RNAi Screen Identifies MTA1 as an Epigenetic Modifier of Differentiation Commitment in Human HSPCs

•RNAi screen targeting 11,000 genes identified regulators of human HSPCs.•MTA1 was identified as a negative regulator of HSPC propagation in vitro.•Knockdown of MTA1 preserves the immature phenotype of human HSPCs in vitro and restricts their engraftment capacity in vivo.•MTA1 is associated with the NuRD complex in HSPCs and mediates H3K27 deacetylation. The molecular mechanisms regulating key fate decisions of hematopoietic stem cells (HSCs) remain incompletely understood. Here, we targeted global shRNA libraries to primary human hematopoietic stem and progenitor cells (HSPCs) to screen for modifiers of self-renewal and differentiation, and identified metastasis-associated 1 (MTA1) as a negative regulator of human HSPC propagation in vitro. Knockdown of MTA1 by independent shRNAs in primary human cord blood (CB) HSPCs led to a cell expansion during culture and a relative accumulation of immature CD34+CD90+ cells with perturbed in vitro differentiation potential. Transplantation experiments in immunodeficient mice revealed a significant reduction in human chimerism in both blood and bone marrow from HSPCs with knockdown of MTA1, possibly caused by reduced maturation of blood cells. We further found that MTA1 associates with the nucleosome remodeling deacetylase (NuRD) complex in human HSPCs, and on knockdown of MTA1, we observed an increase in H3K27Ac marks coupled with a downregulation of genes linked to differentiation toward the erythroid lineage. Together, our findings identify MTA1 as a novel regulator of human HSPCs in vitro and in vivo with critical functions for differentiation commitment. The molecular mechanisms regulating key fate decisions of hematopoietic stem cells (HSCs) remain incompletely understood. Here, we targeted global shRNA libraries to primary human hematopoietic stem and progenitor cells (HSPCs) to screen for modifiers of self-renewal and differentiation, and identified metastasis-associated 1 (MTA1) as a negative regulator of human HSPC propagation in vitro. Knockdown of MTA1 by independent shRNAs in primary human cord blood (CB) HSPCs led to a cell expansion during culture and a relative accumulation of immature CD34+CD90+ cells with perturbed in vitro differentiation potential. Transplantation experiments in immunodeficient mice revealed a significant reduction in human chimerism in both blood and bone marrow from HSPCs with knockdown of MTA1, possibly caused by reduced maturation of blood cells. We further found that MTA1 associates with the nucleosome remodeling deacetylase (NuRD) complex in human HSPCs, and on knockdown of MTA1, we observed an increase in H3K27Ac marks coupled with a downregulation of genes linked to differentiation toward the erythroid lineage. Together, our findings identify MTA1 as a novel regulator of human HSPCs in vitro and in vivo with critical functions for differentiation commitment. Hematopoietic stem cells (HSCs) are characterized by their capacity to self-renew and differentiate into all blood cell types. HSCs can restore the entire blood system and are used as a cornerstone therapy in life-saving transplantation procedures for leukemias and lymphomas, as well as inherited diseases of the hematopoietic system [1Csaszar E Kirouac DC Yu M et al.Rapid expansion of human hematopoietic stem cells by automated control of inhibitory feedback signaling.Cell Stem Cell. 2012; 10: 218-229Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar,2Wagner JE Brunstein CG Boitano AE et al.Phase I/II trial of StemRegenin-1 expanded umbilical cord blood hematopoietic stem cells supports testing as a stand-alone graft.Cell Stem Cell. 2016; 18: 144-155Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar]. The human hematopoietic system is maintained by a relatively small number of HSCs where the balance between self-renewal and differentiation is tightly regulated to preserve the stem cell pool [3Notta F Doulatov S Laurenti E Poeppl A Jurisica I Dick JE. Isolation of single human hematopoietic stem cells capable of long-term multilineage engraftment.Science. 2011; 333: 218-221Crossref PubMed Scopus (633) Google Scholar]. Recent studies have highlighted the importance of the epigenetic landscape and the chromatin state of HSCs in controlling cell identity, lineage priming, and fate decisions [4Farlik M Halbritter F Muller F et al.DNA Methylation dynamics of human hematopoietic stem cell differentiation.Cell Stem Cell. 2016; 19: 808-822Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar,5Buenrostro JD Corces MR Lareau CA et al.Integrated single-cell analysis maps the continuous regulatory landscape of human hematopoietic differentiation.Cell. 2018; 173 (e1516): 1535-1548Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar]. Yet, the more precise details of the molecular programs governing the first critical commitment steps of HSCs have remained incompletely defined. We have previously reported the feasibility of using RNAi screens to identify regulators of renewal and differentiation in primary human HSCs [6Galeev R Baudet A Kumar P et al.Genome-wide RNAi screen identifies cohesin genes as modifiers of renewal and differentiation in human HSCs.Cell Rep. 2016; 14: 2988-3000Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. Using screening paradigms based on pooled lentiviral shRNA libraries targeted to cord blood (CB)-derived hematopoietic stem and progenitor cells (HSPCs), we have successfully identified genes with key roles in both normal and malignant hematopoiesis, including MAPK14 [7Baudet A Karlsson C Safaee Talkhoncheh M Galeev R Magnusson M Larsson J RNAi screen identifies MAPK14 as a druggable suppressor of human hematopoietic stem cell expansion.Blood. 2012; 119: 6255-6258Crossref PubMed Scopus (35) Google Scholar], JARID2 [8Kinkel SA Galeev R Flensburg C et al.Jarid2 regulates hematopoietic stem cell function by acting with polycomb repressive complex 2.Blood. 2015; 125: 1890-1900Crossref PubMed Scopus (36) Google Scholar], and members of the cohesin complex [6Galeev R Baudet A Kumar P et al.Genome-wide RNAi screen identifies cohesin genes as modifiers of renewal and differentiation in human HSCs.Cell Rep. 2016; 14: 2988-3000Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. In this study, we further built on this approach by employing next-generation short hairpin (sh)RNA libraries from The Broad Institute RNAi Consortium (TRC). Using selection assays for enhanced maintenance of the HSPC phenotype in vitro, we identified metastasis-associated 1 (MTA1) as the top-scoring candidate gene in the screen and a potent modifier of human HSPC differentiation. Knockdown (KD) of MTA1 perturbed differentiation of HSPCs, leading to an accumulation of immature cells in vitro and a profound engraftment defect in xenograft transplantation assays. Moreover, we found that MTA1 associates with the nucleosome remodeling deacetylase (NuRD) complex in human HSPCs and regulates the deacetylation of histone H3 lysine 27 (H3K27). Human CB samples were obtained from maternity wards in Helsingborg General Hospital and Skåne University Hospital in Lund and Malmö, Sweden. Samples were collected following regulations set by the regional ethics committee, which includes a written consent. CB samples were processed within 24 hours by isolating mononuclear cells using the density-gradient method (Abbott, Chicago, IL USA). CD34+ cells were enriched using magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's protocol. The shRNA screen was performed according to a previously published protocol [9Galeev R Karlsson C Baudet A Larsson J. Forward RNAi screens in human hematopoietic stem cells.Methods Mol Biol. 2017; 1622: 29-50Crossref PubMed Scopus (3) Google Scholar], using TRC (The RNAi Consortium) shRNA lentivirus libraries TRC 1.5 and TRC 2.0 (Nos. SHPH15 and SHPH2, Merck/Sigma-Aldrich, St. Louis, MO). In short, 70 million CB-derived CD34+ cells collected from more than 100 umbilical cord units were divided through seven biological replicates. Cells were transduced with six shRNA library pools (from TRC1.5 and TRC 2.0 libraries), each consisting of around 7,000 shRNAs. With given library titers, we aimed at around 20%–30% transduction efficacy and coverage of the library between 200 and 300 CD34+ HSPCs per hairpin. As selection pressure, growth advantage under standard culture conditions was used to identify genes that affect self-renewal and differentiation in primary HSPCs. Therefore, cells were maintained in serum-free expansion medium (SFEM, Stem Cell Technologies, Vancouver, BC, Canada), supplemented with stem cell factor (SCF), thrombopoietin (TPO), and FLT3-ligand (FLT3L) (each at 100 ng/mL, Peprotech/Thermo Fisher Scientific, Waltham, MA). To evaluate the initial library distribution, cell culture samples were collected 3 days after transduction. Then, other samples were collected at week 3 followed by purification of CD34+ cells and gDNA extraction. Sample collection and processing for next-generation sequencing (NGS) analysis was done according to a previously published protocol [9Galeev R Karlsson C Baudet A Larsson J. Forward RNAi screens in human hematopoietic stem cells.Methods Mol Biol. 2017; 1622: 29-50Crossref PubMed Scopus (3) Google Scholar]. Reads from NGS were annotated to the shRNA library. The median counts for each technical replicate for a given pool were calculated. Next, by following the edgeR guidelines, after converting the raw counts to counts per million by using the cpm function in edgeR, we retained only those hairpins that are represented as at least 1-cpm reads in at least five replicates [10Robinson MD McCarthy DJ Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.Bioinformatics. 2010; 26: 139-140Crossref PubMed Scopus (23339) Google Scholar]. The data then were normalized by the trimmed mean of M-values (TMM) method. After estimating the dispersion parameters, the glmFit and glmLRT functions were used to identify subsets of differentially represented hairpins between day 3 and week 3. The described analysis was performed for each pool independently, and the results were pooled. The hairpins were ordered by their respective log2 fold change median estimates. To visualize the shRNA ranking, the log2 fold change values of the raw counts were used. For gene ranking, the log2 fold change values of the hairpins for each given gene were aggregated by taking their median value, along with a statistical test to decide if the majority of the log2 fold change values were larger than zero for that gene, and reporting the two metrics together. A one-sided Fisher's exact test was performed on the hairpins of the same gene to decide if their log2 fold change value was greater than zero. p Values and false discovery rates (FDRs) were reported along with the median log2 fold change values. CB-Derived human CD34+ cells were cultured in tissue-treated plates. Cells were maintained in SFEM supplemented with SCF, TPO, and FLT3L at 100 ng/mL (Miltenyi Biotec, Bergisch Gladbach, Germany). Penicillin (100 U/mL) and streptomycin (100 μg/mL) were added to the cultures to prevent infections (Cytiva, Marlborough, MA USA). pLKO1 vectors were used to clone a pLKO1_GFP backbone, where the puromycin-resistance gene was to MTA1 shRNA as well as control were into pLKO1_GFP is a shRNA by were in the human cell according to a previously published protocol of to 1 cells by expression of Scopus (3) Google Scholar], where was used for and was using and CD34+ cells were transduced at a of of to transduction cells a to the same of transduction as CD34+ an of was used for cells. CB-Derived CD34+ cells were at and by medium supplemented with Scientific, Waltham, and 100 I Cell more than from the The cells were at for and in with and then with and on cell was performed on a USA). Cells were into SFEM with used for cell cells were and to culture plates. cells were by and the cells in with and with if and then and in with and for cell Cells were and in with and with at for and then cells were in with and for cell analysis was performed on USA). cells were after the transduction into and on at was performed using an Germany) according to the manufacturer's Scientific, Waltham, was used for expression was used to according to the manufacturer's protocol. of gene expression and knockdown were with for MTA1 and normalized to Cells for were at day 3 after cells were into Stem Cell with medium supplemented with ng/mL SCF, ng/mL factor ng/mL and (100 (100 μg/mL) cells per well were to and under standard conditions for and then hematopoietic were CB-Derived CD34+ cells were transduced with After 3 days the cells were and to differentiation Cell supplemented with ng/mL SCF, ng/mL ng/mL ng/mL TPO, ng/mL 100 and 100 streptomycin cell analysis was performed at days and after to differentiation CB-Derived human CD34+ cells were for CD34+ cell to mice were used for of CD34+ cells. cells were for expression after 3 days and through the into blood samples were collected from and at the were after and bone marrow and samples were collected for analysis by maintenance and procedures followed the regional ethics and CB-derived cells were transduced and cultured for were into was using the The was used for the of from followed by and according to the of and was to Human The system was used for and were using an system by the expression were from using the F et and of PubMed Scopus Google in the For value the log2 gene was used. cells were days after transduction with shRNA Cells were collected for cultured days to more cells and then collected for cells were with and on for in supplemented with 1 and The was for at at and was Sample supplemented with and 1 and was added to the at a Samples were at for and then at on were using according to the manufacturer's protocol The system was used to the to a according to the manufacturer's protocol The was in 1 and in for 1 at were at with primary at in were for each with 1 with and in were added to the at a for 1 of at was with 1 and were by according to the manufacturer's protocol and CD34+ cord blood cells were enriched for CD34+ by at day 3. cells were with cells were using supplemented with and were with MTA1 MA for as an Cell was using beads and then with beads was and beads were for using Samples were by the beads in for at Samples were on for at The was with for 1 was with for The were and at for further samples and were on were performed on a at A of and other is in the analysis was done using A test was used to of were with analysis of with a p value were significant values represented in data in this have in the under In our we paradigms for regulators of using pooled lentiviral shRNA screens in primary human HSPCs. To we TRC libraries 1.5 and The lentiviral libraries used in this of a of shRNAs targeting around 11,000 both shRNA genes represented in the of the library and shRNAs targeting around genes that were in TRC We transduced a of 70 million CB-derived CD34+ cells with the shRNA and used the of HSPCs during culture as a for selection of maintenance expansion of the immature The of shRNA was by NGS 3 days after as well as in CD34+ cells following 3 of selection as previously described [9Galeev R Karlsson C Baudet A Larsson J. Forward RNAi screens in human hematopoietic stem cells.Methods Mol Biol. 2017; 1622: 29-50Crossref PubMed Scopus (3) Google Scholar]. After the relative of shRNAs was with day 3 to their to CD34+ cells during the culture analysis on the hairpins that were enriched in the CD34+ cell at week as a selection protocol was and used in our studies [6Galeev R Baudet A Kumar P et al.Genome-wide RNAi screen identifies cohesin genes as modifiers of renewal and differentiation in human HSCs.Cell Rep. 2016; 14: 2988-3000Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar]. the enriched shRNAs after selection we found targeting genes identified in our screens as and hairpins targeting genes as of the gene in and hematopoietic stem cell were which the of the screen To identify the gene we only genes that were represented by at least hairpins in the and were based on the median log2 fold for the shRNAs the candidate we to on MTA1 in this as the and previously in the of human of five shRNAs for MTA1 in the screen and we first the two top-scoring shRNAs by into a lentiviral to knockdown of MTA1 and the screen We MTA1 expression subsets of cord blood HSPCs and found that the two shRNAs knockdown at both the and We transduced CB-derived CD34+ cells with the shRNA vectors and cultured for 3 as well as and MTA1 we observed an increase in the of the immature CD34+CD90+ with the shRNA as well as an cell in culture knockdown of MTA1 led to of HSPCs 3 of culture our findings MTA1 as a novel regulator of human HSPCs. To the of MTA1 in more we transduced enriched cells with the shRNA vectors and expression of along with and is to HSCs during culture I J et expression marks CD34+ cord blood stem 2017; PubMed Scopus Google A Talkhoncheh Magnusson M Larsson J. C expression marks human hematopoietic stem PubMed Scopus Google Scholar]. We observed a increase in both and of and the cells following the knockdown of MTA1 reduced of MTA1 preserve the immature phenotype of cultured HSPCs, a for MTA1 in regulating the balance between renewal and differentiation in vitro. To further the differentiation of HSPCs, we cultured CB-derived CD34+ cells in the of TPO, we an increase in CD34+ cells reduced of cells the for both MTA1 as well as reduced of cells for perturbed differentiation toward both the and erythroid To cell maturation in vitro, we further performed assays and found that CD34+ cells with of the culture 3 days after transduction following 1 week of in vitro culture perturbed differentiation and a reduced capacity to blood cells. In reduced and a of erythroid Together, findings that MTA1 differentiation of human HSPCs during To further the of HSPCs and the of MTA1 in HSPC in we CD34+ cells to mice using two In the first we transduced cells for expression between the transduced and We found that the and in the of was reduced in the MTA1 and transduced The relative for cells from to at in to the which In the transplantation experiments in immunodeficient mice revealed a significant reduction in human blood cell chimerism on of MTA1 in HSPCs. To this in a we cells to In to the cells were based on expression transplantation to from cells The analysis of blood and bone marrow after revealed reduced engraftment of HSPCs, where control cells to chimerism in both a in hematopoietic in cells. our results that MTA1 is for human and differentiation in The molecular function of MTA1 to with the NuRD complex as an epigenetic modifier A et into the of the NuRD of the Full Text Full Text PDF PubMed Scopus Google Kumar R A chromatin remodeling factor global chromatin through of nucleosome Cell. Full Text Full Text PDF PubMed Scopus Google Scholar]. The complex both chromatin remodeling and histone deacetylase and further of that and A et of the NuRD complex into with 2016; PubMed Scopus Google et regulates in and a PubMed Google The NuRD complex can in to a of with and roles and cell M et and two with and Cell Biol. 26: PubMed Scopus Google Scholar]. Next, we the MTA1 to the between MTA1 and other NuRD complex members in human HSPCs. MTA1 was from CB-derived CD34+ followed by In we identified NuRD complex members were identified in the and the associated data the of the complex in CD34+ cells and further that the of MTA1 in human HSPCs is in with the NuRD The deacetylase of the NuRD complex is to H3K27 marks M et deacetylation of H3K27 of to gene J. 2012; PubMed Scopus Google Scholar], and we if this on MTA1 in HSPCs. we observed a global accumulation of H3K27 marks in the MTA1 CD34+ cells in with perturbed of the NuRD H3K27 marks associated with enhanced P DC C S the expansion of cord blood stem PubMed Scopus Google Scholar]. To further into the molecular mechanisms the differentiation of MTA1 HSPCs, we to the of HSPCs. We cells from human CB samples and transduced with MTA1 targeting shRNAs the After the cells were for global gene expression The expression for of the regulated genes was perturbed than Moreover, gene set analysis with the gene genes in both shRNA we identified a set of erythroid lineage genes the genes we were to identify significant in the of MTA1 profound on gene and is that more in gene expression linked to differentiation programs to the HSPC Here, we on an RNAi screen on our previously paradigms for selection of modifiers of renewal and differentiation in primary human HSPCs. the screen identified both enriched and shRNAs from the selection we on candidate genes from the enriched of shRNAs. are more to from Moreover, screens library which is to with primary cells. A shRNA screens is that the of knockdown between shRNAs and that the screening libraries of shRNAs all genes to in the screens in of primary cells as CD34+ cells is to of all shRNAs in the libraries in the cells. we used of primary CD34+ cells for the we that all shRNAs of HSPCs in all replicates to in the assays. our screens as for discovery of novel regulators in primary human HSPCs and can that as screens in of all genes in the of the genes have previously in HSPC and of to on other candidate genes as As of the as and have previously as of cell growth and et by the through targeting the 2016; PubMed Scopus Google J J of by the a that and Full Text Full Text PDF PubMed Scopus Google and of in 2017; 14: PubMed Scopus Google et factor and the growth of 2018; PubMed Scopus Google Scholar]. to those of our study, the findings from this screen can as a of HSPC regulating and we are an for to the raw data with is in the In this study, we to on the gene, MTA1, and we found that associates with the NuRD complex in human HSPCs. MTA1 linked to a in hematopoiesis, the NuRD complex reported to have a regulatory in both stem cells and HSCs by stem cell maintenance and differentiation I J et of the chromatin in hematopoietic stem cell self-renewal and multilineage PubMed Scopus Google M S et nucleosome and deacetylation complex during lineage J. PubMed Scopus Google Scholar]. findings are in with a for in stem cell maintenance and differentiation in human HSPCs as and we observed a profound accumulation of HSPCs on MTA1 cells to enriched for Yet, we that HSCs were as continuous MTA1 in vivo The reduced engraftment to an differentiation capacity of the HSPCs, other as reduced as system to this in more where MTA1 perturbed in HSPCs, followed by assays with we observed a global accumulation of H3K27 marks in cells. that of MTA1 in HSCs prevent of of regulators by H3K27 possibly cells in an immature state and HSPCs were in H3K27Ac marks were reported to as stem cell and the cells J S et preserves epigenetic marks that are reduced in vivo culture of human HSCs of the Stem Cell. Full Text Full Text PDF PubMed Scopus Google J S et a human PubMed Scopus Google Scholar]. of histone deacetylation using successfully used to maintenance and expansion of human HSPCs in culture E M A C and of the of hematopoietic stem cells cultured vivo with a histone deacetylase Cell PubMed Scopus Google Scholar]. previously reported that deacetylation of H3K27 of complex to gene which an in and differentiation M et deacetylation of H3K27 of to gene J. 2012; PubMed Scopus Google Scholar]. the NuRD complex is as a studies have that can gene expression S M et nucleosome remodeling and deacetylation complex chromatin at of to gene Cell. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar]. we observed only gene expression from MTA1 knockdown in HSPCs, the profound from a set of erythroid genes that were we of gene is that the H3K27Ac marks HSPC with only on genes that the molecular mechanisms in HSPCs further to MTA1 in the of malignant are on in MTA1 associated with that MTA1 have a suppressor in based on our the other reported that MTA1 is in and to functions AE et a of PubMed Scopus Google J M et through metastasis-associated 1 2011; PubMed Scopus Google Scholar]. functions as the NuRD complex linked to a of as and cell J et complex to in by PubMed Scopus Google Scholar]. our that the complex is a key regulator of human HSPC with for the of both normal and malignant The We and for their was by from the the the and the under the and to The was further by the and programs at Lund We for the and for and with with with with with with

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