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

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

Gain of DNA methylation is enhanced in the absence of CTCF at the human retinoblastoma gene promoter

<p>Abstract</p> <p>Background</p> <p>Long-term gene silencing throughout cell division is generally achieved by DNA methylation and other epigenetic processes. Aberrant DNA methylation is now widely recognized to be associated with cancer and other human diseases. Here we addressed the contribution of the multifunctional nuclear factor CTCF to the epigenetic regulation of the human <it>retinoblastoma </it>(<it>Rb</it>) gene promoter in different tumoral cell lines.</p> <p>Methods</p> <p>To assess the DNA methylation status of the <it>Rb </it>promoter, genomic DNA from stably transfected human erythroleukemic K562 cells expressing a <it>GFP </it>reporter transgene was transformed with sodium bisulfite, and then PCR-amplified with modified primers and sequenced. Single- and multi-copy integrants with the CTCF binding site mutated were isolated and characterized by Southern blotting. Silenced transgenes were reactivated using 5-aza-2'-deoxycytidine and Trichostatin-A, and their expression was monitored by fluorescent cytometry. <it>Rb </it>gene expression and protein abundance were assessed by RT-PCR and Western blotting in three different glioma cell lines, and DNA methylation of the promoter region was determined by sodium bisulfite sequencing, together with CTCF dissociation and methyl-CpG-binding protein incorporation by chromatin immunoprecipitation assays.</p> <p>Results</p> <p>We found that the inability of CTCF to bind to the <it>Rb </it>promoter causes a dramatic loss of gene expression and a progressive gain of DNA methylation.</p> <p>Conclusions</p> <p>This study indicates that CTCF plays an important role in maintaining the <it>Rb </it>promoter in an optimal chromatin configuration. The absence of CTCF induces a rapid epigenetic silencing through a progressive gain of DNA methylation. Consequently, CTCF can now be seen as one of the epigenetic components that allows the proper configuration of tumor suppressor gene promoters. Its aberrant dissociation can then predispose key genes in cancer cells to acquire DNA methylation and epigenetic silencing.</p

The local and systemic response to SARS-CoV-2 infection in children and adults

While a substantial proportion of adults infected with SARS-CoV-2 progress to develop severe disease, children rarely manifest respiratory complications. Therefore, understanding differences in the local and systemic response to SARS-CoV-2 infection between children and adults may provide important clues about the pathogenesis of SARS-CoV-2 infection. To address this, we first generated a healthy reference multi-omics single cell data set from children (n=30) in whom we have profiled triple matched samples: nasal and tracheal brushings and PBMCs, where we track the developmental changes for 42 airway and 31 blood cell populations from infancy, through childhood to adolescence. This has revealed the presence of naive B and T lymphocytes in neonates and infants with a unique gene expression signature bearing hallmarks of innate immunity. We then contrast the healthy reference with equivalent data from severe paediatric and adult COVID-19 patients (total n=27), from the same three types of samples: upper and lower airways and blood. We found striking differences: children with COVID-19 as opposed to adults had a higher proportion of innate lymphoid and non-clonally expanded naive T cells in peripheral blood, and a limited interferon-response signature. In the airway epithelium, we found the highest viral load in goblet and ciliated cells and describe a novel inflammatory epithelial cell population. These cells represent a transitional regenerative state between secretory and ciliated cells; they were found in healthy children and were enriched in paediatric and adult COVID-19 patients. Epithelial cells display an antiviral and neutrophil-recruiting gene signature that is weaker in severe paediatric versus adult COVID-19. Our matched blood and airway samples allowed us to study the spatial dynamics of infection. Lastly, we provide a user-friendly interface for this data1 as a highly granular reference for the study of immune responses in airways and blood in children

RNA Interference Is Responsible for Reduction of Transgene Expression after Sleeping Beauty Transposase Mediated Somatic Integration

Integrating non-viral vectors based on transposable elements are widely used for genetically engineering mammalian cells in functional genomics and therapeutic gene transfer. For the Sleeping Beauty (SB) transposase system it was demonstrated that convergent transcription driven by the SB transposase inverted repeats (IRs) in eukaryotic cells occurs after somatic integration. This could lead to formation of double-stranded RNAs potentially presenting targets for the RNA interference (RNAi) machinery and subsequently resulting into silencing of the transgene. Therefore, we aimed at investigating transgene expression upon transposition under RNA interference knockdown conditions. To establish RNAi knockdown cell lines we took advantage of the P19 protein, which is derived from the tomato bushy stunt virus. P19 binds and inhibits 21 nucleotides long, small-interfering RNAs and was shown to sufficiently suppress RNAi. We found that transgene expression upon SB mediated transposition was enhanced, resulting into a 3.2-fold increased amount of colony forming units (CFU) after transposition. In contrast, if the transgene cassette is insulated from the influence of chromosomal position effects by the chicken-derived cHS4 insulating sequences or when applying the Forg Prince transposon system, that displays only negligible transcriptional activity, similar numbers of CFUs were obtained. In summary, we provide evidence for the first time that after somatic integration transposon derived transgene expression is regulated by the endogenous RNAi machinery. In the future this finding will help to further improve the molecular design of the SB transposase vector system

Local and systemic responses to SARS-CoV-2 infection in children and adults

It is not fully understood why COVID-19 is typically milder in children1–3. To examine differences in response to SARS-CoV-2 infection in children and adults, we analysed paediatric and adult COVID-19 patients and healthy controls (total n=93) using single-cell multi-omic profiling of matched nasal, tracheal, bronchial and blood samples. In healthy paediatric airways, we observed cells already in an interferon-activated state, that upon SARS-CoV-2 infection was further induced especially in airway immune cells. We postulate that higher paediatric innate interferon-responses restrict viral replication and disease progression. The systemic response in children was characterised by increases in naive lymphocytes and a depletion of natural killer cells, while in adults cytotoxic T cells and interferon-stimulated subpopulations were significantly increased. We provide evidence that dendritic cells initiate interferon signaling in early infection, and identify novel epithelial cell states that associate with COVID-19 and age. Our matching nasal and blood data showed a strong interferon response in the airways with the induction of systemic interferon-stimulated populations, which were massively reduced in paediatric patients. Together, we provide several mechanisms that explain the milder clinical syndrome observed in children

Single-cell multi-omics analysis of the immune response in COVID-19

Analysis of human blood immune cells provides insights into the coordinated response to viral infections such as severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019 (COVID-19). We performed single-cell transcriptome, surface proteome and T and B lymphocyte antigen receptor analyses of over 780,000 peripheral blood mononuclear cells from a cross-sectional cohort of 130 patients with varying severities of COVID-19. We identified expansion of nonclassical monocytes expressing complement transcripts (CD16+C1QA/B/C+) that sequester platelets and were predicted to replenish the alveolar macrophage pool in COVID-19. Early, uncommitted CD34+ hematopoietic stem/progenitor cells were primed toward megakaryopoiesis, accompanied by expanded megakaryocyte-committed progenitors and increased platelet activation. Clonally expanded CD8+ T cells and an increased ratio of CD8+ effector T cells to effector memory T cells characterized severe disease, while circulating follicular helper T cells accompanied mild disease. We observed a relative loss of IgA2 in symptomatic disease despite an overall expansion of plasmablasts and plasma cells. Our study highlights the coordinated immune response that contributes to COVID-19 pathogenesis and reveals discrete cellular components that can be targeted for therapy

A Large Gene Network in Immature Erythroid Cells Is Controlled by the Myeloid and B Cell Transcriptional Regulator PU.1

PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages

Bivalent promoter marks and a latent enhancer may prime the leukaemia oncogene LMO1 for ectopic expression in T-cell leukaemia

LMO1 is a transcriptional regulator and a T-acute lymphoblastic leukaemia (T-ALL) oncogene. Although first identified in association with a chromosomal translocation in T-ALL, the ectopic expression of LMO1 occurs far more frequently in the absence of any known mutation involving its locus. Given that LMO1 is barely expressed in any haematopoietic lineage, and activation of transcriptional drivers in leukaemic cells is not well described, we investigated the regulation of this gene in normal haematopoietic and leukaemic cells. We show that LMO1 has two promoters that drive reporter gene expression in transgenic mice to neural tissues known to express endogenous LMO1. The LMO1 promoters display bivalent histone marks in multiple blood lineages including T-cells, and a 3' flanking region at LMO1 +57 contains a transcriptional enhancer that is active in developing blood cells in transgenic mouse embryos. The LMO1 promoters become activated in T-ALL together with the 3' enhancer, which is bound in primary T-ALL cells by SCL/TAL1 and GATA3. Taken together, our results show that LMO1 is poised for expression in normal progenitors, where activation of SCL/TAL1 together with a breakdown of epigenetic repression of LMO1 regulatory elements induces ectopic LMO1 expression that contributes to the development and maintenance of T-ALL