Genome-scale definition of the transcriptional programme associated with compromised PU.1 activity in acute myeloid leukaemia.

Transcriptional dysregulation is associated with haematological malignancy. Although mutations of the key haematopoietic transcription factor PU.1 are rare in human acute myeloid leukaemia (AML), they are common in murine models of radiation-induced AML, and PU.1 downregulation and/or dysfunction has been described in human AML patients carrying the fusion oncogenes RUNX1-ETO and PML-RARA. To study the transcriptional programmes associated with compromised PU.1 activity, we adapted a Pu.1-mutated murine AML cell line with an inducible wild-type PU.1. PU.1 induction caused transition from leukaemia phenotype to monocytic differentiation. Global binding maps for PU.1, CEBPA and the histone mark H3K27Ac with and without PU.1 induction showed that mutant PU.1 retains DNA-binding ability, but the induction of wild-type protein dramatically increases both the number and the height of PU.1-binding peaks. Correlating chromatin immunoprecipitation (ChIP) Seq with gene expression data, we found that PU.1 recruitment coupled with increased histone acetylation induces gene expression and activates a monocyte/macrophage transcriptional programme. PU.1 induction also caused the reorganisation of a subgroup of CEBPA binding peaks. Finally, we show that the PU.1 target gene set defined in our model allows the stratification of primary human AML samples, shedding light on both known and novel AML subtypes that may be driven by PU.1 dysfunction.X18.1.1 cells were kindly donated by Dr Wendy Cook (LaTrobe University, Melbourne). MSCV-puro-PuER plasmid was kindly donated by Dr Peter Laslo (University of Leeds). ChIP sequencing was performed at the Genomics Core Facility, CRUK Cambridge Institute. Research in the Göttgens laboratory is supported by Leukaemia and Lymphoma Research, the MRC, BBSRC, CRUK, Leukemia and Lymphoma Society, NIHR Cambridge Biomedical Research Centre and core infrastructure support by the Wellcome Trust to the Wellcome Trust and MRC Cambridge Stem Cell Institute and CIMR. JIS is supported by CRUK and the Raymond and Beverly Sackler Foundation.This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/leu.2015.17

Operative Verfahren bei hohen kryptoglandulären Analfisteln: Systematische Übersicht und Metaanalyse

Purpose: Perianal fistulas, and specifically high perianal fistulas, remain a challenge for surgical treatment. Many techniques have been and are still being developed to improve the outcome after surgery. A systematic review and meta-analysis was performed for surgical treatment of high cryptoglandular perianal fistulas. Methods: Medline (Pubmed, Ovid), Embase and The Cochrane Library databases were searched for relevant randomized controlled trials on surgical treatments for high cryptoglandular perianal fistulas. Two independent reviewers selected articles for inclusion based on title, abstract and outcomes described. The main outcome measurement was the recurrence/healing rate. Secondary outcomes were continence status, quality of life and complications. Results: The number of randomized trials available was low. Fourteen studies could be included in the review. A meta-analysis could only be performed for the mucosal advancement flap versus the fistula plug, and did not show a result in favour of either technique in recurrence or complication rate. The mucosal advancement flap was the most investigated technique but did not show any advantage over any other technique. Other techniques identified in randomized studies were seton treatment, medicated seton treatment, fibrin glue, autologous stem cells, island flap anoplasty, rectal wall advancement flap, ligation of the intersphincteric fistula tract, sphincter reconstruction, sphincter-preserving seton and techniques combined with antibiotics. None of these techniques seemed superior to each other. Conclusions: The best surgical treatment for high cryptoglandular perianal fistulas could not be identified. More randomized controlled trials are needed to find the best treatment. The mucosal advancement flap is the most investigated technique available

Endoglin potentiates nitric oxide synthesis to enhance definitive hematopoiesis.

During embryonic development, hematopoietic cells develop by a process of endothelial-to hematopoietic transition of a specialized population of endothelial cells. These hemogenic endothelium (HE) cells in turn develop from a primitive population of FLK1(+) mesodermal cells. Endoglin (ENG) is an accessory TGF-β receptor that is enriched on the surface of endothelial and hematopoietic stem cells and is also required for the normal development of hemogenic precursors. However, the functional role of ENG during the transition of FLK1(+) mesoderm to hematopoietic cells is ill defined. To address this we used a murine embryonic stem cell model that has been shown to mirror the temporal emergence of these cells in the embryo. We noted that FLK1(+) mesodermal cells expressing ENG generated fewer blast colony-forming cells but had increased hemogenic potential when compared with ENG non-expressing cells. TIE2(+)/CD117(+) HE cells expressing ENG also showed increased hemogenic potential compared with non-expressing cells. To evaluate whether high ENG expression accelerates hematopoiesis, we generated an inducible ENG expressing ES cell line and forced expression in FLK1(+) mesodermal or TIE2(+)/CD117(+) HE cells. High ENG expression at both stages accelerated the emergence of CD45(+) definitive hematopoietic cells. High ENG expression was associated with increased pSMAD2/eNOS expression and NO synthesis in hemogenic precursors. Inhibition of eNOS blunted the ENG induced increase in definitive hematopoiesis. Taken together, these data show that ENG potentiates the emergence of definitive hematopoietic cells by modulating TGF-β/pSMAD2 signalling and increasing eNOS/NO synthesis.The authors thank Dr Zúñiga-Pflücker (University of Toronto) for the ENG-/- and +/- murine ES cells. This work was supported by grants from the National Health and Medical Research Council of Australia, Australian Research Council and the Dr Tom Bee Stem Cell Research Fund to JEP, Cancer Research UK to VK and GL and the BBSRC, Leukaemia and Lymphoma Research, The Leukaemia and Lymphoma Society, Cancer Research UK, and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust - MRC Cambridge Stem Cell Institute to BG.This is the final version of the article. It first appeared from the Company of Biologists via http://dx.doi.org/10.1242/​bio.01149

Gata3 targets Runx1 in the embryonic haematopoietic stem cell niche.

Runx1 is an important haematopoietic transcription factor as stressed by its involvement in a number of haematological malignancies. Furthermore, it is a key regulator of the emergence of the first haematopoietic stem cells (HSCs) during development. The transcription factor Gata3 has also been linked to haematological disease and was shown to promote HSC production in the embryo by inducing the secretion of important niche factors. Both proteins are expressed in several different cell types within the aorta-gonads-mesonephros (AGM) region, in which the first HSCs are generated; however, a direct interaction between these two key transcription factors in the context of embryonic HSC production has not formally been demonstrated. In this current study, we have detected co-localisation of Runx1 and Gata3 in rare sub-aortic mesenchymal cells in the AGM. Furthermore, the expression of Runx1 is reduced in Gata3 -/- embryos, which also display a shift in HSC emergence. Using an AGM-derived cell line as a model for the stromal microenvironment in the AGM and performing ChIP-Seq and ChIP-on-chip experiments, we demonstrate that Runx1, together with other key niche factors, is a direct target gene of Gata3. In addition, we can pinpoint Gata3 binding to the Runx1 locus at specific enhancer elements which are active in the microenvironment. These results reveal a direct interaction between Gata3 and Runx1 in the niche that supports embryonic HSCs and highlight a dual role for Runx1 in driving the transdifferentiation of haemogenic endothelial cells into HSCs as well as in the stromal cells that support this process.This work was supported by an Intermediate Fellowship (K.O.) and a Junior Fellowship (S.R.F.) from the Kay Kendall Leukaemia Fund, a British Society for Haematology Early Stage Investigator Fellowship (K.O.) as well as funding from Bloodwise (N.K.W. and B.G.), MRC (N.K.W. and B.G.) and the Wellcome Trust (N.K.W. and B.G.). MdB is funded by a programme in the MRC Molecular Hematology Unit Core award (Grant number: MC_UU_12009/2). Core facilities are supported by Strategic Award WT100140, equipment grant 093026 and centre grant MR/K017047/1

The impact of sex and gender on the multidisciplinary management of care for persons with Parkinson's disease

Contains fulltext : 226157.pdf (publisher's version ) (Open Access)7 p

Runx1 promotes scar deposition and inhibits myocardial proliferation and survival during zebrafish heart regeneration.

Runx1 is a transcription factor that plays a key role in determining the proliferative and differential state of multiple cell types, during both development and adulthood. Here, we report how Runx1 is specifically upregulated at the injury site during zebrafish heart regeneration, and that absence of runx1 results in increased myocardial survival and proliferation, and overall heart regeneration, accompanied by decreased fibrosis. Using single cell sequencing, we found that the wild-type injury site consists of Runx1-positive endocardial cells and thrombocytes that induce expression of smooth muscle and collagen genes. Both these populations cannot be identified in runx1 mutant wounds that contain less collagen and fibrin. The reduction in fibrin in the mutant is further explained by reduced myofibroblast formation and upregulation of components of the fibrin degradation pathway, including plasminogen receptor annexin 2A as well as downregulation of plasminogen activator inhibitor serpine1 in myocardium and endocardium, resulting in increased levels of plasminogen. Our findings suggest that Runx1 controls the regenerative response of multiple cardiac cell types and that targeting Runx1 is a novel therapeutic strategy for inducing endogenous heart repair.BHF, Wellcome, MR

Cohesin-dependent regulation of gene expression during differentiation is lost in cohesin-mutated myeloid malignancies.

Cohesin complex disruption alters gene expression, and cohesin mutations are common in myeloid neoplasia, suggesting a critical role in hematopoiesis. Here, we explore cohesin dynamics and regulation of hematopoietic stem cell homeostasis and differentiation. Cohesin binding increases at active regulatory elements only during erythroid differentiation. Prior binding of the repressive Ets transcription factor Etv6 predicts cohesin binding at these elements and Etv6 interacts with cohesin at chromatin. Depletion of cohesin severely impairs erythroid differentiation, particularly at Etv6-prebound loci, but augments self-renewal programs. Together with corroborative findings in acute myeloid leukemia and myelodysplastic syndrome patient samples, these data suggest cohesin-mediated alleviation of Etv6 repression is required for dynamic expression at critical erythroid genes during differentiation and how this may be perturbed in myeloid malignancies

RUNX1 Reshapes the Epigenetic Landscape at the Onset of Haematopoiesis

Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a $Runx1^{−/−}$ embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPβ and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies

Single-cell approaches identify the molecular network driving malignant hematopoietic stem cell self-renewal.

Recent advances in single-cell technologies have permitted the investigation of heterogeneous cell populations at previously unattainable resolution. Here we apply such approaches to resolve the molecular mechanisms driving disease in mouse hematopoietic stem cells (HSCs), using JAK2V617F mutant myeloproliferative neoplasms (MPNs) as a model. Single-cell gene expression and functional assays identified a subset of JAK2V617F mutant HSCs that display defective self-renewal. This defect is rescued at the single HSC level by crossing JAK2V617F mice with mice lacking TET2, the most commonly comutated gene in patients with MPN. Single-cell gene expression profiling of JAK2V617F-mutant HSCs revealed a loss of specific regulator genes, some of which were restored to normal levels in single TET2/JAK2 mutant HSCs. Of these, Bmi1 and, to a lesser extent, Pbx1 and Meis1 overexpression in JAK2-mutant HSCs could drive a disease phenotype and retain durable stem cell self-renewal in functional assays. Together, these single-cell approaches refine the molecules involved in clonal expansion of MPNs and have broad implications for deconstructing the molecular network of normal and malignant stem cells.MS is the recipient of a BBSRC Industrial CASE PhD Studentship, and CAO and JF are recipients of Wellcome Trust PhD Studentships. Work in the Kent lab is supported by a Bloodwise Bennett Fellowship (15008), a European Hematology Association Non-Clinical Advanced Research Fellowship, and an ERC Starting Grant (ERC-2016-STG–715371). Work in the Green Lab is supported by the Wellcome Trust, Bloodwise, Cancer Research UK, the Kay Kendall Leukaemia Fund, and the Leukemia and Lymphoma Society of America. Dr. Kent, Professor Göttgens, and Professor Green are all supported by a core support grant from the Wellcome Trust and MRC to the Wellcome Trust – Medical Research Council Cambridge Stem Cell Institute the National Institute for Health Research Cambridge Biomedical Research Centre, the Cambridge Experimental Cancer Medicine Centre

Combined Single-Cell Functional and Gene Expression Analysis Resolves Heterogeneity within Stem Cell Populations.

Heterogeneity within the self-renewal durability of adult hematopoietic stem cells (HSCs) challenges our understanding of the molecular framework underlying HSC function. Gene expression studies have been hampered by the presence of multiple HSC subtypes and contaminating non-HSCs in bulk HSC populations. To gain deeper insight into the gene expression program of murine HSCs, we combined single-cell functional assays with flow cytometric index sorting and single-cell gene expression assays. Through bioinformatic integration of these datasets, we designed an unbiased sorting strategy that separates non-HSCs away from HSCs, and single-cell transplantation experiments using the enriched population were combined with RNA-seq data to identify key molecules that associate with long-term durable self-renewal, producing a single-cell molecular dataset that is linked to functional stem cell activity. Finally, we demonstrated the broader applicability of this approach for linking key molecules with defined cellular functions in another stem cell system.Work in the author’s laboratory is supported by grants from the Leukaemia and Lymphoma Research, the Medical Research Council, Cancer Research UK, Biotechnology and Biological Sciences Research Council, Leukemia Lymphoma Society, and the National Institute for Health Research Cambridge Biomedical Research Centre and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research and Wellcome Trust-MRC Cambridge Stem Cell Institute. D.G.K. is the recipient of a Canadian Institutes of Health Research Postdoctoral Fellowship. F.B. and F.J.T. are funded by the European Research Council (starting grant “LatentCauses”). For funding for the open access charge, the core support grant was provided by the Wellcome Trust-MRC Cambridge Stem Cell Institute. We acknowledge the support of the University of Cambridge, Cancer Research UK Institute (core grant C14303/A17197), and Hutchison Whampoa Limited.This is the final published version. It first appeared at http://www.cell.com/cell-stem-cell/abstract/S1934-5909%2815%2900162-9