Invariant natural killer T (iNKT) cells are unconventional ɑβ T cells that respond to lipid-based antigens. They play a vital role in response to infections and are involved in diseases such as allergic asthma and cancer. Despite clear immunological roles across multiple diseases, remarkably few tools exist to study iNKTs in vivo. In this study, we report that the Fgd5ZsGreen/+ reporter mouse widely used to study hematopoietic stem cells (HSCs) can dually serve to identify rare immune cell populations, including a subset of iNKTs. Specifically, we show that a "non-HSC" population of CD45+Fgd5ZsGreen+ cells reside in multiple organs including the lungs, liver, spleen, and thymus. The majority of these Fgd5ZsGreen+ cells do not express canonical HSC markers but instead express CD5. RNA sequencing of CD5+EPCR-Fgd5ZsGreen+ bone marrow cells showed greatest similarity to iNKT cells, and these findings were corroborated by additional cell surface marker analysis of TCRβint CD1d-PBS57+ iNKT cells in reporter mouse tissues. We found that roughly 20% of total thymic iNKT cells were Fgd5-ZsGreen+ and that these cells predominantly coexpressed NK1.1 and CD122, consistent with an iNKT1 cell phenotype. Transcriptomic and proteomic profiling of sorted Fgd5-ZsGreen- and Fgd5-ZsGreen+ iNKT cells revealed a number of key genes involved in cytotoxic responses as distinct between the two iNKT cell populations. These different cytotoxic profiles were further supported by cytokine expression following stimulation, indicating the potential existence of disparate iNKT1 subsets. Together, these data implicate Fgd5 expression as a powerful new tool for phenotyping and tracking cytotoxic iNKT1 cell subsets in vivo.

Despite rapid advances in mapping genetic drivers and gene expression changes in hematopoietic stem cells (HSCs), few studies exist at the protein level. We perform a deep, multi-omics characterization (epigenome, transcriptome, and proteome) of HSCs in a mouse model carrying a loss-of-function mutation in Tet2, a driver of increased self-renewal in blood cancers. Using state-of-the-art, multiplexed, low-input mass spectrometry (MS)-based proteomics, we profile TET2-deficient (Tet2-/-) HSCs, revealing previously unrecognized molecular processes that define the pre-leukemic HSC molecular landscape. Specifically, we obtain more accurate stratification of wild-type and Tet2-/- HSCs than transcriptomic approaches and identify extracellular matrix (ECM) molecules as being dysregulated upon TET2 loss. HSC expansion assays using ECM-functionalized hydrogels confirm a selective effect on the expansion of Tet2-mutant HSCs. Taken together, our study represents a comprehensive molecular characterization of Tet2-mutant HSCs and identifies a previously unanticipated role of ECM molecules in regulating self-renewal of disease-driving HSCs.

Despite rapid advances in mapping genetic drivers and gene expression changes in hematopoietic stem cells (HSCs), there is a relative paucity of studies at the protein level. Here, we perform a deep, multi-omic characterization (epigenome, transcriptome and proteome) of HSCs carrying a loss-of-function mutation in Tet2, a key driver of increased self-renewal in blood cancers. Using state-of-the-art, multiplexed, low-input mass spectrometry (MS)-based proteomics, we profile wildtype (WT) and TET2-deficient (Tet2-/-) HSCs and show that the proteome captures previously unrecognized molecular processes which define the pre-leukemic HSC molecular landscape. Specifically, we obtain more accurate stratification of WT and Tet2-/- HSCs than transcriptomic approaches and identify extracellular matrix (ECM) molecules as novel points of dysregulation upon TET2 loss. HSC expansion assays using ECM-functionalized hydrogels confirm a selective effect on the expansion of Tet2-mutant HSCs. Taken together, our study represents a comprehensive molecular characterization of Tet2-mutant HSCs and identifies a previously unanticipated role of ECM molecules in regulating self-renewal of disease-driving HSCs. ### Competing Interest Statement The DGK lab has received research funding from STEMBOND Inc. (Cambridge, UK) to conduct experiments using hematopoietic cells that were unrelated to this manuscript.

Delineating cell-intrinsic and -extrinsic drivers of hematopoietic stem cell (HSC) self-renewal is critical to improve efforts in ex vivo HSC expansion and to better understand leukemia cell biology. To characterize molecular changes at the proteome level, we applied miniaturized, multiplexed sample preparation protocols in combination with mass spectrometry (MS)-based quantitative proteomics to compare normal and Tet2-deficient HSCs, and data-independent acquisition (DIA)-MS to characterize the extracellular environment of HSCs in vivo and during ex vivo expansion. We show that both the cellular and secreted proteome accurately stratify HSCs based on functional potency and mutational status and identify novel molecular components not captured in transcriptomic analyses. On the pre-leukemia side, we reveal that Tet2-deficient HSCs have altered expression of extracellular matrix (ECM) proteins and that interaction with these proteins in artificial niches affects cellular function. Extracellular proteomic analysis further reveals that Tet2-deficient cells create a microenvironment is pro-inflammatory and pro-thrombotic even in young, asymptomatic animals. In HSC expansion assays, proteomics identifies the requirement for intact DNA repair pathways, specifically mismatch repair proteins, as key components of HSC clones capable of extensive self-renewal compared to unsuccessful expansion cultures. Analysis of the secretome of unsuccessful cultures further identifies mast cell proteins as predictive of failure to expand engraftable HSCs. Collectively these data highlight novel proteins to which transcriptomic studies are blind, and open new opportunities for HSC expansion and preleukemic biology, paving the way for future ex vivo and in vivo modulation of HSC function via manipulation of the cells and their extracellular environment.

The lack of techniques to apply quantifiable forces to non-adherent cells have limited our understating of how forces influence blood stem cell fate. Present technologies can only deform suspended cells one at time by extensional flow (high speed compression) through microfluidic devices. The limitation of this technology is that it subjects individual cells to shear and compressive stresses simultaneously and for a very short amount of time (seconds). However, blood stem and progenitor cells vary in size and mechanical (e.g., viscoelastic) properties, presenting challenges for cell compression devices. We have built a microfluidic chip that addresses these limitations. For the first time, we can trap a population of heterogeneous non-adherent cells (>600 cells) in a compression chamber and apply a range of quantifiable forces to deform cell membranes for prolonged periods of time (>1 hour) without compromising cell viability. We found that the application of compressive forces for 30-60mins are sufficient to alter the differentiation potential of granulocyte macrophage progenitor cells (GMPs) along specific lineages without alteration of growth factors, hormones, or other regulatory molecules. Different mechanical inputs also led to distinct biological outputs dependent on the genetic background of cells (e.g., TET2-deficient GMPs vs wild type GMPs). At higher compression forces both TET2 and WT GMPs produced more neutrophils, however TET2-deficient GMPs showed rapid differentiation (and lower viability) at lower compressive forces compared to uncompressed or mildly compressed cells. These data support the hypothesis that the application of compressive forces can direct the differentiation of progenitor cells towards specific cell lineage and set the stage for utilising mechanical forces in the future to improve blood stem generation outside the body. The lack of techniques to apply quantifiable forces to non-adherent cells have limited our understating of how forces influence blood stem cell fate. Present technologies can only deform suspended cells one at time by extensional flow (high speed compression) through microfluidic devices. The limitation of this technology is that it subjects individual cells to shear and compressive stresses simultaneously and for a very short amount of time (seconds). However, blood stem and progenitor cells vary in size and mechanical (e.g., viscoelastic) properties, presenting challenges for cell compression devices. We have built a microfluidic chip that addresses these limitations. For the first time, we can trap a population of heterogeneous non-adherent cells (>600 cells) in a compression chamber and apply a range of quantifiable forces to deform cell membranes for prolonged periods of time (>1 hour) without compromising cell viability. We found that the application of compressive forces for 30-60mins are sufficient to alter the differentiation potential of granulocyte macrophage progenitor cells (GMPs) along specific lineages without alteration of growth factors, hormones, or other regulatory molecules. Different mechanical inputs also led to distinct biological outputs dependent on the genetic background of cells (e.g., TET2-deficient GMPs vs wild type GMPs). At higher compression forces both TET2 and WT GMPs produced more neutrophils, however TET2-deficient GMPs showed rapid differentiation (and lower viability) at lower compressive forces compared to uncompressed or mildly compressed cells. These data support the hypothesis that the application of compressive forces can direct the differentiation of progenitor cells towards specific cell lineage and set the stage for utilising mechanical forces in the future to improve blood stem generation outside the body.

The capacity to maintain and expand haematopoietic stem cells (HSCs) ex vivo is key to many cell and gene therapies. Despite this importance, expansion of functional HSCs in vitro has remained a challenge. Most efforts have focused on supplementing in vitro cultures with specific recombinant growth factors or small molecules, but relatively few have accounted for the role of physical attachment to cells in the bone marrow microenvironment. We therefore set out to create a more bone-marrow like microenvironment outside the body by utilising STEMBOND hydrogels (Segel et al., Nature 2019) with different mechanical stiffnesses in combination with the ability to functionalise these gels with different extracellular matrix (ECM) proteins. Using both short- and long-term HSC expansion cultures, we explored the impact on retention and/or expansion of HSCs grown on different hydrogels. By cellular phenotyping, we observed an ∼10-fold expansion of HSCs (as measured by %EPCR+Lin-Sca+Kit+, ELSK) using gels that more closely resembled in vivo bone marrow niche stiffness (15-30% ELSK) and a reduction in HSCs with increased stiffness (5% ELSK), in comparison with plastic (2% ELSK). Next, we moved to functionalising gels with different ECM molecules where we observed that vitronectin supported a 50-fold expansion of phenotypic HSCs (with 30% ELSK) which was accompanied by an increase in transplantation efficiency in vivo compared to other molecules and cells cultured on tissue culture plastic. Finally, we utilised proteomic data from TET2 knockout HSCs to identify novel ECM proteins that could alter HSC expansion. Finally, using alternate hydrogels, we explored 2D versus 3D configurations to determine whether the orientation of ECM protein delivery was also involved. Together, this platform sets the stage for new studies that will further our understanding of HSC:niche interactions. The capacity to maintain and expand haematopoietic stem cells (HSCs) ex vivo is key to many cell and gene therapies. Despite this importance, expansion of functional HSCs in vitro has remained a challenge. Most efforts have focused on supplementing in vitro cultures with specific recombinant growth factors or small molecules, but relatively few have accounted for the role of physical attachment to cells in the bone marrow microenvironment. We therefore set out to create a more bone-marrow like microenvironment outside the body by utilising STEMBOND hydrogels (Segel et al., Nature 2019) with different mechanical stiffnesses in combination with the ability to functionalise these gels with different extracellular matrix (ECM) proteins. Using both short- and long-term HSC expansion cultures, we explored the impact on retention and/or expansion of HSCs grown on different hydrogels. By cellular phenotyping, we observed an ∼10-fold expansion of HSCs (as measured by %EPCR+Lin-Sca+Kit+, ELSK) using gels that more closely resembled in vivo bone marrow niche stiffness (15-30% ELSK) and a reduction in HSCs with increased stiffness (5% ELSK), in comparison with plastic (2% ELSK). Next, we moved to functionalising gels with different ECM molecules where we observed that vitronectin supported a 50-fold expansion of phenotypic HSCs (with 30% ELSK) which was accompanied by an increase in transplantation efficiency in vivo compared to other molecules and cells cultured on tissue culture plastic. Finally, we utilised proteomic data from TET2 knockout HSCs to identify novel ECM proteins that could alter HSC expansion. Finally, using alternate hydrogels, we explored 2D versus 3D configurations to determine whether the orientation of ECM protein delivery was also involved. Together, this platform sets the stage for new studies that will further our understanding of HSC:niche interactions.

While numerous tools have now been developed to study the genome and transcriptome at single cell resolution, the low frequency and number of hematopoietic stem cells (HSCs) still precludes global proteomic approaches. Recent advances in ex vivo HSC expansion put us on the cusp of breaking through this decades-old barrier by generating sufficient numbers of functional HSCs to undertake proteomic studies. However, in these cultures, HSCs still represent a minor fraction of the expanded cell pool and considerable heterogeneity exists between single HSCs and their ability to expand functional HSCs. We have recently developed a method to prospectively identify ex vivo expanded functional HSCs using ESAM, EPCR, and LSK markers, allowing us to both identify clones with large numbers of functional HSCs and to sort them for molecular analyses. Here, we have applied mass spectrometry-based quantitative proteomics to characterize the molecular composition of 17 ex vivo-expanded HSC clones with a phenotypic HSC content ranging between 0.1 and 48%. We identified ∼5,000 proteins and derived a protein signature that significantly correlates with the percentage of phenotypic HSCs within expanded clones. Differentially expressed proteins enriched in clones with high HSC content include several proteins previously implicated in HSC self-renewal, such as HMGA2, PARP1, MDH1, RHOA, and CDC42. Strikingly, we found Granzyme B, which has been reported to be involved in an alternative apoptotic pathway in HSCs, to be the top enriched protein in clones with low HSC content. Collectively, this study illustrates the power of moving beyond the transcriptome for molecular analyses of HSCs, shedding further light on the mechanism underlying HSC self-renewal in culture, while also providing a series of targets with the potential to improve HSC expansion ex vivo. While numerous tools have now been developed to study the genome and transcriptome at single cell resolution, the low frequency and number of hematopoietic stem cells (HSCs) still precludes global proteomic approaches. Recent advances in ex vivo HSC expansion put us on the cusp of breaking through this decades-old barrier by generating sufficient numbers of functional HSCs to undertake proteomic studies. However, in these cultures, HSCs still represent a minor fraction of the expanded cell pool and considerable heterogeneity exists between single HSCs and their ability to expand functional HSCs. We have recently developed a method to prospectively identify ex vivo expanded functional HSCs using ESAM, EPCR, and LSK markers, allowing us to both identify clones with large numbers of functional HSCs and to sort them for molecular analyses. Here, we have applied mass spectrometry-based quantitative proteomics to characterize the molecular composition of 17 ex vivo-expanded HSC clones with a phenotypic HSC content ranging between 0.1 and 48%. We identified ∼5,000 proteins and derived a protein signature that significantly correlates with the percentage of phenotypic HSCs within expanded clones. Differentially expressed proteins enriched in clones with high HSC content include several proteins previously implicated in HSC self-renewal, such as HMGA2, PARP1, MDH1, RHOA, and CDC42. Strikingly, we found Granzyme B, which has been reported to be involved in an alternative apoptotic pathway in HSCs, to be the top enriched protein in clones with low HSC content. Collectively, this study illustrates the power of moving beyond the transcriptome for molecular analyses of HSCs, shedding further light on the mechanism underlying HSC self-renewal in culture, while also providing a series of targets with the potential to improve HSC expansion ex vivo.

Hematopoietic stem and progenitor cell (HSPC) transplantation and gene therapy protocols are limited by stem cell numbers. While decades of work into the chemical and molecular modifiers of hematopoietic differentiation have been fruitful, substantially less is known about the impact of physical factors that govern HSPC survival, differentiation and self-renewal, thus limiting our ability to scale-up production of human HSPC numbers ex vivo. HSPC are exposed to potential mediators of mechanical signals in the bone marrow microenvironment via neighbouring cells. To gain a better understanding of how mechanical forces might impact stem cell behaviour in development and regeneration, we developed a novel microfluidic device that is capable of immobilising and mechanically stimulating a range of isolated HSPC fractions. Heterogenous populations of hematopoietic stem cells (HSC) and granulocyte-monocyte progenitor cells (GMP) were subjected to time-varying shear stress independently from controlled static compressive forces by modulating applied gas pressure using PDMS-based pneumatic valves. Live cell monitoring of HSC and GMP from wild-type and TET2 deficient animals allowed measurement of cellular response to mechanical stimulation ranging from mild deformation through to complete cell lysis. Cells were extracted from the device for downstream cellular and molecular assays. Using single cell and bulk liquid culture assays we show, for the first time, that compressive stress can impact the viability, cloning efficiency and the differentiation capacity of HSPCs. Our approach suggests that mechanical signals can alter HSC fate in the absence of other changes and may be harnessed to improve HSC expansion protocols, while also prompting the need for a more detailed understanding of the physical role of niche cells in the context of leukaemia seeding and transformation. Hematopoietic stem and progenitor cell (HSPC) transplantation and gene therapy protocols are limited by stem cell numbers. While decades of work into the chemical and molecular modifiers of hematopoietic differentiation have been fruitful, substantially less is known about the impact of physical factors that govern HSPC survival, differentiation and self-renewal, thus limiting our ability to scale-up production of human HSPC numbers ex vivo. HSPC are exposed to potential mediators of mechanical signals in the bone marrow microenvironment via neighbouring cells. To gain a better understanding of how mechanical forces might impact stem cell behaviour in development and regeneration, we developed a novel microfluidic device that is capable of immobilising and mechanically stimulating a range of isolated HSPC fractions. Heterogenous populations of hematopoietic stem cells (HSC) and granulocyte-monocyte progenitor cells (GMP) were subjected to time-varying shear stress independently from controlled static compressive forces by modulating applied gas pressure using PDMS-based pneumatic valves. Live cell monitoring of HSC and GMP from wild-type and TET2 deficient animals allowed measurement of cellular response to mechanical stimulation ranging from mild deformation through to complete cell lysis. Cells were extracted from the device for downstream cellular and molecular assays. Using single cell and bulk liquid culture assays we show, for the first time, that compressive stress can impact the viability, cloning efficiency and the differentiation capacity of HSPCs. Our approach suggests that mechanical signals can alter HSC fate in the absence of other changes and may be harnessed to improve HSC expansion protocols, while also prompting the need for a more detailed understanding of the physical role of niche cells in the context of leukaemia seeding and transformation.

Hematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved > 200-fold expansion of functional HSCs, but their molecular characterization has not been possible since the majority of cells are non-HSCs and single cell-initiated cultures have substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. By directly linking single-clone functional transplantation data with single-clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to proliferating fetal HSCs and reveals a gene expression signature, including Esam, Prdm16, Fstl1, and Palld, that can identify functional HSCs from multiple cellular states. This "repopulation signature" (RepopSig) also enriches for HSCs in human datasets. Together, these findings demonstrate the power of integrating functional and molecular datasets to better derive meaningful gene signatures and opens the opportunity for a wide range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Advances in the isolation and gene expression profiling of single hematopoietic stem cells (HSCs) have permitted in-depth resolution of their molecular program. However, long-term HSCs can only be isolated to near purity from adult mouse bone marrow, thereby precluding studies of their molecular program in different physiological states. Here, we describe a powerful 7-day HSC hibernation culture system that maintains HSCs as single cells in the absence of a physical niche. Single hibernating HSCs retain full functional potential compared with freshly isolated HSCs with respect to colony-forming capacity and transplantation into primary and secondary recipients. Comparison of hibernating HSC molecular profiles to their freshly isolated counterparts showed a striking degree of molecular similarity, further resolving the core molecular machinery of HSC self-renewal while also identifying key factors that are potentially dispensable for HSC function, including members of the AP1 complex (Jun, Fos, and Ncor2), Sult1a1 and Cish. Finally, we provide evidence that hibernating mouse HSCs can be transduced without compromising their self-renewal activity and demonstrate the applicability of hibernation cultures to human HSCs.

SummaryHematopoietic stem cells (HSCs) cultured outside the body are the fundamental component of a wide range of cellular and gene therapies. Recent efforts have achieved more than 200-fold expansion of functional HSCs, but their molecular characterization has not been possible due to the substantial majority of cells being non-HSCs and single cell-initiated cultures displaying substantial clone-to-clone variability. Using the Fgd5 reporter mouse in combination with the EPCR surface marker, we report exclusive identification of HSCs from non-HSCs in expansion cultures. Linking single clone functional transplantation data with single clone gene expression profiling, we show that the molecular profile of expanded HSCs is similar to actively cycling fetal liver HSCs and shares a gene expression signature with functional HSCs from all sources, including Prdm16, Fstl1 and Palld. This new tool can now be applied to a wide-range of functional screening and molecular experiments previously not possible due to limited HSC numbers.

Single-cell molecular tools have been developed at an incredible pace over the last five years as sequencing costs continue to drop and numerous molecular assays have been coupled to sequencing readouts. This rapid period of technological development has facilitated the delineation of individual molecular characteristics including the genome, transcriptome, epigenome, and proteome of individual cells, leading to an unprecedented resolution of the molecular networks governing complex biological systems. The immense power of single-cell molecular screens has been particularly highlighted through work in systems where cellular heterogeneity is a key feature, such as stem cell biology, immunology, and tumor cell biology. Single-cell-omics technologies have already contributed to the identification of novel disease biomarkers, cellular subsets, therapeutic targets and diagnostics, many of which would have been undetectable by bulk sequencing approaches. More recently, efforts to integrate single-cell multi-omics with single cell functional output and/or physical location have been challenging but have led to substantial advances. Perhaps most excitingly, there are emerging opportunities to reach beyond the description of static cellular states with recent advances in modulation of cells through CRISPR technology, in particular with the development of base editors which greatly raises the prospect of cell and gene therapies. In this review, we provide a brief overview of emerging single-cell technologies and discuss current developments in integrating single-cell molecular screens and performing single-cell multi-omics for clinical applications. We also discuss how single-cell molecular assays can be usefully combined with functional data to unpick the mechanism of cellular decision-making. Finally, we reflect upon the introduction of spatial transcriptomics and proteomics, its complementary role with single-cell RNA sequencing (scRNA-seq) and potential application in cellular and gene therapy.

De-regulation of cell fate choices in adult stem cells has been implicated in ageing and tumorigenesis. Recent advances in single cell technologies have equipped researchers with unprecedented resolution of both the functional properties and transcriptional profile of single adult haematopoietic stem cells (HSCs). Whether these transcriptional programmes are reflected at the protein level remains poorly understood. Moreover, poly-A based capture methods can introduce biases or completely miss critical molecular changes. To date, the low frequency and number of functional HSCs, the inability to prospectively isolate distinct HSC subtypes, and the technical limitations of large scale protein profiling have precluded extensive analyses of molecular networks at the protein level. Here we describe an optimized mass spectrometry-based workflow to characterise the proteome from as few as 10,000 HSCs using tandem mass tag (TMT) labelling across 10 cell fractions. Our approach reduced the required cell input 30-fold when compared to conventional protocols and permitted the identification and quantification of over 4,000 proteins. We applied this technology to probe the HSC compartment from wild-type and TET2 deficient cells, identifying protein networks potentially involved in the regulation of HSC fate choice. In HSC fractions with different self-renewal durability and HSC content (Lin-Kit+CD150+CD48- compared to Lin-Kit+CD150+CD48-EPCR+) we observed progressive re-shaping of protein networks that implicate the physical role of neighbouring cells in the bone marrow niche. Together our results indicate that HSCs obtained from the bone marrow retain molecular features of the niche from which they were extracted and offer a new potential avenue to study HSC fate choice in normal and malignant cells. De-regulation of cell fate choices in adult stem cells has been implicated in ageing and tumorigenesis. Recent advances in single cell technologies have equipped researchers with unprecedented resolution of both the functional properties and transcriptional profile of single adult haematopoietic stem cells (HSCs). Whether these transcriptional programmes are reflected at the protein level remains poorly understood. Moreover, poly-A based capture methods can introduce biases or completely miss critical molecular changes. To date, the low frequency and number of functional HSCs, the inability to prospectively isolate distinct HSC subtypes, and the technical limitations of large scale protein profiling have precluded extensive analyses of molecular networks at the protein level. Here we describe an optimized mass spectrometry-based workflow to characterise the proteome from as few as 10,000 HSCs using tandem mass tag (TMT) labelling across 10 cell fractions. Our approach reduced the required cell input 30-fold when compared to conventional protocols and permitted the identification and quantification of over 4,000 proteins. We applied this technology to probe the HSC compartment from wild-type and TET2 deficient cells, identifying protein networks potentially involved in the regulation of HSC fate choice. In HSC fractions with different self-renewal durability and HSC content (Lin-Kit+CD150+CD48- compared to Lin-Kit+CD150+CD48-EPCR+) we observed progressive re-shaping of protein networks that implicate the physical role of neighbouring cells in the bone marrow niche. Together our results indicate that HSCs obtained from the bone marrow retain molecular features of the niche from which they were extracted and offer a new potential avenue to study HSC fate choice in normal and malignant cells.

Thrombopoietin (TPO) is a critical cytokine regulating hematopoietic stem cell maintenance and differentiation into the megakaryocytic lineage. However, the transcriptional and chromatin dynamics elicited by TPO signaling are poorly understood. Here, we study the immediate early transcriptional and cis-regulatory responses to TPO in hematopoietic stem/progenitor cells (HSPCs) and use this paradigm of cytokine signaling to chromatin to dissect the relationship between cis-regulatory activity and chromatin architecture. We show that TPO profoundly alters the transcriptome of HSPCs, with key hematopoietic regulators being transcriptionally repressed within 30 min of TPO. By examining cis-regulatory dynamics and chromatin architectures, we demonstrate that these changes are accompanied by rapid and extensive epigenome remodeling of cis-regulatory landscapes that is spatially coordinated within topologically associating domains (TADs). Moreover, TPO-responsive enhancers are spatially clustered and engage in preferential homotypic intra- and inter-TAD interactions that are largely refractory to TPO signaling. By further examining the link between cis-regulatory dynamics and chromatin looping, we show that rapid modulation of cis-regulatory activity is largely independent of chromatin looping dynamics. Finally, we show that, although activated and repressed cis-regulatory elements share remarkably similar DNA sequence compositions, transcription factor binding patterns accurately predict rapid cis-regulatory responses to TPO.

Recent descriptions of single cell gene expression datasets from highly purified long-term hematopoietic stem cells (LT-HSCs) have significantly advanced our understanding of the molecular state of HSCs. Emerging concomitantly, however, is the knowledge that LT-HSCs share a significant amount of their transcriptome with cells that do not possess the functional properties of durable self-renewal and multi-lineage cell output. To discern which genes and pathways drive individual HSC properties and which are bystanders is a major outstanding challenge for the field. We have recently demonstrated that single HSCs (EPCR++CD150+CD48-CD45+Sca-1++, >50% LT-HSCs by single cell transplantation) can be cultured without undergoing division for a period of 710 days with minimal cytokine stimulation (21% single cell survival, 99.2% undivided). Limiting dilution assays of HSCs cultured for 7 days (7dHSCs) estimated the durable multi-lineage HSC frequency at 22%. Single cell transplantation experiments had a similar frequency with both primary (3/10) and secondary (3/3) transplantations demonstrating that single 7dHSCs retained HSC function. Since 7dHSCs share the functional properties of freshly isolated HSCs, they represent a robust comparator population to identify which genes are indispensable for the maintenance of HSC self-renewal and quiescence. We generated RNA-sequencing datasets from freshly isolated HSCs and 7dHSCs and identified 960 genes and a number of biological processes that were significantly down-regulated in 7dHSCs, suggesting that these are dispensable for HSC function. Major down-regulated processes included the response to oxidative stress, ligand-independent apoptosis, and cellular senescence. Interestingly, in all 3 mice successfully transplanted with single 7dHSCs, a lymphoid-deficient (or -HSC) program was observed, suggesting that -HSCs are more resilient to the stress induced by cytokine depletion and represent a potential mechanism of obtaining the molecular program of -HSC. Together, these data highlight the importance of studying HSCs in different contexts to identify common molecular features of their functional properties.

MYST histone acetyltransferases have crucial functions in transcription, replication and DNA repair and are hence implicated in development and cancer. Here we characterise Myst2/Kat7/Hbo1 protein interactions in mouse embryonic stem cells by affinity purification coupled to mass spectrometry. This study confirms that in embryonic stem cells Myst2 is part of H3 and H4 histone acetylation complexes similar to those described in somatic cells. We identify a novel Myst2-associated protein, the tumour suppressor protein Niam (Nuclear Interactor of ARF and Mdm2). Human NIAM is involved in chromosome segregation, p53 regulation and cell proliferation in somatic cells, but its role in embryonic stem cells is unknown. We describe the first Niam embryonic stem cell interactome, which includes proteins with roles in DNA replication and repair, transcription, splicing and ribosome biogenesis. Many of Myst2 and Niam binding partners are required for correct embryonic development, implicating Myst2 and Niam in the cooperative regulation of this process and suggesting a novel role for Niam in embryonic biology. The data provides a useful resource for exploring Myst2 and Niam essential cellular functions and should contribute to deeper understanding of organism early development and survival as well as cancer. Data are available via ProteomeXchange with identifier PXD005987.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Pluripotency and self-renewal, the defining properties of embryonic stem cells, are brought about by transcriptional programs involving an intricate network of transcription factors and chromatin remodeling complexes. The Nucleosome Remodeling and Deacetylase (NuRD) complex plays a crucial and dynamic role in the regulation of stemness and differentiation. Several NuRD-associated factors have been reported but how they are organized has not been investigated in detail. Here, we have combined affinity purification and blue native polyacrylamide gel electrophoresis followed by protein identification by mass spectrometry and protein correlation profiling to characterize the topology of the NuRD complex. Our data show that in mouse embryonic stem cells the NuRD complex is present as two distinct assemblies of differing topology with different binding partners. Cell cycle regulator Cdk2ap1 and transcription factor Sall4 associate only with the higher mass NuRD assembly. We further establish that only isoform Sall4a, and not Sall4b, associates with NuRD. By contrast, Suz12, a component of the PRC2 Polycomb repressor complex, associates with the lower mass entity. In addition, we identify and validate a novel NuRD-associated protein, Wdr5, a regulatory subunit of the MLL histone methyltransferase complex, which associates with both NuRD entities. Bioinformatic analyses of published target gene sets of these chromatin binding proteins are in agreement with these structural observations. In summary, this study provides an interesting insight into mechanistic aspects of NuRD function in stem cell biology. The relevance of our work has broader implications because of the ubiquitous nature of the NuRD complex. The strategy described here can be more broadly applicable to investigate the topology of the multiple complexes an individual protein can participate in.