Chromatin structural states and their remodelling, including higher-order chromatin folding and three-dimensional (3D) genome organisation, play an important role in the control of gene expression. The role of 3D genome organisation in the control and execution of lineage-specific transcription programmes during the development and differentiation of multipotent stem cells into specialised cell types remains poorly understood. Here, we show that substantial remodelling of the higher-order chromatin structure of the epidermal differentiation complex (EDC), a keratinocyte lineage-specific gene locus on mouse chromosome 3, occurs during epidermal morphogenesis. During epidermal development, the locus relocates away from the nuclear periphery towards the nuclear interior into a compartment enriched in SC35-positive nuclear speckles. Relocation of the EDC locus occurs prior to the full activation of EDC genes involved in controlling terminal keratinocyte differentiation and is a lineage-specific, developmentally regulated event controlled by transcription factor p63, a master regulator of epidermal development. We also show that, in epidermal progenitor cells, p63 directly regulates the expression of the ATP-dependent chromatin remodeller Brg1, which binds to distinct domains within the EDC and is required for relocation of the EDC towards the nuclear interior. Furthermore, Brg1 also regulates gene expression within the EDC locus during epidermal morphogenesis. Thus, p63 and its direct target Brg1 play an essential role in remodelling the higher-order chromatin structure of the EDC and in the specific positioning of this locus within the landscape of the 3D nuclear space, as required for the efficient expression of EDC genes in epidermal progenitor cells during skin development.

Components of the type II CRISPR–Cas complex in bacteria have been used successfully in eukaryotic cells to facilitate rapid and accurate cell line engineering, animal model generation and functional genomic screens. Such developments are providing new opportunities for drug target identification and validation, particularly with the application of pooled genetic screening. As CRISPR–Cas is a relatively new genetic screening tool, it is important to assess its functionality in a number of different cell lines and to analyse potential improvements that might increase the sensitivity of a given screen. To examine critical aspects of screening quality, we constructed ultra-complex libraries containing sgRNA sequences targeting a collection of essential genes. We examined the performance of screening in both haploid and hypotriploid cell lines, using two alternative guide design algorithms and two tracrRNA variants in a time-resolved analysis. Our data indicate that a simple adaptation of the tracrRNA substantially improves the robustness of guide loss during a screen. This modification minimises the requirement for high numbers of sgRNAs targeting each gene, increasing hit scoring and creating a powerful new platform for successful screening.

Mammalian genomes contain several dozens of large (>0.5 Mbp) lineage-specific gene loci harbouring functionally related genes. However, spatial chromatin folding, organization of the enhancer-promoter networks and their relevance to Topologically Associating Domains (TADs) in these loci remain poorly understood. TADs are principle units of the genome folding and represents the DNA regions within which DNA interacts more frequently and less frequently across the TAD boundary. Here, we used Chromatin Conformation Capture Carbon Copy (5C) technology to characterize spatial chromatin interaction network in the 3.1 Mb Epidermal Differentiation Complex (EDC) locus harbouring 61 functionally related genes that show lineage-specific activation during terminal keratinocyte differentiation in the epidermis. 5C data validated by 3D-FISH demonstrate that the EDC locus is organized into several TADs showing distinct lineage-specific chromatin interaction networks based on their transcription activity and the gene-rich or gene-poor status. Correlation of the 5C results with genome-wide studies for enhancer-specific histone modifications (H3K4me1 and H3K27ac) revealed that the majority of spatial chromatin interactions that involves the gene-rich TADs at the EDC locus in keratinocytes include both intra- and inter-TAD interaction networks, connecting gene promoters and enhancers. Compared to thymocytes in which the EDC locus is mostly transcriptionally inactive, these interactions were found to be keratinocyte-specific. In keratinocytes, the promoter-enhancer anchoring regions in the gene-rich transcriptionally active TADs are enriched for the binding of chromatin architectural proteins CTCF, Rad21 and chromatin remodeler Brg1. In contrast to gene-rich TADs, gene-poor TADs show preferential spatial contacts with each other, do not contain active enhancers and show decreased binding of CTCF, Rad21 and Brg1 in keratinocytes. Thus, spatial interactions between gene promoters and enhancers at the multi-TAD EDC locus in skin epithelial cells are cell type-specific and involve extensive contacts within TADs as well as between different gene-rich TADs, forming the framework for lineage-specific transcription.

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ABCA12 is known to be critical for skin barrier integrity. Mutations in this gene cause the most severe form of Autosomal Recessive Congenital Ichthyosis, Harlequin Ichthyosis (HI). HI patients have marked hyperkeratosis at birth with fissuring, leading to life-threatening complications due to increased risk of infection, trans-epidermal water and heat loss. The aim of this study was to identify essential pathways involved in the pathomechanisms of Harlequin Ichthyosis, responsible for aberrant epidermal differentiation. We performed RNA-seq on calcium induced primary keratinocytes with siRNA knockdown of ABCA12 and identified 118 genes significantly down-regulated and 36 genes significantly up-regulated (FDR < 0.05). Functional annotation clustering analysis showed changes in epidermal differentiation, fatty acid metabolism, cytokine and interferon signaling. The suppressor of cytokine signaling 3 (SOCS3), a negative feedback regulator of the JAK-STAT signaling pathway, was 2.5 fold downregulated whereas Interleukin-1 (IL1A and IL1B) were 2 fold increased. To investigate these findings further we engineered an ABCA12 CRISPR-Cas9 knockout keratinocyte cell line and compared this with a HI patient-derived cell line and wild type controls. Alterations in differentiation and lipid profile in the HI OT models were observed, recapitulating the HI epidermis phenotype. We found that phospho-STAT1 (Y701) was strongly upregulated in the HI model compared to control. In HI patient skin, the STAT1 expression pattern was altered compared to control skin. The secretion of IL-1a was increased in the HI model compared to control. Both STAT1 and IL-1 regulate the Nitric Oxide (NO) pathway upregulating transcription of inducible NO synthase (iNOS), which we found to be significantly upregulated in HI skin. These data provide insights into the pathogenesis of HI suggest that the NO signaling pathway may be a possible therapeutic target in this disorder.

Mutations in tumour suppressors and un-druggable oncogenes dominate the landscape of cancer driver genes. Only a minority of colon cancers have mutations in druggable cancer drivers, such as PIK3CA. Conversely, mutations in tumour suppressors such as APC and TP53 are frequent, as are mutations in the notoriously difficult to drug KRAS target. There is an urgent need for new therapeutics to target tumours driven by these mutations: immune checkpoint approaches are likely to only prove effective in the fraction of patients whose tumours bear high mutation loads, which is colon cancer may be restricted to the minority of mismatch repair deficient cancers. The concepts of non-oncogene addiction and synthetic lethality provide a conceptual framework for finding therapeutic targets in these cancers. We have used arrayed siRNA and pooled CRISPR-Cas9 libraries to screen a panel of isogenic and non-isogenic colon cancer cell lines under conditions designed to increase the cells dependency on oncogenic pathways. This panel contains cell lines with mutations in TP53, APC, KRAS, PIK3CA and/or FBXW7 for which we are aiming to identify co-dependencies that consistently segregate with genotype. Our screens have identified a number of novel targets for which reduced expression imposes specific fitness defects on cells with mutant PIK3CA, mutant TP53 or mutant FBXW7. Few of these targets were identified by both siRNA and CRISPR-Cas9 approaches. Furthermore, isogenic pairs of cell lines have not proved helpful for identifying synthetic lethal targets in the KRAS or PIK3CA genotypes. However, we believe the increased penetrance of the CRIPSR-Cas9 approach has uncovered novel candidate synthetic lethal genes that were not found by RNA interference. Although our current screens are not saturating, we have confirmed several previously reported genetic interactions including the requirements for expression of MDM2 in TP53 wild-type cancers and HK2 in colon cancer lines with KRAS mutations. The screens in the FBXW7 genotype were particularly interesting: in addition to identifying a potential synthetic lethal interaction with a target for which a drug has recently entered the clinic, we also found that FBXW7 itself seemed to be required in many of the colon cancers with FBXW7 mutations. This suggests that FBXW7 may sometimes act as an oncogene, rather than as a tumour suppressor in cancer initiation or progression. Finally, we have attempted to validate some of the candidate synthetic lethal genes identified by RNA interference via ultra-deep CRISPR-CAS9 pooled screening, extending the approach of the Vakoc lab (Nature Biotech. 33, 661-667, 2015). We can confirm this method can highlight functional domains of particular significance to a protein's function, but note that many-fold drop outs of sgRNAs can also occur where they target multiple sites in the genome, presumably via a DNA damage response. Citation Format: Jonathan D. Moore, Chantelle Hudson, Paul Russell, Gaganpreet Tiwana, David Walter, Ceri M. Wiggins, Joanne Yarker. Synthetic lethal CRISPR-Cas9 screen imply an oncogenic role for FBXW7 mutations in colon cancer [abstract]. In: Proceedings of the AACR Precision Medicine Series: Opportunities and Challenges of Exploiting Synthetic Lethality in Cancer; Jan 4-7, 2017; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2017;16(10 Suppl):Abstract nr A25.

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Genetic perturbations mediated by CRISPR-Cas9 are transforming the drug discovery process. In the hunt for novel drug targets, researchers finally have a screening tool with sufficient penetrance and power to identify new interactions, followed by a rapid gene editing technology to streamline validation approaches. At Horizon Discovery, we have now initiated more than 250 CRISPR-Cas9 knockout (KO) screens, including a significant effort to identify novel synthetic lethal interactions in colon and lung cancer genotypes. These screens have identified a reassuring number of known essential interactions, such as MDM2 and PPMID in TP53 wild type cancer cell lines. These results provide confidence in the ability of these screens to also discover novel targets that should make it through the validation process. Indeed, we have discovered novel targets for selective ablation of TP53 mutant colon cancers, several of which are strongly supported by the literature. In FBXW7 mutant colon cancers, CRISPR-Cas9 KO screens identified a set of potential novel druggable targets in addition to MCL1 - a known substrate of FBXW7, which showed a strong dependency in FBXW7 mutant lines. An important step in triaging primary hits ahead of extensive validation work has been secondary screens that target functional domains with high density sgRNA tiling. They allow more targets to be examined than would be feasible by any arrayed approach. Data from such screens, which test tens to hundreds of guides in unison, not only increases hit calling power, but can also yield information regarding important domains and mechanism of action. This was evident in the FBXW7 secondary screen data, where guides targeting the key functional BH domains of MCL1 proved to be the most effective. This information can be used to prioritise individual targets for further validation, using techniques that include an inducible CRISPR-Cas9 system to assess the effect of individual guides in an arrayed format on cell proliferation and survival, and CRISPRi. These data exemplify how CRISPR-Cas9 can enable the discovery of a new, more robust generation of drug and antibody targets. Citation Format: Ceri M. Wiggins, David Walter, Paul Russell, Clare Sheridan, Chantelle Hudson, Joanne Yarker, Carlos le Sage, Nicola McCarthy, Jonathan D. Moore. CRISPR-Cas9: A tool for rapid target discovery and validation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2912.