Background and Objectives: Non-alcoholic fatty liver disease (NAFLD) develops due to impaired hepatic lipid fluxes and is a risk factor for chronic liver disease and atherosclerosis. Lipidomic studies consistently reported characteristic hepatic/VLDL “lipid signatures” in NAFLD; whole plasma traits are more debated. Surprisingly, the HDL lipid composition by mass spectrometry has not been characterised across the NAFLD spectrum, despite HDL being a possible source of hepatic lipids delivered from peripheral tissues alongside free fatty acids (FFA). This study characterises the HDL lipidomic signature in NAFLD, and its correlation with metabolic and liver disease markers.
Methods: We used liquid chromatography-mass spectrometry to determine the whole serum and HDL lipidomic profile in 89 biopsy-proven NAFLD patients and 20 sex and age-matched controls.
Results: in the whole serum of NAFLD versus controls, we report a depletion in polyunsaturated (PUFA) phospholipids (PL) and FFA; with PUFA PL being also lower in HDL, and negatively correlated with BMI, insulin resistance, triglycerides, and hepatocyte ballooning. In the HDL of the NAFLD group we also describe higher saturated ceramides, which positively correlate with insulin resistance and transaminases.
Conclusion: NAFLD features lower serum lipid species containing polyunsaturated fatty acids; the most affected lipid fractions are FFA and (HDL) phospholipids; our data suggest a possible defect in the transfer of PUFA from peripheral tissues to the liver in NAFLD. Mechanistic studies are required to explore the biological implications of our findings addressing if HDL composition can influence liver composition and damage, thus contributing to NAFLD pathophysiology.

Background: This work is aimed at improving the understanding of cardiometabolic syndrome pathophysiology and its relationship with thrombosis by generating a multi-omic disease signature.
Methods/Results: We combined classic plasma biochemistry and plasma biomarkers with the transcriptional and epigenetic characterisation of cell types involved in thrombosis, obtained from two extreme phenotype groups (morbidly obese and lipodystrophy) and lean individuals to identify the molecular mechanisms at play, highlighting patterns of abnormal activation in innate immune phagocytic cells. Our analyses showed that extreme phenotype groups could be distinguished from lean individuals, and from each other, across all data layers. The characterisation of the same obese group, six months after bariatric surgery revealed the loss of the abnormal activation of innate immune cells previously observed. However, rather than reverting to the gene expression landscape of lean individuals, this occurred via the establishment of novel gene expression landscapes. Netosis and its control mechanisms emerge amongst the pathways that show an improvement after surgical intervention.
Conclusions: We showed that the morbidly obese and lipodystrophy groups, despite some differences, shared a common cardiometabolic syndrome signature. We also showed that this could be used to discriminate, amongst the normal population, those individuals with a higher likelihood of presenting with the disease, even when not displaying the classic features.

Blood cells contain functionally important intracellular structures, such as granules, critical to immunity and thrombosis. Quantitative variation in these structures has not been subjected previously to large-scale genetic analysis. We perform genome-wide association studies of 63 flow-cytometry derived cellular phenotypes-including cell-type specific measures of granularity, nucleic acid content and reactivity-in 41,515 participants in the INTERVAL study. We identify 2172 distinct variant-trait associations, including associations near genes coding for proteins in organelles implicated in inflammatory and thrombotic diseases. By integrating with epigenetic data we show that many intracellular structures are likely to be determined in immature precursor cells. By integrating with proteomic data we identify the transcription factor FOG2 as an early regulator of platelet formation and α-granularity. Finally, we show that colocalisation of our associations with disease risk signals can suggest aetiological cell-types-variants in IL2RA and ITGA4 respectively mirror the known effects of daclizumab in multiple sclerosis and vedolizumab in inflammatory bowel disease.

An altered thrombo-hemorrhagic profile has long been observed in patients with myeloproliferative neoplasms (MPNs). We hypothesized that this observed clinical phenotype may result from altered expression of genes known to harbor genetic variants in bleeding, thrombotic, or platelet disorders. Here, we identify 32 genes from a clinically validated gene panel that were also significantly differentially expressed in platelets from MPN patients as opposed to healthy donors. This work begins to unravel previously unclear mechanisms underlying an important clinical reality in MPNs. Knowledge of altered platelet gene expression in MPN thrombosis/bleeding diathesis opens opportunities to advance clinical care by: (1) enabling risk stratification, in particular, for patients undergoing invasive procedures, and (2) facilitating tailoring of treatment strategies for those at highest risk, for example, in the form of antifibrinolytics, desmopressin or platelet transfusions (not current routine practice). Marker genes identified in this work may also enable prioritization of candidates in future MPN mechanistic as well as outcome studies.

BACKGROUND: Assessing inflammatory disease activity in large vessel vasculitis (LVV) can be challenging by conventional measures. OBJECTIVES: We aimed to investigate somatostatin receptor 2 (SST2) as a novel inflammation-specific molecular imaging target in LVV. METHODS: in a prospective, observational cohort study, in vivo arterial SST2 expression was assessed by positron emission tomography/magnetic resonance imaging (PET/MRI) using 68Ga-DOTATATE and 18F-FET-βAG-TOCA. Ex vivo mapping of the imaging target was performed using immunofluorescence microscopy; imaging mass cytometry; and bulk, single-cell, and single-nucleus RNA sequencing. RESULTS: Sixty-one participants (LVV: n = 27; recent atherosclerotic myocardial infarction of ≤2 weeks: n = 25; control subjects with an oncologic indication for imaging: n = 9) were included. Index vessel SST2 maximum tissue-to-blood ratio was 61.8% (P < 0.0001) higher in active/grumbling LVV than inactive LVV and 34.6% (P = 0.0002) higher than myocardial infarction, with good diagnostic accuracy (area under the curve: ≥0.86; P < 0.001 for both). Arterial SST2 signal was not elevated in any of the control subjects. SST2 PET/MRI was generally consistent with 18F-fluorodeoxyglucose PET/computed tomography imaging in LVV patients with contemporaneous clinical scans but with very low background signal in the brain and heart, allowing for unimpeded assessment of nearby coronary, myocardial, and intracranial artery involvement. Clinically effective treatment for LVV was associated with a 0.49 ± 0.24 (standard error of the mean [SEM]) (P = 0.04; 22.3%) reduction in the SST2 maximum tissue-to-blood ratio after 9.3 ± 3.2 months. SST2 expression was localized to macrophages, pericytes, and perivascular adipocytes in vasculitis specimens, with specific receptor binding confirmed by autoradiography. SSTR2-expressing macrophages coexpressed proinflammatory markers. CONCLUSIONS: SST2 PET/MRI holds major promise for diagnosis and therapeutic monitoring in LVV. (PET Imaging of Giant Cell and Takayasu Arteritis [PITA], NCT04071691; Residual Inflammation and Plaque Progression Long-Term Evaluation [RIPPLE], NCT04073810).

Rare genetic diseases affect millions, and identifying causal DNA variants is essential for patient care. Therefore, it is imperative to estimate the effect of each independent variant and improve their pathogenicity classification. Our study of 140,214 unrelated UK Biobank (UKB) participants found each carries a median of 7 variants previously reported as pathogenic or likely pathogenic. We focused on 967 diagnostic-grade genes (DGGs) variants for rare bleeding, thrombotic, and platelet disorders (BTPDs) observed in 12,367 UKB participants. By association analysis, for a subset of these variants, we estimated effect sizes for platelet count and volume, and odds ratios for bleeding and thrombosis. Variants causal of some autosomal recessive platelet disorders revealed phenotypic consequences in carriers. Loss-of-function variants in MPL, which cause chronic amegakaryocytic thrombocytopenia if biallelic, were unexpectedly associated with increased platelet counts in carriers. We also demonstrated that common variants identified by genome-wide association studies (GWAS) for platelet count or thrombosis risk may influence the penetrance of rare variants in BTPD DGGs on their associated hemostasis disorders. Network-propagation analysis applied to an interactome of 18,410 nodes and 571,917 edges showed that GWAS variants with large effect sizes are enriched in DGGs and their first-order interactors. Finally, we illustrate the modifying effect of polygenic scores for platelet count and thrombosis risk on disease severity in participants carrying rare variants in TUBB1, or PROC and PROS1, respectively. Our findings demonstrate the power of association analyses using large population datasets in improving pathogenicity classifications of rare variants.

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific liver disorder affecting 0.5–2% of pregnancies. The majority of cases present in the third trimester with pruritus, elevated serum bile acids and abnormal serum liver tests. ICP is associated with an increased risk of adverse outcomes, including spontaneous preterm birth and stillbirth. Whilst rare mutations affecting hepatobiliary transporters contribute to the aetiology of ICP, the role of common genetic variation in ICP has not been systematically characterised to date. Here, we perform genome-wide association studies (GWAS) and meta-analyses for ICP across three studies including 1138 cases and 153,642 controls. Eleven loci achieve genome-wide significance and have been further investigated and fine-mapped using functional genomics approaches. Our results pinpoint common sequence variation in liver-enriched genes and liver-specific cis-regulatory elements as contributing mechanisms to ICP susceptibility.

Rare hematopoietic stem and progenitor cell (HSPC) pools outside the bone marrow (BM) contribute to blood production in stress and disease but remain ill-defined. Although nonmobilized peripheral blood (PB) is routinely sampled for clinical management, the diagnosis and monitoring potential of PB HSPCs remain untapped, as no healthy PB HSPC baseline has been reported. Here we comprehensively delineate human extramedullary HSPC compartments comparing spleen, PB, and mobilized PB to BM using single-cell RNA-sequencing and/or functional assays. We uncovered HSPC features shared by extramedullary tissues and others unique to PB. First, in contrast to actively dividing BM HSPCs, we found no evidence of substantial ongoing hematopoiesis in extramedullary tissues at steady state but report increased splenic HSPC proliferative output during stress erythropoiesis. Second, extramedullary hematopoietic stem cells/multipotent progenitors (HSCs/MPPs) from spleen, PB, and mobilized PB share a common transcriptional signature and increased abundance of lineage-primed subsets compared with BM. Third, healthy PB HSPCs display a unique bias toward erythroid-megakaryocytic differentiation. At the HSC/MPP level, this is functionally imparted by a subset of phenotypic CD71+ HSCs/MPPs, exclusively producing erythrocytes and megakaryocytes, highly abundant in PB but rare in other adult tissues. Finally, the unique erythroid-megakaryocytic-skewing of PB is perturbed with age in essential thrombocythemia and β-thalassemia. Collectively, we identify extramedullary lineage-primed HSPC reservoirs that are nonproliferative in situ and report involvement of splenic HSPCs during demand-adapted hematopoiesis. Our data also establish aberrant composition and function of circulating HSPCs as potential clinical indicators of BM dysfunction.

Rare variants outside the classical coagulation cascade might cause inherited thrombosis. We aimed to identify the variant(s) causing venous thromboembolism (VTE) in a family with multiple relatives affected with unprovoked VTE and no thrombophilia defects. We identified by whole exome sequencing an extremely rare Arg to Gln variant (Arg89Gln) in the Microtubule Associated Serine/Threonine Kinase 2 (MAST2) gene that segregates with VTE in the family. Free-tissue factor pathway inhibitor (f-TFPI) plasma levels were significantly decreased in affected family members compared to healthy relatives. Conversely, plasminogen activator inhibitor-1 (PAI-1) levels were significantly higher in affected members than in healthy relatives. RNA sequencing analysis of RNA interference experimental data conducted in endothelial cells revealed that, of the 13,387 detected expressed genes, 2,354 have their level of expression modified by MAST2 knockdown, including SERPINE1 coding for PAI-1 and TFPI. In HEK293 cells overexpressing the MAST2 Gln89 variant, TFPI and SERPINE1 promoter activities were respectively lower and higher than in cells overexpressing the MAST2 wild type. This study identifies a novel thrombophilia-causing Arg89Gln variant in the MAST2 gene that is here proposed as a new molecular player in the etiology of VTE by interfering with hemostatic balance of endothelial cells.

Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome-wide transcriptomes (57.8 k mRNAs). For 14.8 k protein-coding transcripts, we assigned the proteins to 21 UniProt-based classes, based on their preferential intracellular localization and presumed function. This classified transcriptome-proteome profile of platelets revealed: (i) Absence of 37.2 k genome-wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein-coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43-0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identified proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma-derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identified proteome of nuclear-related, membrane and signaling proteins, as well proteins with low-level transcripts. We then constructed a prediction model, based on protein function, transcript level and (peri)nuclear localization, and calculated the achievable proteome at ~ 10 k proteins. Model validation identified 1.0 k additional proteins in the predicted classes. Network and database analysis revealed the presence of 2.4 k proteins with a possible role in thrombosis and hemostasis, and 138 proteins linked to platelet-related disorders. This genome-wide platelet transcriptome and (non)identified proteome database thus provides a scaffold for discovering the roles of unknown platelet proteins in health and disease.

Neutrophils play fundamental roles in innate immune response, shape adaptive immunity, and are a potentially causal cell type underpinning genetic associations with immune system traits and diseases. Here, we profile the binding of myeloid master regulator PU.1 in primary neutrophils across nearly a hundred volunteers. We show that variants associated with differential PU.1 binding underlie genetically-driven differences in cell count and susceptibility to autoimmune and inflammatory diseases. We integrate these results with other multi-individual genomic readouts, revealing coordinated effects of PU.1 binding variants on the local chromatin state, enhancer-promoter contacts and downstream gene expression, and providing a functional interpretation for 27 genes underlying immune traits. Collectively, these results demonstrate the functional role of PU.1 and its target enhancers in neutrophil transcriptional control and immune disease susceptibility.

BACKGROUND: Megakaryocytes (MKs) originate from cells immuno-phenotypically indistinguishable from hematopoietic stem cells (HSCs), bypassing intermediate progenitors. They mature within the adult bone marrow and release platelets into the circulation. Until now, there have been no transcriptional studies of primary human bone marrow MKs. OBJECTIVES: to characterize MKs and HSCs from human bone marrow using single-cell RNA sequencing, to investigate MK lineage commitment, maturation steps, and thrombopoiesis. RESULTS: We show that MKs at different levels of polyploidization exhibit distinct transcriptional states. Although high levels of platelet-specific gene expression occur in the lower ploidy classes, as polyploidization increases, gene expression is redirected toward translation and posttranslational processing transcriptional programs, in preparation for thrombopoiesis. Our findings are in keeping with studies of MK ultrastructure and supersede evidence generated using in vitro cultured MKs. Additionally, by analyzing transcriptional signatures of a single HSC, we identify two MK-biased HSC subpopulations exhibiting unique differentiation kinetics. We show that human bone marrow MKs originate from these HSC subpopulations, supporting the notion that they display priming for MK differentiation. Finally, to investigate transcriptional changes in MKs associated with stress thrombopoiesis, we analyzed bone marrow MKs from individuals with recent myocardial infarction and found a specific gene expression signature. Our data support the modulation of MK differentiation in this thrombotic state. CONCLUSIONS: Here, we use single-cell sequencing for the first time to characterize the human bone marrow MK transcriptome at different levels of polyploidization and investigate their differentiation from the HSC.

Platelet-neutrophil interactions are important for innate immunity, but also contribute to the pathogenesis of deep vein thrombosis, myocardial infarction and stroke. Here we report that, under flow, von Willebrand factor/glycoprotein Ibα-dependent platelet 'priming' induces integrin αIIbβ3 activation that, in turn, mediates neutrophil and T-cell binding. Binding of platelet αIIbβ3 to SLC44A2 on neutrophils leads to mechanosensitive-dependent production of highly prothrombotic neutrophil extracellular traps. A polymorphism in SLC44A2 (rs2288904-A) present in 22% of the population causes an R154Q substitution in an extracellular loop of SLC44A2 that is protective against venous thrombosis results in severely impaired binding to both activated αIIbβ3 and VWF-primed platelets. This was confirmed using neutrophils homozygous for the SLC44A2 R154Q polymorphism. Taken together, these data reveal a previously unreported mode of platelet-neutrophil crosstalk, mechanosensitive NET production, and provide mechanistic insight into the protective effect of the SLC44A2 rs2288904-A polymorphism in venous thrombosis.

Gray platelet syndrome (GPS) is a rare recessive disorder caused by biallelic variants in NBEAL2 and characterized by bleeding symptoms, the absence of platelet α-granules, splenomegaly, and bone marrow (BM) fibrosis. Due to the rarity of GPS, it has been difficult to fully understand the pathogenic processes that lead to these clinical sequelae. To discern the spectrum of pathologic features, we performed a detailed clinical genotypic and phenotypic study of 47 patients with GPS and identified 32 new etiologic variants in NBEAL2. The GPS patient cohort exhibited known phenotypes, including macrothrombocytopenia, BM fibrosis, megakaryocyte emperipolesis of neutrophils, splenomegaly, and elevated serum vitamin B12 levels. Novel clinical phenotypes were also observed, including reduced leukocyte counts and increased presence of autoimmune disease and positive autoantibodies. There were widespread differences in the transcriptome and proteome of GPS platelets, neutrophils, monocytes, and CD4 lymphocytes. Proteins less abundant in these cells were enriched for constituents of granules, supporting a role for Nbeal2 in the function of these organelles across a wide range of blood cells. Proteomic analysis of GPS plasma showed increased levels of proteins associated with inflammation and immune response. One-quarter of plasma proteins increased in GPS are known to be synthesized outside of hematopoietic cells, predominantly in the liver. In summary, our data show that, in addition to the well-described platelet defects in GPS, there are immune defects. The abnormal immune cells may be the drivers of systemic abnormalities such as autoimmune disease.

Both poly(A) enrichment and ribosomal RNA depletion are commonly used for RNA sequencing. Either has its advantages and disadvantages that may lead to biases in the downstream analyses. To better access these effects, we carried out both ribosomal RNA-depleted and poly(A)-selected RNA-seq for CD4+ T naive cells isolated from 40 healthy individuals from the Blueprint Project. For these 40 individuals, the genomic and epigenetic data were also available. This dataset offers a unique opportunity to understand how library construction influences differential gene expression, alternative splicing and molecular QTL (quantitative loci) analyses for human primary cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Primary immunodeficiency (PID) is characterized by recurrent and often life-threatening infections, autoimmunity and cancer, and it poses major diagnostic and therapeutic challenges. Although the most severe forms of PID are identified in early childhood, most patients present in adulthood, typically with no apparent family history and a variable clinical phenotype of widespread immune dysregulation: about 25% of patients have autoimmune disease, allergy is prevalent and up to 10% develop lymphoid malignancies1–3. Consequently, in sporadic (or non-familial) PID genetic diagnosis is difficult and the role of genetics is not well defined. Here we address these challenges by performing whole-genome sequencing in a large PID cohort of 1,318 participants. An analysis of the coding regions of the genome in 886 index cases of PID found that disease-causing mutations in known genes that are implicated in monogenic PID occurred in 10.3% of these patients, and a Bayesian approach (BeviMed4) identified multiple new candidate PID-associated genes, including IVNS1ABP. We also examined the noncoding genome, and found deletions in regulatory regions that contribute to disease causation. In addition, we used a genome-wide association study to identify loci that are associated with PID, and found evidence for the colocalization of—and interplay between—novel high-penetrance monogenic variants and common variants (at the PTPN2 and SOCS1 loci). This begins to explain the contribution of common variants to the variable penetrance and phenotypic complexity that are observed in PID. Thus, using a cohort-based whole-genome-sequencing approach in the diagnosis of PID can increase diagnostic yield and further our understanding of the key pathways that influence immune responsiveness in humans.

Most patients with rare diseases do not receive a molecular diagnosis and the aetiological variants and causative genes for more than half such disorders remain to be discovered1. Here we used whole-genome sequencing (WGS) in a national health system to streamline diagnosis and to discover unknown aetiological variants in the coding and non-coding regions of the genome. We generated WGS data for 13,037 participants, of whom 9,802 had a rare disease, and provided a genetic diagnosis to 1,138 of the 7,065 extensively phenotyped participants. We identified 95 Mendelian associations between genes and rare diseases, of which 11 have been discovered since 2015 and at least 79 are confirmed to be aetiological. By generating WGS data of UK Biobank participants2, we found that rare alleles can explain the presence of some individuals in the tails of a quantitative trait for red blood cells. Finally, we identified four novel non-coding variants that cause disease through the disruption of transcription of ARPC1B, GATA1, LRBA and MPL. Our study demonstrates a synergy by using WGS for diagnosis and aetiological discovery in routine healthcare.

Human neutrophilic granulocytes form the largest pool of innate immune cells for host defense against bacterial and fungal pathogens. The dynamic changes that accompany the metamorphosis from a proliferating myeloid progenitor cell in the bone marrow into a mature non-dividing polymorphonuclear blood cell have remained poorly defined. Using mass spectrometry-based quantitative proteomics combined with transcriptomic data, we report on the dynamic changes of five developmental stages in the bone marrow and blood. Integration of transcriptomes and proteome unveils highly dynamic and differential interactions between RNA and protein kinetics during human neutrophil development, which can be linked to functional maturation of typical end-stage blood neutrophil killing activities. Human neutrophils form the largest pool of innate immune cells. Using mass spectrometry-based quantitative proteomics combined with transcriptomics, Hoogendijk et al. report on the dynamic changes of five developmental stages, unveiling highly dynamic RNA and protein kinetics that can be linked to functional maturation of end-stage blood neutrophils.

To identify novel causes of hereditary thrombocytopenia, we performed a genetic association analysis of whole-genome sequencing data from 13 037 individuals enrolled in the National Institute for Health Research (NIHR) BioResource, including 233 cases with isolated thrombocytopenia. We found an association between rare variants in the transcription factor-encoding gene IKZF5 and thrombocytopenia. We report 5 causal missense variants in or near IKZF5 zinc fingers, of which 2 occurred de novo and 3 co-segregated in 3 pedigrees. A canonical DNA-zinc finger binding model predicts that 3 of the variants alter DNA recognition. Expression studies showed that chromatin binding was disrupted in mutant compared with wild-type IKZF5, and electron microscopy revealed a reduced quantity of a granules in normally sized platelets. Proplatelet formation was reduced in megakaryocytes from 7 cases relative to 6 controls. Comparison of RNA-sequencing data from platelets, monocytes, neutrophils, and CD41 T cells from 3 cases and 14 healthy controls showed 1194 differentially expressed genes in platelets but only 4 differentially expressed genes in each of the other blood cell types. In conclusion, IKZF5 is a novel transcriptional regulator of megakaryopoiesis and the eighth transcription factor associated with dominant thrombocytopenia in humans.

In combination with microspotting, whole-blood microfluidics can provide high-throughput information on multiple platelet functions in thrombus formation. Based on assessment of the inter- and intra-subject variability in parameters of microspot-based thrombus formation, we aimed to determine the platelet factors contributing to this variation. Blood samples from 94 genotyped healthy subjects were analyzed for conventional platelet phenotyping: I.e. hematologic parameters, platelet glycoprotein (GP) expression levels and activation markers (24 parameters). Furthermore, platelets were activated by ADP, CRP-XL or TRAP. Parallel samples were investigated for whole-blood thrombus formation (6 microspots, providing 48 parameters of adhesion, aggregation and activation). Microspots triggered platelet activation through GP Ib-V-IX, GPVI, CLEC-2 and integrins. For most thrombus parameters, inter-subject variation was 2-4 times higher than the intra-subject variation. Principal component analyses indicated coherence between the majority of parameters for the GPVI-dependent microspots, partly linked to hematologic parameters, and glycoprotein expression levels. Prediction models identified parameters per microspot that were linked to variation in agonist-induced αIIbb3 activation and secretion. Common sequence variation of GP6 and FCER1G, associated with GPVI-induced αIIbb3 activation and secretion, affected parameters of GPVIand CLEC-2-dependent thrombus formation. Subsequent analysis of blood samples from patients with Glanzmann thrombasthenia or storage pool disease revealed thrombus signatures of aggregation-dependent parameters that were subject-dependent, but not linked to GPVI activity. Taken together, this high-throughput elucidation of thrombus formation revealed patterns of inter-subject differences in platelet function, which were partly related to GPVI-induced activation and common genetic variance linked to GPVI, but also included a distinct platelet aggregation component.

Sphingolipids are fundamental to membrane trafficking, apoptosis, and cell differentiation and proliferation. KDSR or 3-keto-dihy-drosphingosine reductase is an essential enzyme for de novo sphingolipid synthesis, and pathogenic mutations in KDSR result in the severe skin disorder erythrokeratodermia variabilis et progressiva-4. Four of the eight reported cases also had thrombocytopenia but the underlying mechanism has remained unexplored. Here we expand upon the phenotypic spectrum of KDSR deficiency with studies in two siblings with novel compound heterozygous variants associated with thrombocytopenia, anemia, and minimal skin involvement. We report a novel phenotype of progressive juvenile myelofibrosis in the propositus, with spontaneous recovery of anemia and thrombocytopenia in the first decade of life. Examination of bone marrow biopsies showed megakaryocyte hyperproliferation and dysplasia. Megakaryocytes obtained by culture of CD34+ stem cells confirmed hyperproliferation and showed reduced proplatelet formation. The effect of KDSR insufficiency on the sphingolipid profile was unknown, and was explored in vivo and in vitro by a broad metabolomics screen that indicated activation of an in vivo compensatory pathway that leads to normalization of downstream metabolites such as ceramide. Differentiation of propositus-derived induced pluripotent stem cells to megakaryocytes followed by expression of functional KDSR showed correction of the aberrant cellular and biochemical phenotypes, corroborating the critical role of KDSR in proplatelet formation. Finally, Kdsr depletion in zebrafish recapitulated the thrombocytopenia and showed biochemical changes similar to those observed in the affected siblings. These studies support an important role for sphingolipids as regulators of cytoskeletal organization during megakaryopoiesis and proplatelet formation.

Neutrophils are short-lived blood cells that play a critical role in host defense against infections. To better comprehend neutrophil functions and their regulation, we provide a complete epigenetic overview, assessing important functional features of their differentiation stages from bone marrow-residing progenitors to mature circulating cells. Integration of chromatin modifications, methylation, and transcriptome dynamics reveals an enforced regulation of differentiation, for cellular functions such as release of proteases, respiratory burst, cell cycle regulation, and apoptosis. We observe an early establishment of the cytotoxic capability, while the signaling components that activate these antimicrobial mechanisms are transcribed at later stages, outside the bone marrow, thus preventing toxic effects in the bone marrow niche. Altogether, these data reveal how the developmental dynamics of the chromatin landscape orchestrate the daily production of a large number of neutrophils required for innate host defense and provide a comprehensive overview of differentiating human neutrophils. Grassi et al. report that the establishment of transcriptional enhancers drives neutrophil differentiation. Coordinated waves of gene expression establish the cytotoxic capability of these cells at early stages of maturation. A set of super-enhancers is specifically opened at the end of the differentiation process to control neutrophil activation.

In the version of the article published, the surname of author Aaron Isaacs is misspelled as Issacs.

Cell fate transitions in mammalian stem cell systems have often been associated with transcriptional heterogeneity; however, existing data have failed to establish a functional or mechanistic link between the two phenomena. Experiments in unicellular organisms support the notion that transcriptional heterogeneity can be used to facilitate adaptability to environmental changes and have identified conserved chromatin-associated factors that modulate levels of transcriptional noise. Herein, we show destabilization of pluripotency-associated gene regulatory networks through increased transcriptional heterogeneity of mouse embryonic stem cells in which paradigmatic histone acetyl-transferase, and candidate noise modulator, Kat2a (yeast orthologue Gcn5), have been inhibited. Functionally, network destabilization associates with reduced pluripotency and accelerated mesendodermal differentiation, with increased probability of transitions into lineage commitment. Thus, we show evidence of a relationship between transcriptional heterogeneity and cell fate transitions through manipulation of the histone acetylation landscape of mouse embryonic stem cells, suggesting a general principle that could be exploited in other normal and malignant stem cell fate transitions. Stem Cells 2018;36:1828–11.

The DNA repair protein Cockayne syndrome group B (CSB) has been recently identified as a promising anticancer target. Suppression, by antisense technology, of this protein causes devastating effects on tumor cells viability, through a massive induction of apoptosis, while being non-toxic to non-transformed cells. To gain insights into the mechanisms underlying the pro-apoptotic effects observed after CSB ablation, global gene expression patterns were determined, to identify genes that were significantly differentially regulated as a function of CSB expression. Our findings revealed that response to endoplasmic reticulum stress and response to unfolded proteins were ranked top amongst the cellular processes affected by CSB suppression. The major components of the endoplasmic reticulum stressmediated apoptosis pathway, including pro-apoptotic factors downstream of the ATF3-CHOP cascade, were dramatically up-regulated. Altogether our findings add new pieces to the understanding of CSB mechanisms of action and to the molecular basis of CS syndrome.

Background: Autoimmune disease-associated variants are preferentially found in regulatory regions in immune cells, particularly CD4+ T cells. Linking such regulatory regions to gene promoters in disease-relevant cell contexts facilitates identification of candidate disease genes. Results: Within 4 h, activation of CD4+ T cells invokes changes in histone modifications and enhancer RNA transcription that correspond to altered expression of the interacting genes identified by promoter capture Hi-C. By integrating promoter capture Hi-C data with genetic associations for five autoimmune diseases, we prioritised 245 candidate genes with a median distance from peak signal to prioritised gene of 153 kb. Just under half (108/245) prioritised genes related to activation-sensitive interactions. This included IL2RA, where allele-specific expression analyses were consistent with its interaction-mediated regulation, illustrating the utility of the approach. Conclusions: Our systematic experimental framework offers an alternative approach to candidate causal gene identification for variants with cell state-specific functional effects, with achievable sample sizes.

Background Inflammation drives atherosclerotic plaque rupture. Although inflammation can be measured using fluorine-18-labeled fluorodeoxyglucose positron emission tomography ([18F]FDG PET), [18F]FDG lacks cell specificity, and coronary imaging is unreliable because of myocardial spillover. Objectives This study tested the efficacy of gallium-68-labeled DOTATATE (68Ga-DOTATATE), a somatostatin receptor subtype-2 (SST2)-binding PET tracer, for imaging atherosclerotic inflammation. Methods We confirmed 68Ga-DOTATATE binding in macrophages and excised carotid plaques. 68Ga-DOTATATE PET imaging was compared to [18F]FDG PET imaging in 42 patients with atherosclerosis. Results Target SSTR2 gene expression occurred exclusively in “proinflammatory” M1 macrophages, specific 68Ga-DOTATATE ligand binding to SST2 receptors occurred in CD68-positive macrophage-rich carotid plaque regions, and carotid SSTR2 mRNA was highly correlated with in vivo 68Ga-DOTATATE PET signals (r = 0.89; 95% confidence interval [CI]: 0.28 to 0.99; p = 0.02). 68Ga-DOTATATE mean of maximum tissue-to-blood ratios (mTBRmax) correctly identified culprit versus nonculprit arteries in patients with acute coronary syndrome (median difference: 0.69; interquartile range [IQR]: 0.22 to 1.15; p = 0.008) and transient ischemic attack/stroke (median difference: 0.13; IQR: 0.07 to 0.32; p = 0.003). 68Ga-DOTATATE mTBRmax predicted high-risk coronary computed tomography features (receiver operating characteristics area under the curve [ROC AUC]: 0.86; 95% CI: 0.80 to 0.92; p < 0.0001), and correlated with Framingham risk score (r = 0.53; 95% CI: 0.32 to 0.69; p 

Background: a healthy immune system requires immune cells that adapt rapidly to environmental challenges. This phenotypic plasticity can be mediated by transcriptional and epigenetic variability. Results: We apply a novel analytical approach to measure and compare transcriptional and epigenetic variability genome-wide across CD14+CD16-monocytes, CD66b+CD16+neutrophils, and CD4+CD45RA+naïve T cells from the same 125 healthy individuals. We discover substantially increased variability in neutrophils compared to monocytes and T cells. In neutrophils, genes with hypervariable expression are found to be implicated in key immune pathways and are associated with cellular properties and environmental exposure. We also observe increased sex-specific gene expression differences in neutrophils. Neutrophil-specific DNA methylation hypervariable sites are enriched at dynamic chromatin regions and active enhancers. Conclusions: Our data highlight the importance of transcriptional and epigenetic variability for the key role of neutrophils as the first responders to inflammatory stimuli. We provide a resource to enable further functional studies into the plasticity of immune cells, which can be accessed from: http://blueprint-dev.bioinfo.cnio.es/WP10/hypervariability.

Myeloproliferative neoplasms (MPNs) are a group of related clonal hemopoietic stem cell disorders associated with hyperproliferation of myeloid cells. They are driven by mutations in the hemopoietic stem cell, most notably JAK2V617F, CALR, and MPL. Clinically, they have the propensity to progress to myelofibrosis and transform to acute myeloid leukemia. Megakaryocytic hyperplasia with abnormal features are characteristic, and it is thought that these cells stimulate and drive fibrotic progression. The biological defects underpinning this remain to be explained. In this study we examined the megakaryocyte genome in 12 patients with MPNs to determine whether there are somatic variants and whether there is any association with marrow fibrosis. We performed targeted next-generation sequencing for 120 genes associated with myeloid neoplasms on megakaryocytes isolated from aspirated bone marrow. Ten of the 12 patients had genomic defects in megakaryocytes that were not present in nonmegakaryocytic hemopoietic marrow cells from the same patient. The greatest allelic burden was in patients with increased reticulin deposition. The megakaryocyte-unique mutations were predominantly in genes that regulate chromatin remodeling, chromosome alignment, and stability. These findings show that genomic abnormalities are present in megakaryocytes in MPNs and that these appear to be associated with progression to bone marrow fibrosis.

Linking non-coding genetic variants associated with the risk of diseases or disease-relevant traits to target genes is a crucial step to realize GWAS potential in the introduction of precision medicine. Here we set out to determine the mechanisms underpinning variant association with platelet quantitative traits using cell type-matched epigenomic data and promoter long-range interactions. We identify potential regulatory functions for 423 of 565 (75%) non-coding variants associated with platelet traits and we demonstrate, through ex vivo and proof of principle genome editing validation, that variants in super enhancers play an important role in controlling archetypical platelet functions.

Cardiosphere-derived cell (CDC) infusion into damaged myocardium has shown some reparative effect; this could be improved by better selection of patients and cell subtype. CDCs isolated from patients with ischemic heart disease are able to support vessel formation in vitro but this ability varies between patients. The primary aim of our study was to investigate whether the vascular supportive function of CDCs impacts on their therapeutic potential, with the goal of improving patient stratification. A subgroup of patients produced CDCs which did not efficiently support vessel formation (poor supporter CDCs), had reduced levels of proliferation and increased senescence, despite them being isolated in the same manner and having a similar immunophenotype to CDCs able to support vessel formation. In a rodent model of myocardial infarction, poor supporter CDCs had a limited reparative effect when compared to CDCs which had efficiently supported vessel formation in vitro. This work suggests that not all patients provide cells which are suitable for cell therapy. Assessing the vascular supportive function of cells could be used to stratify which patients will truly benefit from cell therapy and those who would be better suited to an allogeneic transplant or regenerative preconditioning of their cells in a precision medicine fashion. This could reduce costs, culture times and improve clinical outcomes and patient prognosis.

Genome-wide association studies have identified a genetic variant at 3p14.3 (SNP rs1354034) that strongly associates with platelet number and mean platelet volume in humans. While originally proposed to be intronic, analysis of mRNA expression in primary human hematopoietic subpopulations reveals that this SNP is located directly upstream of the predominantly expressed ARHGEF3 isoform in megakaryocytes (MK). We found that ARHGEF3, which encodes a Rho guanine exchange factor, is dramatically upregulated during both human and murine MK maturation. We show that the SNP (rs1354034) is located in a DNase I hypersensitive region in human MKs and is an expression quantitative locus (eQTL) associated with ARHGEF3 expression level in human platelets, suggesting that it may be the causal SNP that accounts for the variations observed in human platelet traits and ARHGEF3 expression. In vitro human platelet activation assays revealed that rs1354034 is highly correlated with human platelet activation by ADP. In order to test whether ARHGEF3 plays a role in MK development and/or platelet function, we developed an Arhgef3 KO/LacZ reporter mouse model. Reflecting changes in gene expression, LacZ expression increases during MK maturation in these mice. Although Arhgef3 KO mice have significantly larger platelets, loss of Arhgef3 does not affect baseline MK or platelets nor does it affect platelet function or platelet recovery in response to antibody-mediated platelet depletion compared to littermate controls. In summary, our data suggest that modulation of ARHGEF3 gene expression in humans with a promoter-localized SNP plays a role in human MKs and human platelet function - a finding resulting from the biological follow-up of human genetic studies. Arhgef3 KO mice partially recapitulate the human phenotype.

Hematopoietic stem cells give rise to all blood cells in a differentiation process that involves widespread epigenome remodeling. Here we present genome-wide reference maps of the associated DNA methylation dynamics. We used a meta-epigenomic approach that combines DNA methylation profiles across many small pools of cells and performed single-cell methylome sequencing to assess cell-to-cell heterogeneity. The resulting dataset identified characteristic differences between HSCs derived from fetal liver, cord blood, bone marrow, and peripheral blood. We also observed lineage-specific DNA methylation between myeloid and lymphoid progenitors, characterized immature multi-lymphoid progenitors, and detected progressive DNA methylation differences in maturing megakaryocytes. We linked these patterns to gene expression, histone modifications, and chromatin accessibility, and we used machine learning to derive a model of human hematopoietic differentiation directly from DNA methylation data. Our results contribute to a better understanding of human hematopoietic stem cell differentiation and provide a framework for studying blood-linked diseases.

Large-scale whole-genome sequence data sets offer novel opportunities to identify genetic variation underlying human traits. Here we apply genotype imputation based on whole-genome sequence data from the UK10K and 1000 Genomes Project into 35,981 study participants of European ancestry, followed by association analysis with 20 quantitative cardiometabolic and hematological traits. We describe 17 new associations, including 6 rare (minor allele frequency (MAF) < 1%) or low-frequency (1% < MAF < 5%) variants with platelet count (PLT), red blood cell indices (MCH and MCV) and HDL cholesterol. Applying fine-mapping analysis to 233 known and new loci associated with the 20 traits, we resolve the associations of 59 loci to credible sets of 20 or fewer variants and describe trait enrichments within regions of predicted regulatory function. These findings improve understanding of the allelic architecture of risk factors for cardiometabolic and hematological diseases and provide additional functional insights with the identification of potentially novel biological targets.

DNA methylation and the localization and post-translational modification of nucleosomes are interdependent factors that contribute to the generation of distinct phenotypes from genetically identical cells. With 112 whole-genome bisulfite sequencing datasets from the BLUEPRINT Epigenome Project, we analyzed the global development of DNA methylation patterns during lineage commitment and maturation of a range of immune system effector cells and the cancers that arise from them. We show clear trends in methylation patterns that are distinct in the innate and adaptive arms of the human immune system, both globally and in relation to consistently positioned nucleosomes. Most notable are a progressive loss of methylation in developing lymphocytes and the consistent occurrence of non-CG methylation in specific cell types. Cancer samples from the two lineages are further polarized, suggesting the involvement of distinct lineage-specific epigenetic mechanisms. We anticipate broad utility for this resource as a basis for further comparative epigenetic analyses.

Aging, chronic inflammation, and environmental insults play an important role in a number of disease processes through alterations of the epigenome. In this review we explore how age-related changes in the epigenetic landscape can affect heterogeneity within the haematopoietic stem cell (HSC) compartment and the deriving clinical implications.

Characterizing the multifaceted contribution of genetic and epigenetic factors to disease phenotypes is a major challenge in human genetics and medicine. We carried out high-resolution genetic, epigenetic, and transcriptomic profiling in three major human immune cell types (CD14+ monocytes, CD16+ neutrophils, and naive CD4+ T cells) from up to 197 individuals. We assess, quantitatively, the relative contribution of cis-genetic and epigenetic factors to transcription and evaluate their impact as potential sources of confounding in epigenome-wide association studies. Further, we characterize highly coordinated genetic effects on gene expression, methylation, and histone variation through quantitative trait locus (QTL) mapping and allele-specific (AS) analyses. Finally, we demonstrate colocalization of molecular trait QTLs at 345 unique immune disease loci. This expansive, high-resolution atlas of multi-omics changes yields insights into cell-type-specific correlation between diverse genomic inputs, more generalizable correlations between these inputs, and defines molecular events that may underpin complex disease risk.

The incidence of type 1 diabetes (T1D) has substantially increased over the past decade, suggesting a role for non-genetic factors such as epigenetic mechanisms in disease development. Here we present an epigenome-wide association study across 406,365 CpGs in 52 monozygotic twin pairs discordant for T1D in three immune effector cell types. We observe a substantial enrichment of differentially variable CpG positions (DVPs) in T1D twins when compared with their healthy co-twins and when compared with healthy, unrelated individuals. These T1D-associated DVPs are found to be temporally stable and enriched at gene regulatory elements. Integration with cell type-specific gene regulatory circuits highlight pathways involved in immune cell metabolism and the cell cycle, including mTOR signalling. Evidence from cord blood of newborns who progress to overt T1D suggests that the DVPs likely emerge after birth. Our findings, based on 772 methylomes, implicate epigenetic changes that could contribute to disease pathogenesis in T1D.

The cost of whole-genome bisulfite sequencing (WGBS) remains a bottleneck for many studies and it is therefore imperative to extract as much information as possible from a given dataset. This is particularly important because even at the recommend 30X coverage for reference methylomes, up to 50% of high-resolution features such as differentially methylated positions (DMPs) cannot be called with current methods as determined by saturation analysis. To address this limitation, we have developed a tool that dynamically segments WGBS methylomes into blocks of comethylation (COMETs) from which lost information can be recovered in the form of differentially methylated COMETs (DMCs). Using this tool, we demonstrate recovery of â1/430% of the lost DMP information content as DMCs even at very low (5X) coverage. This constitutes twice the amount that can be recovered using an existing method based on differentially methylated regions (DMRs). In addition, we explored the relationship between COMETs and haplotypes in lymphoblastoid cell lines of African and European origin. Using best fit analysis, we show COMETs to be correlated in a population-specific manner, suggesting that this type of dynamic segmentation may be useful for integrated (epi)genome-wide association studies in the future.

Long-range interactions between regulatory elements and gene promoters play key roles in transcriptional regulation. The vast majority of interactions are uncharted, constituting a major missing link in understanding genome control. Here, we use promoter capture Hi-C to identify interacting regions of 31,253 promoters in 17 human primary hematopoietic cell types. We show that promoter interactions are highly cell type specific and enriched for links between active promoters and epigenetically marked enhancers. Promoter interactomes reflect lineage relationships of the hematopoietic tree, consistent with dynamic remodeling of nuclear architecture during differentiation. Interacting regions are enriched in genetic variants linked with altered expression of genes they contact, highlighting their functional role. We exploit this rich resource to connect non-coding disease variants to putative target promoters, prioritizing thousands of disease-candidate genes and implicating disease pathways. Our results demonstrate the power of primary cell promoter interactomes to reveal insights into genomic regulatory mechanisms underlying common diseases.

Many common variants have been associated with hematological traits, but identification of causal genes and pathways has proven challenging. We performed a genome-wide association analysis in the UK Biobank and INTERVAL studies, testing 29.5 million genetic variants for association with 36 red cell, white cell, and platelet properties in 173,480 European-ancestry participants. This effort yielded hundreds of low frequency (

The International Human Epigenome Consortium (IHEC) coordinates the generation of a catalog of high-resolution reference epigenomes of major primary human cell types. The studies now presented (see the Cell Press IHEC web portal at http://www.cell.com/consortium/IHEC) highlight the coordinated achievements of IHEC teams to gather and interpret comprehensive epigenomic datasets to gain insights in the epigenetic control of cell states relevant for human health and disease. PaperClip

Epigenome-wide association studies (EWAS) provide an alternative approach for studying human disease through consideration of non-genetic variants such as altered DNA methylation. To advance the complex interpretation of EWAS, we developed eFORGE (http://eforge.cs.ucl.ac.uk/), a new standalone and web-based tool for the analysis and interpretation of EWAS data. eFORGE determines the cell type-specific regulatory component of a set of EWAS-identified differentially methylated positions. This is achieved by detecting enrichment of overlap with DNase I hypersensitive sites across 454 samples (tissues, primary cell types, and cell lines) from the ENCODE, Roadmap Epigenomics, and BLUEPRINT projects. Application of eFORGE to 20 publicly available EWAS datasets identified disease-relevant cell types for several common diseases, a stem cell-like signature in cancer, and demonstrated the ability to detect cell-composition effects for EWAS performed on heterogeneous tissues. Our approach bridges the gap between large-scale epigenomics data and EWAS-derived target selection to yield insight into disease etiology.

The CSB protein, a member of the SWI/SNF ATP dependent chromatin remodeling family of proteins, plays a role in a sub-pathway of nucleotide excision repair (NER) known as transcription coupled repair (TCR). CSB is frequently mutated in Cockayne syndrome group B, a segmental progeroid human autosomal recessive disease characterized by growth failure and degeneration of multiple organs. Though initially classified as a DNA repair protein, recent studies have demonstrated that the loss of CSB results in pleiotropic effects. Identification of novel proteins belonging to the CSB interactome may be useful not only for predicting the molecular basis for diverse pathological symptoms of CS-B patients but also for unraveling the functions of CSB in addition to its authentic role in DNA repair. In this study, we performed tandem affinity purification (TAP) technology coupled with mass spectrometry and co-immunoprecipitation studies to identify and characterize the proteins that potentially interact with CSB-TAP. Our approach revealed 33 proteins that were not previously known to interact with CSB. These newly identified proteins indicate potential roles for CSB in RNA metabolism involving repression and activation of transcription process and in the maintenance of chromatin dynamics and integrity.

Monocyte differentiation into macrophages represents a cornerstone process for host defense. Concomitantly, immunological imprinting of either tolerance or trained immunity determines the functional fate of macrophages and susceptibility to secondary infections. We characterized the transcriptomes and epigenomes in four primary cell types: monocytes and in vitro-differentiated naïve, tolerized, and trained macrophages. Inflammatory and metabolic pathways were modulated in macrophages, including decreased inflammasome activation, and we identified pathways functionally implicated in trained immunity, ß-glucan training elicits an exclusive epigenetic signature, revealing a complex network of enhancers and promoters. Analysis of transcription factor motifs in deoxyribonuclease I hypersensitive sites at cell-type-specific epigenetic loci unveiled differentiation and treatment-specific repertoires. Altogether, we provide a resource to understand the epigenetic changes that underlie innate immunity in humans.

Cockayne syndrome (CS) is a progressive developmental and neurodegenerative disorder resulting in premature death at childhood and cells derived from CS patients display DNA repair and transcriptional defects. CS is caused by mutations in csa and csb genes, and patients with csb mutation are more prevalent. A hallmark feature of CSB patients is neurodegeneration but the precise molecular cause for this defect remains enigmatic. Further, it is not clear whether the neurodegenerative condition is due to loss of CSB-mediated functions in adult neurogenesis. In this study, we examined the role of CSB in neurogenesis by using the human neural progenitor cells that have self-renewal and differentiation capabilities. In this model system, stable CSB knockdown dramatically reduced the differentiation potential of human neural progenitor cells revealing a key role for CSB in neurogenesis. Neurite outgrowth, a characteristic feature of differentiated neurons, was also greatly abolished in CSB-suppressed cells. In corroboration with this, expression of MAP2 (microtubule-associated protein 2), a crucial player in neuritogenesis, was also impaired in CSB-suppressed cells. Consistent with reduced MAP2 expression in CSB-depleted neural cells, tandem affinity purification and chromatin immunoprecipitation studies revealed a potential role for CSB in the assembly of transcription complex on MAP2 promoter. Altogether, our data led us to conclude that CSB has a crucial role in coordinated regulation of transcription and chromatin remodeling activities that are required during neurogenesis.

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor l/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine.

Blood cells derive from hematopoietic stem cells through stepwise fating events. To characterize gene expression programs driving lineage choice, we sequenced RNA from eight primary human hematopoietic progenitor populations representing the major myeloid commitment stages and the main lymphoid stage. We identified extensive cell type-specific expression changes: 6711 genes and 10,724 transcripts, enriched in non-protein-coding elements at early stages of differentiation. In addition, we found 7881 novel splice junctions and 2301 differentially used alternative splicing events, enriched in genes involved in regulatory processes. We demonstrated experimentally cell-specific isoform usage, identifying nuclear factor I/B (NFIB) as a regulator of megakaryocyte maturation-the platelet precursor. Our data highlight the complexity of fating events in closely related progenitor populations, the understanding of which is essential for the advancement of transplantation and regenerative medicine. Copyright © 2014, American Association for the Advancement of Science.

The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10-15). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors. © 2013 Nature America, Inc. All rights reserved.

The blood group Vel was discovered 60 years ago, but the underlying gene is unknown. Individuals negative for the Vel antigen are rare and are required for the safe transfusion of patients with antibodies to Vel. To identify the responsible gene, we sequenced the exomes of five individuals negative for the Vel antigen and found that four were homozygous and one was heterozygous for a low-frequency 17-nucleotide frameshift deletion in the gene encoding the 78-amino-acid transmembrane protein SMIM1. A follow-up study showing that 59 of 64 Vel-negative individuals were homozygous for the same deletion and expression of the Vel antigen on SMIM1-transfected cells confirm SMIM1 as the gene underlying the Vel blood group. An expression quantitative trait locus (eQTL), the common SNP rs1175550 contributes to variable expression of the Vel antigen (P = 0.003) and influences the mean hemoglobin concentration of red blood cells (RBCs; P = 8.6 × 10(-15)). In vivo, zebrafish with smim1 knockdown showed a mild reduction in the number of RBCs, identifying SMIM1 as a new regulator of RBC formation. Our findings are of immediate relevance, as the homozygous presence of the deletion allows the unequivocal identification of Vel-negative blood donors.

In the present study we show that a number of cancer cell lines from different tissues display dramatically increased expression of the Cockayne Syndrome group B (CSB) protein, a DNA repair factor, that has recently been shown to be involved in cell robustness. Furthermore, we demonstrated that ablation of this protein by antisense technology causes devastating effects on tumor cells through a drastic reduction of cell proliferation and massive induction of apoptosis, while non-transformed cells remain unaffected. Finally, suppression of CSB in cancer cells makes these cells hypersensitive to a variety of commonly used cancer chemotherapeutic agents. Based on these results, we conclude that cancer cells overexpress CSB protein in order to enhance their anti-apoptotic capacity. The fact that CSB suppression specifically affects only cancerous cells, without harming healthy cells, suggests that CSB may be a very attractive target for the development of new anticancer therapies. © 2013 Elsevier B.V.

The CSB protein plays a role in the transcription coupled repair (TCR) branch of the nucleotide excision repair pathway. CSB is very often found mutated in Cockayne syndrome, a segmental progeroid genetic disease characterized by organ degeneration and growth failure. The tumor suppressor p53 plays a pivotal role in triggering senescence and apoptosis and suppressing tumorigenesis. Although p53 is very important to avoid cancer, its excessive activity can be detrimental for the lifespan of the organism. This is why a network of positive and negative feedback loops, which most likely evolved to fine-tune the activity of this tumor suppressor, modulate its induction and activation. Accordingly, an unbalanced p53 activity gives rise to premature aging or cancer. The physical interaction between CSB and p53 proteins has been known for more than a decade but, despite several hypotheses, nobody has been able to show the functional consequences of this interaction. In this review we resume recent advances towards a more comprehensive understanding of the critical role of this interaction in modulating p53's levels and activity, therefore helping the system find a reasonable equilibrium between the beneficial and the detrimental effects of its activity. This crosstalk re-establishes the physiological balance towards cell proliferation and survival instead of towards cell death, after stressors of a broad nature. Accordingly, cells bearing mutations in the csb gene are unable to re-establish this physiological balance and to properly respond to some stress stimuli and undergo massive apoptosis. © Frontini and Proietti-De-Santis.

CKS proteins are evolutionarily conserved cyclin-dependent kinase (CDK) subunits whose functions are incompletely understood. Mammals have two CKS proteins. CKS1 acts as a cofactor to the ubiquitin ligase complex SCFSKP2 to promote degradation of CDK inhibitors, such as p27. Little is known about the role of the closely related CKS2. Using a Cks2-/- knockout mouse model, we show that CKS2 counteracts CKS1 and stabilizes p27. Unopposed CKS1 activity in Cks2-/- cells leads to loss of p27. The resulting unrestricted cyclin A/CDK2 activity is accompanied by shortening of the cell cycle, increased replication fork velocity, and DNA damage. In vivo, Cks2-/- cortical progenitor cells are limited in their capacity to differentiate into mature neurons, a phenotype akin to animals lacking p27. We propose that the balance between CKS2 and CKS1 modulates p27 degradation, and with it cyclin A/CDK2 activity, to safeguard replicative fidelity and control neuronal differentiation.

Mutations in Cockayne syndrome (CS) a and B genes (CSA and CSB) result in a rare genetic disease that affects the development and homeostasis of a wide range of tissues and organs. We previously correlated the degenerative phenotype of patients to the enhanced apoptotic response, exhibited by CS cells, which is associated with the exceptional induction of p53 protein upon a variety of stress stimuli. Here we show that the elevated and persistent levels of p53 displayed by CS cells are due to the insufficient ubiquitination of p53. We further demonstrate that CSA and CSB proteins are part of a Cullin Ring Ubiquitin Ligase complex with p53 and Mdm2; this interaction greatly stimulates the ubiquitination of p53 in an Mdm2-dependent manner. Finally, we have found that p53 binds to the CSB promoter and transcriptionally controls the expression of csb gene allowing the establishment of a negative feedback loop that causes p53 to return at basal levels. This study identifies CSA and CSB as the key elements of a regulatory mechanism that equilibrate beneficial and detrimental effects of p53 activity upon cellular stress. The deregulation of p53, in absence of either of the CS proteins, can potentially explain the early onset degeneration of tissues and organs observed in CS patients. © 2011 Landes Bioscience.

Pole3 (DPB4/YBL1/CHRAC17) is one of the subunits of the DNA polymerase e. It contains a histone-like domain required for the hererodimerization with its Pole4 (DPB3) partner. In another interaction, Pole3 heterodimerizes with YCL1/CHRAC15 and associates with the ACF1/SNF2H remodelling complex. We find that the Pol3 gene is regulated in starved NIH3T3 fibroblasts upon induction with serum, with a peak at the entry in the S phase. We characterized the Pole3 promoter, which is linked bidirectionally to C9Orf46, a gene of unknown function: it has no CCAAT nor TATA-boxes, and contains an E box and two potential E2F sites. Mutagenesis analysis points to a minimal promoter region as sufficient for activation; the E box and a neighbouring direct repeat are important for regulation. Cell-cycle regulation was reproduced in stable clones and an additional E2F site was found to be important. Chromatin immunoprecipitation analysis indicates that E2F1/4, as well as MYC, are associated with the Pole3 promoter in a phase-specific way. These data highlight coregulation of a histone-like gene with core histones upon DNA synthesis, and represent a first dissection of the interplay between two essential cell-cycle regulators on a bidirectional promoter.

TFIID, composed of the TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs), plays a role in nucleating the assembly of the RNA polymerase II preinitiation complexes on protein coding genes. TAF10 (formerly TAF II30) is shared between TFIID and other transcription regulatory complexes (i.e. SAGA, TFTC, STAGA and PCAF/GCN5). TAF10 is an essential transcription factor during very early stages of mouse embryo development. To study the in vivo function of TAF10 in cellular differentiation and proliferation at later stages, the role of TAF10 was analysed in keratinocytes during skin development and adult epidermal homeostasis. We demonstrate that ablation of TAF10 in keratinocytes of the forming epidermis affects the expression of some, but not all genes, impairs keratinocyte terminal differentiation and alters skin permeability barrier functions. In contrast, loss of TAF10 in keratinocytes of adult epidermis did not (i) modify the expression of tested genes, (ii) affect epidermal homeostasis and (iii) impair acute response to UV irradiation or skin regeneration after wounding. Thus, this study demonstrates for the first time a differential in vivo requirement for a mammalian TAF for the regulation of gene expression depending on the cellular environment and developmental stage of the cell. © 2005 Elsevier Inc. All rights reserved.

TFIID plays a key role in transcription initiation of RNA polymerase II preinitiation complex assembly. TFIID is comprised of the TATA box binding protein (TBP) and 14 TBP-associated factors (TAFs). A second set of transcriptional regulatory multiprotein complexes containing TAFs has been described (called SAGA, TFTC, STAGA, and PCAF/GCN5). Using matrix-assisted laser desorption ionization mass spectrometry, we identified a novel TFTC subunit, human TAF9Like, encoded by a TAF9 paralogue gene. We show that TAF9Like is a subunit of TFIID, and thus, it will be called TAF9b. TFIID and TFTC complexes in which both TAF9 and TAF9b are present exist. In vitro and in vivo experiments indicate that the interactions between TAF9b and TAF6 or TAF9 and TAF6 histone fold pairs are similar. We observed a differential induction of TAF9 and TAF9b during apoptosis that, together with their different ability to stabilize p53, points to distinct requirements for the two proteins in gene regulation. Small interfering RNA (siRNA) knockdown of TAF9 and TAF9b revealed that both genes are essential for cell viability. Gene expression analysis of cells treated with either TAF9 or TAF9b siRNAs indicates that the two proteins regulate different sets of genes with only a small overlap. Taken together, these data demonstrate that TAF9 and TAF9b share some of their functions, but more importantly, they have distinct roles in the transcriptional regulatory process. Copyright © 2005, American Society for Microbiology. All Rights Reserved.

NF-Y is a trimeric activator with histone fold-HFM-subunits that binds to the CCAAT-box and is required for a majority of cell cycle promoters, often in conjuction with E2Fs. In vivo binding of NF-Y is dynamic during the cell cycle and correlates with gene activation. We performed immunofluorescence studies on endogenous, GFP- and Flag-tagged overexpressed NF-Y subunits. NF-YA, NF-YB are nuclear proteins. Unexpectedly, NF-YC localizes both in cytoplamatic and nuclear compartments and its nuclear localization is determined by the interaction with its heterodimerization partner NF-YB. Most importantly, compartmentalization is regulated during the cell cycle of serum restimulated NIH3T3 cells, accumulating in the nucleus at the onset of S phase. These data point to the control of HFM heterodimerization as an important layer of NF-Y regulation during cell cycle progression.

In a variety of Drosophila TATA-less promoters, transcription is directed by initiator (Inr) sequences, which are faithfully and efficiently recognized only when flanked 3′ by the downstream promoter element (DPE). This motif, which is conserved at ∼30 bp from the RNA start site, is viewed as a downstream counterpart to the TATA box, and is recognized by the general transcription factor (TF) IID. By transient expression assays in human embryonic kidney 293 cells, we show that DE1 (distal element 1), a DNA motif located at residues +23 to +29, sustains faithful Inr-dependent transcription as efficiently as the DPE. Transcription significantly increased when DE1 and DPE sequences were adjacently placed on the same template. Results emerging from in vivo RNA analyses matched electrophoretic mobility shift assay data. In agarose-electrophoretic mobility shift assays, retarded DNA-protein complexes resulting from the interaction of human holo-TFIID with either Inr+/DPE+ or Inr+/DE1+ promoters were formed at comparable levels, whereas binding of TFIID to both DE1 and DPE motifs was 2-fold increased. The strict requirement for spacing between the Inr and DPE was not observed for DE1, as locating the motif 4 bp away from the +1 site did not impair transcriptional enhancement. DE1 sequences may be common to many promoters and be overlooked because of their poor sequence homology.

The nuclear factor y (NF-Y) trimer and TFIID contain histone fold subunits, and their binding to the CCAAT and Initiator elements of the major histocompatibility complex class II Ea promoter is required for transcriptional activation. Using agarose-electrophoretic mobility shift assay we found that NF-Y increases the affinity of holo-TFIID for Ea in a CCAAT- and Inr-dependent manner. We began to dissect the interplay between NF-Y- and TBP-associated factors PO1II (TAFIIs)-containing histone fold domains in protein-protein interactions and transfections. hTAFII20, hTAFII28, and hTAFII18-hTAFII28 bind to the NF-Y B-NF-YC histone fold dimer; hTAFII80 and hTAFII31-hTAFII80 interact with the trimer but not with the NF-YB-NF-YC dimer. The histone fold α2 helix of hTAFII80 is not required for NF-Y association, as determined by interactions with the naturally occurring splice variant hTAFII80δ. Expression of hTAFII28 and hTAFII18 in mouse cells significantly and specifically reduced NF-Y activation in GAL4-based experiments, whereas hTAFII20 and hTAFII135 increased it. These results indicate that NF-Y (i) recruits purified holo-TFIID in vitro and (ii) can associate multiple TAFIIs, potentially accommodating different core promoter architectures.

NF-Y is a conserved trimeric transcriptional activator with an extremely high specificity for CCAAT boxes. The NF-YB and NF-YC subunits have histone fold motifs with a high degree of homology to NC2α/β, a TBP-binding repressor. The histone fold is composed of three α helices, α1, α2, α3, separated by short loops. Structural data on core histones showed that α1 are involved in DNA-binding. To understand the molecular basis of NF-Y sequence-specificity, we constructed deletion and swapping mutants, in which the α1 of NC2 and archeal HMfB, a bona fide histonic protein, was placed in NF-YB and NF-YC. Our analysis indicates that (i) subunit interactions are normal; (ii) NF-YB-NF-YC and NC2α/β do not form heterodimers and NC2 cannot associate NF-YA. (iii) None of the NF-Y swaps can complex with TBP on a TATA box. (iv) Specific residues, R47 and K49 in NF-YC and N61 in NF-YB, are crucial for CCAAT-binding. We conclude that specificity of the NF-Y trimer is not due to NF-YA only, but stems in part from the contribution of the histone fold α1, particularly that of NF-YB.