SUMMARY DNA methylation at the 5-position of cytosine (5mC) in the mammalian genome is a key epigenetic event critical for various cellular processes. The Ten-eleven translocation (Tet) family of 5mC-hydroxylases, which convert 5mC to 5-hydroxymethylcytosine (5hmC), offers a way for dynamic regulation of DNA methylation. Here we report that Tet1 binds unmodified C, 5mC- or 5hmC-modified CpG-rich DNA through its CXXC domain. Genome-wide mapping of Tet1 and 5hmC reveals mechanisms by which Tet1 controls 5hmC and 5mC levels in mouse embryonic stem cells (mESCs). We also uncover a comprehensive gene network influenced by Tet1. Collectively, our data suggest that Tet1 controls DNA methylation both by binding to CpG-rich regions to prevent unwanted DNA methyltransferase activity, and by converting 5mC to 5hmC through hydroxylase activity. This Tet1-mediated antagonism of CpG methylation imparts differential maintenance of DNA methylation status at Tet1 targets, ultimately contributing to mESC differentiation and the onset of embryonic development.

KLF1 (formerly known as EKLF) regulates the development of erythroid cells from bi-potent progenitor cells via the transcriptional activation of a diverse set of genes. Mice lacking Klf1 die in utero prior to E15 from severe anemia due to the inadequate expression of genes controlling hemoglobin production, cell membrane and cytoskeletal integrity, and the cell cycle. We have recently described the full repertoire of KLF1 binding sites in vivo by performing KLF1 ChIP-seq in primary erythroid tissue (E14.5 fetal liver). Here we describe the KLF1-dependent erythroid transcriptome by comparing mRNA-seq from Klf1+/+ and Klf1−/− erythroid tissue. This has revealed novel target genes not previously obtainable by traditional microarray technology, and provided novel insights into the function of KLF1 as a transcriptional activator. We define a cis-regulatory module bound by KLF1, GATA1, TAL1, and EP300 that coordinates a core set of erythroid genes. We also describe a novel set of erythroid-specific promoters that drive high-level expression of otherwise ubiquitously expressed genes in erythroid cells. Our study has identified two novel lncRNAs that are dynamically expressed during erythroid differentiation, and discovered a role for KLF1 in directing apoptotic gene expression to drive the terminal stages of erythroid maturation.

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Idiopathic myelofibrosis (IM) is characterized by increased numbers of CD34

Erythropoietin (EPO) acts through the dimeric erythropoietin receptor to stimulate proliferation, survival, differentiation and enucleation of erythroid progenitor cells. We undertook two complimentary approaches to find EPO-dependent pSTAT5 target genes in murine erythroid cells: RNA-seq of newly transcribed (4sU-labelled) RNA, and ChIP-seq for pSTAT5 30 minutes after EPO stimulation. We found 302 pSTAT5-occupied sites: ~15% of these reside in promoters while the rest reside within intronic enhancers or intergenic regions, some >100kb from the nearest TSS. The majority of pSTAT5 peaks contain a central palindromic GAS element, TTCYXRGAA. There was significant enrichment for GATA motifs and CACCC-box motifs within the neighbourhood of pSTAT5-bound peaks, and GATA1 and/or KLF1 co-occupancy at many sites. Using 4sU-RNA-seq we determined the EPO-induced transcriptome and validated differentially expressed genes using dynamic CAGE data and qRT-PCR. We identified known direct pSTAT5 target genes such as Bcl2l1, Pim1 and Cish, and many new targets likely to be involved in driving erythroid cell differentiation including those involved in mRNA splicing (Rbm25), epigenetic regulation (Suv420h2), and EpoR turnover (Clint1/EpsinR). Some of these new EpoR-JAK2-pSTAT5 target genes could be used as biomarkers for monitoring disease activity in polycythaemia vera, and for monitoring responses to JAK inhibitors.

ALK, ROS1 and RET gene fusions are important predictive biomarkers for tyrosine kinase inhibitors in lung cancer. Currently, the gold standard method for gene fusion detection is Fluorescence In Situ Hybridization (FISH) and while highly sensitive and specific, it is also labour intensive, subjective in analysis, and unable to screen a large numbers of gene fusions. Recent developments in highthroughput transcriptome-based methods may provide a suitable alternative to FISH as they are compatible with multiplexing and diagnostic workflows. However, the concordance between these different methods compared with FISH has not been evaluated. In this study we compared the results from three transcriptome-based platforms (Nanostring Elements, Agena LungFusion panel and ThermoFisher NGS fusion panel) to those obtained from ALK, ROS1 and RET FISH on 51 clinical specimens. Overall agreement of results ranged from 86-96% depending on the platform used. While all platforms were highly sensitive, both the Agena panel and Thermo Fisher NGS fusion panel reported minor fusions that were not detectable by FISH. Our proof-of-principle study illustrates that transcriptome-based analyses are sensitive and robust methods for detecting actionable gene fusions in lung cancer and could provide a robust alternative to FISH testing in the diagnostic setting.Lung cancer remains one of the major causes of cancer mortality in both men and women worldwide 1 . Genomic alterations identified in lung cancer have significant predictive value in the treatment of the disease including EGFR mutations found commonly in advanced non-small cell lung cancer (NSCLC) that are associated with a favourable response to tyrosine kinase inhibitors 2,3 .More recently, structural genomic rearrangements involving the anaplastic lymphoma kinase (ALK) gene have been identified in NSCLCs. The most common rearrangement, occurring between ALK and echinoderm microtubule-associated protein-like 4 (EML4), is an inversion event resulting in the fusion of the 5′ end of EML4 to the 3′ end of ALK, leading to constitutive kinase activity and malignant growth 4 . Multiple EML4-ALK variants have subsequently been described, all occurring in the same region of the ALK gene but involving different breakpoints within the EML4 gene. Variants 1 and 3a/b account for the highest proportions, at 33% and 29% respectively 5 . Other fusion partners have also been described such as kinesin family member 5B (KIF5B), TRK-fused gene (TFG) and kinesin light chain 1 (KLC1) [5][6][7][8] . Subsequent studies have reported ALK rearrangements occurring in 3-5% of NSCLCs, where they are associated with younger patients who have a light or no smoking history 9 .The development of kinase inhibitors such as crizotinib has led to a breakthrough in the treatment of NSCLC patients carrying ALK fusions, who gain significant survival benefit following treatment 10 . Crizotinib has recently shown therapeutic efficacy in NSCLC patients carrying other rearrangements and alterations, including those involving ...

Krüppel-like factors (KLFs) are a family of 17 transcription factors characterized by a conserved DNA-binding domain of three zinc fingers and a variable N-terminal domain responsible for recruiting cofactors. KLFs have diverse functions in stem cell biology, embryo patterning, and tissue homoeostasis. KLF1 and related family members function as transcriptional activators via recruitment of co-activators such as EP300, whereas KLF3 and related members act as transcriptional repressors via recruitment of C-terminal Binding Proteins. KLF1 directly activates the Klf3 gene via an erythroid-specific promoter. Herein, we show KLF1 and KLF3 bind common as well as unique sites within the erythroid cell genome by ChIP-seq. We show KLF3 can displace KLF1 from key erythroid gene promoters and enhancers in vivo. Using 4sU RNA labelling and RNA-seq, we show this competition results in reciprocal transcriptional outputs for >50 important genes. Furthermore, Klf3−/− mice displayed exaggerated recovery from anemic stress and persistent cell cycling consistent with a role for KLF3 in dampening KLF1-driven proliferation. We suggest this study provides a paradigm for how KLFs work in incoherent feed-forward loops or networks to fine-tune transcription and thereby control diverse biological processes such as cell proliferation.

Comparison of the inflammatory cytokine profile in bone marrow-derived macrophages (BMDMs) from normal and Src homology domain 2-containing tyrosine phosphatase-1 (SHP-1)–deficient Motheaten (me/me) mice revealed a dramatic suppression of IL-6 transcript and protein in me/me BMDMs after LPS stimulation. Interfering with SHP-1 expression using antisense SHP-1 oligonucleotides led to a significant downregulation of IL-6 in normal BMDMs. Conversely, reconstitution of me/me BMDMs with the SHP-1 gene using adenoviral vectors restored IL-6 production. Expression of only SHP-1 Src homology region 2 domains in normal BMDMs inhibited IL-6 production, confirming that IL-6 regulation depends on SHP-1 phosphatase activity. We further demonstrated that loss of SHP-1 function affects proper phosphorylation of Erk1/2 MAPKs and, to a lesser degree, of NF-κB downstream of TLR4 in BMDMs. Inefficient phosphorylation of Erk1/2 MAPKs abrogated the activation of C/EBPβ transcription factor, which was reversed on restoration of SHP-1 function and led to a concomitant enhancement of IL-6 production. We demonstrate that IL-6 production is regulated by a complex network of signaling pathways that include SHP-1–dependent activation of Erk1/2–C/EBPβ and NF-κB, in addition to SHP-1–independent IκB pathway through the activation of protein tyrosine kinases downstream of TLR4. Taken together, these results revealed for the first time, to our knowledge, a positive and critical role of SHP-1 in IL-6 regulation and dependence of Erk1/2–C/EBPβ pathway in addition to that of IκB on SHP-1 activity required for IL-6 induction after LPS stimulation.