GFI1 proteins regulate stem cell formation in the AGM

In vertebrates, the first haematopoietic stem cells (HSCs) with multi-lineage and long-term repopulating potential arise in the AGM (aorta-gonad-mesonephros) region. These HSCs are generated from a rare and transient subset of endothelial cells, called haemogenic endothelium (HE), through an endothelial-to-haematopoietic transition (EHT). Here, we establish the absolute requirement of the transcriptional repressors GFI1 and GFI1B (growth factor independence 1 and 1B) in this unique trans-differentiation process. We first demonstrate that Gfi1 expression specifically defines the rare population of HE that generates emerging HSCs. We further establish that in the absence of GFI1 proteins, HSCs and haematopoietic progenitor cells are not produced in the AGM, revealing the critical requirement for GFI1 proteins in intra-embryonic EHT. Finally, we demonstrate that GFI1 proteins recruit the chromatin-modifying protein LSD1, a member of the CoREST repressive complex, to epigenetically silence the endothelial program in HE and allow the emergence of blood cells.We thank the staff at the Advanced Imaging, animal facility, Molecular Biology Core facilities and Flow Cytometry of CRUK Manchester Institute for technical support and Michael Lie-A-Ling and Elli Marinopoulou for initiating the DamID-PIP bioinformatics project. We thank members of the Stem Cell Biology group, the Stem Cell Haematopoiesis groups and Martin Gering for valuable advice and critical reading of the manuscript. Work in our laboratory is supported by the Leukaemia and Lymphoma Research Foundation (LLR), Cancer Research UK (CRUK) and the Biotechnology and Biological Sciences Research Council (BBSRC). SC is the recipient of an MRC senior fellowship (MR/J009202/1).This is the author accepted manuscript. The final version is available from NPG via http://dx.doi.org/10.1038/ncb327

A global non-coding RNA system modulates fission yeast protein levels in response to stress

Non-coding RNAs (ncRNAs) are frequent and prevalent across the taxa. Although individual non-coding loci have been assigned a function, most are uncharacterized. Their global biological significance is unproven and remains controversial. Here we investigate the role played by ncRNAs in the stress response of Schizosaccharomyces pombe. We integrate global proteomics and RNA sequencing data to identify a systematic programme in which elevated antisense RNA arising both from ncRNAs and from 3′-overlapping convergent gene pairs is directly associated with substantial reductions in protein levels throughout the genome. We describe an extensive array of ncRNAs with trans associations that have the potential to influence multiple pathways. Deletion of one such locus reduces levels of atf1, a transcription factor downstream of the stress-activated mitogen-activated protein kinase (MAPK) pathway, and alters sensitivity to oxidative stress. These non-coding transcripts therefore regulate specific stress responses, adding unanticipated information-processing capacity to the MAPK signalling system

Targeted genetic dependency screen facilitates identification of actionable mutations in FGFR4, MAP3K9, and PAK5 in lung cancer

Approximately 70% of patients with non–small-cell lung cancer present with late-stage disease and have limited treatment options, so there is a pressing need to develop efficacious targeted therapies for these patients. This remains a major challenge as the underlying genetic causes of ∼50% of non–small-cell lung cancers remain unknown. Here we demonstrate that a targeted genetic dependency screen is an efficient approach to identify somatic cancer alterations that are functionally important. By using this approach, we have identified three kinases with gain-of-function mutations in lung cancer, namely FGFR4, MAP3K9, and PAK5. Mutations in these kinases are activating toward the ERK pathway, and targeted depletion of the mutated kinases inhibits proliferation, suppresses constitutive activation of downstream signaling pathways, and results in specific killing of the lung cancer cells. Genomic profiling of patients with lung cancer is ushering in an era of personalized medicine; however, lack of actionable mutations presents a significant hurdle. Our study indicates that targeted genetic dependency screens will be an effective strategy to elucidate somatic variants that are essential for lung cancer cell viability

Investigation of radiosensitivity gene signatures in cancer cell lines

Intrinsic radiosensitivity is an important factor underlying radiotherapy response, but there is no method for its routine assessment in human tumours. Gene signatures are currently being derived and some were previously generated by expression profiling the NCI-60 cell line panel. It was hypothesised that focusing on more homogeneous tumour types would be a better approach. Two cell line cohorts were used derived from cervix [n = 16] and head and neck [n = 11] cancers. Radiosensitivity was measured as surviving fraction following irradiation with 2 Gy (SF2) by clonogenic assay. Differential gene expression between radiosensitive and radioresistant cell lines (SF2</> median) was investigated using Affymetrix GeneChip Exon 1.0ST (cervix) or U133A Plus2 (head and neck) arrays. There were differences within cell line cohorts relating to tissue of origin reflected by expression of the stratified epithelial marker p63. Of 138 genes identified as being associated with SF2, only 2 (1.4%) were congruent between the cervix and head and neck carcinoma cell lines (MGST1 and TFPI), and these did not partition the published NCI-60 cell lines based on SF2. There was variable success in applying three published radiosensitivity signatures to our cohorts. One gene signature, originally trained on the NCI-60 cell lines, did partially separate sensitive and resistant cell lines in all three cell line datasets. The findings do not confirm our hypothesis but suggest that a common transcriptional signature can reflect the radiosensitivity of tumours of heterogeneous origins

Enhancer activation by pharmacologic displacement of LSD1 from GFI1 induces differentiation in acute myeloid leukemia

Summary: Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1’s histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes. : Maiques-Diaz et al. report that, while LSD1 inhibitors target both scaffolding and enzymatic functions of the protein, drug-induced myeloid leukemia cell differentiation is primarily due to the disruption and release from enhancers of GFI1/CoREST complexes, leading to the activation of subordinate myeloid transcription factor genes. Keywords: LSD1, GFI1, acute myeloid leukemia, MLL, acetylation, methylatio

Characterisation of a head and neck squamous cell carcinoma (HNSCC) cell line cohort.

<p><b>A)</b> Graph showing the mean SF2 (log10) (y-axis) for each of the 11 cervix cancer cell lines (x-axis). Error bars show the standard error of mean of 2–3 independent experiments. <b>B)</b> Graph showing that there is no difference in TP63 expression between the SF2 high and low groups. Bar shows the median expression. <b>C)</b> Unsupervised hierarchical clustering of the top 1000 genes ranked by coefficient of variation (from U133 array data). Heatmap colouring is by log₂ expression value. Rows represent genes and columns are cell lines. x-axis dendrogram (clusters) indicates the similarity of the cell lines and y-axis dendrogram the similarity of genes. Cluster 1 represents two samples with the lowest TP63 values (p63 negative). Cluster 2 shows the grouping of the other p63− cell line, along with low TP63 expressing lines. Cluster 3 groups together all HNSCC lines with >6.0 (log2 expression) TP63 expression. <b>D)</b> Diagram to represent the integrated SF2 analysis of the cervix and HNSCC cell lines. Rank product analysis (FDR <0.05) identified 96 genes in the cervix cohort differentially expressed between SF2 low and high cell lines. An identical analysis in the HNSCC cell lines identifies 97 probesets (42 genes) differentially expressed between SF2 low and high cell lines. PCA of the cervix genes shows that they are capable of separating the cell lines by SF2. PCA of the HNSCC genes is equally capable of separating the samples based on SF2. The Venn diagram shows that only 4/138 genes are common between the two cohorts and of these only 2/138 are “congruent” and associated with the same directionality (high SF2/low SF2 in both HNSCC and cervix). PCA shows probeset expression of these two “common” and “congruent” genes (MGST1 and TFPI) in the NCI-60 dataset. The NCI-60 upper PCA shows data-points coloured for median SF2 and lower PCA coloured for 0.2, used previously to partition radiosensitive and radioresistant cell lines in this cohort.</p