Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists.

Metastasis results from a complex set of traits acquired by tumor cells, distinct from those necessary for tumorigenesis. Here, we investigate the contribution of enhancer elements to the metastatic phenotype of osteosarcoma. Through epigenomic profiling, we identify substantial differences in enhancer activity between primary and metastatic tumors in human patients as well as near-isogenic pairs of high and low lung-metastatic osteosarcoma cells. We term these regions Metastatic Variant Enhancer Loci (Met-VELs). Met-VELs drive coordinated waves of gene expression during metastatic colonization of the lung. Met-VELs cluster non-randomly in the genome, indicating that activity of these enhancers and their associated gene targets are positively selected. As evidence of this causal association, osteosarcoma lung metastasis is inhibited by global interruptions of Met-VEL-associated gene expression via pharmacologic BET inhibition, by knockdown of AP-1 transcription factors that occupy Met-VELs, and by knockdown or functional inhibition of individual genes activated by Met-VELs, such as coagulation factor III/tissue factor (F3). We further show that genetic deletion of a single Met-VEL at the F3 locus blocks metastatic cell outgrowth in the lung. These findings indicate that Met-VELs and the genes they regulate play a functional role in metastasis and may be suitable targets for anti-metastatic therapies.

SNPs associated with disease susceptibility often reside in clusters of gene enhancers, or super enhancers. Constituents of these enhancer clusters cooperate to regulate target genes, and often extend beyond the linkage disequilibrium blocks containing GWAS risk SNPs. We identified “outside variants”, defined as SNPs in weak LD with GWAS risk SNPs that physically interact with risk SNPs as part of the target gene’s regulatory circuitry. These outside variants explain additional target gene expression variation beyond that of GWAS associated SNPs. Additionally, the clinical risk associated with the GWAS SNPs is considerably modified by the genotype of the outside variant. Collectively, these findings suggest a potential model whereby outside variants and GWAS SNPs that physically interact in 3D chromatin collude to influence target transcript levels as well as clinical risk. This model offers an additional hypothesis for the source of missing heritability of complex traits.

Differentiation of B cells into antibody secreting cells induces changes in gene transcription, Igh RNA processing, the unfolded protein response, and cell architecture. The transcription elongation factor ELL2 (eleven nineteen lysine-rich leukemia gene) stimulates the processing of the secreted form of the Igh mRNA from the heavy chain gene. Mice (mus musculus) with the ELL2 gene floxed in either exon 1 or exon 3 were constructed and crossed to CD19 driven cre/ CD19+. The B-cell specific ELL2 conditional knockouts (ell2loxp/loxp CD19cre/+) exhibit curtailed humoral responses both in NP-ficoll and NP-KLH immunized animals; recall responses were also diminished. The number of immature and recirculating B cells in the bone marrow is increased in the conditional knockouts while plasma cells in spleen are reduced relative to control animals. There are fewer IgG1 antibody producing cells in the bone marrow of conditional knockouts. LPS ex vivo stimulated B220loCD138+ cells from ELL2 deficient mouse spleens are 4-fold less abundant than from control splenic B-cells, have a paucity of secreted Igh, and distended, abnormal appearing ER. IRE1alpha is efficiently phosphorylated but the amounts of Ig kappa, ATF6, BiP, Cyclin B2, OcaB (BOB1, Pou2af1), and XBP1 mRNAs, unspliced and spliced, are severely reduced in ELL2 deficient cells. ELL2 enhances the expression of BCMA, important for long term survival. Transcription yields from the cyclin B2 and the canonical UPR promoter elements are up-regulated by ELL2 cDNA. Thus ELL2 is important for many aspects of antibody secretion, XBP1 expression, and the unfolded protein response.

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