Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells.

Pseudouridylation (Ψ) is the most abundant and widespread type of RNA epigenetic modification in living organisms; however, the biological role of Ψ remains poorly understood. Here, we show that a Ψ-driven posttranscriptional program steers translation control to impact stem cell commitment during early embryogenesis. Mechanistically, the Ψ "writer" PUS7 modifies and activates a novel network of tRNA-derived small fragments (tRFs) targeting the translation initiation complex. PUS7 inactivation in embryonic stem cells impairs tRF-mediated translation regulation, leading to increased protein biosynthesis and defective germ layer specification. Remarkably, dysregulation of this posttranscriptional regulatory circuitry impairs hematopoietic stem cell commitment and is common to aggressive subtypes of human myelodysplastic syndromes. Our findings unveil a critical function of Ψ in directing translation control in stem cells with important implications for development and disease.

Origin of monocytes and macrophages in a committed progenitor.

Monocytes, macrophages and dendritic cells (DCs) are developmentally related regulators of the immune system that share the monocyte-macrophage DC progenitor (MDP) as a common precursor. Unlike differentiation into DCs, the distal pathways for differentiation into monocytes and monocyte-derived macrophages are not fully elucidated. We have now demonstrated the existence of a clonogenic, monocyte- and macrophage-restricted progenitor cell derived from the MDP. This progenitor was a Ly6C(+) proliferating cell present in the bone marrow and spleen that generated the major monocyte subsets and macrophages, but not DCs or neutrophils. By in-depth quantitative proteomics, we characterized changes in the proteome during monocyte differentiation, which provided insight into the molecular principles of developing monocytes, such as their functional maturation. Thus, we found that monocytes and macrophages were renewed independently of DCs from a committed progenitor.

The stem cell-supporting small molecule UM171 triggers Cul3-KBTBD4-mediated degradation of ELM2 domain-harboring proteins.

To chemically modulate the ubiquitin-proteasome system for the degradation of specific target proteins is currently emerging as an alternative therapeutic modality. Earlier, we discovered such properties of the stem cell-supporting small molecule UM171 and identified that members of the CoREST complex (RCOR1 and LSD1) are targeted for degradation. UM171 supports the in vitro propagation of hematopoietic stem cells by transiently perturbing the differentiation-promoting effects of CoREST. Here, we employed global proteomics to map the UM171-targeted proteome and identified the additional target proteins, namely RCOR3, RREB1, ZNF217, and MIER2. Further, we discovered that critical elements recognized by Cul3KBTBD4 ligase in the presence of UM171 are located within the EGL-27 and MTA1 homology 2 (ELM2) domain of the substrate proteins. Subsequent experiments identified conserved amino acid sites in the N-terminus of the ELM2 domain that are essential for UM171-mediated degradation. Overall, our findings provide a detailed account on the ELM2 degrome targeted by UM171 and identify critical sites required for UM171-mediated degradation of specific substrates. Given the target profile, our results are highly relevant in a clinical context and point towards new therapeutic applications for UM171.

Epigenetic dissection of human blood group genes reveals regulatory elements and detailed characteristics of KEL and four other loci.

BACKGROUND: Blood typing is essential for safe transfusions and is performed serologically or genetically. Genotyping predominantly focuses on coding regions, but non-coding variants may affect gene regulation, as demonstrated in the ABO, FY and XG systems. To uncover regulatory loci, we expanded a recently developed bioinformatics pipeline for discovery of non-coding variants by including additional epigenetic datasets. METHODS: Multiple datasets including ChIP-seq with erythroid transcription factors (TFs), histone modifications (H3K27ac, H3K4me1), and chromatin accessibility (ATAC-seq) were analyzed. Candidate regulatory regions were investigated for activity (luciferase assays) and TF binding (electrophoretic mobility shift assay, EMSA, and mass spectrometry, MS). RESULTS: In total, 814 potential regulatory sites in 47 blood-group-related genes were identified where one or more erythroid TFs bound. Enhancer candidates in CR1, EMP3, ABCB6, and ABCC4 indicated by ATAC-seq, histone markers, and co-occupancy of 4 TFs (GATA1/KLF1/RUNX1/NFE2) were investigated but only CR1 and ABCC4 showed increased transcription. Co-occupancy of GATA1 and KLF1 was observed in the KEL promoter, previously reported to contain GATA1 and Sp1 sites. TF binding energy scores decreased when three naturally occurring variants were introduced into GATA1 and KLF1 motifs. Two of three GATA1 sites and the KLF1 site were confirmed functionally. EMSA and MS demonstrated increased GATA1 and KLF1 binding to the wild-type compared to variant motifs. DISCUSSION: This combined bioinformatics and experimental approach revealed multiple candidate regulatory regions and predicted TF co-occupancy sites. The KEL promoter was characterized in detail, indicating that two adjacent GATA1 and KLF1 motifs are most crucial for transcription.

Author Correction: BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

In Extended Data Fig. 1a of this Letter, the flow cytometry plot depicting the surface phenotype of AML sample DD08 was a duplicate of the plot for AML sample DD06. Supplementary Data 4 has been added to the Supplementary Information of the original Letter to clarify the proteome data acquisition and presentation. The original Letter has been corrected online.

BCAT1 restricts αKG levels in AML stem cells leading to IDHmut-like DNA hypermethylation.

The branched-chain amino acid (BCAA) pathway and high levels of BCAA transaminase 1 (BCAT1) have recently been associated with aggressiveness in several cancer entities. However, the mechanistic role of BCAT1 in this process remains largely uncertain. Here, by performing high-resolution proteomic analysis of human acute myeloid leukaemia (AML) stem-cell and non-stem-cell populations, we find the BCAA pathway enriched and BCAT1 protein and transcripts overexpressed in leukaemia stem cells. We show that BCAT1, which transfers α-amino groups from BCAAs to α-ketoglutarate (αKG), is a critical regulator of intracellular αKG homeostasis. Further to its role in the tricarboxylic acid cycle, αKG is an essential cofactor for αKG-dependent dioxygenases such as Egl-9 family hypoxia inducible factor 1 (EGLN1) and the ten-eleven translocation (TET) family of DNA demethylases. Knockdown of BCAT1 in leukaemia cells caused accumulation of αKG, leading to EGLN1-mediated HIF1α protein degradation. This resulted in a growth and survival defect and abrogated leukaemia-initiating potential. By contrast, overexpression of BCAT1 in leukaemia cells decreased intracellular αKG levels and caused DNA hypermethylation through altered TET activity. AML with high levels of BCAT1 (BCAT1high) displayed a DNA hypermethylation phenotype similar to cases carrying a mutant isocitrate dehydrogenase (IDHmut), in which TET2 is inhibited by the oncometabolite 2-hydroxyglutarate. High levels of BCAT1 strongly correlate with shorter overall survival in IDHWTTET2WT, but not IDHmut or TET2mut AML. Gene sets characteristic for IDHmut AML were enriched in samples from patients with an IDHWTTET2WTBCAT1high status. BCAT1high AML showed robust enrichment for leukaemia stem-cell signatures, and paired sample analysis showed a significant increase in BCAT1 levels upon disease relapse. In summary, by limiting intracellular αKG, BCAT1 links BCAA catabolism to HIF1α stability and regulation of the epigenomic landscape, mimicking the effects of IDH mutations. Our results suggest the BCAA-BCAT1-αKG pathway as a therapeutic target to compromise leukaemia stem-cell function in patients with IDHWTTET2WT AML.

Quantitative proteomics reveals specific metabolic features of acute myeloid leukemia stem cells.

Acute myeloid leukemia is characterized by the accumulation of clonal myeloid blast cells unable to differentiate into mature leukocytes. Chemotherapy induces remission in the majority of patients, but relapse rates are high and lead to poor clinical outcomes. Because this is primarily caused by chemotherapy-resistant leukemic stem cells (LSCs), it is essential to eradicate LSCs to improve patient survival. LSCs have predominantly been studied at the transcript level, thus information about posttranscriptionally regulated genes and associated networks is lacking. Here, we extend our previous report on LSC proteomes to healthy age-matched hematopoietic stem and progenitor cells (HSPCs) and correlate the proteomes to the corresponding transcriptomes. By comparing LSCs to leukemic blasts and healthy HSPCs, we validate candidate LSC markers and highlight novel and potentially targetable proteins that are absent or only lowly expressed in HSPCs. In addition, our data provide strong evidence that LSCs harbor a characteristic energy metabolism, adhesion molecule composition, as well as RNA-processing properties. Furthermore, correlating proteome and transcript data of the same individual samples highlights the strength of proteome analyses, which are particularly potent in detecting alterations in metabolic pathways. In summary, our study provides a comprehensive proteomic and transcriptomic characterization of functionally validated LSCs, blasts, and healthy HSPCs, representing a valuable resource helping to design LSC-directed therapies.

Early growth response 1 regulates hematopoietic support and proliferation in human primary bone marrow stromal cells.

Human bone marrow stromal cells (BMSC) are key elements of the hematopoietic environment and they play a central role in bone and bone marrow physiology. However, how key stromal cell functions are regulated is largely unknown. We analyzed the role of the immediate early response transcription factor EGR1 as key stromal cell regulator and found that EGR1 was highly expressed in prospectively-isolated primary BMSC, down-regulated upon culture, and low in non-colony-forming CD45neg stromal cells. Furthermore, EGR1 expression was lower in proliferative regenerating adult and fetal primary cells compared to adult steady-state BMSC. Overexpression of EGR1 in stromal cells induced potent hematopoietic stroma support as indicated by an increased production of transplantable CD34+CD90+ hematopoietic stem cells in expansion co-cultures. The improvement in bone marrow stroma support function was mediated by increased expression of hematopoietic supporting genes, such as VCAM1 and CCL28 Furthermore, EGR1 overexpression markedly decreased stromal cell proliferation whereas EGR1 knockdown caused the opposite effects. These findings thus show that EGR1 is a key stromal transcription factor with a dual role in regulating proliferation and hematopoietic stroma support function that is controlling a genetic program to co-ordinate the specific functions of BMSC in their different biological contexts.

Comparative Secretome Analyses of Primary Murine White and Brown Adipocytes Reveal Novel Adipokines.

The adipose organ, including white and brown adipose tissues, is an important player in systemic energy homeostasis, storing excess energy in form of lipids while releasing energy upon various energy demands. Recent studies have demonstrated that white and brown adipocytes also function as endocrine cells and regulate systemic metabolism by secreting factors that act locally and systemically. However, a comparative proteomic analysis of secreted factors from white and brown adipocytes and their responsiveness to adrenergic stimulation has not been reported yet. Therefore, we studied and compared the secretome of white and brown adipocytes, with and without norepinephrine (NE) stimulation. Our results reveal that carbohydrate-metabolism-regulating proteins are preferably secreted from white adipocytes, while brown adipocytes predominantly secrete a large variety of proteins. Upon NE stimulation, an increased secretion of known adipokines is favored by white adipocytes while brown adipocytes secreted higher amounts of novel adipokines. Furthermore, the secretory response between NE-stimulated and basal state was multifaceted addressing lipid and glucose metabolism, adipogenesis, and antioxidative reactions. Intriguingly, NE stimulation drastically changed the secretome in brown adipocytes. In conclusion, our study provides a comprehensive catalogue of novel adipokine candidates secreted from white and brown adipocytes with many of them responsive to NE. Given the beneficial effects of brown adipose tissue activation on its endocrine function and systemic metabolism, this study provides an archive of novel batokine candidates and biomarkers for activated brown adipose tissue.

Treatment With Bupropion and Varenicline for Smoking Cessation and the Risk of Acute Cardiovascular Events and Injuries: a Swedish Case-Crossover Study.

Introduction: Bupropion and varenicline are non-nicotine medications used for smoking cessation that mitigate craving and withdrawal symptoms. We aim to investigate whether these drugs increase the risk of selected acute adverse outcomes when used in medical practice. Methods: Population-based case-crossover design using data from Swedish health and administrative registers. Adult individuals diagnosed with acute myocardial infarction, stroke, suicide, suicide attempt, fall injury, or that suffered a road traffic crash from 01.10.2006 for bupropion, or from 01.03.2008 for varenicline, until 31.12.2013 were included. Different lengths of exposure periods were analyzed within the 12-week hazard period prior to the adverse outcome (1-14, 15-28, and 29-84 days). The control period was matched using the interval preceding the hazard period (85-168 days), and breaking it up into equivalent periods (85-98, 99-112, and 113-168 days). Conditional logistic regression with each case considered as one stratum was used to estimate adjusted odds ratios (OR) and confidence intervals (CI). Results: Neither medication was associated with consistent higher risks for any of the adverse outcomes. For bupropion and varenicline, respectively, in the 1-14 days hazard period, OR (95% CI) were: myocardial infarction 1.14 (0.55 to 2.34) and 1.06 (0.70 to 1.62); stroke 1.16 (0.39 to 3.47) and 1.26 (0.72 to 2.17), and traffic crashes 0.85 (0.39 to 1.85) and 1.48 (0.90 to 2.41). In the other periods, ORs were similar or even lower. For falls and suicidal events ORs were generally below one for both drugs. Conclusion: The available evidence suggests that if prescription guidelines are properly followed regarding potential contraindications both of these medications could be considered relatively safe. Implications: The reliable exposure and diagnosis assessment used in this nationwide register-based study, along with the number of cases gathered makes this sample one of the largest of its type to assess potential side effects associated with the use of these drugs. Neither medication was associated with consistent higher risks for any of the adverse outcomes studied.

Transcription Factor 7 Limits Regulatory T Cell Generation in the Thymus.

Regulatory T cells (Tregs) differentiate in the thymus, but the mechanisms that control this process are not fully understood. We generated a comprehensive quantitative and differential proteome of murine Tregs and conventional T cells. We identified 5225 proteins, 164 of which were differentially expressed in Tregs. Together with the comparative analysis of proteome and gene expression data, we identified TCF7 as a promising candidate. Genetic elimination of transcription factor 7 (TCF7) led to increased fractions of Tregs in the thymus. Reduced levels of TCF7, found in the heterozygote, resulted in a greater potential for Treg precursors to differentiate into the Treg lineage. In contrast, activation of TCF7 through ß-catenin had the opposite effect. TCF7 levels influenced the required TCR signaling strength of Treg precursors, and TCF7 deficiency broadened the repertoire and allowed lower TCR affinities to be recruited into the Treg lineage. FOXP3 was able to repress TCF7 protein expression. In summary, we propose a regulatory role for TCF7 in limiting access to the Treg lineage.

Use of Scandinavian moist smokeless tobacco (snus) and the risk of atrial fibrillation.

BACKGROUND: Snus is a smokeless tobacco product, widely used among Swedish men and increasingly so elsewhere. There is debate as to whether snus is an acceptable "harm-reduction" tobacco product. Since snus use delivers a dose of nicotine equivalent to cigarettes, and has been implicated in cardiac arrhythmia because of associations with sudden cardiovascular death, a relation with atrial fibrillation is plausible and important to investigate. METHODS: To assess the relation between use of snus and risk of atrial fibrillation, we carried out a pooled analysis of 7 prospective Swedish cohort studies. In total, 274,882 men, recruited between 1978 and 2004, were followed via the National Patient Register for atrial fibrillation. Primary analyses were restricted to 127,907 never-smokers. Relative risks were estimated using Cox proportional hazard regression. RESULTS: The prevalence of snus use was 25% among never-smokers. During follow-up, 3,069 cases of atrial fibrillation were identified. The pooled relative risk of atrial fibrillation was 1.07 (95% confidence interval = 0.97-1.19) in current snus users, compared with nonusers. CONCLUSION: Findings from this large national pooling project indicate that snus use is unlikely to confer any important increase in risk of atrial fibrillation.

Proteomic cornerstones of hematopoietic stem cell differentiation: distinct signatures of multipotent progenitors and myeloid committed cells.

Regenerative tissues such as the skin epidermis, the intestinal mucosa or the hematopoietic system are organized in a hierarchical manner with stem cells building the top of this hierarchy. Somatic stem cells harbor the highest self-renewal activity and generate a series of multipotent progenitors which differentiate into lineage committed progenitors and subsequently mature cells. In this report, we applied an in-depth quantitative proteomic approach to analyze and compare the full proteomes of ex vivo isolated and FACS-sorted populations highly enriched for either multipotent hematopoietic stem/progenitor cells (HSPCs, Lin(neg)Sca-1(+)c-Kit(+)) or myeloid committed precursors (Lin(neg)Sca-1(-)c-Kit(+)). By employing stable isotope dimethyl labeling and high-resolution mass spectrometry, more than 5000 proteins were quantified. From biological triplicate experiments subjected to rigorous statistical evaluation, 893 proteins were found differentially expressed between multipotent and myeloid committed cells. The differential protein content in these cell populations points to a distinct structural organization of the cytoskeleton including remodeling activity. In addition, we found a marked difference in the expression of metabolic enzymes, including a clear shift of specific protein isoforms of the glycolytic pathway. Proteins involved in translation showed a collective higher expression in myeloid progenitors, indicating an increased translational activity. Strikingly, the data uncover a unique signature related to immune defense mechanisms, centering on the RIG-I and type-1 interferon response systems, which are installed in multipotent progenitors but not evident in myeloid committed cells. This suggests that specific, and so far unrecognized, mechanisms protect these immature cells before they mature. In conclusion, this study indicates that the transition of hematopoietic stem/progenitors toward myeloid commitment is accompanied by a profound change in processing of cellular resources, adding novel insights into the molecular mechanisms at the interface between multipotency and lineage commitment.

Defective ribosome assembly impairs leukemia progression in a murine model of acute myeloid leukemia.

Despite an advanced understanding of disease mechanisms, the current therapeutic regimen fails to cure most patients with acute myeloid leukemia (AML). In the present study, we address the role of ribosome assembly in leukemia cell function. We apply patient datasets and murine models to demonstrate that immature leukemia cells in mixed-lineage leukemia-rearranged AML are characterized by relatively high ribosome biogenesis and protein synthesis rates. Using a model with inducible regulation of ribosomal subunit joining, we show that defective ribosome assembly extends survival in mice with AML. Single-cell RNA sequencing and proteomic analyses reveal that leukemia cell adaptation to defective ribosome assembly is associated with an increase in ribosome biogenesis and deregulation of the transcription factor landscape. Finally, we demonstrate that defective ribosome assembly shows antileukemia efficacy in p53-deficient AML. Our study unveils the critical requirement of a high protein synthesis rate for leukemia progression and highlights ribosome assembly as a therapeutic target in AML.

A complex interplay of intra- and extracellular factors regulates the outcome of fetal- and adult-derived MLL-rearranged leukemia.

Infant and adult MLL1/KMT2A-rearranged (MLLr) leukemia represents a disease with a dismal prognosis. Here, we present a functional and proteomic characterization of in utero-initiated and adult-onset MLLr leukemia. We reveal that fetal MLL::ENL-expressing lymphomyeloid multipotent progenitors (LMPPs) are intrinsically programmed towards a lymphoid fate but give rise to myeloid leukemia in vivo, highlighting a complex interplay of intra- and extracellular factors in determining disease subtype. We characterize early proteomic events of MLL::ENL-mediated transformation in fetal and adult blood progenitors and reveal that whereas adult pre-leukemic cells are mainly characterized by retained myeloid features and downregulation of ribosomal and metabolic proteins, expression of MLL::ENL in fetal LMPPs leads to enrichment of translation-associated and histone deacetylases signaling proteins, and decreased expression of inflammation and myeloid differentiation proteins. Integrating the proteome of pre-leukemic cells with their secretome and the proteomic composition of the extracellular environment of normal progenitors highlights differential regulation of Igf2 bioavailability, as well as of VLA-4 dimer and its ligandome, upon initiation of fetal- and adult-origin leukemia, with implications for human MLLr leukemia cells' ability to communicate with their environment through granule proteins. Our study has uncovered opportunities for targeting ontogeny-specific proteomic vulnerabilities in in utero-initiated and adult-onset MLLr leukemia.

Lipoprotein metabolism mediates hematopoietic stem cell responses under acute anemic conditions.

Hematopoietic stem cells (HSCs) react to various stress conditions. However, it is unclear whether and how HSCs respond to severe anemia. Here, we demonstrate that upon induction of acute anemia, HSCs rapidly proliferate and enhance their erythroid differentiation potential. In severe anemia, lipoprotein profiles largely change and the concentration of ApoE increases. In HSCs, transcription levels of lipid metabolism-related genes, such as very low-density lipoprotein receptor (Vldlr), are upregulated. Stimulation of HSCs with ApoE enhances their erythroid potential, whereas HSCs in Apoe knockout mice do not respond to anemia induction. VldlrhighHSCs show higher erythroid potential, which is enhanced after acute anemia induction. VldlrhighHSCs are epigenetically distinct because of their low chromatin accessibility, and more chromatin regions are closed upon acute anemia induction. Chromatin regions closed upon acute anemia induction are mainly binding sites of Erg. Inhibition of Erg enhanced the erythroid differentiation potential of HSCs. Our findings indicate that lipoprotein metabolism plays an important role in HSC regulation under severe anemic conditions.

Time-resolved quantitative proteome analysis of in vivo intestinal development.

Postnatal intestinal development is a very dynamic process characterized by substantial morphological changes that coincide with functional adaption to the nutritional change from a diet rich in fat (milk) to a diet rich in carbohydrates on from weaning. Time-resolved studies of intestinal development have so far been limited to investigation at the transcription level or to single or few proteins at a time. In the present study, we elucidate proteomic changes of primary intestinal epithelial cells from jejunum during early suckling (1-7 days of age), middle suckling (7-14 days), and weaning period (14-35 days) in mice, using a label-free proteomics approach. We show differential expression of 520 proteins during intestinal development and a pronounced change of the proteome during the middle suckling period and weaning. Proteins involved in several metabolic processes were found differentially expressed along the development. The temporal expression profiles of enzymes of the glycolysis were found to correlate with the increase in carbohydrate uptake at weaning, whereas the abundance changes of proteins involved in fatty acid metabolism as well as lactose metabolism indicated a nondiet driven preparation for the nutritional change at weaning. Further, we report the developmental abundance changes of proteins playing a vital role in the neonatal acquisition of passive immunity. In addition, different isoforms of several proteins were quantified, which may contribute to a better understanding of the roles of the specific isoforms in the small intestine. In summary, we provide a first, time-resolved proteome profile of intestinal epithelial cells along postnatal intestinal development.

Use of snus and acute myocardial infarction: pooled analysis of eight prospective observational studies.

The use of snus (also referred to as Scandinavian or Swedish moist smokeless tobacco), which is common in Sweden and increasing elsewhere, is receiving increasing attention since considered a tobacco smoke "potential reduction exposure product". Snus delivers a high dose of nicotine with possible hemodynamic effects, but its impact on cardiovascular morbidity and mortality is uncertain. The aim of this study was to investigate whether snus use is associated with risk of and survival after acute myocardial infarction (AMI). Data from eight prospective cohort studies set in Sweden was pooled and reanalysed. The relative risk of first time AMI and 28-day case-fatality was calculated for 130,361 men who never smoked. During 2,262,333 person-years of follow-up, 3,390 incident events of AMI were identified. Current snus use was not associated with risk of AMI (pooled multivariable hazard ratio 1.04, 95 % confidence interval 0.93 to 1.17). The short-term case fatality rate appeared increased in snus users (odds ratio 1.28, 95 % confidence interval 0.99 to 1.68). This study does not support any association between use of snus and development of AMI. Hence, toxic components other than nicotine appear implicated in the pathophysiology of smoking related ischemic heart disease. Case fatality after AMI is seemingly increased among snus users, but this relationship may be due to confounding by socioeconomic or life style factors.

The Opportunity of Proteomics to Advance the Understanding of Intra- and Extracellular Regulation of Malignant Hematopoiesis.

Fetal and adult hematopoiesis are regulated by largely distinct sets of cell-intrinsic gene regulatory networks as well as extracellular cues in their respective microenvironment. These ontogeny-specific programs drive hematopoietic stem and progenitor cells (HSPCs) in fetus and adult to divergent susceptibility to initiation and progression of hematological malignancies, such as leukemia. Elucidating how leukemogenic hits disturb the intra- and extracellular programs in HSPCs along ontogeny will provide a better understanding of the causes for age-associated differences in malignant hematopoiesis and facilitate the improvement of strategies for prevention and treatment of pediatric and adult acute leukemia. Here, we review current knowledge of the intrinsic and extrinsic programs regulating normal and malignant hematopoiesis, with a particular focus on the differences between infant and adult acute leukemia. We discuss the recent advances in mass spectrometry-based proteomics and its opportunity for resolving the interplay of cell-intrinsic and niche-associated factors in regulating malignant hematopoiesis.

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

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.