SCIENCE ADVANCES | RESEARCH ARTICLE EPIGENETICS Copyright © 2020 The Authors, some The Polycomb-associated factor PHF19 controls rights reserved; exclusive licensee hematopoietic stem cell state and differentiation American Association for the Advancement of Science. No claim to Pedro Vizán1*, Arantxa Gutiérrez1*, Isabel Espejo1, Marc García-Montolio1, Martin Lange1†, original U.S. Government Ana Carretero2, Atefeh Lafzi3, Luisa de Andrés-Aguayo1, Enrique Blanco1, Roshana Thambyrajah4, Works. Distributed Thomas Graf1, Holger Heyn3, Anna Bigas4,5, Luciano Di Croce1,6,7‡ under a Creative Commons Attribution Adult hematopoietic stem cells (HSCs) are rare multipotent cells in bone marrow that are responsible for generat- NonCommercial ing all blood cell types. HSCs are a heterogeneous group of cells with high plasticity, in part, conferred by epigenetic License 4.0 (CC BY-NC). mechanisms. PHF19, a subunit of the Polycomb repressive complex 2 (PRC2), is preferentially expressed in mouse hematopoietic precursors. Here, we now show that, in stark contrast to results published for other PRC2 subunits, genetic depletion of Phf19 increases HSC identity and quiescence. While proliferation of HSCs is normally triggered by forced mobilization, defects in differentiation impede long-term correct blood production, eventually leading to aberrant hematopoiesis. At molecular level, PHF19 deletion triggers a redistribution of the histone repressive mark H3K27me3, which notably accumulates at blood lineage–specific genes. Our results provide novel insights into how epigenetic mechanisms determine HSC identity, control differentiation, and are key for proper hematopoiesis. INTRODUCTION The Polycomb group (PcG) of proteins form multiprotein com- Blood regenerates at a high level, providing an ideal platform for plexes that are major epigenetic regulators of gene expression. The studying stem cell function. Hematopoietic stem cells (HSCs) in bone Polycomb repressive complex 2 (PRC2) catalyzes the trimethylation marrow (BM) are rare cells at the top of a hierarchical hematopoietic of lysine-27 in the histone H3 N-terminal tail (H3K27me3), which system; in this model, HSCs are defined by having long-term re- is associated with chromatin compaction and gene repression (5, 6). population capacity and produce the more committed progenitors, PRC2 comprises three core components [Enhancer of zeste homolog 2 which eventually produce all the differentiated cell types. Although (EZH2), suppressor of zeste 12 (SUZ12), and embryonic ectoderm well established in general terms, this model has been recently chal- development (EED)] and several substoichiometrical accessory factors lenged in two ways: (i) HSCs can be already biased toward specific that regulate its function (7, 8). In the past two decades, PRC2 pro- lineages, and (ii) new technologies (particularly single-cell tran- teins have been studied in the context of hematopoiesis and their scriptomics) have demonstrated a high level of heterogeneity among mutations have been linked to hematological disorders (9). Depletion HSCs, although it has been impossible to define discrete categories. of PRC2 core components in murine models lead to marked effects on Thus, it is becoming more accepted that boundaries between cell hematopoiesis at either neonatal or fetal stage, demonstrating the compartments, probably not only within HSCs but also in all undif- crucial role of PRC2 in hematopoietic homeostasis (10–12). Three ferentiated progenitor compartments, are less strictly defined (1, 2). homologs of the Drosophila melanogaster Polycomb-like (Pcl) gene In addition to being functionally defined as a group of cells capable have been described as PRC2-associated factors: Phf1/Pcl1, Mtf2/Pcl2, of long-term repopulation capacity, HSCs can also be molecularly and Phf19/Pcl3. PHF19 has been mainly characterized in mouse em- defined as cells that are able to maintain the highest level of plasticity. bryonic stem cells (mESCs) (13–15), where it plays a role in self-­ Strong gene regulation programs confer irreversibility during the renewal and differentiation. Phf19 expression declines upon mESC differentiation process, and cell types/compartments can be func- differentiation, although adult stem cell compartments within specific tionally defined by their potency capacity after isolation using asso- tissues still retain substantially high expression levels (16, 17). Here, we ciated surface markers. In this context, epigenetic mechanisms are report a murine model devoid of PHF19, which allowed us to inves- likely to play a central role in controlling and perpetuating the tran- tigate its role in hematopoiesis. We have uncovered how PHF19 scriptional programs important for the hematopoietic system. A number controls HSCs identity and long-term contribution to blood pro- of epigenetic alterations have been linked to multiple hematopoietic duction. Furthermore, we identified chromatin alterations in diseases (3, 4). HSCs in the absence of PHF19 and unveiled the key role of H3K27me3 localization in HSCs differentiation and in preventing malignant hematopoiesis. 1 Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology (BIST), Dr. Aiguader 88, Barcelona 08003, Spain. 2Department of Animal Health and Anatomy, School of Veterinary Medicine, Universitat Autònoma de Barcelona, RESULTS Bellaterra, Spain. 3CNAG-CRG, Centre for Genomic Regulation (CRG), Barcelona PHF19 plays a role in long-term hematopoietic reconstitution Institute of Science and Technology, Baldiri Reixac 4, Barcelona 08028, Spain. 4Institut Hospital del Mar d'Investigacions Mèdiques (IMIM), Barcelona 08003, Spain. 5CIBERONC, In the hematopoietic system, Phf19 expression is relatively elevated Barcelona, Spain. 6Universitat Pompeu Fabra (UPF), Barcelona, Spain. 7ICREA, Pg. in undifferentiated progenitors and progressively decreases during Lluis Companys 23, Barcelona 08003, Spain. differentiation (fig. S1A) (18, 19). To study its role in adult homeosta­ *These authors contributed equally to this work. sis, we generated a mouse genetic model by deleting exons 4 and 5, †Present address: Bayer AG, Pharmaceuticals Division, Research and Development, Berlin, Germany. which produces a stop codon in exon 6 (fig. S1, B to E). Phf19-KO ‡Corresponding author. Email: luciano.dicroce@crg.eu (knockout) mice breed normally, and histopathological analysis of Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 1 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE young adult mice (8 to 12 weeks) did not exhibit any major alterations phenotypically defined HSCs (22, 23). Single Lineage −Sca-1 + in the brain, lungs, heart, stomach, intestine, kidney, testis, ovary, or c-Kit+CD150+CD48− HSCs were individually sorted on 96-well uterus. As reported for other Polycomb members (20, 21), Phf19- plates, and the number of cells was monitored for 4 days (Fig. 2B). KO mice presented anterior-to-posterior homeotic transformations, Each individual well was scored according to its division dynamics: such as the presence of rib anlage or extra ribs in ~40% (6 of 14) of proliferating (i.e., sustained cell growth over the 4 days, rendering animals (fig. S1F; note that no alterations in Phf19-Flox mice were colony sizes from five to hundreds of cells), nonproliferating (i.e., detected). Aged Phf19-KO mice (>60 weeks) presented a high not able to divide more than twice during 4 days), and proliferating penetrance of splenomegaly (fig. S1G), suggestive of a defect in the and stop (i.e., division that eventually stopped or reduced the number hematopoietic system. of cells). Under culture conditions, most wells were scored as prolif- We analyzed the hematopoietic hierarchy of fetal liver [embryonic erating, both in Phf19-Flox and Phf19-KO plates, but a significant day 14.5 (E14.5)] and of 8-week-old mouse BM. Lack of PHF19 led increase in the percentage of proliferating colonies in Phf19-KO to an increase in embryonic phenotypically defined HSCs (Lineage− wells was observed, which correlates with a reduction in non- Sca-1+c-Kit+CD150+CD48−), but in adults, we observed a reduction proliferating wells (Fig. 2C). Notably, not only Phf19-KO wells had in HSCs, as well as in LSK (Lineage−Sca-1+c-Kit+) progenitors more proliferating colonies, but also these colonies individually (Fig. 1A). We also documented a significant reduction in cellularity contained a higher number of cells (fig. S2B). We kept colonies in (raw cell count) in BM (Fig. 1B). Furthermore, we investigated the culture for additional 5 days and then calculated colony size by fluores- adult HSC compartment by assessing the proportion of proliferative cence cytometer after propidium iodide (PI) staining. According to Ki67-positive cells (Fig. 1C) and of bromodeoxyuridine (BrdU) in- the division status during the first 4 days, the number of big colonies corporation during 24 hours (Fig. 1D). Our data indicated a significant (>5000 cells) was higher in the Phf19-KO plates than in the Phf19- increase in quiescent cells in Phf19-KO–derived HSCs, which may Flox plates (fig. S2C). Last, after 2 weeks, we measured the expression account for the observed decrease in LSK, HSC cell populations, of lineage and HSCs markers. We observed an impairment of dif- and total cellularity in adults. ferentiation in Phf19-KO condition (Fig. 2D). Moreover, the To determine the role of Phf19 in BM performance, we per- remaining CD150+CD48— population was higher in the Phf19-KO formed transplantation assays. Initially, we transplanted 0.5 million than in the Phf19-Flox cells (fig. S2D). Together, these data demon- cells of whole BM (WBM) obtained from 8-week-old Phf19-Flox or strate that HSCs from Phf19-KO cells have higher proliferation Phf19-KO mice in competition with 0.5 million cells of CD45 con- but lower differentiation capacity compared to control cells. This is genic wild-type WBM cells. No differences were observed in the in agreement with our in vivo data, which indicate that Phf19-KO engraftment capacity or in blood contribution (fig. S1H). We then HSC cells are not able to fully differentiate in serial transplant ex- performed serial noncompetitive repopulation assays by trans- periments despite its increased capacity of generating progenitors planting 1 million cells of Phf19-Flox or Phf19-KO WBM and upon stress. monitoring blood contribution after 3 months of each transplant (Fig. 1E and fig. S1I). Our analysis indicated a slight bias toward PHF19 regulates HSC gene expression through epigenetic myeloid lineage in Phf19-KO transplanted mice, which was only control of PRC2 activity statistically significant after the third transplant (data not shown). To gain insight into how PHF19 is able to control the quiescence We observed a clear decrease in the contribution of Phf19-KO state in homeostasis, we first performed RNA sequencing (RNA-seq) transplanted donors in serial transplants, pointing to a malfunction transcriptomic analysis on HSCs (Lineage−Sca-1+c-Kit+CD150+CD48−) of HSCs. Colony-forming assays from lineage-specific depleted BM isolated from young adult mice (table S1). Term enrichment analysis showed a similar behavior: No differences were found in the type or of common up-regulated genes of two independent replicates showed the total number of colonies after the first plating (fig. S1J), but after that nuclear receptors and, in particular, retinoic acid category were replating, Phf19-KO–derived cells were significantly impaired in their overrepresented. On the other hand, down-regulated genes were capacity to produce new colonies (Fig. 1F). Parallel experiments using enriched for categories related to biosynthesis and energy production progenitor LSK population led to similar results (fig. S1K). (fig. S3A). This is reminiscent of a recently characterized, highly unbiased, and quiescent HSC population, the so-called dormant Phf19-KO HSCs are mobilized upon stress but HSCs (24). are not fully functional We next did a gene set enrichment analysis (GSEA) for specific To determine whether the decrease in BM HSC levels under gene sets. HSC-specific signatures, a merge of differentially expressed homeostatic conditions (Fig. 1A) is the cause of the long-term lower genes provided by Gazit et al. (25) and Chambers et al. (26), were efficiency in blood production (Fig. 1E), we analyzed the total pro- enriched in Phf19-KO (Fig. 3, A and C), indicating that HSC identity is portion of LSK progenitors and HSCs upon different stress conditions enhanced upon PHF19 depletion. Furthermore, the Myc network, that have been described to force HSC mobilization: aging, trans- which is required for HSC differentiation (24, 27), was down-regulated plantation, and 5-fluorouracil (5-FU) injection. The hematopoietic in Phf19-KO HSCs (Fig. 3, B and C). In addition, a decrease of compartment analysis showed that the reduced number of HSCs enrichment in biosynthetic pathways (such as ribosome or mRNA in Phf19-KO animals was compensated upon stress (Fig. 2A and processing) and an increase in positively regulated retinoic acid tar- fig. S2A), indicating that Phf19-KO HSCs are able to proliferate gets (28) were observed (Fig. 3C and fig. S3B), further supporting an upon stress. Therefore, the reduced long-term capacity for re- intensification in HSC quiescence and dormancy (24, 29). We also plenishing the blood system might be related to a differentiation compared our data with an expression signature derived from HSCs defect. depending on their divisional history (30). Up-regulated genes in To assess cell division kinetics and differentiation capacity of HSCs that have undergone several divisions inversely correlate with PHF19-depleted HSCs, we next performed single-cell culture of gene expression of Phf19-KO HSCs and vice versa for down-regulated Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 2 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE A Lin- LSK HSC B ** 60 Million of cells/leg 40 Lineage CD48 20 c-Kit 0 c-Kit Sca-1 CD150 Phf19-Flox Phf19-KO Lin- LSK HSC Fetal liver (E14.5) 20 1.2 0.06 ** C 100 15 0.9 % of living cells 0.04 * % Ki67-pos HSCs 75 10 0.6 0.2 50 5 0.3 25 0 0 0 Phf19-Flox Phf19-KO Phf19-Flox Phf19-KO Phf19-Flox Phf19-KO 0 Phf19-Flox Phf19-KO Lin- LSK HSC ** * D Adult bone marrow 20 0.08 0.4 100 % BrdU-pos HSCs 15 0.3 0.06 75 % of living cells * 10 0.2 0.04 50 5 0.1 0.02 25 0 0 0 0 Phf19-Flox Phf19-KO Phf19-Flox Phf19-KO Phf19-Flox Phf19-KO Phf19-Flox Phf19-KO E Phf19-Flox or Blood Blood Blood F Phf19-KO Normalized number of colonies analysis analysis analysis 1.5 100 ** * 1.0 3 months after transplant 80 % Donor (CD45.2) 60 0.5 40 0 Phf19 Phf19 Phf19 Phf19 20 Flox KO Flox KO 1st plating 2nd plating 0 x O x KO x KO lo o o K Fl Fl -F 9- 9- 9- 9- 9- 9 f1 f1 f1 f1 f1 f1 Ph Ph Ph Ph Ph Ph 1st transplant 2nd transplant 3rd transplant Fig. 1. Characterization of the hematopoietic system in a Phf19-depleted mouse model. (A) Top: BM gating strategy for phenotypically defining lineage negative (Lin−), LSK, and HSCs (Lineage−Sca-1+c-Kit+CD150+CD48−). Middle: Quantification from 13 control floxed (Phf19-Flox) and 12 Phf19-KO E14.5 embryos, with percentages shown for the live cells measured as 4′,6-diamidino-2-phenylindole (DAPI) negative (box plot). Bottom: Quantification from 14 control floxed (Phf19-Flox) and 13 to 15 Phf19-KO mice, with percentages shown for the live cells measured as DAPI negative (box plot). (B) Raw number of WBM cells counted per leg in 13 independent experi- ments (with at least three animals per experiment) (box plot). (C) Percentage of Ki67+ HSC cells in 12 mice for Phf19-Flox and 10 mice for Phf19-KO (box plot). (D) Percentage of BrdU+ HSCs in six Phf19-Flox mice or five Phf19-KO mice at 24 hours after BrdU injection (box plot). (E) Percentage of donor-derived (CD45.2) cells in peripheral blood in serially transplanted recipients 3 months after each transplant (box plot). (F) Normalized number of colonies in the first and second lineage-negative plated cells for colony-forming assay, performed in three independent replicates (means + SEM). *P < 0.05 and **P < 0.01. Unpaired t test (A and C to E). Paired t test (B and F). genes (fig. S3B). Moreover, since Phf19-KO HSCs are proliferative condition; meanwhile, down-regulated genes are enriched in Phf19- but defective in differentiation and long-term repopulation, we Flox transcriptome (fig. S3B). Together, transcriptomics data cor- wondered whether they resembled aged HSCs (31). GSEA demonstrated roborated that Phf19-depleted cells in homeostasis are in a more that up-regulated genes in old animals are enriched in Phf19-KO multipotent and quiescent state and have a decreased capacity upon Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 3 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE A Young Aging Transplant 5-FU crease is global and not only detectable inside the peaks (fig. S3D). n.s. This acquired H3K27me3 deposition pattern resembled the one observed in aged HSCs (31) since genes marked with H3K27me3 in n.s. aged HSCs intensively accumulate H3K27me3 upon depletion of 0.10 PHF19, and conversely, the H3K27me3 genes in young HSCs are * also more intensely marked with H3K27me3 in Phf19-Flox HSCs % of living cells (fig. S3E). At molecular level, Polycomb complexes regulate differ- HSC n.s. 0.05 entiation by restricting lineage-determining gene expression (5). We thus reasoned that in Phf19-KO cells, this mechanism could be implicated in the increased HSC identity observed at a functional and transcriptomic level. As expected, the genomic accumulation of 0 Phf19 Phf19 Phf19 Phf19 Phf19 Phf19 Phf19 Phf19 H3K27me3 levels inversely correlated with expression data (fig. S3F). Flox KO Flox KO Flox KO Flox KO However, despite the global increase in H3K27me3, the HSC gene sets did not show a significant increase in H3K27me3 levels (Fig. 3D, left). Significant increase was observed for genes that are characteristic B Phf19-Flox Cell cycle counts for 96 hours of differentiated lineages (Fig. 3D, middle). Moreover, transcription SCF or HSCs IL11 Colony size at 9 days factors implicated in HSC differentiation into erythroid, granulocyte, Ftl3 lymphoid, or megakaryocyte (20) also showed a significant increase Phf19-KO Inmunophenotipic characterization at 14 days in H3K27me3 (Fig. 3D, right). Increased H3K27me3 deposition in lineage-specific transcription factors could be observed for essential D Phf19-Flox Phf19-KO genes—such as Cebpa (for myeloid) (33); Pax5 and Irf5 (for B cells) C (34, 35); Tcf7 (for T cells) (36); several members of Notch pathway 100 [Dll1 (depicted), Dll4, Jag2, or Hes1], which are well-known regulators of both T and B cells differentiation (37); and Epo (for erythroid cells) * (38)—and could not be observed in HSC fingerprint genes (Vdr Lineage 13.5% 36.4% depicted as an example; fig. S3G). To further validate how the de- % of wells 50 * FSC-A pletion of PHF19 influences chromatin organization, we performed assay for transposase-accessible chromatin and sequencing (ATAC-seq) E 100 and verified that the increase in H3K27me3-containing regions was * accompanied by a loss of chromatin accessibility (fig. S3H). This % of living cells 0 Phf19 Phf19 Lin− decrease could be visualized in most of the master differentiation Flox KO 50 * Lin+ transcription factors mentioned (Fig. 3E). Nonproliferating Differentiation program genes are poorly expressed in HSCs. Proliferating Proliferating and stop Therefore, to evaluate the impact of chromatin changes on gene ex- 0 Phf19 Phf19 pression, we designed two strategies. First, we performed single-­ Flox KO cell RNA-seq. Our first observation was that Phf19-KO HSCs were Fig. 2. Characterization of Phf19-KO HSCs after mobilization. (A) Quantification transcriptionally more similar between each other than Phf19-Flox of phenotypically defined HSCs in young or aged (>60-week-old) mice after trans- HSCs, both using all expressed genes (fig. S3I) and using a HSC plantation (at 6 months after transplant) and at 14 days after 5-FU injection. signature specifically defined from single-cell data (Fig. 3F) (23). The percentage of the live cells measured as DAPI negative is shown (box plot). (B) Schematic representation of a single-cell HSC in vitro functional assay. (C) Propor- Phenotypically defined HSC comprises a heterogeneous population, tion of wells of proliferating, nonproliferating, and proliferating and then stopped which contains cells primed for differentiation. We reasoned that HSCs in three independent experiments (means + SEM). (D) Gating of lineage-­ we could detect transcripts of differentiation genes in individual negative marker versus FSC-A (forward scatter area) of pooled wells after 14 days in HSC cells. We thus computed the number of cells expressing differen- culture. (E) Percentage of lineage-negative and lineage-positive cells from three tiation gene set (Fig. 3D, middle) (25, 26). As expected, some, but not independent replicates (means + SEM). *P < 0.05; n.s., not significant. Paired t test all, of the cells expressed differentiation genes. Interestingly, we ob- (C and E). served we observed a significant decrease in the number of cells that expressed these genes in the Phf19-KO condition (Fig. 3G). Second, induction of differentiation. Last, the role of Jumonji and AT-rich upon forced in vitro differentiation (as depicted in Fig. 2B), we interaction domain containing 2 (JARID2) in the hematopoietic monitored by quantitative polymerase chain reaction (qPCR) the system has been recently investigated (32). A comparative analysis expression of transcription factors that presented an accumulation of of transcriptomic data revealed a similar effect for JARID2 and H3K27me3 mark within their promoter regions. As represented in PHF19 on HSCs gene regulation (fig. S3B). Fig. 3H, all genes showed a decrease in mRNA expression, with the PHF19 is an associated PRC2 factor (7) and controls the trimethyl­ only exception of Cebpa. Since differentiation is not completely ation on lysine-27 of histone H3 (H3K27me3) on chromatin (8). abolished in Phf19-KO cultured HSCs, we hypothesized that We thus performed chromatin immunoprecipitation sequencing myeloid lineage could be enhanced (see next section), which is in (ChIP-seq) experiments on Lineage−Sca-1+c-Kit+CD150+ population, agreement with the increased expression levels of Cebpa. Together, and unexpectedly, we found a global increase in H3K27me3 levels, our data indicate that the absence of PHF19 produces an epigenetic even when spike-in control for quantitative measurements between rewiring of HSCs, leading to an enhancement of stem identity likely conditions was used (fig. S3C). We also corroborated that such in- instructed by an aberrant repression of differentiation programs. Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 4 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE A B C HSC fingerprint gene set Myc targets Rara Retinoic [Gazit et al. (25)/Chambers et al. (26)] (hallmarks) Rarb acid 0.50 Rxra 0.40 0.00 Ltbp3 0.10 NES = –7.5 Prdm16 0.30 q value = 0.0 Npr2 NES = 4.64 0.20 0.20 q value = 0.0 Tle2 0.30 0.10 Vdr 0.40 Mpdz 0.00 Glis2 Col4a2 Col4a1 H19 HSC fingerprint KO > Flox Flox > KO KO > Flox Flox > KO Mecom Eng Fgd5 Clec1a Procr Gata3 D Foxo1 Pdk4 HSC fingerprint Differentiation TF diff Vasn gene set gene set gene set Mad2l1 Hlf [Gazit et al. (25)/Chambers et al. (26)] [Gazit et al. (25)/Chambers et al. (26)] [Yang et al. (20)] H2afz Av H3K27me3 intensity in TSS ± 2 kb ** 1.0 Hspe1 Myc targets 1.0 n.s. 1.0 ** Ppia Atp5k Cycs Ndufa4 Mitoc and resp Ndufb6 0.5 Cox5a electron transport 0.5 0.5 Cox7b Taf15 Rpl38 Rpl39 mRNA Rps24 ribosome synthesis Rps13 0.0 0.0 0.0 Snrpg Phf19 Phf19 Phf19 Phf19 Phf19 Phf19 −2 0 2 3 5 7 Flox KO Flox KO Flox KO Fold change Flox/KO KO/Flox E Cebpa Pax5 Irf5 Tcf7 Dll1 Epo ATAC H3K27me3 Phf19 30- 32 _ 32 _ 25- 32 _ 28- Flox Phf19 30- 32 _ 32 _ 25- 32 _ 28- KO Phf19 8- 2.9 _ 2.9 _ 1.9- 1.5 _ 1.6- Flox 2.9 _ 2.9 _ 1.5 _ Phf19 8- 1.9- 1.6- KO F MolO gene set G H [Wilson et al. (23)] 2.0 Phf19-Flox Normalized relative mRNA expression *** 120 Phf19-KO 90 Phf19-Flox Phf19-KO 60 1.5 Absolute number of cells 0.10 30 * 30 Density 1.0 0.05 0.5 0 0.00 0.0 Phf19 Phf19 pa x5 f7 o ll1 5 Ep Irf 70 75 80 85 90 Tc Flox KO Pa eb D C Pairwise cell distance Fig. 3. HSC gene expression control and epigenetic changes associated with Phf19 depletion. (A) GSEA showing positive enrichment in Phf19-KO transcriptome for HSC gene set [Gazit et al. (25) and Chambers et al. (26)]. (B) GSEA showing negative enrichment in Phf19-KO transcriptome for Myc target gene set. (C) Normalized fold change expression from two independent RNA sequencing (RNA-seq) experiments in relevant genes (means + SD). (D) ChIP-seq levels of H3K27me3 in the transcription start site (TSS) ± 2 kb of specific gene sets: the HSC gene set, the differentiation gene set, and for transcription factors associated with hematopoietic differentiation pro- cesses. (E) University of California Santa Cruz (UCSC) genome browser screenshots for H3K27me3 and ATAC of hematopoiesis master regulators in Phf19-Flox and Phf19- KO. (F) Density plot of pairwise distances of cells based on single cell–based HSC (MolO) signature (23) genes for Phf19-Flox and Phf19-KO cells. (G) Distribution shown by violin plot and box plot of absolute number of cells that expressed differentiation genes for 174 Phf19-Flox and Phf19-KO cells. (H) mRNA expression levels of cultured HSCs after 9 days under growth/differentiation conditions depicted in Fig. 2B, relative to Rplp0 and normalized for Phf19-Flox of two replicates (means + SEM). *P < 0.05, **P < 0.01, and ***P < 0.001. Paired t test (D). Wilcoxon rank-sum test (F and G). NES, normalized enrichment score. Lack of PHF19 causes long-term aberrant hematopoiesis plant (fig. S4B). Moreover, a high incidence of splenomegaly was To study the consequences of the increased proliferation and observed in mice euthanized for WBM extraction for the second impairment of differentiation observed in Phf19-KO HSCs, we per- transplant (Fig. 4A). Moreover, survival of Phf19-KO versus Phf19- formed WBM transplant from aged mice, which already presented Flox transplanted mice was significantly reduced (Fig. 4B). defects in hematopoiesis (as revealed by splenomegaly; fig. S1G), to Flow cytometry characterization of BM from survivor mice irradiated young recipient mice. We again detected a peripheral showed the presence of an uncharacterized population (fig. S4C), blood lineage myeloid bias after serial transplant (fig. S4A), and suggesting anomalous hematopoiesis. We next analyzed spleens of impairment of blood contribution by Phf19-KO donors was accel- the same young transplant survivor mice (four of six with spleno- erated and already evident in the first and second rounds of trans- megaly in Phf19-KO and one of six with splenomegaly in Phf19-Flox) Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 5 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE A B 100 Phf19-Flox 75 % Survival * 50 Phf19-Flox Phf19-KO Phf19-KO 25 0 0 50 100 Time (relative) C Phf19-Flox Phf19-KO D Myel Myel (5.1%) (25.2%) 80 B cell (74.1%) B cell (38.3%) * 60 Phf19-Flox % Cells CD11b/Gr1/B220 Phf19-KO 40 * T cell T cell 20 (12.9%) (9.28%) Unspecific Unspecific (3.2%) (17.5%) 0 Myeloid B cell T cell CD4/CD8/B220 E F G Up in LSC ** [Pabst et al. (39)] 40 0.30 % Ki67+ cells 0.20 NES = 2.42 0.10 q value = 0.0 20 0.00 KO > Flox Flox > KO 0 Phf19 Phf19 Flox KO Fig. 4. Lack of Phf19 causes aberrant hematopoiesis over the long term. (A) Picture depicting splenomegaly at 6 months after transplantation with WBM from aged mice. Photo credit: Pedro Vizán, CRG. (B) Survival curve of all mice (Phf19-Flox versus Phf19-KO) after their last transplant. Time normalized for each experiment to final sacrifice time of all animals. (C) Representative gating of whole spleen analyzed for myeloid, B cell, and T cell proportions (donor CD45.2-positive cells present in the spleen). (D) Quantification of donor CD45.2-positive myeloid, B cell, and T cell proportions in five Phf19-Flox and three Phf19-KO mice. In Phf19-KO conditions, the sum does not reach 100% because of the unspecific population observed in the panel for hematoxylin and eosin staining (means + SEM). (E) Representative hematoxylin and eosin staining of Phf19-KO spleens. Arrowheads indicate mitotic figures. (F) Percentage of Ki67-positive cells in two spleens per condition (more than four white pulp ar- eas per spleen) (means + SEM). (G) GSEA showing positive enrichment in Phf19-KO transcriptome for leukemic stem cell (LSC) differentially up-regulated genes (37). *P < 0.05 and **P < 0.01. Log-rank (Mantel-Cox) test (B). Unpaired t test (D and F). by flow cytometry. When we evaluated donor CD45.2 cell contribu- thermore, a set of differentially up-regulated genes found in a human tions, we observed a decrease in B cell content together with an leukemic stem cell (LSC) population (39) was enriched in Phf19-KO increase in myeloid infiltration (Fig. 4, C and D), as well as the pres- versus Phf19-Flox (Fig. 4G). It has also been observed that LSC tran- ence of an unusual, unspecific splenic cell population (Fig. 4C). scriptional programs are partially shared with stem cell transcription Further characterization of the spleens by histological analysis re- programs, and a reduced gene catalog of 17 genes was reported to vealed that the tissue architecture was compromised in Phf19-KO correlate with poor prognosis and relapse for acute myeloid leukemia transplanted mice, with almost indistinguishable white and red (40). We manually examined these genes in our transcriptomic data. pulps (fig. S4D), together with the presence of a population of large Thirteen of the original 17 genes were expressed [reads per kilo­ pleomorphic cells with irregularly shaped nuclei (Fig. 4E). The base million (RPKM) > 0.1] in our dataset, and 10 (~77%) were up-­ cytological features of these cells are compatible with myeloid dys- regulated. These observations are quite notable since these specific plasia. Many mitotic figures were present, and immunohistochemistry alterations in the transcriptome are already evident in young mice. for the proliferation marker Ki67 corroborated a higher rate of pro- Together, our results indicate that PHF19 is necessary to maintain a liferation in Phf19-KO spleens than in Phf19-Flox spleens (Fig. 4F). correct hematopoietic balance and that its depletion induces an Moreover, the macrophage marker CD68, which is found mostly in aberrant state, which primes HSCs for malignant progression. the red pulp (fig. S4E, left), was diffusely spread in all areas of the Phf19-KO samples (fig. S4E, right), in accordance with our flow cytometry observation of unspecific and increased myeloid cells. DISCUSSION In light of these results, we reevaluated our transcriptomic data. Here, we have shown that PHF19 contributes to maintain the cor- Term enrichment of up-regulated genes in Phf19-KO condition rect levels of H3K27me3 within the HSC compartment, influencing also showed the pathways in cancer category [Kyoto Encyclopedia of their long-term function and differentiation predisposition. Blood Genes and Genomes (KEGG) mouse 2019, P = 9.16 × 10−7]. Fur- production has been classically explained by a hierarchical model in Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 6 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE which HSCs have the capacity to self-renew and differentiate into a genes necessary for differentiation. At the same time, once differ- population of more committed progenitors, which are still able to entiation is triggered, this compaction would reduce the accessibility self-renew and produce all blood types of the organism. In the past of specific transcription factors. few years, this model is being reinterpreted as a continuous change In summary, we have demonstrated that PHF19 depletion increases between cellular states rather than a stepwise process. Within the the dormancy-like HSC state by a mechanism that, at the same time, HSC compartment, variability is given by characteristics that impairs differentiation and reduces durability upon serial trans- are not initially related to their differentiation potential but rather plants. Several studies have used label dilution models to quantify with their cellular state, such as biosynthetic rate or energy meta­b ­ HSC divisional history, which has allowed a link between low olism. Thus, HSCs have been further characterized in dormant and mitotic rate and greatest repopulation ability (30, 43). The tran- active with a continuum among the two states (24). Our results in- scriptional program associated with low divisional history resembles dicate that PHF19 controls the balance between these two conditions. the Phf19-KO expression data (fig. S3B). Thus, although we observed Epigenetics is one of the main factors that govern cell plasticity, a higher number of HSCs in fetal liver, which could be indicative of as well as the transition towards new cell states. In particular, the an increased proliferation, adult HSCs are more quiescent. This PcG of proteins, which main function is transcriptional repression, could be explained by an early exhaustion of highly proliferative have been reported to play a role in HSC performance (9). Loss-of- HSCs during fetal and early life stages or by the fact that label dilu- function studies of PRC2 core components demonstrated their tion models tend to misinterpret label retention and are simply essential role in hematopoiesis (10–12), but the associated lethality labeling HSCs with high repopulation potential, irrespective of has hampered an in-depth characterization of transcriptional path- mitotic history (44). Beyond these strong functional and transcrip- ways and H3K27me3 (re)distribution implicated in HSC identity. tional similarities, the quiescent features in Phf19-KO do not yield Similarly, metal response element binding transcription factor 2 in fully functional dormant HSCs. In this regard, it is remarkable (MTF2) deletion impairs fetal erythropoiesis (41), causing a pre- that Phf19-KO HSCs resemble aged HSCs (fig. S3B), which have birth death that has also impeded further studying of the role of been observed to accumulate during aging, but are less functional at H3K27me3 in adult HSCs. Several studies have unveiled that PCL generating new blood cells (45). Notably, the H3K27me3 increase proteins and JARID2 give rise to two biochemically distinct versions observed upon PHF19 depletion is in line with the same mark dis- of the PRC2 complex: PRC2.1 and PRC2.2 (6). It has been recently tribution reported for aged HSCs (fig. S3E) (31). As for the Phf19- reported that JARID2 depletion in hematopoietic precursors (32) KO mice, the consequences in aged HSCs are a myeloid bias and a affects the expression of self-renewal genes in HSCs and multiple higher propensity to hematological disorders. Moreover, quiescence progenitor precursors. Transcriptional comparison shows that JARID2-­ is the most significant feature in cancer stem cells and the leading depleted HSCs resemble Phf19-KO HSCs (fig. S3B) despite a reduc- cause of relapse. This study underlines the complex balance between tion in H3K27me3 levels. We believe that PRC2 variants control the silencing of differentiation programs and the maintenance of self-renewal and differentiation by complementary mechanisms. elevated levels of plasticity to sustain stem cell identity. Notably, PRC2.2 may be more active than PRC2.1, as previously suggested (42). Thus, PRC2.2 reduction might cause direct derepression of a set of HSC genes, and conversely, PRC2.1 reduction might METHODS allow PRC2.2 to increment H3K27me3 deposition at differentiation Mice genes, producing a more homogeneous HSC-like transcriptome. All animal procedures were approved by the ethical committee of These two distinct molecular mechanisms would paradoxically pro- the Barcelona Biomedical Research Park and by the animal experi- duce a similar phenotype: increase of HSC/dormancy features and mentation commission of the Catalan government (Generalitat de impairment of proper differentiation. Together, this highlights the Catalunya). All animals used for this study had a C57BL/6 back- importance of the proper balance between PRC2 variations and their ground. LoxP sites were introduced by homologous recombination specific role with respect to the control of adult stem cell functionality. around exons 4 and 5, the deletion of which generates a stop codon Unfortunately, nowadays, there are no available data of PRC2 com- in exon 6. Phf19fl/fl animals were crossed with Sox2Cre (mice were ponent localization at genome-wide level, which impedes a thorough provided by A.R. Nebreda, Institute for Research in Biomedicine, analysis of the impact of distinct PRC2 versions in HSC gene regulation. Barcelona). The CRE activity in the Sox2CRE strain has been of ma- As we have demonstrated in this study, Phf19 deletion does not ternal inheritance, irrespective of whether they inherit the transgene affect breeding or life span. Although we observed changes in the (46). Therefore, after backcrossing with new male floxed animals, an- number of HSCs (higher proportion in fetal liver and lower pro- imals with the knocked-out allele without the SoxCre transgene portion in young adults), these alterations do not have fatal conse- were selected. Once generated, mice were maintained as heterozy- quences during development or in adult homeostasis under animal gosis (Phf19−/fl) and as homozygosis (Phf19−/−). In both cases, off- facility conditions. However, under challenging conditions, such as spring were viable and bred normally. Equal numbers of male and aging or serial transplants, we have unveiled the role for PHF19 in female mice were used; no gender biases were noted. For BM trans- controlling HSC functions. In Phf19-KO animals, dormant HSC plants experiments, 8- to 12-week-old congenic CD45.1 and CD45.1/ characteristics are enhanced under resting conditions, such as high CD45.2 mice (bred in-house) were used. quiescence, or low metabolic and biosynthetic activities. Upon in- duced mobilization, proliferation is triggered, but differentiation is BM extraction and splenocytes isolation impaired. This double-edged behavior could be linked to epigenetic For fetal liver analysis, the tissue was harvested from E14.5 embryos states. The main role of PcG is to maintain cell identity by restricting and the dissected liver was crushed with the end of a 1-ml syringe lineage-specific gene transcription. In Phf19-KO HSCs, we observed through a 40-m cell strainer into Iscove’s modified Dulbecco’s an increase in H3K27me3 mark and chromatin compaction at medium and 10% fetal bovine serum. For cell BM analysis of individual Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 7 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE mice, clean femurs and tibias were flushed using a 23-gauge needle tion, mice were euthanized, and BM cells were isolated and stained and a 10-cm3 syringe with cold dissection medium [1× phosphate-­ for cytometry as described above. buffered saline (PBS), 2% fetal calf serum (FCS), and 2 mM EDTA] before cell surface staining (see next section). For pooled mice analyses, BM transplantation the clean bones were crushed with mortar and pestle in cold dissection For competitive transplantation assay, 0.5 × 106 million nucleated medium (1× PBS, 2% FCS, and 2 mM EDTA). For sorting and colony-­ BM cells from femurs and tibias (see next section) of 8- to 12-week-old forming unit assays (see below), lineage depletion was performed CD45.2 mice were mixed with 0.5 × 106 million CD45.1 BM cells using the mouse Lineage Cell Depletion Kit (Miltenyi Biotec) by and transplanted by retro-orbital injection into 8- to 12-week-old magnetic-activated cell sorting (MACS) technique according to the CD45.1/CD45.2 recipient mice that were lethally irradiated using a manufacturer’s instructions. Splenocytes were freshly dissociated split dose of 8 gray (Gy) (two doses of 4 Gy administered at least by mincing the tissue to obtain a single-cell suspension. 4 hours apart). CD45.2 contribution to peripheral blood was assessed 3 months after transplantation. For both young and old serial trans- Flow cytometry plantation assays, 1 × 106 nucleated BM cells from 8- to 12-week-old Before flow cytometry analysis, fetal liver cell suspension, blood, (young) or 80- to 85-week-old (old) CD45.2 mice were transplanted BM, and splenocytes, erythrocytes were lysed with 1× red blood cell by retro-orbital injection into 8- to 12-week-old lethally irradiated lysis buffer (eBioscience) and washed with 1× PBS. For surface (two split doses of 4 Gy, 4 hours apart) CD45.1/CD45.2 recipient staining, single-cell suspensions in fluorescence-activated cell sort- mice. The CD45.2 contribution to peripheral blood was assessed at ing (FACS) buffer (1× PBS, 2% FCS, and 2 mM EDTA) were 3 months after transplantation. For serial transplants, animals were blocked for 10 min with anti-CD16/CD32 (mouse Fc block, BD euthanized after 6 months, and pooled 1 × 106 nucleated BM cells Pharmingen). Samples were stained in FACS buffer for 30 min at were used to transplant new 8- to 12-week-old lethally irradiated 4°C with fluorochrome-­conjugated antibodies and washed before CD45.1/CD45.2 recipient mice. Recipient mice were treated for 4 weeks analysis. Blood and splenocytes were incubated with the following after transplantation with Enrovet (enrofloxacin) at 600 g/ml in fluorochrome-conjugated monoclonal antibodies for lineage their drinking water. analysis: phycoerythrin (PE) anti-mouse CD4, PE anti-mouse CD8, PE anti-mouse B220, PECy7 anti-mouse B220, allophycocyanin (APC) Colony-forming assay anti-mouse CD45.1, fluorescein isothiocyanate (FITC) anti-mouse Lineage-depleted or LSK-sorted BM cells were plated in duplicate CD45.2, PECy7 anti-mouse CD11b, and PECy7 anti-mouse GR-1. in 35-mm tissue culture dishes (STEMCELL Technologies) in Fetal liver and BM were analyzed using the lineage marker (Lin) methylcellulose-based medium MethoCultTM GF M3434 (STEMCELL mixture (BD Biosciences) that include the following V450-conjugated Technologies) at 2000 cells per plate. Plates were incubated 10 to antibodies: CD3 (145-2C11); CD11b (M1/70) (this antibody was 14 days at 37°C, 100% humidity, and 5% CO2. Colonies were scored excluded in fetal liver analyses); CD45R/B220 (RA3-6B2); mouse using an inverted microscope and resuspended and counted in 1× erythroid cells Ly-76 (Ter119), Ly6G, and Ly-6C (RB6-8C5); and the PBS. Cells at 100,000 per plate were used for replating. following fluorochrome-conjugated antibodies for HSC and precur- sors (LSK): PECy7 anti-mouse Sca I, APC-Fluo780 anti-mouse Single-cell HSC culture cKit, APC anti-­mouse CD150, and PE anti-mouse CD48. Cell viability Lineage-depleted BM cells were stained as previously described for was evaluated with 4′,6-diamidino-2-phenylindole (DAPI) at a HSC. Single-cell sorting (FACSAria, BD) was performed in 96-well final concentration of 1:10,000. plates containing 100 l of filtered HSC expansion medium [StemSpan For intracellular staining (Ki67 or BrdU), BM cells were resus- serum-free expansion medium, murine stem cell factor (300 ng/ml), pended in FACS buffer and stained for 10 min at 4°C with anti-­ murine interleukin-11 (20 ng/ml), and human Flt3L (1 ng/ml)]. CD16/CD32 (mouse FC block, BD Pharmingen) for 30 min at 4°C The plates were incubated at 37°C with 5% CO2, and medium was with a cocktail of lineage marker biotin-conjugated monoclonal replaced every 7 days. Cell divisions were visually scored for 96 hours. antibodies against CD2, CD3, CD11b, CD14, CD15, CD16, CD19, At day 9, single wells were stained with PI (1 g/ml) and counted CD56, CD123, and CD235a (Miltenyi Biotec) and, lastly, with using FACSCanto II High Throughput Sampler. In addition, an in- fluorochrome-conjugated antibodies specific for HSCs (as detailed dependent plate was harvested and pooled for RNA extraction. At above). Cells were then washed and incubated with the secondary day 14, cells were harvested, pooled, and stained with BM surface antibody PE-CF594 streptavidin for 10 min at 4°C. For Ki67 immuno­ markers as previously described. staining, surface-stained BM cells were washed and fixed with reagent A (Invitrogen) for 15 min at room temperature and then Real-time PCR washed and permeabilized in reagent B in the presence of FITC RNA was isolated from cultured HSCs with the RNeasy Plus Mini anti-mouse Ki67 for 20 min at room temperature. After two washes, Kit (Qiagen), and cDNA was generated from 1 g of RNA with the cells were resuspended in 500 l of 1× PBS containing ribonuclease qScript cDNA Synthesis Kit (Quantabio), both following the manufac- (RNase; 5 g/ml) and DAPI (2 g/ml). For BrdU (see next section turer’s instructions. Real-time PCR reactions were performed using for BrdU in vivo injection), surface-stained BM cells were washed, SYBR Green I PCR Master Mix (Roche) and the Roche LightCycler 480. and the BrdU incorporation assay was performed using the BrdU Kit from BD Biosciences. Population RNA-seq Two RNA replicates were obtained from 5000 to 10,000 HSCs BrdU incorporation assay (Lineage−Sca-1+c-Kit+CD150+CD48−) sorted from flow cytometry Subject mice were intraperitoneally injected twice (at 12-hour intervals) as described above. RNA extraction was performed using the with BrdU (150 l of 10 mg/ml). At 12 hours after the second injec- RNeasy Micro Kit (Qiagen). Library preparation was performed with Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 8 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE 3 to 5 ng of total RNA using the SMARTer Ultra Low RNA Kit number of normalized reads of H3K27me3 corrected by spike-in at (Clontech) and sequenced [50–base pair (bp) single end] on an Illumina each sample. To discard regions of the genome virtually with no HiSeq 3000. The RNA-seq samples were mapped against the mm9 signal, we focused on the bins in which, at least in one of the condi- mouse genome assembly using TopHat (47) with the option –g 1 to tions, a minimum amount of ChIP signal was detected (threshold: 5). discard those reads that could not be uniquely mapped in just one region. Cufflinks (48) was run to quantify the expression in RPKMs Omni-ATAC-seq of each annotated transcript in RefSeq (49). We applied the follow- Omni-ATAC-seq libraries were generated as previously described ing thresholds to find differentially expressed genes between each (54). Briefly, 10,000 Lineage−Sca-1+c-Kit+CD150+CD48− cells per pair of Phf19-Flox versus Phf19-KO samples [RPKM > 0.5 and KO/ condition were washed in cold PBS and resuspended in 50 l of cold Flox FC ≥ 1.5 or Flox/KO FC ≥ 1.3]. We intersected the up- or lysis buffer 1 [10 mM tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, down-regulated genes in common between duplicates to end up with 0.1% (v/v) Igepal CA-630, 0.01% digitonin, and 0.1% Tween 20]. the final sets of up-regulated (901) and down-regulated (556) genes, Samples were incubated on ice for 3 min and washed out with 1 ml respectively. Reports of functional enrichments of Gene Ontology of cold lysis buffer 2 [10 mM tris-HCl (pH 7.4), 10 mM NaCl, 3 mM and other genomic libraries were generated using the Enrichr tool MgCl2, and 0.1% Tween 20]. Samples were centrifuged for 10 min (44). For specific GSEA, we considered as expressed genes those at 4°C, and the nuclei pellet was resuspended in the transposition with RPKM > 0.1. Next, we rank-ordered them according to fold reaction mix [25 l of 2× transposition reaction buffer from Nextera change and analyzed using the preranked tool of GSEA (50). kit, 2.5 l of Nextera Tn5 transposase from Nextera kit, 16.5 l of PBS, 0.5 l of digitonin (1%), 0.5 l of Tween 20 (10%), and 5 l of Chromatin immunoprecipitation sequencing nuclease-free water] and incubated at 37°C for 1 hour. Samples Lineage−Sca-1+c-Kit+CD150+ cells (10,000) were sorted by flow were purified using the Qiagen MinElute PCR Purification Kit. cytometry as described above and freshly cross-linked with 1% formal- Transposed DNA was eluted in 10 l of elution buffer and subjected dehyde. Low Cell ChIP-seq kit (Active Motif) was used following the to 5 cycles of PCR before amplification using barcoded primers manufacturer’s instructions for histone H3 lysine-27 trimethylated and NEBNext High-Fidelity PCR Master Mix. We determined the ChIP grade antibody (39155, Active Motif). For ChIP experiments optimal total number of PCR cycles for each Omni-ATAC-seq li- with spike-in control, a ratio of 1:2 (Drosophila:mouse) S2 cells was brary using qPCR. Omni-ATAC-seq libraries were purified using added prior cell lysis. Total chromatin was sheared with a Covaris 1.8× volumes of AMPure XP beads to remove fragments below sonication system for 20 min at intensity 8 and 20% duty cycle. 100 bp and sequenced (50-bp single end) on an Illumina HiSeq 3000. ChIP-seq libraries were prepared with total recovered chromatin, The ATAC-seq samples were mapped against the mm9 mouse ge- using the Next Gen DNA Library Kit and the Next Gen Indexing Kit nome assembly using Bowtie with the option –m 1 to discard those (Active Motif) as per the manufacturer’s instructions. Libraries were reads that could not be uniquely mapped to just one region (51). sequenced (50-bp single end) on a HiSeq 3000 platform (Illumina). Mitochondrial reads were removed from each resulting map, and ChIP-seq samples were mapped against the mm9 mouse genome down-sampling was applied to obtain the same number of mapped assembly using Bowtie with the option –m 1 to discard those reads fragments per sample. Box plots showing the ATAC-seq level distri- that could not be uniquely mapped to just one region (51). ChIP-seq bution particular sets of genes were calculated by determining the samples normalized by spike-in were mapped against a synthetic average value at transcript level on the region ±2 kbp around each genome constituted by the mouse and the fruit fly chromosomes TSS. The UCSC genome browser was used to generate the screen- (mm9 and dm3) using Bowtie with the option –m 1 to discard reads shots depicted in the manuscript (53). that did not map uniquely to one region. MACS was run with the default parameters but with the shift size adjusted to 100 bp to Single-cell RNA-seq perform the peak calling against the corresponding control sample Library preparation (MARSseq) (52). Each set of target genes was retrieved by matching the ChIP- Single-cell libraries from polyadenylate-tailed RNA were constructed, seq peaks in the region 2.5 kbp upstream of the transcription start applying massively parallel single-cell RNA-seq (MARSseq) (55, 56). site (TSS) until the end of the transcripts as annotated in RefSeq Briefly, single HSC (Lineage−Sca-1+c-Kit+CD150+CD48−) cells were annotations (49). The heat maps displaying the density of ChIP-seq FACS-sorted into 384-well plates, containing lysis buffer [0.2% Triton reads around the TSS of each target gene set were generated by (Sigma-Aldrich) and RNase inhibitor (Invitrogen)] and reverse counting the number of reads in this region for each individual gene transcription (RT) primers. The RT primers contained the single-­ and normalizing this value with the total number of mapped reads cell barcodes and unique molecular identifiers (UMIs) for subsequent of the sample. Genes on each ChIP heat map were ranked by the demultiplexing and correction for amplification biases, respectively. logarithm of the average number of reads in the same genomic Single-cell lysates were denatured and immediately placed on ice. region. Box plots showing the ChIP-seq level distribution for The RT reaction mix containing SuperScript III reverse transcrip- H3K27me3 for particular sets of genes were calculated by determin- tase (Invitrogen) was added to each sample. In the RT reaction, ing the average value at transcript level on the region ±2 kbp around spike-in artificial transcripts (External RNA Controls Consortium, each TSS. The values of the samples including spike-in were corrected Ambion) were included at a dilution of 1:16 × 106 per cell. After RT, the by the number of fly reads mapped of the sequencing experiment. cDNA was pooled using an automated pipeline (epMotion, The University of California Santa Cruz (UCSC) genome browser Eppendorf). Unbound primers were eliminated by incubating the was used to generate the screenshots depicted in the manuscript cDNA with exonuclease I [New England Biolabs (NEB)]. A second (53). To confirm the general increase of H3K27me3 ChIP-seq levels pooling was performed through cleanup with solid-phase reversible from Phf19-Flox to Phf19-KO conditions, we segmented the mouse immobilization (SPRI) magnetic beads (Beckman Coulter). Sub- genome into bins of 100 bp. Next, we counted within each bin the sequently, pooled cDNAs were converted into double-stranded DNA Vizán et al., Sci. Adv. 2020; 6 : eabb2745 5 August 2020 9 of 12 SCIENCE ADVANCES | RESEARCH ARTICLE with the Second Strand Synthesis enzyme (NEB), followed by cleanup eosin standard protocols were done using a CoverStainer (Dako-­ and linear amplification by T7 in vitro transcription overnight. Agilent) following the manufacturer’s procedures. Immunohisto- Afterward, the DNA template was removed by Turbo deoxyribo- chemistry was performed using an Autostainer Plus (Dako-Agilent). nuclease I (Ambion), and the RNA was purified with SPRI beads. Before immunohistochemistry, sections were dewaxed for Ki67 as Amplified RNA was chemically fragmented with Zn2+ (Ambion) part of the antigen retrieval process using the low-pH EnVision and then purified with SPRI beads. The fragmented RNA was ligated FLEX Target Retrieval Solutions (Dako, Burlington). For CD68, with ligation primers containing a pool barcode and partial Illumina samples were dewaxed and rehydrated, and thereafter, antigen re- Read1 sequencing adapter using T4 RNA ligase I (NEB). Ligated trieval was performed using an autoclave for 20 min at 121°C with products were reversed transcribed using the AffinityScript RT citrate buffer (pH 6). Samples were blocked with Peroxidase-Blocking enzyme (Agilent Technologies) and a primer complementary to the Solution (S2023, Dako-Agilent), 5% normal goat serum, and 2.5% ligated adapter, partial Read1. The cDNA was purified with SPRI bovine serum albumin. Primary antibodies rabbit polyclonal anti-­ beads. Libraries were completed through a PCR step using the Ki67 (Abcam) and rabbit polyclonal anti-CD68 (orb47985, Biorbyt) KAPA HiFi HotStart ReadyMix (Kapa Biosystems) and a forward were diluted at 1:2000 and 1:4000, respectively, with EnVision primer that contains Illumina P5-Read1 sequence and a reverse primer FLEX Antibody Diluent (Dako, Agilent) and incubated for 60 min. containing the P7-Read2 sequence. The final library was purified The secondary antibody used was a BrightVision poly–horseradish with SPRI beads to remove excess primers. Library concentration and peroxidase anti-rabbit immunoglobulin G (IgG) biotin-free, ready molecular size were determined with High Sensitivity DNA Chip to use (Immunologic, DPVR-110HRP). Antigen-antibody complexes (Agilent Technologies). The libraries consist of 192 single-cell were visualized with 3-3′-diaminobenzidine (Dako). Sections were pools. Multiplexed pools (2) were run in one Illumina HiSeq 2500 counterstained with hematoxylin (Dako, S202084) and mounted with Rapid two-lane flow cell following the manufacturer’s protocol. Primary Toluene-Free Mounting Medium (CS705, Dako) using the Dako data analysis was carried out with the standard Illumina pipeline. CoverStainer. Specificity of staining was confirmed with a rabbit Data preprocessing IgG polyclonal isotype control (Abcam). Bright-field images were Sequencing was carried out as paired-end reads, wherein the first acquired with a NanoZoomer-2.0 HT C9600 digital scanner read contains the transcript sequence and the second read contains (Hamamatsu) equipped with a 20× objective. All images were visualized the cell barcode and UMIs. Data analyses were performed as de- with a gamma correction set to 1.8 in the image control panel of the scribed previously (56). Briefly, quality check of the generated reads NDP.view2 U123888-01 software (Hamamatsu Photonics, France). was performed with the FastQC quality control suite. Samples that reached the quality standards were then processed to deconvolute Quantification and statistical analysis the reads to single-cell level by demultiplexing according to the cell Animal data are presented as box plots produced using Prism 6 and pool barcodes. Reads were filtered to remove polyT sequences. software (GraphPad, San Diego, CA). Quantified data are presented Sequencing reads were mapped to the mouse reference genome as means ± SEM. Statistical tests between groups are detailed in the (Gencode release M11, assembly GRCm38) with the RNA pipeline figure legends. Student’s paired or unpaired t test, depending on the of the GEMTools 1.7.0 suite using default parameters (6% of mis- experimental design, was broadly used in all figures. In addition, matches, minimum of 80% matched bases, and minimum quality Wilcoxon rank-sum test for single-cell data (Fig. 3, F and G, and fig. S3I) threshold of 26). Cells with less than 60% of reads mapping to the and log-rank (Mantel-Cox) test (Fig. 4B) were used. Significance is de- reference genome or more than 2 × 106 total reads were discarded. picted throughout the figures as *P < 0.05, **P < 0.01, and ***P < 0.001. Gene quantification was performed using UMI-corrected transcript information to correct for amplification biases, collapsing read Data and software availability counts for reads mapping on a gene with the same UMI (allowing Raw data and processed information of the ChIP-seq, RNA-seq, an edit distance up to two nucleotides in UMI comparisons). Only and ATAC-seq experiments generated here were deposited in the unambiguously mapped reads were considered. Thresholds were National Center for Biotechnology Information Gene Expression set not only to reduce technical noise but also to conserve the sensitivity Omnibus (58) repository under the accession number GSE135283. to identify low-frequency outlier cell populations and to capture differences between fresh and cryopreserved cells. SUPPLEMENTARY MATERIALS Supplementary material for this article is available at http://advances.sciencemag.org/cgi/ Skeletal staining content/full/6/32/eabb2745/DC1 Skeletal whole mounts were performed as previously described (57). View/request a protocol for this paper from Bio-protocol. Briefly, the completely eviscerated and skinned animals were fixed in 96% ethanol for 5 days and transferred to acetone for 2 days. Staining was performed in 0.005% Alizarin Red S, 0.015% Alcian REFERENCES AND NOTES blue 8GS in 5% acetic acid, 5% H2O, and 90% ethanol for 5 days at room 1. S. Haas, A. Trumpp, M. D. 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Kitts, T. D. Murphy, K. D. Pruitt, Reference sequence (RefSeq) database at (BFU2016-75008-P), “Fundación Vencer El Cancer” (VEC), the European Regional Development NCBI: Current status, taxonomic expansion, and functional annotation. Nucleic Acids Res. Fund (FEDER), Fundació “La Marató de TV3,” and from AGAUR. The laboratory of A.B. is 44, D733–D745 (2016). supported by SAF2016-75613-R from the Ministerio de Ciencia, Innovación y Universidades. H.H. 50. A. Subramanian, P. Tamayo, V. K. Mootha, S. Mukherjee, B. L. Ebert, M. A. Gillette, is a Miguel Servet (CP14/00229) researcher funded by the Spanish Institute of Health Carlos III A. Paulovich, S. L. Pomeroy, T. R. Golub, E. S. Lander, J. P. Mesirov, Gene set enrichment (ISCIII) and received funding from the European Union’s Horizon 2020 research and innovation analysis: A knowledge-based approach for interpreting genome-wide expression programme (MSCA-ITN-2015-675752) and the Ministerio de Ciencia, Innovación y Universidades profiles. Proc. Natl. Acad. Sci. U.S.A. 102, 15545–15550 (2005). (SAF2017-89109-P; AEI/FEDER, UE). P.V. was supported by “Fundación Científica de la Asociación 51. B. Langmead, C. Trapnell, M. Pop, S. L. Salzberg, Ultrafast and memory-efficient alignment Española Contra el Cáncer.” I.E. was supported by La Caixa INPhINIT program. M.G.-M. was of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009). supported by the FPI Program, and R.T. was supported by Generalitat de Catalunya (Beatriu de 52. Y. Zhang, T. Liu, C. A. Meyer, J. Eeckhoute, D. S. Johnson, B. E. Bernstein, C. Nusbaum, Pinós program; 2016 BP 00021). We acknowledge support from the Spanish MEIC through the R. M. Myers, M. Brown, W. Li, X. S. Liu, Model-based analysis of ChIP-Seq (MACS). Instituto de Salud Carlos III and the EMBL partnership, Centro de Excelencia Severo Ochoa, Genome Biol. 9, R137 (2008). CERCA Programme/Generalitat de Catalunya, the Generalitat de Catalunya through 53. W. J. Kent, C. W. Sugnet, T. S. Furey, K. M. Roskin, T. H. Pringle, A. M. Zahler, D. Haussler, Departament de Salut and Departament d’Empresa i Coneixement, and the Co-financing by the The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002). Spanish Ministry of Economy, Industry and Competitiveness (MEIC) with funds from the 54. M. R. Corces, A. E. Trevino, E. G. Hamilton, P. G. Greenside, N. A. Sinnott-Armstrong, European Regional Development Fund (ERDF) corresponding to the 2014-2020 Smart Growth S. Vesuna, A. T. Satpathy, A. J. Rubin, K. S. Montine, B. Wu, A. Kathiria, S. W. Cho, Operating Program. Author contributions: P.V. and A.G. conceived and planned this project M. R. Mumbach, A. C. Carter, M. Kasowski, L. A. Orloff, V. I. Risca, A. Kundaje, P. A. Khavari, and collected, analyzed, and interpreted data. I.E. and M.G.-M. collected, analyzed, and T. J. Montine, W. J. Greenleaf, H. Y. Chang, An improved ATAC-seq protocol reduces interpreted data. M.L. and L.D.C. conceived and planned this project. A.C. performed skeletal background and enables interrogation of frozen tissues. Nat. Methods 14, 959–962 (2017). staining and interpreted data. A.L. and H.H. performed and analyzed single-cell transcriptomic 55. D. A. Jaitin, E. Kenigsberg, H. Keren-Shaul, N. Elefant, F. Paul, I. Zaretsky, A. Mildner, data. L.d.A.-A. and T.G. conceived and planned transplant experiments and provided insightful N. Cohen, S. Jung, A. Tanay, I. Amit, Massively parallel single-cell RNA-seq for marker-free advice during the execution of the project. E.B. performed the bioinformatic analyses. R.T. and decomposition of tissues into cell types. Science 343, 776–779 (2014). A.B. performed and analyzed fetal liver analyses and provided insightful advice during the 56. A. Guillaumet-Adkins, G. Rodríguez-Esteban, E. Mereu, M. Mendez-Lago, D. A. Jaitin, execution of the project. P.V. and L.D.C. wrote the manuscript, with the assistance and final A. Villanueva, A. Vidal, A. Martinez-Marti, E. Felip, A. Vivancos, H. Keren-Shaul, S. Heath, approval of all authors. Competing interests: The authors declare that they have no competing M. Gut, I. Amit, I. Gut, H. Heyn, Single-cell transcriptome conservation in cryopreserved interests. Data and materials availability: All data needed to evaluate the conclusions in the cells and tissues. Genome Biol. 18, 45 (2017). paper are present in the paper and/or the Supplementary Materials. Additional data related to 57. N. M. van der Lugt, J. Domen, K. Linders, M. van Roon, E. Robanus-Maandag, H. te Riele, this paper may be requested from the authors. M. van der Valk, J. Deschamps, M. Sofroniew, M. van Lohuizen, Posterior transformation, neurological abnormalities, and severe hematopoietic defects in mice with a targeted Submitted 12 February 2020 deletion of the bmi-1 proto-oncogene. Genes Dev. 8, 757–769 (1994). Accepted 19 June 2020 58. T. Barrett, S. E. Wilhite, P. Ledoux, C. Evangelista, I. F. Kim, M. Tomashevsky, K. A. Marshall, Published 5 August 2020 K. 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