Person:

Marto, Jarrod

Background: Motivated by the precarious state of the world's coral reefs, there is currently a keen interest in coral transcriptomics. By identifying changes in coral gene expression that are triggered by particular environmental stressors, we can begin to characterize coral stress responses at the molecular level, which should lead to the development of more powerful diagnostic tools for evaluating the health of corals in the field. Furthermore, the identification of genetic variants that are more or less resilient in the face of particular stressors will help us to develop more reliable prognoses for particular coral populations. Toward this end, we performed deep mRNA sequencing of the cauliflower coral, Pocillopora damicornis, a geographically widespread Indo-Pacific species that exhibits a great diversity of colony forms and is able to thrive in habitats subject to a wide range of human impacts. Importantly, P. damicornis is particularly amenable to laboratory culture. We collected specimens from three geographically isolated Hawaiian populations subjected to qualitatively different levels of human impact. We isolated RNA from colony fragments ("nubbins") exposed to four environmental stressors (heat, desiccation, peroxide, and hypo-saline conditions) or control conditions. The RNA was pooled and sequenced using the 454 platform. Description: Both the raw reads (n = 1, 116, 551) and the assembled contigs (n = 70, 786; mean length = 836 nucleotides) were deposited in a new publicly available relational database called PocilloporaBase http://www.PocilloporaBase.org. Using BLASTX, 47.2% of the contigs were found to match a sequence in the NCBI database at an E-value threshold of (\leq).001; 93.6% of those contigs with matches in the NCBI database appear to be of metazoan origin and 2.3% bacterial origin, while most of the remaining 4.1% match to other eukaryotes, including algae and amoebae. Conclusions: P. damicornis now joins the handful of coral species for which extensive transcriptomic data are publicly available. Through PocilloporaBase http://www.PocilloporaBase.org, one can obtain assembled contigs and raw reads and query the data according to a wide assortment of attributes including taxonomic origin, PFAM motif, KEGG pathway, and GO annotation.

Curated gene sets from databases such as KEGG Pathway and Gene Ontology are often used to systematically organize lists of genes or proteins derived from high-throughput data. However, the information content inherent to some relationships between the interrogated gene sets, such as pathway crosstalk, is often underutilized. A gene set network, where nodes representing individual gene sets such as KEGG pathways are connected to indicate a functional dependency, is well suited to visualize and analyze global gene set relationships. Here we introduce a novel gene set network construction algorithm that integrates gene lists derived from high-throughput experiments with curated gene sets to construct co-enrichment gene set networks. Along with previously described co-membership and linkage algorithms, we apply the co-enrichment algorithm to eight gene set collections to construct integrated multi-evidence gene set networks with multiple edge types connecting gene sets. We demonstrate the utility of approach through examples of novel gene set networks such as the chromosome map co-differential expression gene set network. A total of twenty-four gene set networks are exposed via a web tool called MetaNet, where context-specific multi-edge gene set networks are constructed from enriched gene sets within user-defined gene lists. MetaNet is freely available at http://blaispathways.dfci.harvard.edu/metanet/.

The small genome of polyomaviruses encodes a limited number of proteins that are highly dependent on interactions with host cell proteins for efficient viral replication. The SV40 large T antigen (LT) contains several discrete functional domains including the LXCXE or RB-binding motif, the DNA binding and helicase domains that contribute to the viral life cycle. In addition, the LT C-terminal region contains the host range and adenovirus helper functions required for lytic infection in certain restrictive cell types. To understand how LT affects the host cell to facilitate viral replication, we expressed full-length or functional domains of LT in cells, identified interacting host proteins and carried out expression profiling. LT perturbed the expression of p53 target genes and subsets of cell-cycle dependent genes regulated by the DREAM and the B-Myb-MuvB complexes. Affinity purification of LT followed by mass spectrometry revealed a specific interaction between the LT C-terminal region and FAM111A, a previously uncharacterized protein. Depletion of FAM111A recapitulated the effects of heterologous expression of the LT C-terminal region, including increased viral gene expression and lytic infection of SV40 host range mutants and adenovirus replication in restrictive cells. FAM111A functions as a host range restriction factor that is specifically targeted by SV40 LT.

The nuclear lamina is a major obstacle encountered by herpesvirus nucleocapsids in their passage from the nucleus to the cytoplasm (nuclear egress). We found that the human cytomegalovirus (HCMV)-encoded protein kinase UL97, which is required for efficient nuclear egress, phosphorylates the nuclear lamina component lamin A/C in vitro on sites targeted by Cdc2/cyclin-dependent kinase 1, the enzyme that is responsible for breaking down the nuclear lamina during mitosis. Quantitative mass spectrometry analyses, comparing lamin A/C isolated from cells infected with viruses either expressing or lacking UL97 activity, revealed UL97-dependent phosphorylation of lamin A/C on the serine at residue 22 (Ser[super]22). Transient treatment of HCMV-infected cells with maribavir, an inhibitor of UL97 kinase activity, reduced lamin A/C phosphorylation by approximately 50%, consistent with UL97 directly phosphorylating lamin A/C during HCMV replication. Phosphorylation of lamin A/C during viral replication was accompanied by changes in the shape of the nucleus, as well as thinning, invaginations, and discrete breaks in the nuclear lamina, all of which required UL97 activity. As Ser[super]22 is a phosphorylation site of particularly strong relevance for lamin A/C disassembly, our data support a model wherein viral mimicry of a mitotic host cell kinase activity promotes nuclear egress while accommodating viral arrest of the cell cycle.

Many human diseases, arising from mutations of disease susceptibility genes (genetic diseases), are also associated with viral infections (virally implicated diseases), either in a directly causal manner or by indirect associations. Here we examine whether viral perturbations of host interactome may underlie such virally implicated disease relationships. Using as models two different human viruses, Epstein-Barr virus (EBV) and human papillomavirus (HPV), we find that host targets of viral proteins reside in network proximity to products of disease susceptibility genes. Expression changes in virally implicated disease tissues and comorbidity patterns cluster significantly in the network vicinity of viral targets. The topological proximity found between cellular targets of viral proteins and disease genes was exploited to uncover a novel pathway linking HPV to Fanconi anemia.

Cell migration is essential for embryonic development and tissue formation in all animals. cacn-1 is a conserved gene of unknown molecular function identified in a genome-wide screen for genes that regulate distal tip cell migration in the nematode worm Caenorhabditis elegans. In this study we take a proteomics approach to understand CACN-1 function. To isolate CACN-1−interacting proteins, we used an in vivo tandem-affinity purification strategy. Tandem-affinity purification−tagged CACN-1 complexes were isolated from C. elegans lysate, analyzed by mass spectrometry, and characterized bioinformatically. Results suggest significant interaction of CACN-1 with the C. elegans spliceosome. All of the identified interactors were screened for distal tip cell migration phenotypes using RNAi. Depletion of many of these factors led to distal tip cell migration defects, particularly a failure to stop migrating, a phenotype commonly seen in cacn-1 deficient animals. The results of this screen identify eight novel regulators of cell migration and suggest CACN-1 may participate in a protein network dedicated to high-fidelity gonad development. The composition of proteins comprising the CACN-1 network suggests that this critical developmental module may exert its influence through alternative splicing or other post-transcriptional gene regulation.

Loss of the chromatin remodeling ATPase CHD5 has been linked to the progression of neuroblastoma tumors, yet the underlying mechanisms behind the tumor suppressor role of CHD5 are unknown. In this study, we purified the human CHD5 complex and found that CHD5 is a component of the full NuRD transcriptional repressor complex, which also contains methyl-CpG binding proteins and histone deacetylases. The CHD5/NuRD complex appears mutually exclusive with the related CHD4/NuRD complex as overexpression of CHD5 results in loss of the CHD4 protein in cells. Following a search for genes that are regulated by CHD5 in neuroblastoma cells, we found that CHD5 binds to and represses the G2/M checkpoint gene WEE1. Reintroduction of CHD5 into neuroblastoma cells represses WEE1 expression, demonstrating that CHD5 can function as a repressor in cells. A catalytically inactive mutant version of CHD5 is able to associate with a NuRD cofactor but fails to repress transcription. Our study shows that CHD5 is a NuRD-associated transcriptional repressor and identifies WEE1 as one of the CHD5-regulated genes that may link CHD5 to tumor suppression.

E-type cyclins (cyclins E1 and E2) are components of the cell cycle machinery that has been conserved from yeast to humans. The major function of E-type cyclins is to drive cell division. It is unknown whether in addition to their ‘core’ cell cycle functions, E-type cyclins also perform unique tissue-specific roles. Here, we applied high-throughput mass spectrometric analyses of mouse organs to define the repertoire of cyclin E protein partners in vivo. We found that cyclin E interacts with distinct sets of proteins in different compartments. These cyclin E interactors are highly enriched for phosphorylation targets of cyclin E and its catalytic partner, the cyclin-dependent kinase 2 (Cdk2). Among cyclin E interactors we identified several novel tissue-specific substrates of cyclin E-Cdk2 kinase. In proliferating compartments, cyclin E-Cdk2 phosphorylates Lin proteins within the DREAM complex. In the testes, cyclin E-Cdk2 phosphorylates Mybl1 and Dmrtc2, two meiotic transcription factors that represent key regulators of spermatogenesis. In embryonic and adult brains cyclin E interacts with proteins involved in neurogenesis, while in adult brains also with proteins regulating microtubule-based processes and microtubule cytoskeleton. We also used quantitative proteomics to demonstrate re-wiring of the cyclin E interactome upon ablation of Cdk2. This approach can be used to study how protein interactome changes during development or in any pathological state such as aging or cancer.

Activating mutations in genes encoding G protein α (Gα) subunits occur in 4-5% of all human cancers, but oncogenic alterations in Gβ subunits have not been defined. Here we demonstrate that recurrent mutations in the Gβ proteins GNB1 and GNB2 confer cytokine-independent growth and activate canonical G protein signaling. Multiple mutations in GNB1 affect the protein interface that binds Gα subunits as well as downstream effectors and disrupt Gα interactions with the Gβγ dimer. Different mutations in Gβ proteins clustered partly on the basis of lineage; for example, all 11 GNB1 K57 mutations were in myeloid neoplasms, and seven of eight GNB1 I80 mutations were in B cell neoplasms. Expression of patient-derived GNB1 variants in Cdkn2a-deficient mouse bone marrow followed by transplantation resulted in either myeloid or B cell malignancies. In vivo treatment with the dual PI3K-mTOR inhibitor BEZ235 suppressed GNB1-induced signaling and markedly increased survival. In several human tumors, mutations in the gene encoding GNB1 co-occurred with oncogenic kinase alterations, including the BCR-ABL fusion protein, the V617F substitution in JAK2 and the V600K substitution in BRAF. Coexpression of patient-derived GNB1 variants with these mutant kinases resulted in inhibitor resistance in each context. Thus, GNB1 and GNB2 alterations confer transformed and resistance phenotypes across a range of human tumors and may be targetable with inhibitors of G protein signaling.

Tumor oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state1, but direct pharmacological inhibition of transcription factors has thus far proven difficult2. However, the transcriptional machinery contains various enzymatic co-factors that can be targeted for development of new therapeutic candidates3, including cyclin-dependent kinases (CDKs)4. Here we present the discovery and characterization of the first covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell line profiling indicates that a subset of cancer cell lines, including T-ALL, exhibit exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and this transcription factor’s key role in the core transcriptional regulatory circuitry of these tumor cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumor types exhibiting extreme dependencies on transcription for maintenance of the oncogenic state.