BET protein inhibition shows efficacy against JAK2V617F-driven neoplasms.

Small molecule inhibition of the BET family of proteins, which bind acetylated lysines within histones, has been shown to have a marked therapeutic benefit in pre-clinical models of mixed lineage leukemia (MLL) fusion protein-driven leukemias. Here, we report that I-BET151, a highly specific BET family bromodomain inhibitor, leads to growth inhibition in a human erythroleukemic (HEL) cell line as well as in erythroid precursors isolated from polycythemia vera patients. One of the genes most highly downregulated by I-BET151 was LMO2, an important oncogenic regulator of hematopoietic stem cell development and erythropoiesis. We previously reported that LMO2 transcription is dependent upon Janus kinase 2 (JAK2) kinase activity in HEL cells. Here, we show that the transcriptional changes induced by a JAK2 inhibitor (TG101209) and I-BET151 in HEL cells are significantly over-lapping, suggesting a common pathway of action. We generated JAK2 inhibitor resistant HEL cells and showed that these retain sensitivity to I-BET151. These data highlight I-BET151 as a potential alternative treatment against myeloproliferative neoplasms driven by constitutively active JAK2 kinase.The Kouzarides laboratory was supported by Cancer Research UK, Leukaemia and Lymphoma Research, GlaxoSmithKline and BBSRC. The Green laboratory was supported by Cancer Research UK and Leukaemia and Lymphoma Research, UK. The Gottgens laboratory was supported by Cancer Research UK and Leukaemia and Lymphoma Research, UK. The Huntly laboratory was supported by Cancer Research UK and Leukaemia and Lymphoma Research, UK. M. A Dawson, E Cannizzaro and M. Wiese are funded by the Wellcome Trust Beit Fellowship.This is the accepted manuscript version of the article. The final version is available from http://www.nature.com/leu/journal/v28/n1/full/leu2013234a.html

Mammalian Transcription Factor Networks: Recent Advances in Interrogating Biological Complexity

Transcription factor (TF) networks are a key determinant of cell fate decisions in mammalian development and adult tissue homeostasis and are frequently corrupted in disease. However, our inability to experimentally resolve and interrogate the complexity of mammalian TF networks has hampered the progress in this field. Recent technological advances, in particular large-scale genome-wide approaches, single-cell methodologies, live-cell imaging, and genome editing, are emerging as important technologies in TF network biology. Several recent studies even suggest a need to re-evaluate established models of mammalian TF networks. Here, we provide a brief overview of current and emerging methods to define mammalian TF networks. We also discuss how these emerging technologies facilitate new ways to interrogate complex TF networks, consider the current open questions in the field, and comment on potential future directions and biomedical applications.ACW is funded by a Bloodwise Visiting Fellowship. HN is funded by the Japan Science and Technology Agency, the California Institute of Regenerative Medicine and Ludwig Foundation. BG is funded by Bloodwise, Cancer Research UK, the Wellcome Trust, the MRC, NIH-NIDDK and core funding from the Wellcome Trust to the Cambridge Stem Cell Institute

Mbd3/NuRD controls lymphoid cell fate and inhibits tumorigenesis by repressing a B cell transcriptional program

Differentiation of lineage-committed cells from multipotent progenitors requires establishment of accessible chromatin at lineage-specific transcriptional enhancers and promoters, which is mediated by pioneer transcription factors that recruit activating chromatin remodeling complexes. Here we show that the Mbd3/NuRD chromatin remodeling complex opposes this transcriptional pioneering during B cell programming of multipotent lymphoid progenitors by restricting chromatin accessibility at B cell enhancers and promoters. Mbd3/NuRD-deficient lymphoid progenitors therefore prematurely activate a B cell transcriptional program and are biased towards overproduction of pro-B cells at the expense of T cell progenitors. The striking reduction in early thymic T cell progenitors results in compensatory hyperproliferation of immature thymocytes and development of T cell lymphoma. Our results reveal that Mbd3/NuRD can regulate multilineage differentiation by constraining the activation of dormant lineage-specific enhancers and promoters. In this way, Mbd3/NuRD protects the multipotency of lymphoid progenitors, preventing B cell-programming transcription factors from prematurely enacting lineage commitment. Mbd3/NuRD therefore controls the fate of lymphoid progenitors, ensuring appropriate production of lineage-committed progeny and suppressing tumour formation.F.C. was supported by an EMBO long-term fellowship (1305-2015 and Marie Curie Actions LTFCOFUND2013/GA-2013-609409). Work in the Green lab is supported Cancer Research UK (grants refs. C1163/A12765 and C1163/A12526), Bloodwise (grant ref. 13003), and core support grants by the Wellcome Trust to the Cambridge Institute for Medical Research (100140/Z/12/Z) and Wellcome Trust-MRC Cambridge Stem Cell Institute (097922/Z/11/Z)

HOX-mediated LMO2 expression in embryonic mesoderm is recapitulated in acute leukaemias

The Lim Domain Only 2 (LMO2) leukaemia oncogene encodes an LIM domain transcriptional cofactor required for early haematopoiesis. During embryogenesis, LMO2 is also expressed in developing tail and limb buds, an expression pattern we now show to be recapitulated in transgenic mice by an enhancer in LMO2 intron 4. Limb bud expression depended on a cluster of HOX binding sites, while posterior tail expression required the HOX sites and two E-boxes. Given the importance of both LMO2 and HOX genes in acute leukaemias, we further demonstrated that the regulatory hierarchy of HOX control of LMO2 is activated in leukaemia mouse models as well as in patient samples. Moreover, Lmo2 knock-down impaired the growth of leukaemic cells, and high LMO2 expression at diagnosis correlated with poor survival in cytogenetically normal AML patients. Taken together, these results establish a regulatory hierarchy of HOX control of LMO2 in normal development, which can be resurrected during leukaemia development. Redeployment of embryonic regulatory hierarchies in an aberrant context is likely to be relevant in human pathologies beyond the specific example of ectopic activation of LMO2

Single-cell RNA-sequencing reveals a distinct population of proglucagon-expressing cells specific to the mouse upper small intestine

Objectives: To identify sub-populations of intestinal preproglucagon-expressing (PPG) cells producing Glucagon-like Peptide-1, and their associated expression profiles of sensory receptors, thereby enabling the discovery of therapeutic strategies that target these cell populations for the treatment of diabetes and obesity. Methods: We performed single cell RNA sequencing of PPG-cells purified by flow cytometry from the upper small intestine of 3 GLU-Venus mice. Cells from 2 mice were sequenced at low depth, and from the third mouse at high depth. High quality sequencing data from 234 PPG-cells were used to identify clusters by tSNE analysis. qPCR was performed to compare the longitudinal and crypt/villus locations of cluster-specific genes. Immunofluorescence and mass spectrometry were used to confirm protein expression. Results: PPG-cells formed 3 major clusters: a group with typical characteristics of classical L-cells, including high expression of Gcg and Pyy (comprising 51% of all PPG-cells); a cell type overlapping with Gip-expressing K-cells (14%); and a unique cluster expressing Tph1 and Pzp that was predominantly located in proximal small intestine villi and co-produced 5-HT (35%). Expression of G-protein coupled receptors differed between clusters, suggesting the cell types are differentially regulated, and would be differentially targetable. Conclusions: Our findings support the emerging concept that many enteroendocrine cell populations are highly overlapping, with individual cells producing a range of peptides previously assigned to distinct cell types. Different receptor expression profiles across the clusters highlight potential drug targets to increase gut hormone secretion for the treatment of diabetes and obesity.Research in the FR/FMG lab is funded by a Wellcome joint investigator award (106262/Z/14/Z and 106263/Z/14/Z) and a joint MRC programme within the Metabolic Diseases Unit (MRC_MC_UU_12012/3). Single cell collection and analysis was supported through MRC Clinical Research Infrastructure funds for the Cambridge Single Cell Analysis Clinical Core Facility. Work in the Göttgens laboratory is supported by grants from the Wellcome Trust, Bloodwise, Cancer Research UK, NIDDK and core support grants by the Wellcome Trust to the Wellcome Trust-MRC Cambridge Stem Cell Institute

Neurogenin3 phosphorylation controls reprogramming efficiency of pancreatic ductal organoids into endocrine cells

β-cell replacement has been proposed as an effective treatment for some forms of diabetes, and in vitro methods for β-cell generation are being extensively explored. A potential source of β-cells comes from fate conversion of exocrine pancreatic cells into the endocrine lineage, by overexpression of three regulators of pancreatic endocrine formation and β-cell identity, Ngn3, Pdx1 and MafA. Pancreatic ductal organoid cultures have recently been developed that can be expanded indefinitely, while maintaining the potential to differentiate into the endocrine lineage. Here, using mouse pancreatic ductal organoids, we see that co-expression of Ngn3, Pdx1 and MafA are required and sufficient to generate cells that express insulin and resemble β-cells transcriptome-wide. Efficiency of β-like cell generation can be significantly enhanced by preventing phosphorylation of Ngn3 protein and further augmented by conditions promoting differentiation. Taken together, our new findings underline the potential of ductal organoid cultures as a source material for generation of β-like cells and demonstrate that post-translational regulation of reprogramming factors can be exploited to enhance β-cell generation

Defining Lineage Potential and Fate Behavior of Precursors during Pancreas Development

Pancreas development involves a coordinated process in which an early phase of cell segregation is followed by a longer phase of lineage restriction, expansion, and tissue remodeling. By combining clonal tracing and whole-mount reconstruction with proliferation kinetics and single-cell transcriptional profiling, we define the functional basis of pancreas morphogenesis. We show that the large-scale organization of mouse pancreas can be traced to the activity of self-renewing precursors positioned at the termini of growing ducts, which act collectively to drive serial rounds of stochastic ductal bifurcation balanced by termination. During this phase of branching morphogenesis, multipotent precursors become progressively fate-restricted, giving rise to self-renewing acinar-committed precursors that are conveyed with growing ducts, as well as ductal progenitors that expand the trailing ducts and give rise to delaminating endocrine cells. These findings define quantitatively how the functional behavior and lineage progression of precursor pools determine the large-scale patterning of pancreatic sub-compartments