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The ETS transcription factor ERG has a crucial role in hematopoiesis. It is essential for hematopoietic stem cell maintenance and megakaryopoiesis. Increased expression of ERG is associated with poor prognosis in acute myeloid leukemia. Previous studies by us and others established ERG as an oncogene in human leukemia. The PNT domain of ERG is a conserved domain that, in some other ETS factors, is required for protein-protein interactions, but its function for ERG is unknown. To examine whether the PNT domain of ERG is essential for hematopoietic stem and progenitor cells (HSPC) transformation, murine fetal liver-derived-HSPC were transduced with human WT and deleted PNT (DelPNT) domain ERG and cultured in methylcellulose. In re-plating assays, the PNT domain was not essential for HSPC self-renewal yet, DelPNT-ERG-transduced cells demonstrated increased proliferation capacity. Flow cytometry analysis showed decreased expression of hematopoietic stem markers for DelPNT-ERG-transduced cells. Consistent with these findings, RNA sequencing revealed enrichment of genes associated with myeloid differentiation and loss of hematopoietic stem cell signature in HSPC transduced with DelPNT-ERG. Transduction-transplantation assays in C57B6 mice suggested that the PNT domain is not essential for leukemia development. Nevertheless, time to leukemia development was significantly delayed for delPNT-ERG-transduced mice. Gene expression analysis showed enrichment for cell cycle and Myc target genes in leukemia induced by WT-ERG as compared to DelPNT-ERG. The proximity map of ERG-interacting protein suggested that the PNT domain is essential for ERG association with the SWI-SNF complex, which was previously shown to mediate the viability and proliferation of leukemia cells, at least in part by the maintenance of Myc expression. To further validate this finding, we conducted ChIP-sequencing on myeloid progenitor cell line and revealed decreased intensities of histone modifications associated with an active enhancer at the Myc locus in cells transduced with DelPNT-ERG as compared to WT-ERG. These results suggest that in pre-leukemic conditions, the PNT domain has a role in preserving the stem cell phenotype associated with ERG, thereby preventing premature differentiation. Furthermore, ERG interaction with the SWI-SNF complex was significantly dependent on the PNT domain and may be essential for leukemia maintenance.

CTCF binding sites are frequently mutated in cancer, but how these mutations accumulate and whether they broadly perturb CTCF binding are not well understood. Here, we report that skin cancers exhibit a highly specific asymmetric mutation pattern within CTCF motifs attributable to ultraviolet irradiation and differential nucleotide excision repair (NER). CTCF binding site mutations form independently of replication timing and are enriched at sites of CTCF/cohesin complex binding, suggesting a role for cohesin in stabilizing CTCF-DNA binding and impairing NER. Performing CTCF ChIP-seq in a melanoma cell line, we show CTCF binding site mutations to be functional by demonstrating allele-specific reduction of CTCF binding to mutant alleles. While topologically associating domains with mutated CTCF anchors in melanoma contain differentially expressed cancer-associated genes, CTCF motif mutations appear generally under neutral selection. However, the frequency and potential functional impact of such mutations in melanoma highlights the need to consider their impact on cellular phenotype in individual genomes.

Aberrant expression of the SNAIL & ZEB families of Epithelial to Mesenchymal Transition (EMT) modulators is an emerging theme in acute leukemia biology. EMT regulation has long been implicated in epithelial tumor pathogenesis, however a role in hematopoietic malignancy has only recently been explored. In this study, we describe a novel mechanism of AML pathogenesis involving SNAI1 induced epigenetic dysregulation via corruption of the histone demethylase LSD1. SNAI1 is expressed ∼10-fold higher in AML blasts compared to normal hematopoietic cells, suggesting a functional role for SNAI1 in AML. Indeed, SNAI1 knockdown induces differentiation in human AML cells, while Snai1 deletion delays tumour onset in mouse AML models. Furthermore, transgenic Snai1 expression perturbs myeloid development, resulting in accumulation of immature myeloid cells with enhanced self-renewal capacity - ultimately predisposing mice to AML. Intriguingly, impaired myeloid development by Snai1 absolutely requires its interaction with Lsd1, a H3K4 histone demethylase that has emerged as a novel therapeutic target in AML. Mechanistically, this interaction results in corruption of Lsd1 function via aberrant recruitment to Snai1 gene targets, and sequestration away from its own targets. Ultimately, this leads to changes in H3K4me1/2 methylation at specific promoters and enhancers, and subsequently altered gene expression. This model provides insight into the mechanisms by which LSD1 can be perturbed in AML, and offers a possible explanation for why LSD1 inhibition/loss has somewhat dichotomous effects on normal and malignant hematopoietic stem cells. Furthermore, as other members of the SNAIL and ZEB families are known to interact with LSD1, it is likely that this model will be more broadly applicable to other EMT modulators in AML, representing a possible new focus for future therapeutic studies.

Summary Glucocorticoids play a critical role in the treatment of lymphoid malignancies. While glucocorticoid efficacy can be largely attributed to lymphocyte-specific apoptosis, its molecular basis remains elusive. Here, we studied genome-wide lymphocyte-specific open chromatin domains (LSOs), and integrated LSOs with glucocorticoid-induced RNA transcription and chromatin modulation using an in vivo patient-derived xenograft model of acute lymphoblastic leukemia (ALL). This led to the identification of LSOs critical for glucocorticoid-induced apoptosis. Glucocorticoid receptor cooperated with CTCF at these LSOs to mediate DNA looping, which was inhibited by increased DNA methylation in glucocorticoid-resistant ALL and non-lymphoid cell types. Our study demonstrates that lymphocyte-specific epigenetic modifications pre-determine glucocorticoid resistance in ALL and may account for the lack of glucocorticoid sensitivity in other cell types.

Interlinked gene regulatory networks (GRNs) are vital for the spatial and temporal control of gene expression during development. The hematopoietic transcription factors (TFs) Scl, Gata2 and Fli1 form one such densely connected GRN which acts as a master regulator of embryonic hematopoiesis. This triad has been shown to direct the specification of the hemogenic endothelium and emergence of hematopoietic stem cells (HSCs) in response to Notch1 and Bmp4–Smad signaling. Here we employ previously published data to construct a mathematical model of this GRN network and use this model to systematically investigate the network dynamical properties. Our model uses a statistical-thermodynamic framework to describe the combinatorial regulation of gene expression and reconciles, mechanistically, several previously published but unexplained results from different genetic perturbation experiments. In particular, our results demonstrate how the interactions of Runx1, an essential hematopoietic TF, with components of the Bmp4 signaling pathway allow it to affect triad activation and acts as a key regulator of HSC emergence. We also explain why heterozygous deletion of this essential TF, Runx1, speeds up the network dynamics leading to accelerated HSC emergence. Taken together our results demonstrate that the triad, a master-level controller of definitive hematopoiesis, is an irreversible bistable switch whose dynamical properties are modulated by Runx1 and components of the Bmp4 signaling pathway.

SUMMARY Combinatorial transcription factor (TF) interactions control cellular phenotypes and, therefore, underpin stem cell formation, maintenance, and differentiation. Here, we report the genome-wide binding patterns and combinatorial interactions for ten key regulators of blood stem/progenitor cells (SCL/ TAL1, LYL1, LMO2, GATA2, RUNX1, MEIS1, PU.1, ERG, FLI-1, and GFI1B), thus providing the most comprehensive TF data set for any adult stem/ progenitor cell type to date. Genome-wide computational analysis of complex binding patterns, followed by functional validation, revealed the following: first, a previously unrecognized combinatorial interaction between a heptad of TFs (SCL, LYL1, LMO2, GATA2, RUNX1, ERG, and FLI-1). Second, we implicate direct protein-protein interactions between four key regulators (RUNX1, GATA2, SCL, and ERG) in stabilizing complex binding to DNA. Third, Runx1 +/� ::Gata2 +/� compound heterozygous mice are not viable with severe hematopoietic defects at midgestation. Taken together, this study demonstrates the power of genome-wide analysis in generating novel functional insights into the transcriptional control of stem and progenitor cells.

Plasma von Willebrand factor (vWF) is a multimeric protein that mediates adhesion of platelets to sites of vascular injury, however only the very large vWF multimers are haemostatically competent. Plasma vWF derives predominantly from the vascular endothelium and is of a smaller average multimer size to that found in the sub-endothelial matrix. The existence of plasma factors that control the size of vWF multimers and account for this difference has long been suspected. vWF cleaving protease (vWFCP), a metalloproteinase that regulates vWF multimer size by proteolytic cleavage, and thrombospondin-1 (TSP-1), a trimeric glycoprotein that uncouples multimers by reducing the disulfide-bonds which link individual subunits, are two such factors that have recently been identified. The large and ultra large vWF multimers play a central role in arterial thrombosis and agents that regulate their size hold promise as novel anti-thrombotic drugs.