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ABSTRACT The timely mobilization of hematopoietic stem and progenitor cells (HSPCs) is essential for maintaining hematopoietic and tissue leukocyte homeostasis. Understanding how HSPCs

migrate between bone marrow (BM) and peripheral tissues is of great significance in the clinical setting, where therapeutic strategies for modulating their migration capacity determine the

clinical outcome. Here, we identify an epigenetic regulator, Phc2, as a critical modulator of HSPC trafficking. The genetic ablation of _Phc2_ in mice causes a severe defect in HSPC

mobilization through the derepression of _Vcam1_ in bone marrow stromal cells (BMSCs), ultimately leading to a systemic immunodeficiency. Moreover, the pharmacological inhibition of VCAM-1

in _Phc2_-deficient mice reverses the symptoms. We further determine that Phc2-dependent _Vcam1_ repression in BMSCs is mediated by the epigenetic regulation of H3K27me3 and H2AK119ub.

Together, our data demonstrate a cell-extrinsic role for Phc2 in controlling the mobilization of HSPCs by finely tuning their bone marrow niche. SIMILAR CONTENT BEING VIEWED BY OTHERS CD63

ACTS AS A FUNCTIONAL MARKER IN MAINTAINING HEMATOPOIETIC STEM CELL QUIESCENCE THROUGH SUPPORTING TGFΒ SIGNALING IN MICE Article 06 August 2021 EPIGENETIC PROGRAMMING DEFINES HAEMATOPOIETIC

STEM CELL FATE RESTRICTION Article 01 May 2023 CLONAL HEMATOPOIESIS RELATED TET2 LOSS-OF-FUNCTION IMPEDES IL1Β-MEDIATED EPIGENETIC REPROGRAMMING IN HEMATOPOIETIC STEM AND PROGENITOR CELLS

Article Open access 07 December 2023 INTRODUCTION Hematopoietic stem and progenitor cells (HSPCs) are rare populations of cells that can sustain the hematopoietic system by continuously

replenishing blood cells1,2. Postnatally, most HSPCs engraft and reside in the BM niche, but a small fraction of these cells is mobilized and migrate into the peripheral blood (PB) to

reconstitute the hematopoietic system under specific signaling cues1,3,4. Therefore, identifying key regulators that critically control HSPC mobilization and understanding their underlying

mechanisms have been of significant interest to facilitate BM transplantation efficiency and to therapeutically inhibit the malignant migration of the transformed hematopoietic cells into

peripheral tissues5. Indeed, several BM niche proteins, including stem cell factor (kit ligand), CXCL12 (SDF-1α), and β1 integrin have been identified as key signaling components to control

the maturation of HSPCs, their migration into the circulation and their homing to the BM by mediating the interaction between HSPCs and bone marrow stromal cells (BMSCs)2,5,6,7. However, the

molecular pathways that regulate the expression of these BM niche proteins remain unclear. Polycomb group (PcG) proteins function as transcriptional repressors of target genes by mainly

modulating histone methylation8. These PcG proteins can be divided into two functionally distinct complexes: polycomb repressor complex 1 (PRC1) and polycomb repressor complex 2 (PRC2). PRC2

induces the formation of trimethylated histone H3 at lysine 27 (H3K27me3) through methyltransferase activity of Ezh proteins which are component proteins in PRC2 complex9. The canonical

PRC1 then recognizes and binds to H3K27me3 to sustain the transcriptional repression of a target gene8,10,11. Both canonical and noncanonical PRC1 complexes can cause additional

transcriptional silencing as E3 ubiquitin ligases by forming monoubiquitylated histone H2A at lysine 119 (H2AK119ub)8,12,13. In this study, we demonstrate that Phc2, a component of the

canonical PRC1, regulates HSPC mobilization through the repression of _Vcam1_ expression by enhancing both H3K27me3 and H2AK119ub in BMSCs. Therefore, Phc2 deficiency causes a severe HSPC

mobilization defect via the derepression of _Vcam1_ in BMSCs, and the pharmacological inhibition of VCAM-1 in BMSCs significantly reverses the symptoms of Phc2-deficient mice. These data

demonstrate the critical cell-extrinsic role of Phc2 in controlling HSPC mobilization and provide the first evidence of epigenetic control over HSPC mobilization. RESULTS PHC2 DEFICIENCY

LEADS TO A DEFECT IN HSPC MOBILIZATION As an initial step to elucidate the functional role of Phc2 during hematopoiesis, we characterized immune phenotypes of _Phc2_−/− (KO) mice14 compared

to those of _Phc2_+/+ (wild-type, WT) mice. We observed macroscopic abnormalities in the thymus and peripheral lymphoid organs of KO mice compared to those of WT mice. Both the thymus and

spleen of the KO mice maintained normal architecture, but these organs were smaller than those of the WT mice (Fig. 1a, b). Consistent with this observation, the absolute numbers of immune

cells and their precursors in the PB, thymus, spleen, and liver of the KO mice were significantly reduced compared to those of the WT mice (Tables 1 and 2, Fig. 1c). Notably, the absolute

number of early T-cell precursors (ETPs) in the thymus or immature B cells in the spleen was significantly lower in the KO mice relative to the WT mice (Table 1). Unexpectedly, the absolute

numbers and relative ratios of HSPCs, early progenitor cells, and immune cells in the BM were similar among all examined mice (Table 1 and Fig. 1c, d). Additionally, the loss of _Phc2_ did

not influence the cell cycle progression or apoptosis of HSPCs (Fig. 1e, f), and both the WT and KO BM-resident HSPCs exhibited no difference in terms of their ability to generate

multipotential or myeloid progenitor cells (Fig. 1g). The absolute numbers of WT and KO B cells in the BM at each developmental stage were not significantly different either (Fig. 1h). Given

that no functional defect was evident in the HSPCs of the BM but that there was a shortage of ETPs and immature B cells from the BM, we postulated that a Phc2 deficiency could lead to a

systemic immunodeficiency due to a defect in HSPC migration into the circulation. To test this hypothesis, we examined the numbers of circulating HSPCs from WT and KO mice using a

colony-forming unit (CFU) assay. The absolute numbers of clonogenic progenitors in the PB and spleen were decreased by approximately half in the KO mice compared to the WT mice, whereas the

absolute numbers of clonogenic progenitors in the BM were comparable between the WT and KO mice (Supplementary Fig. 1). To confirm this result, we performed in vivo HSPC mobilization assays

using two commonly used mobilization regimens for therapy, granulocyte colony-stimulating factor (G-CSF) and AMD3100 (CXCR4 antagonist)15,16,17,18. Five days after G-CSF treatment, the

absolute numbers of white blood cells (WBCs) and splenocytes from the KO mice remained significantly lower than those from the WT mice (Fig. 2a, b). The spleen size of the KO mice also

remained much smaller than that of the WT mice after G-CSF treatment (Fig. 2b). Consistent with these results, both the frequencies and absolute numbers of Lin−Sca-1+c-kit+ cells (LSK cells)

in the PB and spleen were significantly lower in the KO mice than in the WT mice (Supplementary Fig. 2). Likewise, the absolute numbers of clonogenic progenitors in the PB and spleen of the

KO mice were significantly reduced compared to those of the WT mice (Fig. 2c). However, the frequencies and absolute numbers of LSK cells in the BM were comparable between the WT and KO

mice (Supplementary Fig. 2). Moreover, the absolute numbers of clonogenic progenitors in the BM were not significantly different between the WT and KO mice (Fig. 2c). When we used an AMD3100

as a mobilizing agent of LSK, we still observed that the migration of KO LSK into the periphery is statistically decreased compared with that of WT LSK (Fig. 2d, e). However, the defective

migration of KO LSK after treatment of AMD3100 is relatively modest when we compared with results from G-CSF treatment (Fig. 2d, e). To remove circulating HSPCs and visualize neo-HSPC

mobilization from BM, we further treated WT and KO mice with 5-fluorouracil (5-FU), a chemotherapeutic agent that depletes circulating HSPCs and promotes HSPC release from BM during

hematopoietic repopulation19,20. The absolute numbers of WBCs and splenocytes recovered from KO mice were lower than those recovered from WT mice starting on day 8 after 5-FU treatment (Fig.

 2f, g). The most prominent differences in the absolute numbers of WBCs and splenocytes or the size of the spleen between WT and KO mice was observed on day 16 after 5-FU treatment (Fig. 2f,

g). On day 16 after 5-FU treatment, the frequency and the absolute number of recovered LSK cells and the absolute number of recovered clonogenic progenitors in the PB and spleen were

significantly lower in KO mice than in WT mice, whereas those in the BM were comparable between the WT and KO mice (Fig. 2h, Supplementary Fig. 3). Taken together, these results indicate

that Phc2 deficiency causes a defect in HSPC mobilization from the BM into the periphery. _PHC2_ _−/_− BMSCS CAUSE A DEFECT IN HSPC MOBILIZATION To identify which cell type in the BM niche

contributed to the defect in HSPC release into the circulation of KO mice, we adoptively transferred LSK cells isolated from WT CD45.1 mice into irradiated WT or KO CD45.2 recipient mice

(Fig. 3a). KO recipients showed impaired repopulation of donor-derived immune cells in the thymus and spleen (Fig. 3b, c). However, the absolute numbers of LSK cells and lineage-committed

progenitors (Lin−c-kit+ cells; LK cells) in the BM of KO recipients were comparable to those in the WT recipients (Fig. 3b–d). To confirm these observations, irradiated WT CD45.1 mice were

reconstituted with LSK cells isolated from WT or KO CD45.2 mice (Fig. 3e). The origin of the donor LSK cells did not affect the repopulation of leukocytes in WT recipients (Fig. 3f, g). The

origin of the donor LSK cells did not affect the absolute number of LSK cells or LK cells in the BM of WT recipients either (Fig. 3h). We also performed a serial competitive BM repopulation

assay to further determine whether KO HSPCs had an intrinsic defect. We found that KO HSPCs had the same ability to reconstitute immune cells as WT HSPCs (Supplementary Fig. 4). This

strongly suggests that the defect in HSPC release into the circulation in KO mice is caused by an extrinsic factor from BMSCs within BM niches. To confirm this result, we performed an in

vivo homing assay with CFSE-labeled WT and KO LSK cells. The number of CFSE+ LSK cells homing to the BM was higher among the KO recipients than among the WT recipients (Fig. 4a). Such an

increase in the short-term migration of CFSE+ LSK cells into the BM of KO recipients led to fewer CFSE+ LSK cells homing to the PB and spleen of the KO recipients (Fig. 4a). Accordingly, the

frequency of clonogenic progenitors in the BM of the KO recipients was increased, whereas the frequencies of clonogenic progenitors in the PB and spleen of the KO recipients were decreased

compared to those of the WT recipients (Fig. 4b, Supplementary Fig. 5). We next measured the capacity of WT and KO LSK cells to transmigrate across their BMSCs using a trans-stromal

migration assay. The number of LSK cells transmigrating across KO BMSCs was smaller than the number of LSK cells transmigrating across WT BMSCs (Fig. 4c, Supplementary Fig. 6). In contrast,

the number of LSK cells adhering to KO BMSCs was greater than the number of LSK cells adhering to WT BMSCs (Fig. 4d, Supplementary Fig. 6). Collectively, these data demonstrate that an

extrinsic factor from BMSCs causes HSPCs to adhere more strongly to BMSCs, ultimately leading to the defective mobilization of HSPCs from the BM into the periphery in KO mice. PCG PROTEINS

REPRESS _VCAM1_ GENE EXPRESSION IN BMSCS To understand the molecular mechanisms through which PcG strengthens the adhesion properties of HSPCs to BMSCs, we first investigated the

compositions and architectures of BM niches in WT and KO mice and measured the expression levels of candidate genes that are known to be involved in these processes. Endothelial cells,

mesenchymal stem cells, and osteoblasts are major BMSCs found in BM niches21,22,23. Importantly, neither a change in BMSC composition nor an abnormal architecture was found in the KO BM

niches (Supplementary Figs. 7 and 8). Next, we examined the expression patterns of soluble factors and cell surface molecules that might regulate HSPC mobilization in WT and KO BMSCs2,7

(Fig. 5a, Supplementary Fig. 9). Intriguingly, both the mRNA and protein levels of VCAM-1 were significantly increased in KO BMSCs compared to WT BMSCs (Fig. 5a, b). Flow cytometry analysis

further confirmed that the expression of VCAM-1 was significantly higher in KO BMSCs than in WT BMSCs (Fig. 5c). Consistent with this observation, _Vcam1_ mRNA levels were significantly

elevated in activated KO peritoneal macrophages compared to those of WT (Supplementary Fig. 10). Taken together, these results indicate that Phc2 may repress _Vcam1_ expression in BMSCs. To

further examine Phc2-dependent _Vcam1_ repression, we knocked down Phc2 expression in BMSCs (OP9 cells) and concurrently monitored _Vcam1_ expression levels under treatment with TNF-α, a

well-known inducer of _Vcam1_ transcription24 (Fig. 5d). As expected, TNF-α treatment induced the expression of _Vcam1_ at 3 h post-treatment. However, this was significantly exacerbated

when Phc2 was depleted, and thus, the VCAM-1 level was not dampened up to 12 h post-TNF-α treatment (Fig. 5e, f). Overall, these results suggest that Phc2 represses the expression of _Vcam1_

in BMSCs at the transcriptional level. To demonstrate a role for Phc2 in the transcriptional repression of _Vcam1_, we performed a chromatin immunoprecipitation (ChIP) assay with seven

primer pairs covering the putative Phc2 binding sites located in the _Vcam1_ gene (Fig. 6a). This analysis revealed the strong binding of Phc2 to the promoter region of the _Vcam1_ locus

(Fig. 6b, primers #2 and #3). Interestingly, direct binding by other members of the canonical PRC1 family, such as Bmi1, Ring1b, and Cbx7, was also found around the same region covered by

primers #2 and #3 (Fig. 6c). Consistent with this, Bmi1, Ring1b, and Cbx7 binding to these regions were significantly decreased in the absence of Phc2 (Fig. 6c). The acetylation of histone

H3 at lysine 9/14 (H3K9ac), trimethylation of histone H3 at lysine 4 (H3K4me3), H3K27me3, and H2AK119ub were observed around the same region covered by primers #2 and #3 (Fig. 6c). H3K9ac

and H3K4me3 were increased, whereas H3K27me3 and H2AK119ub were decreased in KO BM cells compared to WT BM cells (Fig. 6c). Furthermore, the treatment of BMSCs (OP9 cells) with GSK126, an

Ezh2 inhibitor25, led to the upregulation of _Vcam1_ expression in a dose-dependent manner (Fig. 6d, e and Supplementary Fig. 11). Consistent with these observations, the binding of Phc2,

Bmi1, and Ring1b to the regions covered by primers #2 and #3 was decreased in GSK126-treated BMSCs (OP9 cells) (Fig. 6f). Next, we then asked whether GSK126 treatment could mimic Phc2

deficiency in vivo. Different dose of GSK126 was administered intraperitoneally into WT mice. Sixteen hours after GSK injection, BMSCs were isolated and analyzed for VCAM-1 expression. We

also measured the capacity of WT LSK cells to transmigrate across their GSK126-treated BMSCs using a trans-stromal migration assay. Results clearly showed that GSK126 treatment increased

both VCAM-1 expression in BMSCs and adherence of LSKs onto BMSCs in a dose-dependent manner (Supplementary Fig. 12). Therefore, the canonical PRC1 containing Phc2 can repress _Vcam1_

expression through histone modifications, such as by enhancing H3K27me3 and H2AK119ub, in BMSCs. ANTI-VCAM-1 AB TREATMENT RESTORES _PHC2_ _−/_− HSPC MOBILIZATION Next, we examined whether

blocking the interaction between VCAM-1 and VLA-4 could restore HSPC mobilization in KO mice. We first attempted to measure the mobilization capacity of WT LSK cells across KO BMSCs after

treatment with neutralizing antibodies (Abs) against VCAM-1 and/or VLA-4. Treatment with either Ab restored the trans-stromal migration activity of LSK cells across KO BMSCs to a level

similar to that of LSK cells across WT BMSCs (Fig. 7a). Additionally, there was no significant difference in the relative ratio of LSK cell adhesion to KO or WT BMSCs after treatment with

the neutralizing Abs (Fig. 7b). The in vivo homing assay further confirmed that blocking the interaction between VCAM-1 and VLA-4 could restore LSK cell mobilization from the KO BM into the

PB and spleen (Fig. 7c, d and Supplementary Fig. 13). We then tested whether treatment of the neutralizing VCAM-1 Ab could rescue steady-state HSPC mobilization in KO mice. Results clearly

demonstrated that the administration of the neutralizing VCAM-1 Ab in KO mice successfully reconstitutes HSPC mobilization from BM into periphery (Fig. 7e–g). We also performed a G-CSF-,

AMD3100-, or 5-FU-induced LSK cell mobilization assay using WT and KO mice treated with the neutralizing VCAM-1 Ab. There were no significant differences in the absolute numbers of WBCs and

splenocytes between the WT and KO mice treated with anti-VCAM-1 on day 5 after the G-CSF treatment and on day 16 following the 5-FU challenge (Fig. 8a, b, f, g). Similarly, the absolute

number of LSK cells and the clonogenic progenitor frequency in the BM, PB, and spleen were comparable between WT and KO recipients treated with the neutralizing Ab on day 5 after the G-CSF

treatment and on day 16 following the 5-FU challenge (Fig. 8c, h, Supplementary Figs. 14 and 15). Consistent with these results, the defective HSPC migration into the PB of the KO mice was

reversed by treating the mice with anti-VCAM-1, as observed in the HSPC mobilization assay using AMD3100 alone or in combination with G-CSF (Fig. 8d, e and Supplementary Fig. 16). To further

reinforce these observations, we assessed the significance of VCAM-1 blockage when the migration defect of HSPCs are driven by extrinsic microenvironment which mimics the phenotype

associated with Phc2 KO mice. First, we adoptively transferred LSK cells isolated from WT CD45.1 mice into irradiated WT or KO CD45.2 recipient mice and then performed the same G-CSF-induced

LSK cell mobilization assay using recipient mice treated with the neutralizing VCAM-1 Ab (Supplementary Fig. 17). Consistent to the HSPC mobilization assay in KO mice, WT cells

reconstituted in irradiated KO recipient cells were only partially rescued the mobilization defects when the single G-CSF agent was treated (Supplementary Fig. 17). However, the

administration of the neutralizing VCAM-1 Ab in combination with G-CSF in KO recipient mice completely reconstituted WT donor HSPC mobilization from BM into periphery, the same degree to WT

recipient mice (Supplementary Fig. 17). Taken together, these results demonstrate that blocking the interaction between VCAM-1 and VLA-4 can restore HSPC migration from the BM into the

periphery of KO mice. DISCUSSION In this study, we demonstrated that Phc2 binds to the _Vcam1_ locus to repress its transcription by enhancing H3K27me3 and H2AK119ub in BMSCs. In the absence

of Phc2, the increased _Vcam1_ expression in BMSCs strengthened the interaction between HSPCs and BMSCs, compromising timely HSPC mobilization from the BM into the periphery, which in turn

led to a systemic immunodeficiency. Consistent with these observations, treatment with a neutralizing Ab against VCAM-1 in Phc2-deficient mice restored HSPC mobilization from the BM into the

periphery. These data firmly establish a causative role for the epigenetic regulation of VCAM-1 in regulating HSPC mobilization, as a key downstream mediator of Phc2 functions. Recent

genetic evidence from conditional VCAM-1- or VLA-4-deficient mice further supports our model of the VLA-4/VCAM-1 pathway as downstream of Phc2 function, and as expected from our results with

Phc2-deficient mice overexpressing VCAM-1, VCAM-1- or VLA-4 deficient mice exhibit increased circulating progenitors and immature B cells, and impaired HSPC homing to BM26,27,28,29,30. The

BM homing defect of HSPCs in these mice became obvious when the mice were treated with 5-FU or G-CSF26,27. However, the Phc2-deficient mice still displayed the migration defect of HSPCs from

the BM into the periphery after treatment with these mobilization agents. The identification of a single key mediator of Phc2 function may have important clinical implications. Similar to

previous observations with conditional VCAM-1- or VLA-4-deficient mice26,27,28,29,30, we found an abnormality of only steady-state HSPC mobilization in Phc2-deficient mice. Additionally,

treatment with the anti-VCAM-1 Ab or anti-VLA-4 Ab in primates or mice does not cause any other physiological change31,32. Combined evidence may suggest a dominant role for VCAM-1 in

steady-state HSPC migration due to the VCAM-1 expression pattern. In steady-state conditions, the BMSC is the only cell type that constitutively expresses VCAM-1 at physiological levels,

although vascular endothelial cells and other immune cell populations express minimally detectable levels of VCAM-133,34,35,36,37,38. Furthermore, with a proof-of-concept experiment, we

successfully demonstrated that blocking VCAM-1 in BM niches critically reverses the HSPC mobilization defect in Phc2-deficient mice. Therefore, along with other recent data39,40,41,42,43,

our results suggest that modulating the interaction between VCAM-1 and VLA-4 in BM niches provides potential strategies to develop HSPC mobilizing agents or anti-leukemia drugs. Therefore,

it will be interesting to observe whether a specific inhibitor that blocks Phc2 function is useful for keeping malignant cells from hematopoietic lineages within the BM. To our knowledge, we

are the first to provide evidence that a systemic immunodeficiency can be caused by a defect in a particular epigenetic regulator that represses the expression of a certain cell adhesion

molecule in BMSCs. Our study also demonstrates the molecular mechanism through which the epigenetic regulation of extrinsic factors can help finely tune the interaction between HSPCs and

BMSCs within BM niches during HSPC mobilization. Although several genes that are involved in hematopoiesis have been identified as targets for epigenetic regulation, the epigenetic effect on

HSPC mobilization has not been elucidated. Notably, the vast majority of previously identified genes play important roles in defining the intrinsic property of HSCs and the development of

leukemia8,44,45. Like deficiencies in other epigenetic regulators, a single PcG gene deficiency in mice mostly causes early embryonic lethality or a limited life span with severe

developmental defects, including an intrinsic defect in HSCs8,46,47. This characteristic hinders the search for additional roles of polycomb proteins in the regulation of immune phenomena

aside from their known functions in HSC differentiation. Similar to other members of the PcG protein family, Phc2 is expressed in various tissues and cells and regulates the transcription of

_Cdkn2a_ (_p16__INK4a_ and _p19__ARF_) and _hox_ genes through direct associations with chromatin8,14. However, Phc2-deficient mice are fertile and exhibit less severe defects associated

with the abnormal axial skeleton and Peyer’s patch14,48. Moreover, the systemic immunodeficiency observed in Phc2-deficient mice is not caused by an intrinsic defect in HSC function, but

instead, the Phc2 deficiency causes a defect in HSPC mobilization by enhancing the expression of VCAM-1 in BMSCs. This may suggest that most of the Phc2 functions in mammals are dispensable

or can be replaced by other components of the canonical PRC1 due to a structural heterogeneity of PRC1 or functional redundancy of PcG proteins. In fact, each of the mammalian PRC2 or PRC1

subunits has several paralogs8,9,10,11,12,13. For example, Phc2 has two paralogs, Phc1 and Phc38,9,10,11,12,13. Previous observation revealed that Phc1 deficiency causes a defect in HSC’s

self-renewal activity49,50. Therefore, Phc1, but not phc2, might be critical for HSC maintenance and function. Consistent with this idea, the expression and epigenetic pattern of _Cdkn2a_

(_p16__INK4a_ and _p19__ARF_) in KO BM cells showed no significant differences compared to those in WT BM cells (Supplementary Fig. 18). Another example of nonredundant function by each

paralog within PRC2 or PRC1 components might be found in Ezh proteins or Cbx proteins, respectively51,52,53,54,55. Among Ezh proteins, Ezh1 is critical for self-renewal activity of HSCs,

whereas Ezh2 is dispensable for proper HSC function51,52. Also, Cbx7 is the only paralog which is necessary for self-renewal activity of HSC among Cbx proteins53,54. In our molecular model,

PRC2 containing Ezh2 with methyltransferase activity initiates H3K27me3 on the _Vcam1_ locus in BMSCs because the Ezh2 inhibitor (GSK126) treatment of BMSCs increases the _Vcam1_ mRNA levels

by negating the recognition of H3K27me3 by the canonical PRC1 complex containing Phc2 on the same locus. The canonical PRC1 then recognizes and binds to H3K27me3 to maintain _Vcam1_ gene

suppression. The recognition of H3K27me3 on the _Vcam1_ locus by the canonical PRC1 is dependent on _Phc2_ expression. Consistently, we observed reductions in the binding of other canonical

PRC1 components to the _Vcam1_ locus when Phc2 was not expressed. Our study also revealed that the H2AK119ub activity of the canonical PRC1 on the _Vcam1_ locus was dependent on _Phc2_

expression, which is consistent with a recent observation that the interaction between Bmi1 and Phc2 is critical for the H2AK119ub activity of the canonical PRC155. In conclusion, the

results of our study demonstrated Phc2-regulated HSPC mobilization through the direct repression of _Vcam1_ gene expression in BMSCs. These findings may offer a novel approach to

manipulating the epigenetic patterns of genes involved in HSPC mobilization, leading to new therapeutic strategies for BM transplantation and leukemia treatment. METHODS EXPERIMENTAL MICE

AND PHENOTYPIC ANALYSIS _Phc2_+/− (_Phc2_ heterozygote) mice with a C57BL/6 background were used in this study14. C57BL/6 and B6 CD45.1 mice were obtained from The Jackson Laboratory. WT and

KO mice were bred from _Phc2_+/− mice. All animals received proper care in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The study

protocol was approved by the Institutional Animal Care and Use Committee of Korea University (protocol numbers: KUIACUC-20110104-3, KUIACUC-20141002-4, and KUIACUC-20160927-3). CELL CULTURE

BMSCs were obtained and maintained from WT and KO mice56. Briefly, bone marrow (BM) cells were isolated from the femurs and tibias of 8-week-old mice and cultured in Dulbecco’s modified

Eagle medium (Welgene LM 001-05) supplemented with 10% fetal bovine serum (FBS; Welgene S 001–01), 10% horse serum (Welgene S 104–01), 2 mM L-glutamine (Welgene LS 002–01), 2 mM sodium

pyruvate (Welgene LS 002–02), and 100 U ml−1 penicillin/streptomycin (Welgene LS 202–02) at 37 °C and 5% CO2. Non-adherent cells were removed by washing with PBS after overnight incubation.

Adherent cells were cultured and fresh medium was added every 2 days. After 4 weeks of culture, cells were harvested by incubation with trypsin/EDTA (Welgene LS 015–01) for 2 min at 37 °C

and plated onto culture dishes. At two weeks after the first passage, cells were harvested and cultured under the same conditions for subsequent passages (passages 2–7). OP9 BMSC line (ATCC

CRL-2749) was maintained in alpha MEM (Welgene LM 008-02) supplemented with 20% FBS and 1% penicillin/streptomycin at 37 °C and 5% CO2. For induction of VCAM-1, OP9 cells were treated with

TNF-α (100 ng ml−1; R&D Systems 410-MT) for the indicated time period. To inhibit Ezh activity, OP9 cells were treated with various concentrations of GSK126, an Ezh2 inhibitor (Xcessbio

Biosciences M60071-2), for 48 h. PB COUNT, FLOW CYTOMETRY ANALYSIS, AND CELL SORTING PB samples from each mouse were collected by retro-orbital bleeding and analyzed using a Coulter LH 780

Hematology Analyzer (Beckman Coulter). For flow cytometry, single cell suspensions were prepared from the BM, PB, thymus, and spleen of each mouse. After removing red blood cells, cells were

stained with MACS buffer at 4 °C in the presence of Fc Block Ab (BD Biosciences 553141, dilution 1:100). Abs purchased from BD Biosciences, Biolegend or Thermo Fisher Scientific were used

to detect the following cell surface markers by flow cytometry: B220 (BD Biosciences 553090, dilution 1:100), CD3ε (Thermo Fisher Scientific 11-0031-63, dilution 1:100), CD4 (BD Biosciences

553729, dilution 1:100), CD8 (BD Biosciences 553030, dilution 1:100), CD11b (Thermo Fisher Scientific 11-0112-41, dilution 1:100), CD11c (BD Biosciences 553802, dilution 1:100), CD16:32

(Thermo Fisher Scientific 14-0161-81, dilution 1:100), CD25 (Thermo Fisher Scientific 17-0251-81, dilution 1:100), CD31 (BD Biosciences 561814, dilution 1:100), CD34 (BD Biosciences 560238,

dilution 1:100), CD44 (BD Biosciences 561861, dilution 1:100), CD45 (BD Biosciences 550539, dilution 1:100), CD48 (Biolegend 103412, dilution 1:100), CD51 (BD Biosciences 551187, dilution

1:100), CD106 (BD Biosciences 553332, dilution 1:100), CD127 (Thermo Fisher Scientific 45-1271-80, dilution 1:100), CD150 (Biolegend 115922, dilution 1:100), c-kit (Thermo Fisher Scientific

17-1171-81, dilution 1:100), F4/80 (Thermo Fisher Scientific MF48004, dilution 1:100), Flt3 (Thermo Fisher Scientific 46-1351-80, dilution 1:100), IgD (BD Biosciences 562022, dilution

1:100), IgM (BD Biosciences 550676, dilution 1:100), NK1.1 (BD Biosciences 557391, dilution 1:100), and Sca-1 (BD Biosciences 558162, dilution 1:100). After washing several times with PBS,

stained cells were resuspended in PBS and analyzed by flow cytometry using a FACSCalibur with CellQuest software (BD Biosciences). Analysis and sorting of HSPCs (Lin–Sca-1+c-kit+ cells; LSK

cells) and early progenitor cells were performed using MACS cell separation system and FACSAria Fusion Cell Sorter (BD Biosciences)57. Depletion of specific lineage cells was achieved with a

lineage-specific Ab cocktail and anti-biotin microbeads (Miltenyi Biotec 130-090-858, dilution 1:5). Gating strategy for LSK cell sorting58 was depicted in Supplementary Fig. 19.

HISTOLOGICAL ANALYSIS AND IMMUNOBLOTTING For histological analysis59, tissue samples were fixed with 10% formalin (Sigma, HT5011) and embedded in paraffin. Then, sections measuring 5 μm were

cut using a Leica CM1800 cryostat (Leica Microsystems), and air dried at room temperature. After dry, slides containing each tissue sample were stained with hematoxylin (Merck 1.05174.0500)

and eosin (Merck 109844) per the manufacturer’s protocol. For immunoblot analysis59, cells were lysed with RIPA lysis buffer containing 50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP-40 (Sigma,

74385), 0.5% sodium deoxycholate (Sigma, D6750), 0.1% SDS (Sigma, L4509) with 200 μg ml−1 of phenylmethylsulfonyl fluoride (Sigma, P7626), phosphatase inhibitor cocktail (Sigma, P0044) and

protease inhibitor cocktail (Millipore, 535140). The cell lysates were then resolved by 12% SDS-polyacrylamide gel electrophoresis, transferred onto Immobilon P membranes (Millipore,

IPVH00010), and immunoblotted with anti-VCAM-1 Ab (Santa Cruz Biotechnology, Inc. sc-8304, dilution 1:1000), anti-H3K27me3 Ab (EMD Millipore 07-449, dilution 1:1000), or anti-histone H3 Ab

(Cell Signaling Technology 9715, dilution 1:1000) coupled with goat anti-rabbit IgG-HRP (Santa Cruz Biotechnology, Inc. sc-2004, dilution 1:2500), and anti-Phc2 Ab (mouse IgG, dilution

1:200)14 or anti-β-actin Ab (Sigma-Aldrich A5441, dilution 1:25,000) coupled with goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, Inc. sc-2005, dilution 1:2500). Immunoreactive bands were

visualized using an ECL solution (Thermo Fisher Scientific 34080). To quantify the relative protein expression, immunoreactive bands were normalized to levels of β-actin. The relative band

intensity of protein expression was quantified using ImageJ software (National Institutes of Health). Uncropped blots can be found in the Source Data. CELL CYCLE ANALYSIS AND MEASUREMENT OF

APOPTOSIS BM-resident LSK cells from 8-week-old mice were isolated and incubated in 70% ethanol overnight at 4 °C. After washing with PBS, cells were stained with propidium iodide

(Sigma-Aldrich P4170) for cell cycle analysis. To measure apoptosis, LSK cells were stained with 7-AAD (BD Biosciences 559925, dilution 1:20) and Annexin V-FITC (BD Biosciences 556547,

dilution 1:20). After washing with PBS, stained cells were analyzed by flow cytometry. CFU ASSAY Mononuclear cells isolated from the BM, PB, and spleen were plated onto Methocult GF M3434

(StemCell Technologies 03444) in 35 mm cell culture dishes. Cells were then incubated at 37 °C and 5% CO2. After 7 days of incubation, type and number of colonies were determined60. IN VIVO

HSPC MOBILIZATION ASSAYS For G-CSF-induced mobilization assays15, recombinant human G-CSF (Amgen Filgrastim Neupogen) was diluted in PBS with 0.1% BSA (GenDEPOT A0100-050). G-CSF or vehicle

was administered by daily subcutaneous injection to 8-week-old mice at a dose of 250 μg kg−1 day−1 for 5 days. For AMD3100-induced mobilization assays17, AMD3100 (Sigma-Aldrich A5602) was

diluted in PBS and administered subcutaneously to 8-week-old mice at 5 mg kg−1. For G-CSF and AMD3100-induced mobilization assays61, G-CSF or vehicle was administered by daily subcutaneous

injection to 8-week-old mice at a dose of 250 μg kg−1 day−1 for 5 days. One h before final G-CSF dose, AMD3100 administered subcutaneously to the same group of mice at 5 mg kg−1. Three h

after the final G-CSF or AMD3100 dose, mice were sacrificed to assess the absolute numbers of nucleated cells in the PB and spleen. The absolute numbers of LSK cells in the BM, PB, and

spleen were determined by flow cytometry and CFU assays. To block the VLA-4 and VCAM-1 interaction, anti-VCAM-1 (BD Biosciences 553330) Ab or the respective isotype control (2 mg kg−1 day−1;

BD Biosciences 553926) was administered intravenously every day. For 5-FU-induced mobilization assays62, 5-FU (Sigma-Aldrich F6627) was administered intravenously to 8-week-old mice at 200 

mg kg−1. The absolute numbers of nucleated cells in the PB and spleen were counted on days 4, 8, 12, and 16 after 5-FU treatment. On day 16 after 5-FU administration, mice were sacrificed,

and the absolute numbers of Lin−Sca-1+c-kit+ cells (LSK cells) in the BM, PB, and spleen were determined by flow cytometry and CFU assays. To block the VLA-4 and VCAM-1 interaction, mice

were injected with anti-VCAM-1 Ab or the respective isotype control (2 mg kg−1 day−1) from day 8 to day 15 after 5-FU administration. For anti-VCAM-1 Ab-induced mobilization assays63,

anti-VCAM-1 Ab or the respective isotype control administered by daily subcutaneous injection to 8-week-old mice at a dose of 2 mg kg−1 day−1 for 3 days. Eight hours after the final Ab dose,

mice were sacrificed, and the absolute numbers of nucleated cells in the PB and spleen were counted. The absolute numbers of LSK cells in the BM, PB, and spleen were determined by CFU

assays described above. LSK TRANSPLANTATION LSK cells (1 × 105) isolated from 8-week-old donor mice were injected intravenously into lethally irradiated (10 Gy) 8-week-old recipient mice.

Twelve weeks after the LSK transfer, recipient mice were sacrificed and analyzed for reconstitution of donor immune cells. Donor cells and recipient cells were discriminated by flow

cytometry using anti-CD45.2 (104)-PerCP-Cy5.5 Ab (BD 552950, dilution 1:100) for congenic strain (CD45.1) discrimination. For serial competitive LSK repopulation assays58, LSK cells (5 × 

104) isolated from WT or KO mice (CD45.2) were mixed with an equal number of LSK cells isolated from competitor mice (WT in CD45.1). The cell mixture was then intravenously injected into

lethally irradiated (10 Gy) 8-week-old recipient mice (WT in CD45.1). Secondary transplantation was performed at 12 weeks after primary engraftment. LSK cells (1 × 105) harvested from

primary transplants were intravenously injected into lethally irradiated (10 Gy) 8-week-old recipient mice (WT in CD45.1). The ratio of CD45.1 to CD45.2 positive cells in the BM, PB, thymus,

and spleen of recipient mice was measured by flow cytometry. IN VIVO HOMING ASSAY For in vivo homing assay64, recipient mice were lethally irradiated (10 Gy) 24 h before LSK

transplantation. Freshly isolated LSK cells from 8-week-old mice were labeled with 1 μM CFSE (Thermo Fisher Scientific C34554) per the manufacturer’s instructions. CFSE-labeled LSK cells (2 

× 105) were then resuspended in PBS and intravenously injected into recipient mice. Sixteen hours after injection, mice were sacrificed, and the frequencies of CFSE+ cells in the BM, PB, and

spleen were determined by flow cytometry. To determine the frequency of clonogenic progenitors, mononuclear cells from the BM, PB, and spleen were subjected to CFU assays as described

above. To block the interaction between VLA-4 and VCAM-1, CFSE-labeled LSK cells were incubated with an anti-VCAM-1 Ab (2 mg kg−1) or an isotype control, and the cells were intravenously

injected into recipients 1 h before transplantation. TRANS-STROMAL MIGRATION AND ADHESION ASSAY For the trans-stromal migration assay65, LSK cells (1 × 105 cells chamber−1) were loaded into

the top chamber of a 24-well transwell plate containing a monolayer of BMSCs. Recombinant mouse CXCL12 (100 ng mL−1; R&D Systems P40224) was loaded into the bottom chamber. The plate was

then incubated at 37 °C in 5% CO2 for 16 h. After incubation, the number of migrating LSK cells in the bottom chamber was calculated. To perform the adhesion assay, CFSE-labeled LSK cells

(5 × 104) were co-cultured with BMSCs (2 × 105) in 24-well cell culture plates. Three h after co-culture, nonadherent LSK cells were removed via three gentle washes with PBS, and adherent

LSK cells were harvested. The frequency of CFSE+ LSK cells was determined by flow cytometry. To block the VLA-4 and VCAM-1 interaction, LSK cells were preincubated with anti-VLA-4 (10 μg 

mL−1; Merck CBL1304) Ab or an isotype control (10 μg ml−1; BD Biosciences 559478) for 30 min, and BMSCs were preincubated with anti-VCAM-1 Ab (10 μg mL−1) or an isotype control for 30 min.

MRNA-SEQ DATA ANALYSIS Total RNAs from WT and KO BM cells were extracted using TRIzol (Thermo Fisher Scientific 15596026) according to the manufacturer’s protocol. Subsequently, 1 μg of

total RNA was used to construct cDNA libraries using TruSeq RNA library kit (Illumina, RS-122-2001) according to the manufacturer’s protocol. The size distribution and quality of cDNA

libraries were monitored by Agilent 2100 Bioanalyzer (Agilent Technologies) and quantitative PCR using Kapa Library Quantification Kit (Kapa Biosystems KK4824). After the quality check, cDNA

libraries were sequenced using an Illumina HiSeq4000 sequencer (Illumina). After removing low quality and adapter sequences, the raw reads were aligned to the reference genome _Mus

musculus_ (mm10) (genome assembly information access, GCF_000001635.20) using HISAT v2.0.566. After aligning reads to the genome, StringTie v1.3.3b was used to assemble transcripts and

estimate their abundance measured in fragments per kilobase of exon per million fragments mapped (FPKM)67,68. False discovery rate (FDR) was controlled by adjusting _p_-value cutoff of 0.05

using Benjamini–Hochberg algorithm69. Subsequently, hierarchical clustering analysis was performed to analyze differentially expressed gene (DEG) set using complete linkage and Euclidean

distance as a measure of similarity. Gene set enrichment analysis including functional annotation and pathway analyses were performed based on Gene Ontology (www.geneontology.org/) and KEGG

pathway (http://www.genome.jp/kegg/pathway.html). An overview of the gene expression data was deposited at NCBI’s Gene Expression Omnibus (GEO). It is accessible through GEO series accession

number of GSE128705. REAL-TIME QUANTITATIVE RT-PCR ANALYSIS Total RNA was purified from BMSCs using Trizol reagent (Thermo Fisher Scientific 15596026) according to the manufacturer’s

instructions; it was then reverse transcribed into cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific K1621). After cDNA synthesis, quantitative PCR (qPCR) was

conducted to measure mRNA levels of mouse _Alcam_, _Angpt1_, _Cdh2_, _Ctsk_, _Cxcl12_, _Fn1_, _Kitl_, _Icam1_, _p16__INK4a_, _p19__ARF_, _Selp_, _Vcam1,_ and _Gapdh_ using a 7500 Real-time

PCR system (Thermo Fisher Scientific). _Gapdh_ was used as an internal control to quantify the levels of other mRNA transcripts. The primer pairs used for qRT-PCR are listed in Supplementary

Table 1. INHIBITION OF _PHC2_ EXPRESSION BY SHRNA Lentiviral plasmids expressing shRNAs directed against several distinct regions of _Phc2_ (SHCLNG-NM_018774) and control shRNA plasmids

(SHC001) were purchased from Sigma-Aldrich. OP9 cells (mouse BMSC line, ATCC CRL-2749) were transfected with each shRNA-containing plasmid using Lipofectamine 2000 transfection reagent

(Thermo Fisher Scientific 11668019) according to the manufacturer’s instructions. After transfection, inhibition of _Phc2_ expression was determined by qRT-PCR and immunoblotting. Primer

sequences for _Phc2_ for qRT-PCR are listed in Supplementary Table 1. CHIP ASSAY The lysates of BM cells (2 × 106) from 8-week-old mice were prepared for the ChIP assay70. BM cell lysates

were sonicated and immunoprecipitated with the anti-Bmi1 (Santa Cruz Biotechnology, Inc. sc-10745, 2 μg sample−1), anti-Cbx7 (Santa Cruz Biotechnology, Inc. sc-70232, 2 μg sample−1),

anti-H2AK119ub (Cell Signaling Technology 8240, 2 μg sample−1), anti-H3K9ac (Merck 06-942, 2 μg sample−1), anti-H3K4me3 (Abcam ab12209, 2 μg sample−1), anti-H3K27me3 (Merck 07-449, 2 μg

sample−1), anti-Phc2 (Santa Cruz Biotechnology, Inc. sc-160664, 2 μg sample−1), and anti-Ring1b (Cell Signaling Technology 5694, 2 μg sample−1) Abs. Additionally, GSK126-treated OP9 cell

lysates were sonicated and immunoprecipitated with the anti-Bmi1, anti-Phc2, and anti-Ring1b Abs. After immunoprecipitation, immune complexes were collected with protein A agarose (Merck

GE17-0963-03) and extracted with an extraction buffer (1% SDS, 0.1 M NaHCO2). DNA cross-links were reversed by heating to 65 °C for 8 h. DNA was extracted with phenol/chloroform and

precipitated with ethanol. DNA isolated from an aliquot of total nuclear extract was used as the loading control for PCR (input control). qPCR was performed as described above. Data are

presented after normalizing each immunoprecipitated DNA Ct value to 10% of the input DNA Ct value. IN VIVO ADMINISTRATION OF GSK126 WT mice were intraperitoneally injected with 50 or 100 mg 

kg−1 of DMSO-dissolved GSK126. Mice were sacrificed at 16 h after GSK126 injection and the expression of VCAM-1 from BMSCs of sacrificed mice were analyzed by immunoblotting and flow

cytometry. In addition, trans-stromal migration and adhesion assays using LSK cells and BMSCs of sacrificed mice were performed as described above. STATISTICAL ANALYSIS Mean values among

more than three experimental groups were compared by one-way ANOVA with Tukey HSD analysis and comparison between two groups was analyzed by two-tailed Student’s _t_-test using SPSS

statistics software, ver. 24.0 (SPSS Inc). All data are presented as the mean ± standard error of mean (SEM). In each graph, significant differences as determined by _p_-values less than

0.05 and 0.01 are indicated by asterisks (* and **, respectively). REPORTING SUMMARY Further information on research design is available in the Nature Research Reporting Summary linked to

this article. DATA AVAILABILITY The raw mRNA-seq data were deposited into the Sequence Read Archive (SRA) of National Center for Biotechnology Information (NCBI) with the following accession

numbers: GSM3683312, GSM3683313, GSM3683314, GSM3683315, GSM3683316, and GSM3683317. An overview of the gene expression data was deposited at NCBI’s Gene Expression Omnibus (GEO). It is

accessible through GEO series accession number of GSE128705. Source data underlying Figs. 1–8, Tables 1–2, Supplementary Figs. 1–7, and Supplementary Figs. 10–18 are provided as a Source

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Amgen Inc. (Thousand Oaks, CA, USA) for providing G-CSF. We also thank Eun-Kyeong Jo (Chungnam National University School of Medicine), Kyung-Mi Lee (Korea University College of Medicine),

Seungkwon You (Korea University School of Life Sciences and Biotechnology), and Seok-Ho Hong (Kangwon National University College of Medicine) for their critical reading of our manuscript or

helpful suggestions. This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science &

ICT (2017M3A9C8060392), and an intramural research program funded by Korea University grant (2016). AUTHOR INFORMATION Author notes * These authors contributed equally: Joonbeom Bae,

Sang-Pil Choi. AUTHORS AND AFFILIATIONS * Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul, 02841, Republic of Korea Joonbeom Bae, Sang-Pil

Choi, Si-Won Park, Chang-Yong Choi, Jihye Han, Sang-Hoon Kim, Han-Hyoung Lee, Kyungmin Park, Young Sik Lee & Taehoon Chun * Laboratory Animal Center, Wakayama Medical University,

Wakayama, 641-8509, Japan Kyoichi Isono * Department of Biomedical Science, CHA University, Seongnam, Gyounggi-do, 13488, Republic of Korea Ji Yoon Lee * Department of Urology, School of

Medicine, University of California, San Francisco, San Francisco, CA, 94158, USA Hyun Yong Jin * Department of Biomedical Laboratory Science, College of Health Science, Cheongju University,

Cheongju-si, 28503, Republic of Korea Suk Jun Lee * Department of Microbiology and Immunology, Cancer Research Institute and Xenotransplantation Research Center, Seoul National University

College of Medicine, Seoul, 03087, Republic of Korea Chung-Gyu Park * Laboratory for Developmental Genetics, RIKEN Center for Integrative Medical Sciences, Yokohama, 230-0045, Japan Haruhiko

Koseki Authors * Joonbeom Bae View author publications You can also search for this author inPubMed Google Scholar * Sang-Pil Choi View author publications You can also search for this

author inPubMed Google Scholar * Kyoichi Isono View author publications You can also search for this author inPubMed Google Scholar * Ji Yoon Lee View author publications You can also search

for this author inPubMed Google Scholar * Si-Won Park View author publications You can also search for this author inPubMed Google Scholar * Chang-Yong Choi View author publications You can

also search for this author inPubMed Google Scholar * Jihye Han View author publications You can also search for this author inPubMed Google Scholar * Sang-Hoon Kim View author publications

You can also search for this author inPubMed Google Scholar * Han-Hyoung Lee View author publications You can also search for this author inPubMed Google Scholar * Kyungmin Park View author

publications You can also search for this author inPubMed Google Scholar * Hyun Yong Jin View author publications You can also search for this author inPubMed Google Scholar * Suk Jun Lee

View author publications You can also search for this author inPubMed Google Scholar * Chung-Gyu Park View author publications You can also search for this author inPubMed Google Scholar *

Haruhiko Koseki View author publications You can also search for this author inPubMed Google Scholar * Young Sik Lee View author publications You can also search for this author inPubMed 

Google Scholar * Taehoon Chun View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS T.C. conceived, designed, and supervised this study. J.B.,

S.-P.C., K.I., J.Y.L., S.-W.P., C.-Y.C., J.H., S.-H.K., H.-H.L., K.P. and S.J.L. performed experiments. J.B., S.-P.C., C.-Y.C., C.-G.P. and T.C. analyzed and interpreted data. J.B., S.-P.C.,

H.Y.J., C.-G.P., H.K., Y.S.L. and T.C. discussed, wrote and edited the manuscript. All authors reviewed and approved the manuscript. CORRESPONDING AUTHOR Correspondence to Taehoon Chun.

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SP., Isono, K. _et al._ Phc2 controls hematopoietic stem and progenitor cell mobilization from bone marrow by repressing _Vcam1_ expression. _Nat Commun_ 10, 3496 (2019).

https://doi.org/10.1038/s41467-019-11386-4 Download citation * Received: 18 April 2018 * Accepted: 12 July 2019 * Published: 02 August 2019 * DOI: https://doi.org/10.1038/s41467-019-11386-4

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