In vivo CRISPR/Cas9 screening identifies Pbrm1 as a regulator of myeloid leukemia development in mice.

CRISPR/Cas9 screening approaches are powerful tool for identifying in vivo cancer dependencies. Hematopoietic malignancies are genetically complex disorders in which the sequential acquisition of somatic mutations generates clonal diversity. Over time, additional cooperating mutations may drive disease progression. Using an in vivo pooled gene editing screen of epigenetic factors in primary murine hematopoietic stem and progenitor cells (HSPCs), we sought to uncover unrecognized genes that contribute to leukemia progression. We, first, modeled myeloid leukemia in mice by functionally abrogating both Tet2 and Tet3 in HSPCs, followed by transplantation. We, then, performed pooled CRISPR/Cas9 editing of genes encoding epigenetic factors and identified Pbrm1/Baf180, a subunit of the polybromo BRG1/BRM-associated factor SWItch/Sucrose Non-Fermenting chromatin-remodeling complex, as a negative driver of disease progression. We found that Pbrm1 loss promoted leukemogenesis with a significantly shortened latency. Pbrm1-deficient leukemia cells were less immunogenic and were characterized by attenuated interferon signaling and reduced major histocompatibility complex class II (MHC II) expression. We explored the potential relevance to human leukemia by assessing the involvement of PBRM1 in the control of interferon pathway components and found that PBRM1 binds to the promoters of a subset of these genes, most notably IRF1, which in turn regulates MHC II expression. Our findings revealed a novel role for Pbrm1 in leukemia progression. More generally, CRISPR/Cas9 screening coupled with phenotypic readouts in vivo has helped identify a pathway by which transcriptional control of interferon signaling influences leukemia cell interactions with the immune system.

Mapping the evolving landscape of super-enhancers during cell differentiation.

BACKGROUND: Super-enhancers are clusters of enhancer elements that play critical roles in the maintenance of cell identity. Current investigations on super-enhancers are centered on the established ones in static cell types. How super-enhancers are established during cell differentiation remains obscure. RESULTS: Here, by developing an unbiased approach to systematically analyze the evolving landscape of super-enhancers during cell differentiation in multiple lineages, we discover a general trend where super-enhancers emerge through three distinct temporal patterns: conserved, temporally hierarchical, and de novo. The three types of super-enhancers differ further in association patterns in target gene expression, functional enrichment, and 3D chromatin organization, suggesting they may represent distinct structural and functional subtypes. Furthermore, we dissect the enhancer repertoire within temporally hierarchical super-enhancers, and find enhancers that emerge at early and late stages are enriched with distinct transcription factors, suggesting that the temporal order of establishment of elements within super-enhancers may be directed by underlying DNA sequence. CRISPR-mediated deletion of individual enhancers in differentiated cells shows that both the early- and late-emerged enhancers are indispensable for target gene expression, while in undifferentiated cells early enhancers are involved in the regulation of target genes. CONCLUSIONS: In summary, our analysis highlights the heterogeneity of the super-enhancer population and provides new insights to enhancer functions within super-enhancers.

Phase separation of the LINE-1 ORF1 protein is mediated by the N-terminus and coiled-coil domain.

Long interspersed nuclear element-1 (L1) is a retrotransposable element that autonomously replicates in the human genome, resulting in DNA damage and genomic instability. Activation of L1 in senescent cells triggers a type I interferon response and age-associated inflammation. Two open reading frames encode an ORF1 protein functioning as messenger RNA chaperone and an ORF2 protein providing catalytic activities necessary for retrotransposition. No function has been identified for the conserved, disordered N-terminal region of ORF1. Using microscopy and NMR spectroscopy, we demonstrate that ORF1 forms liquid droplets in vitro in a salt-dependent manner and that interactions between its N-terminal region and coiled-coil domain are necessary for phase separation. Mutations disrupting blocks of charged residues within the N-terminus impair phase separation, whereas some mutations within the coiled-coil domain enhance phase separation. Demixing of the L1 particle from the cytosol may provide a mechanism to protect the L1 transcript from degradation.

Support for removal of point-of-purchase tobacco advertising and displays: findings from the International Tobacco Control (ITC) Canada survey.

BACKGROUND: Although most countries now have at least some restrictions on tobacco marketing, the tobacco industry meet these restrictions by re-allocating expenditure to unregulated channels, such as at point-of-purchase. METHODS: Longitudinal data from 10 Canadian provinces in the International Tobacco Control Survey was analysed to examine adult smokers' support for a ban on tobacco advertising and displays in stores and whether this support is associated with noticing either advertising or displays in stores, and quit intentions, over time. In total, there were 4580 respondents in wave 5 (October 2006 to February 2007), wave 6 (September 2007 to February 2008) and wave 7 (October 2008 to June 2009). The surveys were conducted before, during and in some cases after the implementation of display bans in most Canadian provinces and territories. RESULTS: Smokers in all provinces showed strong support for a ban on tobacco displays over the study period. Levels of support for an advertising and display ban were comparable between Canadian provinces over time, irrespective of whether they had been banned or not. Noticing tobacco displays and signs in-store was demonstrably less likely to predict support for display (OR=0.73, p=0.005) and advertising (OR=0.78, p=0.02) ban, respectively. Smokers intending to quit were more likely to support advertising and display bans over time. CONCLUSION: This study serves as a timely reminder that the implementation of tobacco control measures, such as the removal of tobacco displays, appear to sustain support among smokers, those most likely to oppose such measures.