Bereshchenko et al. Clinical Epigenetics (2019) 11:121 https://doi.org/10.1186/s13148-019-0724-z RESEARCH Open Access Deficiency and haploinsufficiency of histone macroH2A1.1 in mice recapitulate hematopoietic defects of human myelodysplastic syndrome Oxana Bereshchenko1*, Oriana Lo Re2,3, Fedor Nikulenkov2,3, Sara Flamini1, Jana Kotaskova4,5, Tommaso Mazza6, Marguerite-Marie Le Pannérer7,8, Marcus Buschbeck7,8, Cesarina Giallongo9, Giuseppe Palumbo10, Giovanni Li Volti11, Valerio Pazienza12, Libor Cervinek5, Carlo Riccardi1, Lumir Krejci2,3, Sarka Pospisilova4,5, A. Francis Stewart13 and Manlio Vinciguerra2* Abstract Background: Epigenetic regulation is important in hematopoiesis, but the involvement of histone variants is poorly understood. Myelodysplastic syndromes (MDS) are heterogeneous clonal hematopoietic stem cell (HSC) disorders characterized by ineffective hematopoiesis. MacroH2A1.1 is a histone H2A variant that negatively correlates with the self-renewal capacity of embryonic, adult, and cancer stem cells. MacroH2A1.1 is a target of the frequent U2AF1 S34F mutation in MDS. The role of macroH2A1.1 in hematopoiesis is unclear. Results: MacroH2A1.1 mRNA levels are significantly decreased in patients with low-risk MDS presenting with chromosomal 5q deletion and myeloid cytopenias and tend to be decreased in MDS patients carrying the U2AF1 S34F mutation. Using an innovative mouse allele lacking the macroH2A1.1 alternatively spliced exon, we investigated whether macroH2A1.1 regulates HSC homeostasis and differentiation. The lack of macroH2A1.1 decreased while macroH2A1.1 haploinsufficiency increased HSC frequency upon irradiation. Moreover, bone marrow transplantation experiments showed that both deficiency and haploinsufficiency of macroH2A1.1 resulted in enhanced HSC differentiation along the myeloid lineage. Finally, RNA-sequencing analysis implicated macroH2A1.1-mediated regulation of ribosomal gene expression in HSC homeostasis. Conclusions: Together, our findings suggest a new epigenetic process contributing to hematopoiesis regulation. By combining clinical data with a discrete mutant mouse model and in vitro studies of human and mouse cells, we identify macroH2A1.1 as a key player in the cellular and molecular features of MDS. These data justify the exploration of macroH2A1.1 and associated proteins as therapeutic targets in hematological malignancies. Keywords: Hematopoiesis, MacroH2A1, Myelodysplastic syndrome * Correspondence: oxana.bereshchenko@unipg.it; manlio.vinciguerra@fnusa.cz 1 Department of Medicine, Department of Philosophy, Social Sciences and Education, University of Perugia, Perugia, Italy 2 International Clinical Research Center, St’Anne University Hospital, Brno, Czech Republic Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 2 of 14 Background its function in HSC homeostasis and differentiation in The healthy hematopoietic stem cell (HSC) pool is mice. Moreover, we found that macroH2A1 isoforms’ maintained by genetically and epigenetically regulated mRNA levels are significantly decreased in MDS patients gene expression [1, 2]. When these systems go awry, ab- with a 5q deletion compared to other MDS groups and errant hematopoietic stem and progenitor cells may healthy individuals. emerge (reviewed in [3]), which are implicated in the de- velopment of a heterogeneous group of disorders called Results myelodysplastic syndromes (MDS). MDS are character- Mice lacking macroH2A1.1 exhibit blood abnormalities ized by ineffective hematopoiesis, leading to variable per- under a steady state ipheral blood cytopenias and risk of transformation to As macroH2A1.1 expression is decreased in U2AF1 S34F acute myeloid leukemia (AML) [4]. MDS can remain MDS patients and its knockdown in vitro perturbs eryth- stable for many years with few symptoms or can rapidly roid and granulomonocytic differentiation [6], we sought progress into a more aggressive MDS subtype [5]; to investigate macroH2A1.1 function in mice using a gene current treatment options, however, are limited and KO approach. We first generated a mouse line carrying a often ineffective. Studies have started to identify com- conditional macroH2A1 allele that permits selective elim- mon mutations underlying HSC dysregulation in MDS, ination of one of the two macroH2A protein isoforms but more progress is needed to identify the molecules using either Cre or Dre recombinase (Fig. 1a). and pathways contributing to disease mechanisms. Mice carrying the targeted (floxed; fl) allele exhibited Recent data have suggested a possible role for histone no phenotype compared to wild-type (WT) mice and variants, in particular, the macroH2A1 splice variant were viable and fertile (data not shown). When crossed macroH2A1.1, in the pathological processes underlying to deleter HPRT-Cre mice (129S1/Sv-Hprttm1(CAG-- cre)Mnn MDS development [6]. Histone macroH2A1 exists as two /J) that ubiquitously express Cre in early develop- mutually exclusive exon-spliced isoforms: macroH2A1.1 ment [23], the loxP-flanked cassette and exon 6b were and macroH2A1.2. These isoforms display largely divergent removed thereby generating macroH2A1.1fl/− mice. functions in cell differentiation and tumorigenesis [7, 8]. These mice were then intercrossed to generate MacroH2A1.1 is a potent transcriptional modulator associ- macroH2A1.1−/− (KO) mice. MacroH2A1.1fl/− and ated with favorable outcomes in several solid tumor types, macroH2A1.1−/− mice were morphologically and histo- restriction of cancer stem-cell emergence, and PARP1 (Poly logically indistinguishable from their WT and macro- (ADP-ribose) Polymerase-1)-dependent chromatin remod- H2A1.1fl/fl littermates under normal conditions, with no eling [8–20]. The gene encoding human macroH2A1.1 and overt metabolic, reproductive, or behavioral abnormal- macroH2A1.2, H2AFY, is found on the long arm of ities. To confirm the absence of macroH2A1.1 in the chromosome 5 (at 5q31.1), which is deleted in ~ 20% of KO cells, we isolated murine embryonic fibroblasts MDS patients. Between 45 and 85% of MDS patients ex- (MEFs) from macroH2A1.1fl/fl and macroH2A1.1−/− hibit mutations in various components of the RNA spliceo- mice, which exhibited comparable cell morphology some machinery, including U2AF1, ZRSR2, SRSF2, and (Fig. 1b, left panel), and analyzed extracted protein ly- SF3B1 [21]. Recapitulation of the common U2AF1 S34F sates by Western blotting. MacroH2A1.1, but not mutation in mice reproduces aspects of human MDS and macroH2A1.2, protein expression was abolished in induces dysregulated splicing of multiple gene products, in- macroH2A1.1−/− KO MEFs (Fig. 1b). We then confirmed cluding H2AFY [22]. Yip et al. recently demonstrated that undetectable macroH2A1.1 mRNA levels in a panel of tis- bone marrow (BM) progenitor cells from MDS patients sues isolated from adult (3-month-old) macroH2A1.1−/− with an endogenous U2AF1 S34F mutation showed aber- mice, compared to macroH2A1.1fl/fl littermates, and reduced rantly spliced H2AFY, resulting in reduced macroH2A1.1, macroH2A1.1 mRNA levels by 50% in macroH2A1.1fl/− het- but not macroH2A1.2, isoform expression in both erythroid erozygous (HET) mice (Fig. 1c). MacroH2A1.2 transcript and granulomonocytic derived colonies [6]. shRNA-medi- abundance was comparable between all macroH2A1.1 geno- ated silencing of macroH2A1.1 in BM progenitor cells led types (Fig. 1c). These results confirmed efficient Cre-medi- to increased apoptosis and decreased differentiation when ated macroH2A1.1 allele targeting. cells were cultured under erythroid and granulomonocytic Blood counts and biochemical tests identified a low conditions [6]. hematocrit and reduced neutrophil counts in macro- Here, we report for the first time that loss of H2A1.1fl/− HET and macroH2A1.1−/− KO mice com- macroH2A1.1 expression in mice led to the dysregulation pared to macroH2A1.1fl/fl and WT littermates; an of myeloid differentiation. RNA-sequencing identified de- opposite trend was observed for lymphocyte and eosino- fective production of ribosomal mRNAs as a potential phil cell counts (Additional file 1: Figure S2A and B). No mechanism for disrupted HSC homeostasis following difference in cholesterol or triglyceride levels was ob- macroH2A1.1 depletion. Thus, macroH2A1.1 delineates served between groups, but decreased liver transaminase Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 3 of 14 Fig. 1 Conditional macroH2A1.1 knockout (KO) in mice. a Upper panel. Targeting construct containing the sequence encoding mouse macroH2A1 (H2AFY), a loxP-flanked neomycin (neo) cassette 3′ of exon 6b (included in macroH2A1.2), and a rox-flanked cassette 3′ of exon 6a (included in macroH2A1.1). Lower panel. Targeting construct upon Cre-mediated excision. b Left: phase-contrast of first passage mouse embryonic fibroblasts (MEFs) after 3 days in culture. Right: protein lysates from MEFs of macroH2A1.1fl/fl and macroH2A1.1−/− (KO) mice were immunoblotted with anti-macroH2A1.1 and macroH2A1.2 antibodies. Histone H2A was used as a loading control. c macroH2A1.1 and macroH2A1.2 mRNA levels in adult mice tissues. RNA was extracted from the lung, liver, kidney, seminal gland, intestine, testis, brain, skeletal muscle, heart, and spleen of 3-month-old macroH2A1.1fl/fl, macroH2A1.1fl/− (HET), and macroH2A1.1−/− (KO) mice, and analyzed by qRT-PCR using isoform-specific primers. N = 5 mice/group. **p < 0.01; ***p < 0.001 relative to macroH2A1.1fl/fl levels were observed in macroH2A1.1fl/− HET and MacroH2A1.1 haploinsufficiency or deficiency perturbs macroH2A1.1−/− KO mice (Additional file 1: Figure hematopoiesis following DNA damage S2C). The blood count abnormalities are reminiscent of An expansion of phenotypically primitive HSCs is often those observed in human patients with MDS. We also observed in patients with MDS [3]. Murine HSCs are detected lower alanine transaminase (ALT) and aspar- enriched in the bone marrow cell population that do not tate transaminase (AST) levels in macroH2A1.1fl/− express mature hematopoietic cell lineage markers (Lin−), HET, and to a greater extent in macroH2A1.1−/− KO such as B220, CD4, CD8, Gr-1, Mac-1, and Ter-119, but mice (Additional file 1: Figure S2C), which in humans do express c-Kit and Sca-1, collectively termed LSK (Lin−- is associated with higher all-cause mortality [24, 25]. Sca-1+c-Kit+) cells [26]. HSC and early progenitors can be Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 4 of 14 further characterized within the LSK population based on macroH2A1.1 might be involved in regulating the the surface expression of SLAM (signaling lymphocyte ac- hematopoietic compartment following genotoxic insult in a tivation molecule) markers CD150 and CD48 [27]. Given dose-dependent and time-dependent manner. We analyzed the enhanced susceptibility of macroH2A1.1 KO mice to ra- HSCs in the BM of all three macroH2A1.1 genotypes at 1 diation-induced death and cell damage, we tested the effects day (early time point) and 7 days (late time point) after sys- of haploinsufficiency and absence of the macroH2A1.1 on temic exposure to 600 (low dose) or 1200 rad (high dose) ir- the hematopoietic compartment at a steady state and under radiation. Downregulation of c-Kit cell surface expression DNA damaging conditions. At a steady state, total bone on functional HSCs has been reported in the situation of marrow cellularity (Fig. 2a) as well as the frequency of the distress, such as 5-FU treatment and Myc gene deletion LSK CD48−CD150+ HSC did not vary significantly among [29]. Since all Lin−Sca-1+CD150+ cells in control animals macroH2A1.1fl/fl, HET and KO mice (Fig. 2b). Because are c-Kit+, the Lin−Sca-1+CD150+CD48− marker combin- macroH2A1 isoforms regulate self-renewal and differenti- ation can also be used to identify HSCs [29]. Since in ation of induced pluripotent stem cells, embryonic stem irradiated mice, we also observed a dramatic reduction in cells and cancer stem cells [11, 16, 28], we hypothesized that c-Kit+ cells (data not shown), we used the Lin−Sca- Fig. 2 Assessment of the long-term hematopoietic stem-cell (LT-HSC) pool at a steady state and upon irradiation in macroH2A1.1fl/− (HET) and macroH2A1.1−/− (KO) mice. Live, Lin− cells were gated and analyzed for c-Kit and Sca-1 surface marker expression. Lin−c-Kit+Sca-1+ (LSK) cells were gated and further analyzed for CD150+ CD48− surface marker expression. Evaluation of the absolute cell number (a) and frequencies (percentage relative to whole bone marrow cell count) (b) of LT-HSCs in the bone marrow (BM) of macroH2A1.1fl/fl, macroH2A1.1fl/− (HET), and macroH2A1.1−/− (KO) mice, at a steady state. N = 5–6 mice per genotype. c, d Evaluation of the absolute cell number and frequencies of LT-HSCs in the BM of irradiated [(600 rad (low dose, c) or 1200 rad (high dose, d)] macroH2A1.1fl/fl, macroH2A1.1fl/− (HET), and macroH2A1.1−/− (KO) mice. Analyses were performed on days 1 (early time point) or 7 (late time point) after irradiation. Error bars represent the SD. N = 3–4 mice per genotype. *p < 0.05, **p < 0.01 relative to macroH2A1.1fl/fl Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 5 of 14 1+CD150+CD48− marker combination to identify HSC in (RNA-Seq) to identify differentially expressed genes (DEGs) WT, HET, and KO mice following different doses of ir- between HPCs (CD150−CD48+ LSK) isolated by cell sorting radiation in comparison to the same population in un- from macroH2A1.1−/− KO and macroH2A1.1fl/fl mice. treated mice. We observed that the macroH2A1.1 Using a 1.5-fold change threshold, we identified 599 DEGs, haploinsufficiency caused a significant increase in HSC of which 225 were upregulated and 374 were downregulated frequency at late time point following low-dose (Fig. 2c) in macroH2A1.1−/− compared to macroH2A1.1fl/fl controls and high-dose (Fig. 2d) irradiation compared to WT mice. (Additional file 2: Table S1). KEGG analysis revealed that Conversely, the macroH2A1.1−/− mice showed a decrease these genes significantly over-represented the ribosome in HSC frequency, caused by low (Fig. 2c, early time point) pathway, protein processing in the endoplasmic reticulum, and high (Fig. 2d) doses of irradiation, compared to un- and the cysteine/methionine metabolism pathways (Fig. 4a). treated and similarly treated WT mice. Altogether, these The ribosome pathway was the most significantly enriched data demonstrate that the macroH2A1.1 isoform regulates pathway in our experiment (Fig. 4b), with 16 differentially HSC response to irradiation in a time- and dose- expressed ribosomal proteins with their functional location dependent manner. within the large (Rpl) and small (Rps) subunits. Further- more, we found the broad functions of chromatin modifica- MacroH2A1.1 haploinsufficiency or deficiency in HSCs tion/remodeling, transcription, redox cell metabolism, leads to a myeloid differentiation bias in transplantation cytoskeleton homeostasis, and cellular response to DNA assays damage stimulus among the top 20 most represented bio- A myeloid differentiation bias is found in human patients logical processes (Gene Ontology, GO) in macroH2A1.1−/− with MDS [3]. To understand the downstream effects of HPCs (Fig. 4c). As defective ribosome biogenesis has been macroH2A1.1 haploinsufficiency or deficiency on HSC/ reported in MDS [30], we next asked whether hematopoietic progenitor cells (HPC) populations, we ana- macroH2A1.1-deficiency-driven changes in ribosomal pro- lyzed the BM from macroH2A1.1fl/fl, macroH2A1.1fl/− tein gene expression underpin pathology in hematopoietic HET, and macroH2A1.1−/− KO mice by flow cytometry. cells. We stably knocked down (KD) macroH2A1.1 mRNA Higher frequencies of myeloid (Mac-1+) cells were found in in human promyelocytic leukemia HL-60 and monocytic macroH2A1.1−/− KO mice compared to macroH2A1.1fl/fl THP-1 cell lines grown in suspension [31], using lentivi- mice, and lower frequencies of B (B220+) cells were found rally transduced shRNAs against the H2AFY gene. Upon in both macroH2A1.1fl/− HET and in macroH2A1.1fl/fl mice efficient specific silencing of the macroH2A1.1 transcript (Fig. 3a). To demonstrate that the cause of this myeloid bias (without altering the levels of macroH2A1.2 isoform) observed in macroH2A1.1−/− KO mice is intrinsic to the (Additional file 1: Figure S3A, B), we measured transcript hematopoietic system, we performed an in vivo transplant- levels for a subset of Rpl (19, 29, 38) and Rps (15a, 21) ation study. Here, flow cytometry analysis confirmed higher genes, whose expression displayed > twofold change in our frequencies of myeloid cells and lower frequencies of B cells RNA-Seq analysis (Additional file 2: Table S1). in the peripheral blood of mice receiving macroH2A1.1fl/− MacroH2A1.1 KD led to decreased Rpl19 (fivefold), Rpl29 HET or macroH2A1.1−/− KO BM cells, compared to mice (twofold), Rpl38 (fivefold), Rps15a (sixfold), and Rps21 receiving macroH2A1.1fl/fl BM cells (Fig. 3b–d). The mye- (twofold) mRNA levels in both HL-60 and THP-1 cells loid differentiation bias was also observed in HET mice in (Additional file 1: Figure S3C, D), consistent with our noncompetitive BM transplantation assay (Fig. 3c). More- RNA-Seq findings made in macroH2A1.1−/− mouse HPCs over, the analysis of the frequencies of myeloid progenitor (Fig. 4). In order to obtain evidence that macroH2A1.1 de- sub-populations revealed a tendency increase in GMPs ficiency-dependent decreased ribosomal protein gene ex- (Lin/IL7R/Sca-1−c-Kit+CD34+CD16/32+) and a tendency pression may lead to defective ribosome biogenesis and decrease in megakaryocyte-erythrocyte progenitors (MEPs; protein synthesis, we assessed the steady-state 47S pre- Lin−/IL7R/Sca-1−c-Kit+CD34−CD16/32−) in the BM of rRNA level in macroH2A1.1−/− HPCs, and in human HL- macroH2A1.1fl/− HET and macroH2A1.1−/− KO mice com- 60 and THP-1 cells KD for macroH2A1.1. Under these pared to macroH2A1.1fl/fl mice (Fig. 3e). These data show conditions, a ~ 85% decrease in the relative amount of 47S that decreased macroH2A1.1 levels has a profound impact pre-rRNA was observed in macroH2A1.1−/− HPCs on HSC differentiation in the BM, resulting in a myeloid compared to macroH2A1.1fl/fl HPCs (Fig. 5a). Similarly, a skewing similar to that observed in human MDS. ~ 70% and ~ 60% decrease in the relative amount of 47S pre-rRNA was observed in HL-1 and in THP-1 cells, re- Downregulation of ribosomal protein genes after spectively (Fig. 5b). In addition, we performed a protein macroH2A1.1 depletion synthesis inhibition assay using puromycin, an antibiotic To gain a mechanistic insight into the hematopoietic de- that competes by acting as an analog of the three-terminal rangements associated with decreased macroH2A1.1 expres- end of aminoacyl-tRNA, disrupting protein synthesis. To sion, we performed transcriptome-wide RNA sequencing this purpose, we performed a [3H]-leucine incorporation Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 6 of 14 Fig. 3 MacroH2A1.1 deficiency causes myeloid bias upon bone marrow (BM) transplantation. a Peripheral blood (PB) analysis from macroH2A1.1fl/fl, macroH2A1.1fl/− (HET), and macroH2A1.1−/− (KO) mice showing the percentage of myeloid-cell (Mac-1+) and B-cell (B220+) gated populations. b, c Dot plots of PB analysis from animals transplanted with macroH2A1.1fl/fl, macroH2A1.1fl/− (HET), and macroH2A1.1−/− (KO) BM cells in a competitive (b) or noncompetitive (c) transplantation assay. Both plots show the percentage of myeloid cells (Mac-1+) within engrafted CD45.2+ cells. d Representative plots indicating in each quadrant the frequency of gated cell populations within CD45.2+ cells. Graphs show the frequency of [Mac-1+, B (B220+), and T (CD4+/CD8+) cells] within CD45.2+ cells. e Frequencies of CMPs, MEPs, and GMPs in the BM of irradiated macroH2A1.1fl/fl, macroH2A1.1fl/− (HET), and macroH2A1.1−/− (KO) mice. Graphs represent CMP (Lin−c-Kit+Sca-1−CD34+CD16/32), MEP (Lin−c- Kit+Sca-1−CD34−CD16/32−), and GMP (Lin−c-Kit+Sca-1−CD34+CD16/32+) frequencies in the BM. Results are presented as the means ± SEM of three independent experiments. N = 6–10/group. *p < 0.05, ***p < 0.001, NS = not significant Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 7 of 14 Fig. 4 RNA-Seq and bio-informatic analyses on multipotent progenitor cells (CD150−CD48+ LSK) isolated from bone marrow and using flow cytometry. a KEGG pathways significantly represented by the differentially expressed genes. Enrichment-log (p values) is reported on the x-axis. The dashed red line marks the significant enrichment threshold (p value = 0.05). b Mouse KEGG map (mmu03010) of the Ribosome pathway. c Top 20 most enriched Gene Ontology biological processes. The blue line marks the significance enrichment threshold (p value = 0.05), and text within a bar refers to the number of significantly expressed genes belonging to a process and the overall enrichment score (into brackets) assay upon treatment with puromycin (0, 0.5, 2 μg/ml) for cells (Fig. 6a, b). This decrease was due to increased 72 h in HL-60 and THP-1 cells; our findings show that apoptosis, as a 48-h pre-incubation of the cells with the protein synthesis was significantly inhibited in HL-1 and pan-caspase inhibitor Z-VAD-FMK (carbobenzoxy-valyl- THP1 cells even in the absence of puromycin, and it was alanyl-aspartyl-[O-methyl]-fluoromethylketone; 10 μM) further inhibited upon treatment with 0.5 μg/ml puro- completely rescued viability (Fig. 6a, b). Conversely, stable mycin (Fig. 5c). Two micrograms per milliliter puromycin lentivirus-mediated overexpression of macroH2A1.1 fused was lethal in both cell lines (Fig. 5c). to GFP(9) in HL-60 cells (Fig. 7a) led to increased cell pro- In fact, macroH2A1.1 KD was associated with a ~ 60– liferation (Fig. 7b). Treating HL-60 cells with phorbol 12- 80% decrease in cell viability of both HL-60 and THP-1 myristate 13-acetate (PMA) drives their differentiation Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 8 of 14 Fig. 5 MacroH2A1.1 deficiency decreases rRNA transcription and protein translation in mouse HPCs (a) and in human HL-60 and THP-1 cells (b), respectively. a, b Total RNA was extracted and 47S pre-rRNA levels were measured by qPCR in macroH2A1.1fl/fl versus macroH2A1.1−/− HPCs and in HL-60 or THP-1 control (CTL) cells compared to KD cells for macroH2A1.1, respectively. Data were normalized to the amount of GAPDH mRNA and to the amount of 47S pre-rRNA in macroH2A1.1fl/fl or in CTL cells, respectively. c Global protein synthesis in HL-60 and THP-1 cells treated for 72 h with puromycin (0, 0.5, 2 μγ/ml) was measured by [3H]-leucine incorporation assay and shown as a percentage of inhibition of control in CTL and macroH2A1.1 KD HL-60 and THP-1 cells. n = 3. ***p < 0.001 relative to the respective CTL (0 μg/ml puromycin); #p < 0.05, ###p < 0.001 relative to the respective 0.5 μg/ml puromycin condition toward a monocytic lineage [32]; however, here, we found were categorized as either having a normal karyotype that this process was apparently unaffected by macroH2A1.1 (NK; n = 4); a 5q deletion (del(5q); n = 15), of which overexpression, with over-expressing cells successfully up- seven also additional cytogenetic changes; or a non- regulating CD11b expression (Fig. 7c) and becoming adher- del5q abnormal karyotype (AK; n = 5) (Additional file 3: ent (Fig. 7d, e). PMA-driven differentiation was also Table S2). As expected, del(5q) patients expressed signifi- unaffected by KD of macroH2A1.1 in the population of cells cantly lower macroH2A1.1 and macroH2A1.2 mRNA levels that remained viable (Fig. 7e). compared to healthy controls (p < 0.05), due to the loss of H2AFY located at 5q31.1 within the commonly-deleted 5q MacroH2A1 isoforms’ transcript levels are decreased in region (Fig. 8a, b). By contrast, NK and AK MDS patients the BM of MDS patients carrying a 5q deletion or U2AF1 showed normal macroH2A1.1/macroH2A1.2 mRNA levels S34F mutation (Fig. 8a, b). Aberrant H2AFY splicing causing specifically re- We first compared the BM expression levels of duced macroH2A1.1, but not macroH2A1.2, expression macroH2A1.1 or macroH2A1.2 between sub-groups of has been associated with the U2AF1 S34F mutation in MDS patients with different genetic abnormalities and MDS [6]. However, only three MDS patients in this cohort healthy controls (n = 5). MDS patients (n = 24 total) carried this mutation, and they were not del(5q) carriers Fig. 6 a, b MacroH2A1.1 knockdown (KD) affects cell viability in HL-60 and THP-1 cells. a, b Cell viability assay in HL-60 (a) and THP-1 (b) cells, incubated or not for 48 h in the presence of 10 μM pan caspase inhibitor Z-VAD-FMK. Data are presented as means relative to CTL cells, +/− SD, n = 4. ***p < 0.001 relative to CTL Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 9 of 14 Fig. 7 Effect of macroH2A1.1-GFP transgene overexpression, or macroH2A1.1 KD, on cell proliferation and differentiation. a Histones were extracted from HL-60 cells stably overexpressing a GFP vector or a stable macroH2A1-GFP transgene [9], and lysates were processed for immunoblotting with an anti-GFP antibody. Representative images are shown. b Cell proliferation assay in HL-60 cells, overexpressing GFP or macroH2A1.1-GFP, using labeling with carboxyfluorescein diacetate succinimidyl ester (CFSE) dye dilution by flow cytometry, 4 days upon adding the dye. Data are presented as the percentage of GFP cells, +/− SD, n = 4. **p < 0.01 relative to GFP. c FACS staining for pan-macrophage marker CD11b in HL-60 cells, overexpressing GFP or macroH2A1.1-GFP, upon treatment with Phorbol 12-myristate 13-acetate (PMA, 25 nM) or an equal amount of vehicle (DMSO) for 24 h. Representative gating is shown. d Representative images of HL-60 cells in suspension or adherent upon addition of PMA or DMSO as in c. e Adherent HL-60 cells, overexpressing GFP or macroH2A1.1-GFP, or KD for macroH2A1.1, upon treatment with PMA or DMSO as in c, were counted. Data are presented as the percentage of total cells (in suspension + adherent). Data are presented as means +/− SD, n = 4 (Additional file 3: Table S2). When comparing these HPCs provides a critical link between this epigenetic U2AF1 mutants versus healthy subjects, we detected a de- regulator and the molecular pathologies typical of MDS. creased, albeit not significant, macroH2A1.1 but not Our findings are in line with a recent report from Yip macroH2A1.2 mRNA expression (Fig. 8c, d), according to et al. [6]. These authors analyzed the role of the muta- previous studies [6]. These data provide evidence that tion S34F in the splicing factor U2AF1, which is fre- macroH2A1.1 mRNA tends to decrease in the BM of MDS quent in MDS: U2AF1 S34F altered mRNA splicing of patients carrying the U2AF1 S34F mutation and that both many transcripts and among those H2AFY (coding for macroH2A1.1 isoforms’ transcript levels are decreased in macroH2A1.1 and macroH2A1.2). Only 3 out of 29 pa- the BM of MDS patients who are del(5q) carriers. tients in our cohort displayed U2AF1 S34F mutation; in those individuals, we observed a not statistically signifi- cant tendency toward a decrease in macroH2A1.1, but Conclusions not macroH2A1.2 mRNA level: analyses on larger co- Here, we show that the histone variant macroH2A1.1, horts are required to corroborate these data [6]. which is downregulated in a subset of patients with H2AFY is physically localized on the long arm of MDS, profoundly affects the survival and differentiation chromosome 5, implying a connection with the most of murine HSCs/HPCs in vivo and human leukemic cell common karyotypic abnormality in MDS—del(5q). As lines in vitro. We provide evidence that macroH2A1.1 is expected, both macroH2A1.1 and macroH2A1.2 mRNA required for normal ribosomal protein gene expression levels were found decreased in the BM of del(5q) MDS in HPCs. Without proper expression of this histone vari- patients, compared to healthy subjects. ant, abundant cell death occurred in the hematopoietic MacroH2A1.1 but not macroH2A1.2, which is deficient in cells. Uncovering the relationship between macroH2A1.1 poly-ADP-ribose binding, is required for PARP-1 activation depletion and defective ribosomal biogenesis in HSCs/ [16, 28, 33, 34]. MacroH2A1 and PARP1 activities have been Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 10 of 14 A B C D Fig. 8 Expression analysis of two macroH2A1 transcript variants in MDS cells. a, b Relative mRNA expression of macroH2A1.1 (a) or macroH2A1.2 (b) in bone marrow samples from healthy individuals (n = 5), MDS patients with a 5q deletion (del5q; n = 15), or without del5q (NK, normal karyotype; n = 4), and MDS patients carrying other different chromosomal aberrations (AK, abnormal karyotype; n = 5). c, d MacroH2A1.1 and macroH2A1.2 mRNA levels were also compared between healthy individuals, MDS patients without the U2AF1 S34F mutation (wild type, wt), and MDS patients with the U2AF1 S34F mutation (mut). The p values are indicated, compared to healthy individuals independently implicated in ribosome assembly [35–37]. epigenetic regulator of hematopoiesis. Silencing Mutational [38] and transcriptomic [39] approaches have macroH2A1.1 expression in human progenitor cells alters not fully clarified yet the role of del(5q) in MDS pathogen- erythroid and granulomonocytic differentiation, and the re- esis; however, CSNK1A1 [40] and RPS14 [41] have been introduction of macroH2A1.1 in U2AF1 mutant BM cells identified as strong promising therapeutic targets. rescues cell death and their differentiation potential [6]. Hematopoietic differences observed in macroH2A1.1 hap- Consistently, we found that in lineage-committed HL-60 loinsufficient and macroH2A1.1-deleted mice have been (neutrophils) and THP-1 (monocytes) human myelomono- similarly reported in a murine model displaying conditional cytic leukemia cell lines, KD for macroH2A1.1 led to inactivation of CSNK1A1 [40]. In this model, CSNK1A1 massive reduction in proliferation and death, and that haploinsufficiency induced HSC expansion, whereas homo- macroH2A1.1 haploinsufficiency, and to a lesser extent, its zygous deletion caused HSC failure [40]. If CSNK1A1 and full deficiency, tends to decrease the frequency of CMPs and macroH2A1.1 functionally interact in regulating, MEPs, and to increase GMPs in mice. Expanded GMPs hematopoiesis remains to be established. The phenotype of characterize high-risk MDS [44]. Compared to in vitro our newly generated macroH2A1.1 haploinsufficient mouse shRNA-mediated silencing or full in vivo KO, we believe recapitulates several features of human MDS, including that the features of our newly generated macroH2A1.1 hap- anemia, neutropenia, expansion of the LT-HSC pool, and a loinsufficient mouse might be more akin to the pathological marked level of susceptibility to irradiation and defective changes observed in MDS patients. In fact, we also detected myelopoiesis [3]. a myeloid bias in mice that lack the full dose of Various transcriptional and epigenetic mechanisms con- macroH2A1.1: the hematopoietic cell-autonomous enrich- tribute to HSC maintenance and stepwise differentiation to ment of myeloid Mac-1+ cells parallels the myeloid differen- produce distinct hematopoietic lineages [42, 43], and tiation bias that is commonly observed in other MDS macroH2A1.1 has only recently emerged as a novel murine models and in human MDS. MDS is characterized Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 11 of 14 on one hand by dysplastic myeloid expansion, with myeloid By combining clinical data with a discrete mutant mouse cells that have reduced ability to differentiate, and on the model and in vitro studies of human cells, we identify other hand by neutropenia, thrombocytopenia, and anemia macroH2A1.1 as a key determinant of the cellular and [45–47]: we propose macroH2A1.1−/− mouse as a useful molecular features of MDS. These data justify the explor- tool for further mechanistic studies on MDS. ation of macroH2A1.1 and associated proteins as thera- By characterizing the phenotype of macroH2A1.1-insuf- peutic targets in hematological malignancies. ficient mice, we report also another primary in vivo role for macroH2A1.1 in hematopoiesis: our RNA-Seq analysis Methods of HPCs from macroH2A1.1−/− mice identified a substan- Clinical and laboratory data of MDS patients tial depletion of many transcripts [small ribosomal pro- Bone marrow aspirates, clinical information, and routine teins (Rps) and large ribosomal proteins (Rpl)] involved in laboratory data were collected from 24 patients diag- ribosome assembly, and this was confirmed in HL-60 and nosed with MDS according to the revised World Health THP-1 myeloid cell lines. Moreover, we found that Organization criteria [52] and prior to commencement macroH2A1.1−/− HPCs and HL-60/THP-1 KD for of treatment, at the University Hospital Brno. Basic macroH2A1.1 display decreased levels of 47S pre-rRNA, a MDS patients and cytogenetic characteristics are shown long primary transcript that is the precursor of three of in Additional file 3: Table S2. U2AF1 mutation analyses the four ribosomal RNAs: 18S, 5.8S, and 28S. Within ribo- were performed as described previously [6]. some structure, the 18S rRNA assembles with 33 Rps to form the 40S ribosomal subunit or small subunit, while Human bone marrow cell isolation and sample processing the 5S, 5.8S, and 28S rRNAs associate with 47 Rpl to as- Red blood cells were depleted using ACK lysing buffer semble the 60S or large subunit. 47S pre-rRNA and Rps/ (NH4Cl 150 mM, KHCO3, 10 mM, Na2EDTA 0.1 mM, Rpl levels might thus be connected events leading to de- pH 7.2). White blood cells were further processed for fective ribosome biogenesis in macroH2A1.1−/− HPCs. Al- RNA isolation. Total RNA was isolated using TriReagent though the causal role for ribosomal biogenesis in HSC (MRC, USA) according to the manufacturer’s instruc- maintenance is not fully understood, impaired ribosome tions and the quality of RNA was assayed with an Agi- biogenesis-induced nuclear stress, for instance, due to lent 2100 Bioanalyser (RNA 6000 Nano Assay; Agilent hemizygosity for genes encoding ribosomal proteins, is Technologies, USA). associated with the development of clinical entities col- lectively known as “ribosomopathies” which include sev- Animal models eral bone marrow failure syndromes, including MDS [48]. Mice lacking macroH2A1.1 were generated as follows: a We speculate that macroH2A1.1 depletion in the 12 kb segment of the murine H2AFY gene (introns 5–8) hematopoietic system might have two deleterious effects. was subcloned from a BAC by recombineering into First, it alters HSC homeostasis through defective riboso- p15A-HSV tk-DTA-amp. A lacZ-neo cassette [53], mal production [30]: adult HSCs display low levels of glo- flanked by loxP and rox sites at the 5′ and 3′ ends, re- bal protein synthesis relative to HPCs and genetic spectively, was inserted into the intron between exons 6a perturbations that alter the dynamics of protein synthesis and 6b, also by recombineering [54]. Another rox site impair HSC function [49]. Our data suggesting that was inserted upstream of exon 6a and another loxP site macroH2A1.1-dependent impairment in ribosome bio- inserted downstream of exon 6b so that Dre/rox recom- genesis relates to impaired HSC differentiation are con- bination [55] would remove exon 6a and the lacZ-neo sistent with Signer et al., who demonstrated that reduced cassette, and Cre/loxP recombination would remove ribosome function in Rpl24 mice reduced protein synthe- exon 6b and the lacZ-neo cassette; thus, Cre recombin- sis of 30% in HSCs and impaired HSC function [50]. ation will eliminate macroH2A1.1 expression. Southern Second, given the extensive Rpl and Rps expression blotting of genomic NheI-digested DNA from individual heterogeneity in the hematopoietic system [51], ES-cell-derived clones with a 3′ probe was used to identify macroH2A1.1−/− might disrupt the regulation of homologous recombinants (Additional file 1: Figure S1). A hematopoietic lineage-specific ribosomal proteins that 12.3-kb DNA fragment corresponds to the wild-type might be involved in lineage differentiation. Predicting macroH2A1.1 locus; integration of the loxP-flanked neo- the phenotypes of perturbed Rpl and Rps expression pat- mycin cassette 3′ of exon 6b introduced an additional terns remains challenging and is an area that warrants NheI site, thus increasing the size of the NheI DNA frag- future research. ment to 16.2 kb in the targeted allele (Additional file 1: In summary, our study shows that a loss of Figure S1). Cre-mediated recombination resulted in a 3.9- macroH2A1.1, which affects a subset of MDS patients, has kb NheI DNA fragment recognized by the 3′ probe, which a critical role in the defective hematopoiesis and perturbed is diagnostic of the macroH2A1.1 allele. The targeting of ribosomal biogenesis that are central to MDS pathology. the macroH2A1.1 allele was performed by electroporation Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 12 of 14 of A9 ES cells, which were then injected into C57BL/6 site, thus increasing the size of the NheI DNA fragment to 16.2-kb in the eight cell-stage embryos. The targeted macroH2A1fl/fl targeted allele. Figure S2. Peripheral blood counts and biochemical mice were crossed to deleter HPRT-Cre mice (129S1/Sv- parameters in macroH2A1.1 Fl/- and 1 KO mice. Blood samples were collected into heparinized containers from wild type (WT), macroH2A1.1 Hprttm1(CAG-cre)Mnn/J), purchased from Jackson Laborator- Fl/Fl, macroH2A1.1 Fl/- and KO mice via tail vein. Data are expressed as ies, USA, to remove the loxP-flanked neomycin cassette mean ± SEM. * p < 0.05; **p < 0.01; ***p < 0.001 as compared to and generate macroH2A1.1fl/− mice (heterozygous, HET), macroH2A1.1 Fl/Fl. N=12-15 per each group. Figure S3. Effect of macroH2A1.1 knock/down (KD) on ribosomal protein gene expression in respectively. Mice heterozygous for the macroH2A1.1 al- HL-60 and THP-1 cells. A, B. RNA was extracted from HL-60 (A) or THP-1 lele were further crossed to deleter Cre mice to generate (B) cells stably overexpressing a vector carrying scrambled shRNA (CTL) or the macroH2A1.1−/− (knockout, KO) mice, respectively. a vector carrying shRNA for macroH2A1.1 (KD), and processed for qPCR using specific primers against macroH2A1.1 or macroH2A1.2 transcripts. All mice used were obtained after eight generations of C, D. RNA was extracted from HL-60 (C) or THP-1 (D) cells stably back crossing on a C57Bl/6 genetic background. Mice overexpressing a lentiviral vector carrying scrambled shRNA (CTL) or a were bred and maintained at the EMBL Mouse Biology vector carrying shRNA for macroH2A1 (KD), and processed for qPCR using specific primers against Rpl19, Rpl29, Rpl38, Rps15a and Rps21 Unit, Monterotondo, or at Plaisant Srl (Rome, Italy), in ac- transcripts. Data are presented as means relative to CTL cells, +/- SD, n = cordance with current Italian legislation (article 9, 27 4. *** P < 0.001 relative to CTL. (DOCX 680 kb) January 1992, number 116) under a license from the Ital- Additional file 2: Table S1. Basic MDS patient/sample characteristics. ian Health Ministry. The congenic C57BL/6-Ly5.1 mice Patients are classified according to the WHO classification 2016 13. Excel file provided separately. MDS-SLD - MDS with single lineage dysplasia. were purchased from Charles River Laboratory. C57BL/6- MDS-MLD - MDS with multi-lineage dysplasia. MDS-RS-MLD - MDS with Ly5.1/2 recipients were generated by intercrossing ring sideroblasts and multi-lineage dysplasia. MDS-EB-1 - MDS with C57BL/6-Ly5.1 and C57Bl/6-Ly5.2 mice (Harlan, Italy). excess blasts-1. MDS-EB-2 - MDS with excess blasts-2. 5q- syndrome - MDS with isolated del(5q). (XLSX 56 kb) Additional file 3: Table S2. List of 599 transcripts displaying >1.5 fold Bone marrow transplantation assays change in hematopoietic progenitor cells (HPC) isolated from bone Radiation chimeras were generated as described [56]. For marrow of macroH2A1.1 KO versus Fl/Fl mice. (PDF 295 kb) competitive transplantation assays, a 1:3 mixture of BM cells from the donor macroH2A1.1fl/fl, macroH2A1.1 Abbreviations HET, or macroH2A1.1 KO mice (CD45.2+) and helper BM: Bone marrow; CMP: Common myeloid progenitor; GMP : Cranulocyte- WT (CD45.1+) mice, respectively, was injected into the tail macrophage progenitor; HSC: Hematopoietic stem cells; KO: Knockout; MDS : Myelodysplastic syndrome; MEP: Erythroid progenitor; RNA-Seq : RNA- vein of the recipient (CD45.1/2+) mice. For noncompeti- sequencing tive transplantation assays, 1 × 106 BM cells from macro- H2A1.1fl/fl, macroH2A1.1 HET, or macroH2A1.1 KO Acknowledgements mice (CD45.2+) were injected into the recipient mice. The We are grateful to Madeleine Teucher, Sant’Agata, Sara Cannito, Kamilla Malinowska-Ozdowy, Takis Biotech (Rome, Italy), and the HistoPathology of recipient mice were treated with Baytril (Enrofloxacin, the VBCF (Vienna, Austria) for the technical help. We thank Prof. Andreas Bayer) in drinking water (17 mg/ml) for the duration of Ladurner (LMU, Germany) and Prof. Nadia Rosenthal (Jackson Laboratories, the experiment. US) for the mentorship, and Insight Editing London for the professional edit- ing of the manuscript. Statistical analyses Authors’ contributions Data are shown as means ± standard error of the mean OB, OLR, and SF performed HSC extraction, processing, and flow cytometry (SEM). Groups were compared with either Student’s t analyses. FN performed in vitro irradiation/DNA damage experiments and test or the non-parametric Mann-Whitney U test, as ap- immunofluorescence. TM performed bioinformatics analyses. JK performed gene expression analyses on patient bone marrow samples. AFS designed propriate, using GraphPad Prism Software (version 5.00 the construct to generate KO mice. MMLP and MB performed in vitro gene for Windows, San Diego, CA, USA): significance was expression experiments. CG, GP, GLV, VP, and LC assisted with clinical and p ≤ 0.05. Survival analyses of mice employed the Kaplan- patient data analyses. OB, CR, LK, SP, AFS, and MV conceived experiments, analyzed data, supervised experiments, and provided essential reagents, Meier estimator. equipment, and infrastructure. MV conceived the study, directed the study, and wrote the manuscript. All authors read and approved the final manuscript. Additional files Additional file 1: Supplemental Material and Methods. Figure S1. Funding This work was supported by the European Social Fund and European Conditional KO of macroH2A1.1 in mice. A. Upper panel. Targeting construct containing the sequence encoding mouse macroH2A1 Regional Development Fund - Projects MAGNET (No. CZ.02.1.01/0.0/0.0/ (H2AFY), a loxP-flanked neomycin (neo) cassette 3′ of exon 6b (included 15_003/0000492) (M.V.) and EATRIS-CZ (No. CZ.02.1.01/0.0/0.0/16_013/ in macroH2A1.2), and a rox-flanked cassette 3’of exon 6a (included in 0001818) (S.P.), by Italian Ministry of Education and Research grants to O.B., and by Italian Ministry of Health grant (GR-2010-2311017) to V.P. and M.V.. macroH2A1.1). Lower panel. Target construct upon Cre-mediated excision. B. Southern Blot strategy to screen for positive clones: genomic We acknowledge also the CF Genomics CEITEC MU support by the NCMG re- NheI-digested DNA from individual ES-cell-derived clones with a 3′ probe search infrastructure (LM2015091 funded by MEYS CR) to J.K., and the Univer- was used to identify homologous recombinants. A 12.3-kb DNA fragment sity Hospital Brno support by MH CZ - DRO (FNBr, 65269705) to J.K. and L.C. The work in L.K. group was supported by Wellcome Trust Collaborative Grant corresponds to the wild-type macroH2A1.1 locus; integration of the loxP- flanked neomycin cassette 3′ of exon 6b introduced an additional NheI 206292/E/17/Z, the Czech Science Foundation (GACR 17-17720S), and the project LQ1605 from MEYS CR. Bereshchenko et al. Clinical Epigenetics (2019) 11:121 Page 13 of 14 Availability of data and materials 11. Chen H, Ruiz PD, McKimpson WM, Novikov L, Kitsis RN, Gamble MJ. The datasets used and/or analyzed during the current study are available MacroH2A1 and ATM play opposing roles in paracrine senescence and the from the corresponding author on reasonable request. senescence-associated secretory phenotype. Molecular cell. 2015;59(5):719–31. 12. Lo Re O, Fusilli C, Rappa F, Van Haele M, Douet J, Pindjakova J, et al. 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