BloodChIP Xtra: an expanded database of comparative genome-wide transcription factor binding and gene-expression profiles in healthy human stem/progenitor subsets and leukemic cells.

The BloodChIP Xtra database (http://bloodchipXtra.vafaeelab.com/) facilitates genome-wide exploration and visualization of transcription factor (TF) occupancy and chromatin configuration in rare primary human hematopoietic stem (HSC-MPP) and progenitor (CMP, GMP, MEP) cells and acute myeloid leukemia (AML) cell lines (KG-1, ME-1, Kasumi1, TSU-1621-MT), along with chromatin accessibility and gene expression data from these and primary patient AMLs. BloodChIP Xtra features significantly more datasets than our earlier database BloodChIP (two primary cell types and two cell lines). Improved methodologies for determining TF occupancy and chromatin accessibility have led to increased availability of data for rare primary cell types across the spectrum of healthy and AML hematopoiesis. However, there is a continuing need for these data to be integrated in an easily accessible manner for gene-based queries and use in downstream applications. Here, we provide a user-friendly database based around genome-wide binding profiles of key hematopoietic TFs and histone marks in healthy stem/progenitor cell types. These are compared with binding profiles and chromatin accessibility derived from primary and cell line AML and integrated with expression data from corresponding cell types. All queries can be exported to construct TF-gene and protein-protein networks and evaluate the association of genes with specific cellular processes.

Demethylation and Up-Regulation of an Oncogene after Hypomethylating Therapy.

BACKGROUND: Although hypomethylating agents are currently used to treat patients with cancer, whether they can also reactivate and up-regulate oncogenes is not well elucidated. METHODS: We examined the effect of hypomethylating agents on SALL4, a known oncogene that plays an important role in myelodysplastic syndrome and other cancers. Paired bone marrow samples that were obtained from two cohorts of patients with myelodysplastic syndrome before and after treatment with a hypomethylating agent were used to explore the relationships among changes in SALL4 expression, treatment response, and clinical outcome. Leukemic cell lines with low or undetectable SALL4 expression were used to study the relationship between SALL4 methylation and expression. A locus-specific demethylation technology, CRISPR-DNMT1-interacting RNA (CRISPR-DiR), was used to identify the CpG island that is critical for SALL4 expression. RESULTS: SALL4 up-regulation after treatment with hypomethylating agents was observed in 10 of 25 patients (40%) in cohort 1 and in 13 of 43 patients (30%) in cohort 2 and was associated with a worse outcome. Using CRISPR-DiR, we discovered that demethylation of a CpG island within the 5' untranslated region was critical for SALL4 expression. In cell lines and patients, we confirmed that treatment with a hypomethylating agent led to demethylation of the same CpG region and up-regulation of SALL4 expression. CONCLUSIONS: By combining analysis of patient samples with CRISPR-DiR technology, we found that demethylation and up-regulation of an oncogene after treatment with a hypomethylating agent can indeed occur and should be further studied. (Funded by Associazione Italiana per la Ricerca sul Cancro and others.).

Contribution of mutant HSC clones to immature and mature cells in MDS and CMML, and variations with AZA therapy.

Myelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of individual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.

Genome-wide transcription factor-binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs.

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.

p57Kip2 regulates embryonic blood stem cells by controlling sympathoadrenal progenitor expansion.

Hematopoietic stem cells (HSCs) are of major clinical importance, and finding methods for their in vitro generation is a prime research focus. We show here that the cell cycle inhibitor p57Kip2/Cdkn1c limits the number of emerging HSCs by restricting the size of the sympathetic nervous system (SNS) and the amount of HSC-supportive catecholamines secreted by these cells. This regulation occurs at the SNS progenitor level and is in contrast to the cell-intrinsic function of p57Kip2 in maintaining adult HSCs, highlighting profound differences in cell cycle requirements of adult HSCs compared with their embryonic counterparts. Furthermore, this effect is specific to the aorta-gonad-mesonephros (AGM) region and shows that the AGM is the main contributor to early fetal liver colonization, as early fetal liver HSC numbers are equally affected. Using a range of antagonists in vivo, we show a requirement for intact ß2-adrenergic signaling for SNS-dependent HSC expansion. To gain further molecular insights, we have generated a single-cell RNA-sequencing data set of all Ngfr+ sympathoadrenal cells around the dorsal aorta to dissect their differentiation pathway. Importantly, this not only defined the relevant p57Kip2-expressing SNS progenitor stage but also revealed that some neural crest cells, upon arrival at the aorta, are able to take an alternative differentiation pathway, giving rise to a subset of ventrally restricted mesenchymal cells that express important HSC-supportive factors. Neural crest cells thus appear to contribute to the AGM HSC niche via 2 different mechanisms: SNS-mediated catecholamine secretion and HSC-supportive mesenchymal cell production.

Acute myeloid leukemia requires Hhex to enable PRC2-mediated epigenetic repression of Cdkn2a.

Unlike clustered HOX genes, the role of nonclustered homeobox gene family members in hematopoiesis and leukemogenesis has not been extensively studied. Here we found that the hematopoietically expressed homeobox gene Hhex is overexpressed in acute myeloid leukemia (AML) and is essential for the initiation and propagation of MLL-ENL-induced AML but dispensable for normal myelopoiesis, indicating a specific requirement for Hhex for leukemic growth. Loss of Hhex leads to expression of the Cdkn2a-encoded tumor suppressors p16(INK4a) and p19(ARF), which are required for growth arrest and myeloid differentiation following Hhex deletion. Mechanistically, we show that Hhex binds to the Cdkn2a locus and directly interacts with the Polycomb-repressive complex 2 (PRC2) to enable H3K27me3-mediated epigenetic repression. Thus, Hhex is a potential therapeutic target that is specifically required for AML stem cells to repress tumor suppressor pathways and enable continued self-renewal.

Analysing high-throughput sequencing data in Python with HTSeq 2.0.

SUMMARY: HTSeq 2.0 provides a more extensive application programming interface including a new representation for sparse genomic data, enhancements for htseq-count to suit single-cell omics, a new script for data using cell and molecular barcodes, improved documentation, testing and deployment, bug fixes and Python 3 support. AVAILABILITY AND IMPLEMENTATION: HTSeq 2.0 is released as an open-source software under the GNU General Public License and is available from the Python Package Index at https://pypi.python.org/pypi/HTSeq. The source code is available on Github at https://github.com/htseq/htseq. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Disruption of a GATA2-TAL1-ERG regulatory circuit promotes erythroid transition in healthy and leukemic stem cells.

Changes in gene regulation and expression govern orderly transitions from hematopoietic stem cells to terminally differentiated blood cell types. These transitions are disrupted during leukemic transformation, but knowledge of the gene regulatory changes underpinning this process is elusive. We hypothesized that identifying core gene regulatory networks in healthy hematopoietic and leukemic cells could provide insights into network alterations that perturb cell state transitions. A heptad of transcription factors (LYL1, TAL1, LMO2, FLI1, ERG, GATA2, and RUNX1) bind key hematopoietic genes in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and have prognostic significance in acute myeloid leukemia (AML). These factors also form a densely interconnected circuit by binding combinatorially at their own, and each other's, regulatory elements. However, their mutual regulation during normal hematopoiesis and in AML cells, and how perturbation of their expression levels influences cell fate decisions remains unclear. In this study, we integrated bulk and single-cell data and found that the fully connected heptad circuit identified in healthy HSPCs persists, with only minor alterations in AML, and that chromatin accessibility at key heptad regulatory elements was predictive of cell identity in both healthy progenitors and leukemic cells. The heptad factors GATA2, TAL1, and ERG formed an integrated subcircuit that regulates stem cell-to-erythroid transition in both healthy and leukemic cells. Components of this triad could be manipulated to facilitate erythroid transition providing a proof of concept that such regulatory circuits can be harnessed to promote specific cell-type transitions and overcome dysregulated hematopoiesis.

Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys.

Mouse kidney parvovirus (MKPV) is a member of the provisional genus Chapparvovirus that causes renal disease in immune-compromised mice, with a disease course reminiscent of polyomavirus-associated nephropathy in immune-suppressed kidney transplant patients. Here we map four major MKPV transcripts, created by alternative splicing, to a common initiator region, and use mass spectrometry to identify "p10" and "p15" as novel chapparvovirus accessory proteins produced in MKPV-infected kidneys. p15 and the splicing-dependent putative accessory protein NS2 are conserved in all near-complete amniote chapparvovirus genomes currently available (from mammals, birds and a reptile). In contrast, p10 may be encoded only by viruses with >60% amino acid identity to MKPV. We show that MKPV is kidney-tropic and that the bat chapparvovirus DrPV-1 and a non-human primate chapparvovirus, CKPV, are also found in the kidneys of their hosts. We propose, therefore, that many mammal chapparvoviruses are likely to be nephrotropic.

The EMT modulator SNAI1 contributes to AML pathogenesis via its interaction with LSD1.

Modulators of epithelial-to-mesenchymal transition (EMT) have recently emerged as novel players in the field of leukemia biology. The mechanisms by which EMT modulators contribute to leukemia pathogenesis, however, remain to be elucidated. Here we show that overexpression of SNAI1, a key modulator of EMT, is a pathologically relevant event in human acute myeloid leukemia (AML) that contributes to impaired differentiation, enhanced self-renewal, and proliferation of immature myeloid cells. We demonstrate that ectopic expression of Snai1 in hematopoietic cells predisposes mice to AML development. This effect is mediated by interaction with the histone demethylase KDM1A/LSD1. Our data shed new light on the role of SNAI1 in leukemia development and identify a novel mechanism of LSD1 corruption in cancer. This is particularly pertinent given the current interest surrounding the use of LSD1 inhibitors in the treatment of multiple different malignancies, including AML.

Differential DNA repair underlies mutation hotspots at active promoters in cancer genomes.

Promoters are DNA sequences that have an essential role in controlling gene expression. While recent whole cancer genome analyses have identified numerous hotspots of somatic point mutations within promoters, many have not yet been shown to perturb gene expression or drive cancer development. As such, positive selection alone may not adequately explain the frequency of promoter point mutations in cancer genomes. Here we show that increased mutation density at gene promoters can be linked to promoter activity and differential nucleotide excision repair (NER). By analysing 1,161 human cancer genomes across 14 cancer types, we find evidence for increased local density of somatic point mutations within the centres of DNase I-hypersensitive sites (DHSs) in gene promoters. Mutated DHSs were strongly associated with transcription initiation activity, in which active promoters but not enhancers of equal DNase I hypersensitivity were most mutated relative to their flanking regions. Notably, analysis of genome-wide maps of NER shows that NER is impaired within the DHS centre of active gene promoters, while XPC-deficient skin cancers do not show increased promoter mutation density, pinpointing differential NER as the underlying cause of these mutation hotspots. Consistent with this finding, we observe that melanomas with an ultraviolet-induced DNA damage mutation signature show greatest enrichment of promoter mutations, whereas cancers that are not highly dependent on NER, such as colon cancer, show no sign of such enrichment. Taken together, our analysis has uncovered the presence of a previously unknown mechanism linking transcription initiation and NER as a major contributor of somatic point mutation hotspots at active gene promoters in cancer genomes.

A novel role for Lyl1 in primitive erythropoiesis.

Stem cell leukemia (Scl or Tal1) and lymphoblastic leukemia 1 (Lyl1) encode highly related members of the basic helix-loop-helix family of transcription factors that are co-expressed in the erythroid lineage. Previous studies have suggested that Scl is essential for primitive erythropoiesis. However, analysis of single-cell RNA-seq data of early embryos showed that primitive erythroid cells express both Scl and Lyl1 Therefore, to determine whether Lyl1 can function in primitive erythropoiesis, we crossed conditional Scl knockout mice with mice expressing a Cre recombinase under the control of the Epo receptor, active in erythroid progenitors. Embryos with 20% expression of Scl from E9.5 survived to adulthood. However, mice with reduced expression of Scl and absence of Lyl1 (double knockout; DKO) died at E10.5 because of progressive loss of erythropoiesis. Gene expression profiling of DKO yolk sacs revealed loss of Gata1 and many of the known target genes of the SCL-GATA1 complex. ChIP-seq analyses in a human erythroleukemia cell line showed that LYL1 exclusively bound a small subset of SCL targets including GATA1. Together, these data show for the first time that Lyl1 can maintain primitive erythropoiesis.

Label-Free Isolation and Single Cell Biophysical Phenotyping Analysis of Primary Cardiomyocytes Using Inertial Microfluidics.

To advance the understanding of cardiomyocyte (CM) identity and function, appropriate tools to isolate pure primary CMs are needed. A label-free method to purify viable CMs from mouse neonatal hearts is developed using a simple particle size-based inertial microfluidics biochip achieving purities of over 90%. Purified CMs are viable and retained their identity and function as depicted by the expression of cardiac-specific markers and contractility. The physico-mechanical properties of sorted cells are evaluated using downstream real-time deformability cytometry. CMs exhibited different physico-mechanical properties when compared with non-CMs. Taken together, this CM isolation and phenotyping method could serve as a valuable tool to progress the understanding of CM identity and function, and ultimately benefit cell therapy and diagnostic applications.

Shared roles for Scl and Lyl1 in murine platelet production and function.

The stem cell leukemia (Scl or Tal1) protein forms part of a multimeric transcription factor complex required for normal megakaryopoiesis. However, unlike other members of this complex such as Gata1, Fli1, and Runx1, mutations of Scl have not been observed as a cause of inherited thrombocytopenia. We postulated that functional redundancy with its closely related family member, lymphoblastic leukemia 1 (Lyl1) might explain this observation. To determine whether Lyl1 can substitute for Scl in megakaryopoiesis, we examined the platelet phenotype of mice lacking 1 or both factors in megakaryocytes. Conditional Scl knockout (KO) mice crossed with transgenic mice expressing Cre recombinase under the control of the mouse platelet factor 4 (Pf4) promoter generated megakaryocytes with markedly reduced but not absent Scl These Pf4Sclc-KO mice had mild thrombocytopenia and subtle defects in platelet aggregation. However, Pf4Sclc-KO mice generated on an Lyl1-null background (double knockout [DKO] mice) had severe macrothrombocytopenia, abnormal megakaryocyte morphology, defective pro-platelet formation, and markedly impaired platelet aggregation. DKO megakaryocytes, but not single-knockout megakaryocytes, had reduced expression of Gata1, Fli1, Nfe2, and many other genes that cause inherited thrombocytopenia. These gene expression changes were significantly associated with shared Scl and Lyl1 E-box binding sites that were also enriched for Gata1, Ets, and Runx1 motifs. Thus, Scl and Lyl1 share functional roles in platelet production by regulating expression of partner proteins including Gata1. We propose that this functional redundancy provides one explanation for the absence of Scl and Lyl1 mutations in inherited thrombocytopenia.

Srsf2P95H initiates myeloid bias and myelodysplastic/myeloproliferative syndrome from hemopoietic stem cells.

Mutations in SRSF2 occur in myelodysplastic syndromes (MDS) and MDS/myeloproliferative neoplasms (MPN). SRSF2 mutations cluster at proline 95, with the most frequent mutation being a histidine (P95H) substitution. They undergo positive selection, arise early in the course of disease, and have been identified in age-related clonal hemopoiesis. It is not clear how mutation of SRSF2 modifies hemopoiesis or contributes to the development of myeloid bias or MDS/MPN. Two prior mouse models of Srsf2P95H mutation have been reported; however, these models do not recapitulate many of the clinical features of SRSF2-mutant disease and relied on bone marrow (BM) transplantation stress to elicit the reported phenotypes. We describe a new conditional murine Srsf2P95H mutation model, where the P95H mutation is expressed physiologically and heterozygously from its endogenous locus after Cre activation. Using multiple Cre lines, we demonstrate that during native hemopoiesis (ie, no BM transplantation), the Srsf2P95H mutation needs to occur within the hemopoietic stem-cell-containing populations to promote myelomonocytic bias and expansion with corresponding transcriptional and RNA splicing changes. With age, nontransplanted Srsf2P95H animals developed a progressive, transplantable disease characterized by myeloid bias, morphological dysplasia, and monocytosis, hallmarks of MDS/MPN in humans. Analysis of cooccurring mutations within the BM demonstrated the acquisition of additional mutations that are recurrent in humans with SRSF2 mutations. The tractable Srsf2P95H/+ knock-in model we have generated is highly relevant to human disease and will serve to elucidate the effect of SRSF2 mutations on initiation and maintenance of MDS/MPN.

Jak2V617F and Dnmt3a loss cooperate to induce myelofibrosis through activated enhancer-driven inflammation.

Myeloproliferative neoplasms (MPNs) are a group of blood cancers that arise following the sequential acquisition of genetic lesions in hematopoietic stem and progenitor cells (HSPCs). We identify mutational cooperation between Jak2V617F expression and Dnmt3a loss that drives progression from early-stage polycythemia vera to advanced myelofibrosis. Using in vivo, clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated protein 9 (Cas9) disruption of Dnmt3a in Jak2V617F knockin HSPC, we show that Dnmt3a loss blocks the accumulation of erythroid elements and causes fibrotic infiltration within the bone marrow and spleen. Transcriptional analysis and integration with human data sets identified a core DNMT3A-driven gene-expression program shared across multiple models and contexts of Dnmt3a loss. Aberrant self-renewal and inflammatory signaling were seen in Dnmt3a-/- Jak2V617F HSPC, driven by increased chromatin accessibility at enhancer elements. These findings identify oncogenic cooperativity between Jak2V617F-driven MPN and Dnmt3a loss, leading to activation of HSPC enhancer-driven inflammatory signaling.

DKC1 is a transcriptional target of GATA1 and drives upregulation of telomerase activity in normal human erythroblasts.

Telomerase is a ribonucleoprotein complex that maintains the length and integrity of telomeres, and thereby enables cellular proliferation. Understanding the regulation of telomerase in hematopoietic cells is relevant to the pathogenesis of leukemia, in which telomerase is constitutively activated, as well as bone marrow failure syndromes that feature telomerase insufficiency. Past studies showing high levels of telomerase in human erythroblasts and a prevalence of anemia in disorders of telomerase insufficiency provide the rationale for investigating telomerase regulation in erythroid cells. Here it is shown for the first time that the telomerase RNA-binding protein dyskerin (encoded by DKC1) is dramatically upregulated as human hematopoietic stem and progenitor cells commit to the erythroid lineage, driving an increase in telomerase activity in the presence of limiting amounts of TERT mRNA. It is also shown that upregulation of DKC1 was necessary for expansion of glycophorin A+ erythroblasts and sufficient to extend telomeres in erythroleukemia cells. Chromatin immunoprecipitation and reporter assays implicated GATA1-mediated transcriptional regulation of DKC1 in the modulation of telomerase in erythroid lineage cells. Together these results describe a novel mechanism of telomerase regulation in erythroid cells which contrasts with mechanisms centered on transcriptional regulation of TERT that are known to operate in other cell types. This is the first study to reveal a biological context in which telomerase is upregulated by DKC1 and to implicate GATA1 in telomerase regulation. The results from this study are relevant to hematopoietic disorders involving DKC1 mutations, GATA1 deregulation and/or telomerase insufficiency.

Transcriptional networks in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a complex disease characterized by a diverse range of recurrent molecular aberrations that occur in many different combinations. Components of transcriptional networks are a common target of these aberrations, leading to network-wide changes and deployment of novel or developmentally inappropriate transcriptional programs. Genome-wide techniques are beginning to reveal the full complexity of normal hematopoietic stem cell transcriptional networks and the extent to which they are deregulated in AML, and new understandings of the mechanisms by which AML cells maintain self-renewal and block differentiation are starting to emerge. The hope is that increased understanding of the network architecture in AML will lead to identification of key oncogenic dependencies that are downstream of multiple network aberrations, and that this knowledge will be translated into new therapies that target these dependencies. Here, we review the current state of knowledge of network perturbation in AML with a focus on major mechanisms of transcription factor dysregulation, including mutation, translocation, and transcriptional dysregulation, and discuss how these perturbations propagate across transcriptional networks. We will also review emerging mechanisms of network disruption, and briefly discuss how increased knowledge of network disruption is already being used to develop new therapies.