An expanded universe of cancer targets.

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation.

Core Components of the Nuclear Pore Bind Distinct States of Chromatin and Contribute to Polycomb Repression.

Interactions between the genome and the nuclear pore complex (NPC) have been implicated in multiple gene regulatory processes, but the underlying logic of these interactions remains poorly defined. Here, we report high-resolution chromatin binding maps of two core components of the NPC, Nup107 and Nup93, in Drosophila cells. Our investigation uncovered differential binding of these NPC subunits, where Nup107 preferentially targets active genes while Nup93 associates primarily with Polycomb-silenced regions. Comparison to Lamin-associated domains (LADs) revealed that NPC binding sites can be found within LADs, demonstrating a linear binding of the genome along the nuclear envelope. Importantly, we identified a functional role of Nup93 in silencing of Polycomb target genes and in spatial folding of Polycomb domains. Our findings lend to a model where different nuclear pores bind different types of chromatin via interactions with specific NPC sub-complexes, and a subset of Polycomb domains is stabilized by interactions with Nup93.

Cell cycle-dependent differences in nuclear pore complex assembly in metazoa.

In metazoa, nuclear pore complexes (NPCs) assemble from disassembled precursors into a reforming nuclear envelope (NE) at the end of mitosis and into growing intact NEs during interphase. Here, we show via RNAi-mediated knockdown that ELYS, a nucleoporin critical for the recruitment of the essential Nup107/160 complex to chromatin, is required for NPC assembly at the end of mitosis but not during interphase. Conversely, the transmembrane nucleoporin POM121 is critical for the incorporation of the Nup107/160 complex into new assembly sites specifically during interphase. Strikingly, recruitment of the Nup107/160 complex to an intact NE involves a membrane curvature-sensing domain of its constituent Nup133, which is not required for postmitotic NPC formation. Our results suggest that in organisms with open mitosis, NPCs assemble via two distinct mechanisms to accommodate cell cycle-dependent differences in NE topology.

Metazoan Nuclear Pores Provide a Scaffold for Poised Genes and Mediate Induced Enhancer-Promoter Contacts.

Nuclear pore complex components (Nups) have been implicated in transcriptional regulation, yet what regulatory steps are controlled by metazoan Nups remains unclear. We identified the presence of multiple Nups at promoters, enhancers, and insulators in the Drosophila genome. In line with this binding, we uncovered a functional role for Nup98 in mediating enhancer-promoter looping at ecdysone-inducible genes. These genes were found to be stably associated with nuclear pores before and after activation. Although changing levels of Nup98 disrupted enhancer-promoter contacts, it did not affect ongoing transcription but instead compromised subsequent transcriptional activation or transcriptional memory. In support of the enhancer-looping role, we found Nup98 to gain and retain physical interactions with architectural proteins upon stimulation with ecdysone. Together, our data identify Nups as a class of architectural proteins for enhancers and supports a model in which animal genomes use the nuclear pore as an organizing scaffold for inducible poised genes.

Sparse dictionary learning recovers pleiotropy from human cell fitness screens.

In high-throughput functional genomic screens, each gene product is commonly assumed to exhibit a singular biological function within a defined protein complex or pathway. In practice, a single gene perturbation may induce multiple cascading functional outcomes, a genetic principle known as pleiotropy. Here, we model pleiotropy in fitness screen collections by representing each gene perturbation as the sum of multiple perturbations of biological functions, each harboring independent fitness effects inferred empirically from the data. Our approach (Webster) recovered pleiotropic functions for DNA damage proteins from genotoxic fitness screens, untangled distinct signaling pathways upstream of shared effector proteins from cancer cell fitness screens, and predicted the stoichiometry of an unknown protein complex subunit from fitness data alone. Modeling compound sensitivity profiles in terms of genetic functions recovered compound mechanisms of action. Our approach establishes a sparse approximation mechanism for unraveling complex genetic architectures underlying high-dimensional gene perturbation readouts.

YAP1 and PRDM14 converge to promote cell survival and tumorigenesis.

The transcriptional co-activator YAP1 oncogene is the downstream effector of the Hippo pathway, which regulates tissue homeostasis, organ size, regeneration, and tumorigenesis. Multiple cancers are dependent on sustained expression of YAP1 for cell proliferation, survival, and tumorigenesis, but the molecular basis of this oncogene dependency is not well understood. To identify genes that can functionally substitute for YAP1, we performed a genome-scale genetic rescue screen in YAP1-dependent colon cancer cells expressing an inducible YAP1-specific shRNA. We found that the transcription factor PRDM14 rescued cell proliferation and tumorigenesis upon YAP1 suppression in YAP1-dependent cells, xenografts, and colon cancer organoids. YAP1 and PRDM14 individually activated the transcription of calmodulin 2 (CALM2) and a glucose transporter SLC2A1 upon YAP1 suppression, and CALM2 or SLC2A1 expression was required for the rescue of YAP1 suppression. Together, these findings implicate PRDM14-mediated transcriptional upregulation of CALM2 and SLC2A1 as key components of oncogenic YAP1 signaling and dependency.

DNA sequence and analysis of human chromosome 18.

Chromosome 18 appears to have the lowest gene density of any human chromosome and is one of only three chromosomes for which trisomic individuals survive to term. There are also a number of genetic disorders stemming from chromosome 18 trisomy and aneuploidy. Here we report the finished sequence and gene annotation of human chromosome 18, which will allow a better understanding of the normal and disease biology of this chromosome. Despite the low density of protein-coding genes on chromosome 18, we find that the proportion of non-protein-coding sequences evolutionarily conserved among mammals is close to the genome-wide average. Extending this analysis to the entire human genome, we find that the density of conserved non-protein-coding sequences is largely uncorrelated with gene density. This has important implications for the nature and roles of non-protein-coding sequence elements.

SMAD4 represses FOSL1 expression and pancreatic cancer metastatic colonization.

Metastasis is a complex and poorly understood process. In pancreatic cancer, loss of the transforming growth factor (TGF)-ß/BMP effector SMAD4 is correlated with changes in altered histopathological transitions, metastatic disease, and poor prognosis. In this study, we use isogenic cancer cell lines to identify SMAD4 regulated genes that contribute to the development of metastatic colonization. We perform an in vivo screen identifying FOSL1 as both a SMAD4 target and sufficient to drive colonization to the lung. The targeting of these genes early in treatment may provide a therapeutic benefit.

Nuclear envelope and genome interactions in cell fate.

The eukaryotic cell nucleus houses an organism's genome and is the location within the cell where all signaling induced and development-driven gene expression programs are ultimately specified. The genome is enclosed and separated from the cytoplasm by the nuclear envelope (NE), a double-lipid membrane bilayer, which contains a large variety of trans-membrane and associated protein complexes. In recent years, research regarding multiple aspects of the cell nucleus points to a highly dynamic and coordinated concert of efforts between chromatin and the NE in regulation of gene expression. Details of how this concert is orchestrated and how it directs cell differentiation and disease are coming to light at a rapid pace. Here we review existing and emerging concepts of how interactions between the genome and the NE may contribute to tissue specific gene expression programs to determine cell fate.

POM121 and Sun1 play a role in early steps of interphase NPC assembly.

Nuclear pore complexes (NPCs) assemble at the end of mitosis during nuclear envelope (NE) reformation and into an intact NE as cells progress through interphase. Although recent studies have shown that NPC formation occurs by two different molecular mechanisms at two distinct cell cycle stages, little is known about the molecular players that mediate the fusion of the outer and inner nuclear membranes to form pores. In this paper, we provide evidence that the transmembrane nucleoporin (Nup), POM121, but not the Nup107-160 complex, is present at new pore assembly sites at a time that coincides with inner nuclear membrane (INM) and outer nuclear membrane (ONM) fusion. Overexpression of POM121 resulted in juxtaposition of the INM and ONM. Additionally, Sun1, an INM protein that is known to interact with the cytoskeleton, was specifically required for interphase assembly and localized with POM121 at forming pores. We propose a model in which POM121 and Sun1 interact transiently to promote early steps of interphase NPC assembly.

Single bead affinity detection (SINBAD) for the analysis of protein-protein interactions.

We present a miniaturized pull-down method for the detection of protein-protein interactions using standard affinity chromatography reagents. Binding events between different proteins, which are color-coded with quantum dots (QDs), are visualized on single affinity chromatography beads by fluorescence microscopy. The use of QDs for single molecule detection allows the simultaneous analysis of multiple protein-protein binding events and reduces the amount of time and material needed to perform a pull-down experiment.

The genome of M. acetivorans reveals extensive metabolic and physiological diversity.

Methanogenesis, the biological production of methane, plays a pivotal role in the global carbon cycle and contributes significantly to global warming. The majority of methane in nature is derived from acetate. Here we report the complete genome sequence of an acetate-utilizing methanogen, Methanosarcina acetivorans C2A. Methanosarcineae are the most metabolically diverse methanogens, thrive in a broad range of environments, and are unique among the Archaea in forming complex multicellular structures. This diversity is reflected in the genome of M. acetivorans. At 5,751,492 base pairs it is by far the largest known archaeal genome. The 4524 open reading frames code for a strikingly wide and unanticipated variety of metabolic and cellular capabilities. The presence of novel methyltransferases indicates the likelihood of undiscovered natural energy sources for methanogenesis, whereas the presence of single-subunit carbon monoxide dehydrogenases raises the possibility of nonmethanogenic growth. Although motility has not been observed in any Methanosarcineae, a flagellin gene cluster and two complete chemotaxis gene clusters were identified. The availability of genetic methods, coupled with its physiological and metabolic diversity, makes M. acetivorans a powerful model organism for the study of archaeal biology. [Sequence, data, annotations and analyses are available at http://www-genome.wi.mit.edu/.]