33Eukaryotes use a temporally regulated process, known as the replication timing program, to 34 ensure that their genomes are fully and accurately duplicated during S phase. Replication timing 35 programs are predictive of genomic features and activity, and considered to be functional 36 readouts of chromatin organization. Although replication timing programs have been described 37 for yeast and animal systems, much less is known about the temporal regulation of plant DNA 38 replication or its relationship to genome sequence and chromatin structure. We used the 39 thymidine analog, 5-ethynyl-2'-deoxyuridine, in combination with flow sorting and Repli-Seq to 40 describe, at high-resolution, the genome-wide replication timing program for Arabidopsis 41 thaliana Col-0 suspension cells. We identified genomic regions that replicate predominantly 42 during early, mid and late S phase, and correlated these regions with genomic features and with 43 data for chromatin state, accessibility and long-distance interaction.

One sentence summary: DNA replication initiation sites in plants associate most strongly with AT-rich and highly accessible chromatin, and not with genes or a particular epigenetic signature.

Immune evasion is a significant contributor to tumor evolution, and the immunoinhibitory axis PD-1/PD-L1 is a frequent mechanism employed to escape tumor immune surveillance. To identify cancer drivers involved in immune evasion, we performed a CRISPR-Cas9 screen of tumor suppressor genes regulating the basal and interferon (IFN)-inducible cell surface levels of PD-L1. Multiple regulators of PD-L1 were identified, including IRF2, ARID2, KMT2D, and AAMP. We also identified CTCF and the cohesin complex proteins, known regulators of chromatin architecture and transcription, among the most potent negative regulators of PD-L1 cell surface expression. Additionally, loss of the cohesin subunit RAD21 was shown to up-regulate PD-L2 and MHC-I surface expression. PD-L1 and MHC-I suppression by cohesin were shown to be conserved in mammary epithelial and myeloid cells. Comprehensive examination of the transcriptional effect of STAG2 deficiency in epithelial and myeloid cells revealed an activation of strong IFN and NF-κB expression signatures. Inhibition of JAK-STAT or NF-κB pathways did not result in rescue of PD-L1 up-regulation in RAD21-deficient cells, suggesting more complex or combinatorial mechanisms at play. Discovery of the PD-L1 and IFN up-regulation in cohesin-mutant cells expands our understanding of the biology of cohesin-deficient cells as well as molecular regulation of the PD-L1 molecule.

Splicing modulation is a promising treatment strategy pursued to date only in splicing-factor mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in cancer, including myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. Here, we show that cohesin mutations are biomarkers of sensitivity to drugs targeting the splicing-factor SF3B1 (H3B-8800 and E-7107). We identify drug-induced alterations in splicing and corresponding reduced gene expression of a large number of DNA repair genes, including BRCA1 and BRCA2, as the mechanism underlying this sensitivity in cell line models, primary patient samples and patient-derived xenograft (PDX) models of AML. We find that DNA damage repair genes are particularly sensitive to exon skipping induced by SF3B1 modulators given their long length and large number of exons per transcript. Furthermore, we demonstrate that treatment of cohesin-mutant cells with SF3B1 modulators not only results in impaired DNA damage response and accumulation of DNA damage, but it significantly sensitizes cells to subsequent killing by PARP inhibitors and chemotherapy, and leads to improved overall survival of PDX models of cohesin-mutant AML in vivo. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS and AML, and we propose this as a broader strategy for therapeutic targeting of other DNA damage-repair deficient cancers.

No abstract

<div>Abstract<p>DNA methyltransferase inhibitors (DNMTI) like 5-Azacytidine (5-Aza) are the only disease-modifying drugs approved for the treatment of higher-risk myelodysplastic syndromes (MDS), however less than 50% of patients respond, and there are no predictors of response with clinical utility. Somatic mutations in the DNA methylation regulating gene <i>tet-methylcytosine dioxygenase 2</i> (<i>TET2</i>) are associated with response to DNMTIs, however the mechanisms responsible for this association remain unknown. Using bisulfite padlock probes, mRNA sequencing, and hydroxymethylcytosine pull-down sequencing at several time points throughout 5-Aza treatment, we show that <i>TET2</i> loss particularly influences DNA methylation (5mC) and hydroxymethylation (5hmC) patterns at erythroid gene enhancers and is associated with downregulation of erythroid gene expression in the human erythroleukemia cell line TF-1. 5-Aza disproportionately induces expression of these down-regulated genes in TET2KO cells and this effect is related to dynamic 5mC changes at erythroid gene enhancers after 5-Aza exposure. We identified differences in remethylation kinetics after 5-Aza exposure for several types of genomic regulatory elements, with distal enhancers exhibiting longer-lasting 5mC changes than other regions. This work highlights the role of 5mC and 5hmC dynamics at distal enhancers in regulating the expression of differentiation-associated gene signatures, and sheds light on how 5-Aza may be more effective in patients harboring <i>TET2</i> mutations.</p>Implications:<p>TET2 loss in erythroleukemia cells induces hypermethylation and impaired expression of erythroid differentiation genes which can be specifically counteracted by 5-Azacytidine, providing a potential mechanism for the increased efficacy of 5-Aza in TET2-mutant patients with MDS.</p>Visual Overview:<p><a href="http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg" target="_blank">http://mcr.aacrjournals.org/content/molcanres/19/3/451/F1.large.jpg</a>.</p></div>

Supplementary table from Integrative RNA-omics Discovers <i>GNAS</i> Alternative Splicing as a Phenotypic Driver of Splicing Factor–Mutant Neoplasms

Supplementary table from Integrative RNA-omics Discovers <i>GNAS</i> Alternative Splicing as a Phenotypic Driver of Splicing Factor–Mutant Neoplasms