissues in a comparable manner, indicating that the reconstitution defect of Hira-KO FL was not due to a deficiency in homing (Figure 5L). These results show that although HIRA is not required for hematopoiesis in FL, it is critically required for HSC self-renewal and progenitor differentiation. HIRA Defines HSC Transcriptomes: RNA-Seq Analyses To delineate transcriptome profiles of BM HSCs, we performed RNA-seq analyses for LSK cells, FACS-sorted from WT and Hira-KO BM cells. Differentially expressed genes were identified by the cutoff of >2-fold with a false discovery rate (FDR) of <0.0.5. The volcano plot in Figure 6A shows that 519 genes were downregulated in Hira-KO BM LSK cells relative to WT

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cells, while 234 genes were upregulated in Hira-KO LSK cells (marked in red in Figure 6A). As shown in the heatmap in Figure 6B, a series of transcription factors were strongly downregulated in Hira-KO cells, including those of the bZip family (Tsc22d, Hlf, Fosb, Jun, and Junb), zinc finger proteins (Egr1, Kfl2, and Mecom), and Hox-family factors (Hoxa9 and Meis1). Some of these transcription factors are expressed selectively in HSCs and indicated to be involved in shaping HSC transcriptomes (Olsson et al., 2016; Wilson et al., 2010). Additional transcription factors downregulated in Hira-KO HSCs include Stat4, Irf4, and Rel, which are activated by immune signaling. Also downregulated are Btg1and Btg2, involved in the regulation of proliferation (Farioli-Vecchioli et al., 2012; Olsson et al., 2016). A complete list of downregulated and upregulated genes and

Figure 5. HIRA Is Dispensable for Fetal Hematopoiesis, and Hira-KO FL HSCs Lack Long-Term Reconstitution Capacity (A) Representative photographs of WT and HiraKO embryos at E14.5. (B) The number of nucleated cells in WT and HiraKO FL. (C) The percentage and absolute number of LK (Lin
Sca1
cKit+), LSK (Lin
Sca1+cKit+), and LTHSC (Lin
Sca1+cKit+CD150+D48
) cells in WT and Hira-KO FL. (D) The percentage and absolute number of erythroid lineage cells at various stages of differentiation in WT and Hira-KO FL cells are shown: P1 = CD71+Ter119
; P2 = CD71+Ter119
; P3 = CD71+Ter119+; P4 = CD71
Ter119+. (E) The percentage and absolute number of T cells (CD5+CD3+), B cells (B220+ CD19+), and myeloid cells (CD11b+Gr1+) in FL, detected using FACS (n = 6 mice per group). In (B)–(E), all data represent average values from six mice ± SD; ns, not significant. (F–K) FL cells from E14.5 WT or Hira-KO embryos (CD45.2) were mixed with the same number of competitor BM cells (CD45.1), which were injected into lethally irradiated CD45.1 recipient mice. (F–I) The percentage of CD45.2+ donor-derived cells in total blood (F), B220+ (G), CD3+ (H), and CD11b+ (I) cells in peripheral blood is shown over indicated weeks. (J) The percentages of CD45.2+ cells in LK, LSK, B220+, and CD3+ cells in BM at 16 weeks are shown. (K) The percentages of CD45.2+ cells in B220+, CD11b+, CD4+, and CD8+ cells in spleen at 16 weeks. In (F)–(K), values are the average percentages of donor cells from five mice ± SD. (L) Homing assay for Hira-KO FL cells. CFSElabeled WT and Hira-KO LSK cells were injected into lethally irradiated recipients as above. CFSEstained cells in BM were detected 24 h later using FACS analysis (n = 6 mice per group). Data represent the average percentages of CFSE+ cells from three independent experiments ± SD. **p < 0.01; ns, not significant.

an extended heatmap are shown in Table S1 and Figure S6A. qRT-PCR analyses of separate RNA preparations confirmed downregulation of these genes in Hira-KO BM LSK cells (Figure S6B). Other downregulated genes include growth factors, signaling molecules, and cytokines and chemokines active in HSCs, such as Flt3, Lif, Socs3, Tlr7, Gbp4, Gbp6, Gbp8, CCl4, Il7, and Il18 (extended heatmap in Figure S6A; gene list in Table S1). It is worth noting here that Mpl, which encodes the receptor for thrombopoietin, a well-known marker for HSCs, was also noticeably downregulated in Hira-KO cells. MPL is a major driver of Mkp differentiation leading to platelet formation (Olsson et al., 2016)(Geddis, 2010). In contrast to downregulation of HSC-specific genes, genes upregulated in Hira-KO cells tended to be those of differentiated myeloid and lymphoid cells (e.g., Cd74, Cxcl9, Cxcl12, and Mpo). In keeping with these observations, Gene Ontology (GO) analysis of downregulated genes highlighted categories such as control of he-

matopoiesis, cytokines, and signal transduction (Figure 6C). However, GO terms enriched in upregulated genes were unrelated to hematopoiesis (Figure 6D). HIRA Opens Chromatin Accessibility Sites Important for Establishing HSC Transcriptomes: ATAC-Seq Analyses To study an underlying basis of dysregulated transcriptomes in Hira-KO LSK cells, we examined the state of chromatin accessibility using ATAC-seq (Buenrostro et al., 2015). Global chromatin accessibility sites in Figure 7A showed a single peak of similar reads per kilobase of transcript per million mapped reads (RPKM) values in WT and Hira-KO LSK cells. When realigned to the genic regions, the majority peaked at the transcription start site (TSS) with a trail toward the gene body (Figure S7A). Data in Figure 7B showed that although many peaks were shared in WT and Hira-KO cells, 1,928 peaks that were present in WT HSCs were lost in Hira-KO cells.

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Figure 6. HIRA Defines HSC Transcriptome Identity: RNA-Seq Analyses (A) Volcano plot for differentially expressed genes. The x axis indicates log2(fold change), and the y axis indicates
log10(p value). Red dots represent significantly downregulated or upregulated genes (519 versus 234) in Hira-KO LSK cells (p < 0.05, fold change > 2). (B) Heatmap showing representative differentially expressed genes. The color bar indicates the Z score. (C and D) Network of enriched GO terms for upregulated genes (C) and downregulated genes (D) in Hira-KO cells. Each circle node represents one enriched term, where its size is proportional to the number of genes falling into that term. The nodes are colored by cluster ID, where nodes that share the same cluster ID are usually close to each other. Terms with similarity > 0.3 are connected by edges.

Conversely, Hira-KO cells gained 1,318 new peaks not present in WT cells, indicating that HIRA has a large impact on setting chromatin accessibility. Motif analysis in Figure 7C showed that the SPIB/PU.1/IRF sites containing a TTCC element were most significantly deceased in Hira-KO cells. PU.1 is an ETS family of transcription factor, important for HSC development (Olsson et al., 2016; Wilson et al., 2010). In contrast, GATA and related motifs were increased in HIRAKO cells (Figure 7D). Additional motifs gained and lost in Hira-KO cells are presented in Figures S7B and S7C. Functional annotation of the lost peaks pointed to a close association with hematopoiesis and hematopoietic processes (Figure 7E). On the other hand, gained peaks were associated with translation, RNA processing, and so on, distinct from

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those for lost peaks (Figure 7F). GO terms enriched in the lost or gained peaks are clearly related to those found in transcriptome analyses above. We further asked how chromatin accessibility relates to transcriptome profiles and found that significant numbers of genes downregulated in HIRA-KO cells were near ATAC-seq peaks lost in Hira-KO cells. Conversely, genes upregulated in Hira-KO cells were associated more strongly with gained ATAC-seq peaks (Figure 7G; number of peaks and genes in Figure S7D). Table S2 presents the list of downregulated genes with lost peaks, and Table S3 provides the list of upregulated genes with gained peaks. These analyses illustrate an excellent concordance between chromatin accessibility and transcript expression. Integrative Genomics Viewer (IGV) tracks in Figures 7H and 7I show examples of

Figure 7. HIRA Confers Open Chromatin Sites upon HSCs: ATAC-Seq Analyses (A) Average profiles of chromatin accessibility in WT and Hira-KO LSK cells at pooled ATAC-seq peaks. The y axis indicates the RPKM values, and the x axis represents distance from the center (kb). (B) MA plot showing alternations of chromatin accessibility sites in Hira-KO LSK cells relative to WT cells. The numbers of peaks lost and gained in Hira-KO cells are indicated with differential peaks marked in red. (C and D) Motif analysis: the two most significantly enriched DNA binding motifs for lost peaks (C) and gained peaks (D) in Hira-KO cells. (E and F) Representative GO terms enriched for lost (E) and gained (F) peaks in Hira-KO cells. (G) Bar plots showing the fraction of downregulated and upregulated genes that are associated with lost and gained peaks, respectively. A peak was considered associated with a gene, when its peak summit was located within 10 kb of the gene (at the TSS). p values from Fisher’s exact test are indicated. (H and I) Representative IGV tracks showing the ATAC-seq and RNA-seq profiles for downregulated genes (Cd69, Flt3) with decreased chromatin accessibility in the promoter regions (H) and upregulated genes (Tspo2, Tnfaip2) with increased accessibility (I).

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genes associated with loss or gain of ATAC-seq peaks along with RNA-seq peaks (see Figure S7E for additional examples, Irf4 and Stat4). In sum, HIRA confers proper chromatin accessibility on BM LSK cells, thus guiding HSC-specific gene expression. DISCUSSION Mice with Vav-Cre-driven Hira deletion were afflicted with broad hematologic and immunologic defects, ranging from anemia and thrombopenia to combined immune cell deficiency. The latter immune cell deficiency included a severe loss of B cells, shortage of peripheral T cells, and reduced monocytes, dendritic cells, and morphologically abnormal neutrophils. These defects were traced largely to the inability of Hira-KO mice to sustain HSCs in BM. Hira-KO HSCs were prone to DNA damage and apoptosis. Accordingly, Hira-KO mice had fewer LK and LSK cells, as well as fewer LT-HSCs and lineage progenitors. Cell transfer experiments pointed to the failure in self-renewal and lineage progression capacity to be a primary defect of Hira-KO HSCs rather than a defect in BM niche (Ng and Alexander, 2017; Pouzolles et al., 2016). Hira-KO mice displayed phenotypes reminiscent of some clinical features of DiGeorge syndromes, including thrombopenia
kova´ et al., and defects in B and T cells (Davies, 2013; Fron 2014; Lawrence et al., 2003; Patel et al., 2012). That Hira-KO mice exhibited compound immune cell deficiency also suggests a link, as DiGeorge syndromes are a part of newborn SCID screen (Kwan et al., 2013). We did not observe gross defects in thymocytes in Hira-KO mice, even though athymic conditions are noted in DiGeorge syndrome patients. This difference may not be consequential, as thymic deficiency in DiGeorge syndromes is attributed to defects in thymic stroma cells rather than thymocytes themselves (Davies, 2013). It should be noted here that DiGeorge syndromes are complex, likely governed by the deletion of multiple genes and regulatory sequences mapped to chromosome 22q11. In contrast to the dramatic failure of BM hematopoiesis, embryonic hematopoiesis in FL was unaffected in Hira-KO mice, in that erythroid, lymphoid, and myeloid cells were present in FL in normal numbers. The dichotomy between adult and embryonic hematopoiesis may not be surprising, given that hematopoiesis involves developmentally distinct mechanisms. There are factors differentially required for fetal and adult hematopoiesis (Babovic and Eaves, 2014). For example, Tel/Etv6, C/EBPa, and others are needed for adult BM hematopoiesis, but not for fetal hematopoiesis. Conversely, Sox17 is required for fetal, but not adult hematopoiesis (Kim et al., 2007, 2018). On the other hand, factors such as BRD4 are required for both fetal and adult hematopoiesis (Dey et al., 2019). HIRA apparently belongs to the former group. We noted that HSCs in Hira-KO FL exhibited a defect when tested for a LT self-renewal capacity, as evidenced by a failure of reconstitution in irradiated mice. We also noted that HIRA regulates not only HSCs but has a role in later stages of immune cell differentiation. This was revealed by the pronounced blockade in B cell development specifically from pro-B-to-pre-B transition, as well as the failure of post-thymic T cell differentiation in Hira-KO mice.

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RNA-seq analyses revealed that transcription factors of diverse families were downregulated in Hira-KO LSK cells, including Meis, Mecom, Fos, Jun, and Hoxa9, some of which are central to HSC development (Olsson et al., 2016; Wilson et al., 2010). Furthermore, genes in the signaling pathways active in immune cell lineages were downregulated in Hira-KO cells, such as Irf4, Stat4, and rel. For example, IRF4 is required for development of both T and B lymphocytes (Biswas et al., 2012; Wang et al., 2015). Because transcription factors and signaling pathways influence many downstream target genes, their deficiency would cause an amplifying impact on gene expression. Downregulation of Mpl, conspicuous in Hira-KO cells, may also result in broader consequences, as MPL is a receptor for thrombopoietin, a major driver of Mkp differentiation and platelet formation (Geddis, 2010). Overall, RNA-seq data revealed that HIRA helps implement the proper transcription program necessary for HSCs. ATAC-seq experiments revealed that HIRA plays a fundamental role in opening chromatin sites necessary for BM HSCs. Accordingly, many chromatin accessibility sites found in WT that were linked to hematopoiesis in GO terms were not present in Hira-KO cells. Reinforcing the significance of this result, many genes downregulated in Hira-KO cells were near the ATAC-seq sites lost in Hira-KO cells. SPIB/PU.1/IRF sites and related sites were most strongly lost in Hira-KO cells. The sites contain a conspicuous TTCC element, a well-known binding site for SPIB/PU.1. SPIB/PU.1 is one of representative transcription factors that shape HSCs (Olsson et al., 2016; Wilson et al., 2010). SPIB/PU.1 is also known as a pioneer factor that sets the chromatin and enhancer sites in myeloid and lymphoid progenitors (Zaret and Mango, 2016). SPIB/PU.1 interacts with IRFs and binds additional composite sites similar to CTTT, as found in the SPIB/PU.1/IRF motif here (Kurotaki et al., 2013; Mancino et al., 2015). It seems that the accessibility of the SPIB/PU.1/IRF sites is critically dependent on HIRA. In summary, our study demonstrates that HIRA confers upon BM HSCs the DNA-accessible sites essential for HSC-specific transcriptome programs. In this way HIRA promotes selfrenewal and lineage specification, thereby governing the entire hematopoietic processes. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d d d d

KEY RESOURCES TABLE LEAD CONTACT AND MATERIALS AVAILABILITY EXPERIMENTAL MODEL AND SUBJECT DETAILS B Mouse studies METHOD DETAILS B Flow cytometry (FACS analysis) and cell sorting B Blood cell count and morphology B Bone marrow and fetal liver cell transfer experiments B Megakaryocyte colony formation assay (CFU-MK) B Quantitative (q)RT-PCR and Immunoblot assay

B

RNA-seq analysis ATAC-seq analysis QUANTIFICATION AND STATISTICAL ANALYSIS DATA AND CODE AVAILABILITY B

d d

SUPPLEMENTAL INFORMATION Supplemental Information can be found online at https://doi.org/10.1016/j. celrep.2020.01.062. ACKNOWLEDGMENTS We thank D.-M. Sin (Seoul University, Korea), K. Ge, D. Singer, A. Gegonne, and S. Chauhan (NIH) for discussions and technical help and advice. We acknowledge GenOway for constructing Hiraf/f mice with germline transmission. This work was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) Intramural Programs (ZIA HD001310-32) and National Institute on Aging (NIA, P01 AG031862). AUTHOR CONTRIBUTIONS C.C. performed all experiments and organized the work flow. C.W., T.W., and A.D. advised and assisted in cell transfer, related logistics, and flow cytometry experiments. M.S. and M.B. led Illumina-based DNA sequencing and performed bioinformatics analyses. P.A supplied critical reagents along with procedural guidance. P.L. and T.M. provided intellectual input on data interpretation and required experiments. K.O. conceived and steered the project. C.C. and K.O. wrote the manuscript. DECLARATION OF INTERESTS The authors declare no competing interests. Received: May 20, 2019 Revised: December 5, 2019 Accepted: January 21, 2020 Published: February 18, 2020

Buenrostro, J.D., Wu, B., Chang, H.Y., and Greenleaf, W.J. (2015). ATAC-seq: a method for assaying chromatin accessibility genome-wide. Curr. Protoc. Mol. Biol. 109, 21.29.1–21.29.9. Challen, G.A., Boles, N., Lin, K.K., and Goodell, M.A. (2009). Mouse hematopoietic stem cell identification and analysis. Cytometry A 75, 14–24. Chen, C., Qiu, H., Gong, J., Liu, Q., Xiao, H., Chen, X.W., Sun, B.L., and Yang, R.G. (2012). (-)-Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C virus. Arch. Virol. 157, 1301–1312. Cullen, S.M., Mayle, A., Rossi, L., and Goodell, M.A. (2014). Hematopoietic stem cell development: an epigenetic journey. Curr. Top Dev. Biol. 107, 39–75. D’Antoni, S., Mattina, T., Di Mare, P., Federico, C., Motta, S., and Saccone, S. (2004). Altered replication timing of the HIRA/Tuple1 locus in the DiGeorge and Velocardiofacial syndromes. Gene 333, 111–119. Davies, E.G. (2013). Immunodeficiency in DiGeorge syndrome and options for treating cases with complete athymia. Front. Immunol. 4, 322. Dey, A., Yang, W., Gegonne, A., Nishiyama, A., Pan, R., Yagi, R., Grinberg, A., Finkelman, F.D., Pfeifer, K., Zhu, J., et al. (2019). BRD4 directs hematopoietic stem cell development and modulates macrophage inflammatory responses. EMBO J. 38, e100293. Dilg, D., Saleh, R.N., Phelps, S.E., Rose, Y., Dupays, L., Murphy, C., Mohun, T., Anderson, R.H., Scambler, P.J., and Chapgier, A.L. (2016). HIRA is required for heart development and directly regulates Tnni2 and Tnnt3. PLoS ONE 11, e0161096. Dutta, D., Ray, S., Home, P., Saha, B., Wang, S., Sheibani, N., Tawfik, O., Cheng, N., and Paul, S. (2010). Regulation of angiogenesis by histone chaperone HIRA-mediated incorporation of lysine 56-acetylated histone H3.3 at chromatin domains of endothelial genes. J. Biol. Chem. 285, 41567–41577. Farioli-Vecchioli, S., Micheli, L., Saraulli, D., Ceccarelli, M., Cannas, S., Scardigli, R., Leonardi, L., Cina`, I., Costanzi, M., Ciotti, M.T., et al. (2012). Btg1 is required to maintain the pool of stem and progenitor cells of the dentate gyrus and subventricular zone. Front. Neurosci. 6, 124.
kova´, E., Klocperk, A., Svaton
, M., Nova´kova´, M., Kotrova´, M., Kayserova´, Fron J., Kalina, T., Keslova´, P., Votava, F., Vinohradska´, H., et al. (2014). The TREC/ KREC assay for the diagnosis and monitoring of patients with DiGeorge syndrome. PLoS ONE 9, e114514.

REFERENCES

Geddis, A.E. (2010). Megakaryopoiesis. Semin. Hematol. 47, 212–219.

Adam, S., Polo, S.E., and Almouzni, G. (2013). Transcription recovery after DNA damage requires chromatin priming by the H3.3 histone chaperone HIRA. Cell 155, 94–106.

Goldberg, A.D., Banaszynski, L.A., Noh, K.M., Lewis, P.W., Elsaesser, S.J., Stadler, S., Dewell, S., Law, M., Guo, X., Li, X., et al. (2010). Distinct factors control histone variant H3.3 localization at specific genomic regions. Cell 140, 678–691.

Aggarwal, R., Lu, J., Pompili, V.J., and Das, H. (2012). Hematopoietic stem cells: transcriptional regulation, ex vivo expansion and clinical application. Curr. Mol. Med. 12, 34–49. Akar, N.A., and Adekile, A.D. (2007). Chromosome 22q11.2 deletion presenting with immune-mediated cytopenias, macrothrombocytopenia and platelet dysfunction. Med. Princ. Pract. 16, 318–320. Allis, C.D., and Jenuwein, T. (2016). The molecular hallmarks of epigenetic control. Nat. Rev. Genet. 17, 487–500. Anders, S., Pyl, P.T., and Huber, W. (2015). HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31, 166–169. Babovic, S., and Eaves, C.J. (2014). Hierarchical organization of fetal and adult hematopoietic stem cells. Exp. Cell Res. 329, 185–191. Banaszynski, L.A., Wen, D., Dewell, S., Whitcomb, S.J., Lin, M., Diaz, N., Elsa¨sser, S.J., Chapgier, A., Goldberg, A.D., Canaani, E., et al. (2013). Hiradependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells. Cell 155, 107–120. Biswas, P.S., Gupta, S., Stirzaker, R.A., Kumar, V., Jessberger, R., Lu, T.T., Bhagat, G., and Pernis, A.B. (2012). Dual regulation of IRF4 function in T and B cells is required for the coordination of T-B cell interactions and the prevention of autoimmunity. J. Exp. Med. 209, 581–596.

Gordon, A.R., Outram, S.V., Keramatipour, M., Goddard, C.A., Colledge, W.H., Metcalfe, J.C., Hager-Theodorides, A.L., Crompton, T., and Kemp, P.R. (2008). Splenomegaly and modified erythropoiesis in KLF13-/- mice. J. Biol. Chem. 283, 11897–11904. Hall, C., Nelson, D.M., Ye, X., Baker, K., DeCaprio, J.A., Seeholzer, S., Lipinski, M., and Adams, P.D. (2001). HIRA, the human homologue of yeast Hir1p and Hir2p, is a novel cyclin-cdk2 substrate whose expression blocks S-phase progression. Mol. Cell. Biol. 21, 1854–1865. Hammond, C.M., Strømme, C.B., Huang, H., Patel, D.J., and Groth, A. (2017). Histone chaperone networks shaping chromatin function. Nat. Rev. Mol. Cell Biol. 18, 141–158. Hardy, R.R., and Hayakawa, K. (2001). B cell development pathways. Annu. Rev. Immunol. 19, 595–621. Kataoka, Y., Bindokas, V.P., Duggan, R.C., Murley, J.S., and Grdina, D.J. (2006). Flow cytometric analysis of phosphorylated histone H2AX following exposure to ionizing radiation in human microvascular endothelial cells. J. Radiat. Res. (Tokyo) 47, 245–257. Kim, I., Saunders, T.L., and Morrison, S.J. (2007). Sox17 dependence distinguishes the transcriptional regulation of fetal from adult hematopoietic stem cells. Cell 130, 470–483.

Cell Reports 30, 2136–2149, February 18, 2020 2147

Kim, H., Lee, S., and Lee, S.W. (2018). TRAF6 distinctly regulates hematopoietic stem and progenitors at different periods of development in mice. Mol. Cells 41, 753–761.

Oguro, H., Ding, L., and Morrison, S.J. (2013). SLAM family markers resolve functionally distinct subpopulations of hematopoietic stem cells and multipotent progenitors. Cell Stem Cell 13, 102–116.


s
mova´, K., Kayserova´, J., Fron
kova´, E., and Klocperk, A., Grecova´, J., Si
´ , A. (2014). Helios expression in T-regulatory cells in patients with di Sediva George Syndrome. J. Clin. Immunol. 34, 864–870.

Olsson, A., Venkatasubramanian, M., Chaudhri, V.K., Aronow, B.J., Salomonis, N., Singh, H., and Grimes, H.L. (2016). Single-cell analysis of mixed-lineage states leading to a binary cell fate choice. Nature 537, 698–702.

Kurotaki, D., Osato, N., Nishiyama, A., Yamamoto, M., Ban, T., Sato, H., Nakabayashi, J., Umehara, M., Miyake, N., Matsumoto, N., et al. (2013). Essential role of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation. Blood 121, 1839–1849. Kwan, A., Church, J.A., Cowan, M.J., Agarwal, R., Kapoor, N., Kohn, D.B., Lewis, D.B., McGhee, S.A., Moore, T.B., Stiehm, E.R., et al. (2013). Newborn screening for severe combined immunodeficiency and T-cell lymphopenia in California: results of the first 2 years. J. Allergy Clin. Immunol. 132, 140–150. Lawrence, S., McDonald-McGinn, D.M., Zackai, E., and Sullivan, K.E. (2003). Thrombocytopenia in patients with chromosome 22q11.2 deletion syndrome. J. Pediatr. 143, 277–278.

Patel, K., Akhter, J., Kobrynski, L., Benjamin Gathmann, M.A., Davis, O., and Sullivan, K.E.; International DiGeorge Syndrome Immunodeficiency Consortium (2012). Immunoglobulin deficiencies: the B-lymphocyte side of DiGeorge Syndrome. J. Pediatr. 161, 950–953. Pchelintsev, N.A., McBryan, T., Rai, T.S., van Tuyn, J., Ray-Gallet, D., Almouzni, G., and Adams, P.D. (2013). Placing the HIRA histone chaperone complex in the chromatin landscape. Cell Rep. 3, 1012–1019. Pouzolles, M., Oburoglu, L., Taylor, N., and Zimmermann, V.S. (2016). Hematopoietic stem cell lineage specification. Curr. Opin. Hematol. 23, 311–317.

Li, Y., and Jiao, J. (2017). Histone chaperone HIRA regulates neural progenitor cell proliferation and neurogenesis via b-catenin. J. Cell Biol. 216, 1975–1992.

Psaila, B., Barkas, N., Iskander, D., Roy, A., Anderson, S., Ashley, N., Caputo, V.S., Lichtenberg, J., Loaiza, S., Bodine, D.M., et al. (2016). Single-cell profiling of human megakaryocyte-erythroid progenitors identifies distinct megakaryocyte and erythroid differentiation pathways. Genome Biol. 17, 83.

Li, L., Jothi, R., Cui, K., Lee, J.Y., Cohen, T., Gorivodsky, M., Tzchori, I., Zhao, Y., Hayes, S.M., Bresnick, E.H., et al. (2011). Nuclear adaptor Ldb1 regulates a transcriptional program essential for the maintenance of hematopoietic stem cells. Nat. Immunol. 12, 129–136.

Rai, T.S., Cole, J.J., Nelson, D.M., Dikovskaya, D., Faller, W.J., Vizioli, M.G., Hewitt, R.N., Anannya, O., McBryan, T., Manoharan, I., et al. (2014). HIRA orchestrates a dynamic chromatin landscape in senescence and is required for suppression of neoplasia. Genes Dev. 28, 2712–2725.

Lindsay, E.A. (2001). Chromosomal microdeletions: dissecting del22q11 syndrome. Nat. Rev. Genet. 2, 858–868. Liu, Y., Elf, S.E., Miyata, Y., Sashida, G., Liu, Y., Huang, G., Di Giandomenico, S., Lee, J.M., Deblasio, A., Menendez, S., et al. (2009). p53 regulates hematopoietic stem cell quiescence. Cell Stem Cell 4, 37–48.

Ricketts, M.D., Frederick, B., Hoff, H., Tang, Y., Schultz, D.C., Singh Rai, T., Grazia Vizioli, M., Adams, P.D., and Marmorstein, R. (2015). Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex. Nat. Commun. 6, 7711.

Loppin, B., Bonnefoy, E., Anselme, C., Laurenc¸on, A., Karr, T.L., and Couble, P. (2005). The histone H3.3 chaperone HIRA is essential for chromatin assembly in the male pronucleus. Nature 437, 1386–1390.

Roberts, C., Sutherland, H.F., Farmer, H., Kimber, W., Halford, S., Carey, A., Brickman, J.M., Wynshaw-Boris, A., and Scambler, P.J. (2002). Targeted mutagenesis of the Hira gene results in gastrulation defects and patterning abnormalities of mesoendodermal derivatives prior to early embryonic lethality. Mol. Cell. Biol. 22, 2318–2328.

Love, M.I., Huber, W., and Anders, S. (2014). Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550.

Rossi, D.J., Bryder, D., Seita, J., Nussenzweig, A., Hoeijmakers, J., and Weissman, I.L. (2007). Deficiencies in DNA damage repair limit the function of haematopoietic stem cells with age. Nature 447, 725–729.

Mancino, A., Termanini, A., Barozzi, I., Ghisletti, S., Ostuni, R., Prosperini, E., Ozato, K., and Natoli, G. (2015). A dual cis-regulatory code links IRF8 to constitutive and inducible gene expression in macrophages. Genes Dev. 29, 394–408.

Stadtfeld, M., and Graf, T. (2005). Assessing the role of hematopoietic plasticity for endothelial and hepatocyte development by non-invasive lineage tracing. Development 132, 203–213.

McDonald-McGinn, D.M., and Sullivan, K.E. (2011). Chromosome 22q11.2 deletion syndrome (DiGeorge syndrome/velocardiofacial syndrome). Medicine (Baltimore) 90, 1–18. McGrath, K.E., Frame, J.M., Fegan, K.H., Bowen, J.R., Conway, S.J., Catherman, S.C., Kingsley, P.D., Koniski, A.D., and Palis, J. (2015). Distinct sources of hematopoietic progenitors emerge before HSCs and provide functional blood cells in the mammalian embryo. Cell Rep. 11, 1892–1904. Meshorer, E., Yellajoshula, D., George, E., Scambler, P.J., Brown, D.T., and Misteli, T. (2006). Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells. Dev. Cell 10, 105–116.

Sullivan, K.E. (2019). Chromosome 22q11.2 deletion syndrome and DiGeorge syndrome. Immunol. Rev. 287, 186–201. Takizawa, H., Fritsch, K., Kovtonyuk, L.V., Saito, Y., Yakkala, C., Jacobs, K., Ahuja, A.K., Lopes, M., Hausmann, A., Hardt, W.D., et al. (2017). Pathogeninduced TLR4-TRIF innate immune signaling in hematopoietic stem cells promotes proliferation but reduces competitive fitness. Cell Stem Cell 21, 225–240.e5. Talbert, P.B., and Henikoff, S. (2017). Histone variants on the move: substrates for chromatin dynamics. Nat. Rev. Mol. Cell Biol. 18, 115–126. Tong, W., and Lodish, H.F. (2004). Lnk inhibits Tpo-mpl signaling and Tpomediated megakaryocytopoiesis. J. Exp. Med. 200, 569–580.

Mitroulis, I., Ruppova, K., Wang, B., Chen, L.S., Grzybek, M., Grinenko, T., Eugster, A., Troullinaki, M., Palladini, A., Kourtzelis, I., et al. (2018). Modulation of myelopoiesis progenitors is an integral component of trained immunity. Cell 172, 147–161.e12.

Tripathi, S., Pohl, M.O., Zhou, Y., Rodriguez-Frandsen, A., Wang, G., Stein, D.A., Moulton, H.M., DeJesus, P., Che, J., Mulder, L.C., et al. (2015). Metaand orthogonal integration of influenza ‘‘OMICs’’ data defines a role for UBR4 in virus budding. Cell Host Microbe 18, 723–735.

Nashun, B., Hill, P.W., Smallwood, S.A., Dharmalingam, G., Amouroux, R., Clark, S.J., Sharma, V., Ndjetehe, E., Pelczar, P., Festenstein, R.J., et al. (2015). Continuous histone replacement by Hira is essential for normal transcriptional regulation and de novo DNA methylation during mouse oogenesis. Mol. Cell 60, 611–625.

Walter, D., Lier, A., Geiselhart, A., Thalheimer, F.B., Huntscha, S., Sobotta, M.C., Moehrle, B., Brocks, D., Bayindir, I., Kaschutnig, P., et al. (2015). Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells. Nature 520, 549–552.

Ng, A.P., and Alexander, W.S. (2017). Haematopoietic stem cells: past, present and future. Cell Death Discov. 3, 17002.

2148 Cell Reports 30, 2136–2149, February 18, 2020

Wang, S., He, Q., Ma, D., Xue, Y., and Liu, F. (2015). Irf4 regulates the choice between T lymphoid-primed progenitor and myeloid lineage fates during embryogenesis. Dev. Cell 34, 621–631.

€tte, J., Kaimakis, P., Wilson, N.K., Foster, S.D., Wang, X., Knezevic, K., Schu Chilarska, P.M., Kinston, S., Ouwehand, W.H., Dzierzak, E., et al. (2010). Combinatorial transcriptional control in blood stem/progenitor cells: genome-wide analysis of ten major transcriptional regulators. Cell Stem Cell 7, 532–544. Yang, J.H., Song, T.Y., Jo, C., Park, J., Lee, H.Y., Song, I., Hong, S., Jung, K.Y., Kim, J., Han, J.W., et al. (2016). Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch. Exp. Mol. Med. 48, e252.

Zaret, K.S., and Mango, S.E. (2016). Pioneer transcription factors, chromatin dynamics, and cell fate control. Curr. Opin. Genet. Dev. 37, 76–81. Zhou, T., Kinney, M.C., Scott, L.M., Zinkel, S.S., and Rebel, V.I. (2015). Revisiting the case for genetically engineered mouse models in human myelodysplastic syndrome research. Blood 126, 1057–1068. Zhu, Z., Li, C., Zeng, Y., Ding, J., Qu, Z., Gu, J., Ge, L., Tang, F., Huang, X., Zhou, C., et al. (2017). PHB associates with the HIRA complex to control an epigenetic-metabolic circuit in human ESCs. Cell Stem Cell 20, 274–289.e7.

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STAR+METHODS KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies PE anti-mouse Lineage Cocktail

BioLegend

Cat# 133303

Biotin anti-mouse Lineage Panel

BioLegend

Cat# 133307

PE-Cy7-Sca-1

BD Biosciences

Cat# 558162

PerCP-Sca-1

BD Biosciences

Cat# 108122

APC-c-Kit

BioLegend

Cat# 105812

APC-Fluor 780-c-Kit

eBioscience

Cat# 47-1171-82

FITC-CD48

eBioscience

Cat# 11-0481-82

PerCP/Cy5.5-CD48

BioLegend

Cat# 103422

APC-CD150

BioLegend

Cat# 115909

Alexa Fluor 647-CD34

BD Biosciences

Cat# 560230 Cat# 553144

FITC-CD16/32

BD Biosciences

Purified anti-mouse CD16/32 Antibody

BioLegend

Cat# 101302

PE-CD127

BioLegend

Cat# 135009

PerCP/Cy5.5-CD41

BioLegend

Cat# 133917

Brilliant Violet 605-Ki67

BioLegend

Cat# 652413

Alexa Fluor 488-H2A.X

BioLegend

Cat# 613405

FITC-CD43

BioLegend

Cat# 143203

PerCP-B220

BioLegend

Cat# 103234

APC-IgM

BioLegend

Cat# 406509

PE-IgD

BioLegend

Cat# 405705

PE-CD4

BioLegend

Cat# 100408

PerCP-CD8

BioLegend

Cat# 100732

APC-CD25

BioLegend

Cat# 101909

FITC-CD44

BioLegend

Cat# 103006

PerCP/Cy5.5-CD11b

BioLegend

Cat# 101228

FITC-Ly-6G/Ly-6C (Gr-1)

BioLegend

Cat# 108406

APC-Ly-6G

BioLegend

Cat# 127614

PE-Ly-6C

BioLegend

Cat# 128007

APC-F4/80

BioLegend

Cat# 123116

PE-CD11c

BioLegend

Cat# 117308

APC/Cy7-CD3

BioLegend

Cat# 100222

PE/Cy7-CD19

BioLegend

Cat# 115519

FITC-CD5

BioLegend

Cat# 100605

APC-Ter119

BioLegend

Cat# 116212

PE-CD71

BioLegend

Cat# 113808

APC-CD45.1

BioLegend

Cat# 110714

PE-CD45.2

BioLegend

Cat# 109808

Hira

Peter Adams lab

N/A

TFIIB

Abcam

ab109518

Chemicals, Peptides, and Recombinant Proteins rh TPO

STEMCELL Technologies

Cat# 02522

rh IL11

R&D systems

Cat# 218-IL-005

rh IL-6

Pepro Technology

Cat# 200-06 (Continued on next page)

e1 Cell Reports 30, 2136–2149.e1–e4, February 18, 2020

Continued REAGENT or RESOURCE

SOURCE

IDENTIFIER

rm IL-3

Pepro Technology

Cat# 213-13

MegaCult-C Collagen and Medium

STEMCELL Technologies

Cat# 04960

SYBR Green PCR Master Mix

Thermo Fisher

Cat# 4309155

SuperScript II Reverse Transcriptase

Thermo Fisher

Cat# 18064014

TRIzol Reagent

Thermo Fisher

Cat# 15596026

Nextera Tn5 Transposase

illumina

Cat# FC-121-1030

CellTrace CFSE

Thermo Fisher

Cat# C34554

Wright-Giemsa Stain Kit

Thermo Fisher

Cat# 9990710

Lineage Cell Depletion Kit, mouse

Miltenyi Biotec

Cat# 130-090-858

Fixation/Permeabilization Solution Kit

BD biosciences

Cat# 554714

Annexin V-FITC Apoptosis Kit

BD biosciences

Cat# K101-100

Ovation RNA-Seq System V2

Nugen

Cat# 7102-A01

Nextera XT DNA Library Preparation Kit

illumina

Cat# FC-131-1024

RNA-seq

GEO

GSE125154

ATAC-seq

GEO

GSE125154

Mouse: Hiraf/f mice

Generated in this study

N/A

Mouse: Vav-Cre mice

Jackson lab

Cat# 008610

Mouse: B6.SJL-Ptprca/BoyAiTac (CD45.1)

Taconic

Cat# 4007

Critical Commercial Assays

Deposited Data

Experimental Models: Organisms/Strains

Oligonucleotides mHiraExon7-F: 50 -AACTCTGAGAGGTCATTCTG-30

Synthesized in this study

N/A

mHiraExon7-R: 50 - CTTGGTGATGCTAGTCTCTA-30

Synthesized in this study

N/A

mHiraExon9-F: 50 -TCCGGCTTAGTTGGTCACCTG-30

Synthesized in this study

N/A

Synthesized in this study

N/A

Graphad Prism 7

https://www.graphpad.com

Version 7

FlowJo

https://www.flowjo.com

Version 7 and 10

RNA seq data of LSK cells from WT or Hira KO mice

This paper

GEO: GSE125154

ATAC seq data of LSK cells from WT or Hira KO mice

This paper

GEO: GSE125154

0

mHiraExon9-R: 5 - CACAACAGTCACAGCTTTCC-3

0

Software and Algorithms

Other

LEAD CONTACT AND MATERIALS AVAILABILITY Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Keiko Ozato ([email protected]). Mouse line generated in this study are available from the Lead Contact upon request with a completed Materials Transfer Agreement. EXPERIMENTAL MODEL AND SUBJECT DETAILS Mouse studies All mice used in this study were bred and maintained in the NICHD animal facility in accordance with the specifications of the Association for Assessment and Accreditation of Laboratory Animal Care. Animal procedures were all performed under an animal study protocol (ASP# 014-044 and ASP# 17-044) approved by the NICHD animal care and use committee (ACUC). A targeting vector carrying LoxP sites flanking exon 7 of the Hira gene and a neomycin selection cassette flanked by Flip recombinase target sites were inserted in the sequence between exon 6 and exon 8 of the endogenous allele (Figure S1A). The targeting vector was linearized and electroporated into C57BL/6 ES cells. ES cells carrying the targeted locus were constructed by homologous recombination, and injected into C57BL/6 female mice and mice with Hiraf/+ germline transmission were selected and intercrossed to generate Hiraf/f mice.

Cell Reports 30, 2136–2149.e1–e4, February 18, 2020 e2

The neomycin cassette was removed by crossing to FlpE transgenic mice (Figure S1A). These procedures were performed by GenOway. Hiraf/f mice were bred with Vav-Cre to generate Hira KO mice as described (Dey et al., 2019). Mice of the same age (8-16-week-old), sex, and genotype were randomly grouped for subsequent experiments. No gender differences in gene deletion associated phenotypes were found in Hiraf/f. Vav-Cre mice. METHOD DETAILS Flow cytometry (FACS analysis) and cell sorting Single cell suspensions prepared from BM, spleen, thymus, lymph node, peripheral blood, and E14.5 fetal livers were tested after removal of red blood cells by ACK lysis buffer. Cells were blocked with purified anti-mouse CD16/32 and then stained with appropriate fluorochrome-conjugated antibodies (See key resources table above) for 30 min on ice in staining buffer (phosphate buffered saline with 2% fetal bovine serum). Details of antibody combinations used to identify each population are given in the figure legends. After staining, the cells were washed with staining buffer. BD FACS Calibur and Beckman CytoFLEX were used for analysis. Data were analyzed with Flowjo software. For the intracellular staining (Ki67 and gH2AX staining), BM cells were stained with HSC cell surface maker first, then fixed and permeabilized using the Fixation/Permeabilization Solution Kit, followed by Brilliant Violet 605-Ki67 (cell cycles) and Alexa Fluor 488-H2A.X (DNA damage) staining, and further incubated with DAPI. For cell sorting, BM cells were depleted using the mouse hematopoietic lineage depletion kit (Miltenyi Biotec) to enrich Lin- cells. The enriched Lin- cells were then stained with anti-lineage cocktail, anti-Scal-1, and anti-cKit antibodies for further sorting to enrich HSPCs (LSK cells). BD FACS Aria was used for cell sorting. Blood cell count and morphology Peripheral bloods were analyzed for cell number and morphology as follows. Blood was collected from the superficial facial vein into EDTA-treated tubes. The complete blood cell analysis was performed by the Animal Testing Program of the Department of Laboratory Medicine, Clinical Center at NIH. Morphology of granulocytes was evaluated for air-dried smear samples stained with WrightGiemsa and light microscopy. Bone marrow and fetal liver cell transfer experiments For competitive bone marrow transplantation experiment, sorted LSK cells from WT CD45.2+ or littermate Hira KO CD45.2+ mice were mixed with the same number of WT CD45.1+ competitor LSK cells, and the mixture was injected into lethally irradiated (950 cGy) CD45.1+ recipients by the retro-orbital route. For competitive fetal liver transplantation experiment, fetal liver cells from WT CD45.2+ or littermate Hira KO CD45.2+ embryos were mixed with the same number of WT CD45.1+ competitor bone marrow cells, and the mixture was injected into lethally irradiated (950 cGy) CD45.1+ recipients by the retro-orbital route. Reconstituted cell populations were determined by FACS analyses in peripheral blood every 4 weeks (week 0, 4, 8, and 16 after cell transfer). Reconstituted cell populations were further analyzed for BM, spleen, and thymus at 16 weeks. For homing assay, BM or FL LSK cells were labeled with CellTrace CFSE (Thermofisher), and then CFSE stained cells were injected retro-orbital into lethally irradiated CD45.1+ recipients. Twenty-four hours after injection, BM cells were analyzed by FACS to detect CFSE positive cells. Megakaryocyte colony formation assay (CFU-MK) CFU-MKs assays were performed with fresh bone marrow cells using MegaCult-C medium (Stem Cell Technologies) supplemented with 50 ng/ ml rhTPO, 20 ng/ml rhIL-6, 10 ng/ ml rmIL-3, and 50 ng/ ml rhIL-11 according to the manufacturer’s instruction. After 8 days of incubation, cultures were fixed, and megakaryocyte colonies were stained for acetylcholinesterase activity. Colonies containing more than three megakaryocytes were scored as CFU-MKs. Quantitative (q)RT-PCR and Immunoblot assay Total RNA was isolated with TRIzol RNA Isolation Reagents (Thermo Fisher) and treated with DNaseI. Reverse transcription was performed with Superscript II Reverse Transcriptase with random primers (Thermo Fisher). qRT-PCR was performed using SYBR Green PCR master mix on an ABI Prism 7000 with appropriate primers. The primers of Hira mRNA detection are: mHiraExon7-F: 50 -AA CTCTGAGAGGTCATTCTG-30 ; mHiraExon7-R: 50 - CTTGGTGATGCTAGTCTCTA-30 ; mHiraExon9-F: 50 -TCCGGCTTAGTTGGTCA CCTG-30 ; mHiraExon9-R: 50 - CACAACAGTCACAGCTTTCC-30 . The primers of other genes are purchased from Sigma. Relative expression values were calculated by the 66CT method, and normalized by Hprt. Immunoblot assays were performed using 20 ug of nuclear extracts as in (Chen et al., 2012). The primary antibodies used were mouse mAb Hira clone WC115 (Hall et al., 2001) and TF2B (Abcam). RNA-seq analysis Total RNA was isolated from sorted LSK cells from WT or Hira KO mice. The RNA sequencing library was prepared from approximately 50 ng of RNA using the Ovation RNA-seq System V2 (NuGEN) and Nextera XT DNA Library Preparation Kit (illumina), and sequencing was done using an illumina HiSeq 2000.The mouse reference genome (GRCm38) and corresponding gene annotation

e3 Cell Reports 30, 2136–2149.e1–e4, February 18, 2020

were obtained from illumina iGenome. Reads were aligned to the reference genome (GRCm38) using STAR 2.5.3a with parameters:– outSAMstrandField intronMotif–outFilterType BySJout outFilterIntronMotifs RemoveNoncanonical. To identify differentially expressed genes, The read counts were obtained for each gene with htseq-count (Anders et al., 2015), then identified differential genes as fold Change > 2 and FDR < 0.05 using DESeq2 (Love et al., 2014). To visualize the expression profile of representative genes, gene expression was first quantified using RSEM 1.3.0 with parameters:–bowtie2–calc-ci–ci-memory 1024. The calculated TPM (transcript per million) values were normalized using trimmed mean of M-values normalization (TMM) method. The normalized TPM values were used for visualization. Gene ontology enrichment analysis for differentially expressed genes was performed using metascape (Tripathi et al., 2015). The raw sequence reads from RNA-seq experiments have been submitted to the NCBI Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) and are accessible with the accession #: GSE125154. ATAC-seq analysis ATAC-seq was performed as described (Buenrostro et al., 2015). In brief, transposed DNA fragments were amplified by PCR for 11 cycles after the transposition reaction and purification. The library quality was check by gel electrophoresis. Libraries were sequenced using paired-end sequencing on an Illumina Next seq500. Reads were first processed using Trim Galore 0.4.5 (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/), then aligned to mouse reference genome (GRCm38) using bowtie 2-2.2.9. Peak calling was performed by using MACS 2.1.1 with default settings. To identify differential ATAC-seq peaks between WT and Hira KO samples, peak calling was first performed by using MACS 2.1.1 after merging all related data, then counted the number of reads located within ± 125 bp of peak summit with the multicov function of BEDTools. Finally, differential peak calling was carried out by using DESeq2 (Love et al., 2014) with threshold: foldChange > 2 and FDR < 0.05. Gene ontology enrichment analysis of the differential peaks was performed by using GREAT. Motif analysis for differential peaks was performed by MEME-ChIP, with sequences of ± 100 bp of peak summit used as input. All sequence data were analyzed using GraphPad Prism 6.0 and R Programming Language. P values comparing two means were calculated using the two-tailed unpaired Student’s t test. p < 0.05 was considered statistically significant. Heatmaps were generated by using the R package pheatmap. Visualization of RNA-Seq and ATAC-Seq tracks were performed by using Integrative Genomic Viewer (IGV). Raw sequence information obtained from ATAC-seq experiments have been submitted to the NCBI Gene Expression Omnibus (GEO. https://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series with the accession number GSE125154. QUANTIFICATION AND STATISTICAL ANALYSIS All data are presented as mean+/
S.D, and the statistical significant of differences was determined by the two-tailed Student’s t test for comparison between two experimental groups. Statistical analyses were performed using GraphPad Prism 7. p < 0.05 was considered statistically significant. DATA AND CODE AVAILABILITY Raw sequence information obtained from RNA-seq and ATAC-seq experiments have been submitted to the NCBI Gene Expression Omnibus (GEO. https://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO series with the accession number GSE125154.

Cell Reports 30, 2136–2149.e1–e4, February 18, 2020 e4