Kyle L. Johnson

ING4 Mediates Crosstalk between Histone H3 K4 Trimethylation and H3 Acetylation to Attenuate Cellular Transformation

Aberrations in chromatin dynamics play a fundamental role in tumorigenesis, yet relatively little is known of the molecular mechanisms linking histone lysine methylation to neoplastic disease. ING4 (Inhibitor of Growth 4) is a native subunit of an HBO1 histone acetyltransferase (HAT) complex and a tumor suppressor protein. Here we show a critical role for specific recognition of histone H3 trimethylated at lysine 4 (H3K4me3) by the ING4 PHD finger in mediating ING4 gene expression and tumor suppressor functions. The interaction between ING4 and H3K4me3 augments HBO1 acetylation activity on H3 tails and drives H3 acetylation at ING4 target promoters. Further, ING4 facilitates apoptosis in response to genotoxic stress and inhibits anchorage-independent cell growth, and these functions depend on ING4 interactions with H3K4me3. Together, our results demonstrate a mechanism for brokering crosstalk between H3K4 methylation and H3 acetylation and reveal a molecular link between chromatin modulation and tumor suppressor mechanisms.

Nodaviruses of Insects

The study of nodaviruses began with the isolation of nodamura virus (NOV) from mosquitoes in 1956 (Scherer and Hurlbut, 1967; Scherer et al., 1968). The virus drew immediate attention because it uniquely combined the biological property of arthropod transmission to vertebrates with the physical property of resistance to lipid solvents, a characteristic that is now known to indicate the absence of a viral envelope. Molecular studies established that NOV was also unique in its genome structure: two molecules of single-stranded, positive-sense RNA copackaged in spherical virus particles (Fig. 1) (Newman and Brown, 1973, 1977; Clewley et al., 1982). Despite this combination of unusual features, however, the lack of a convenient cell culture system for growing NOV and the absence of antibodies to the virus in human sera, which suggested that it was not naturally transmitted to man, diverted most investigators to more pressing and tractable systems.

The crystal structure of the ING5 PHD finger in complex with an H3K4me3 histone peptide

Chromatin dynamics regulates diverse nuclear processes and influences cellular viability and tumorigenesis. The discrete chromatin states are linked to covalent histone modifications that control the extent of DNA accessibility to transacting factors. One of the most common epigenetic modifications is methylation of histone H3 at lysine 4 (H3K4). Lys4 can be mono-, di-, or tri-methylated, and the tri-methylated mark (H3K4me3) is normally associated with euchromatin and active gene transcription.1,2 The inhibitor of growth (ING) family of tumor suppressors contain a C-terminal plant homeodomain (PHD) finger. This conserved zinc-binding module is found in many nuclear proteins including transcription factors, histone modifying enzymes, and ATP dependent chromatin remodeling complexes.3-5 A subset of PHD fingers has recently been shown to bind methylated and unmodified histone tails,5-9 with the H3K4me3 mark being specifically recognized by ING proteins. Unlike ING1 and ING2, which have been identified as components of histone deacetylase (HDAC) complexes, ING5 associates with histone acetyltransferase (HAT) complexes containing MOZ (monocytic leukemia zinc finger protein)/MORF (MOZ-related factor) and HBO1.10 To establish the structural basis of chromatin targeting by the HAT associated ING proteins, we determined the crystal structure of the ING5 PHD finger in complex with its histone target (H3K4me3). We also measured binding affinities for unmodified, mono-, di-, and tri-methylated histone peptides, and showed that both full-length ING5 and methylated H3K4 are essential for the acetyltransferase activity of the MOZ/MORF and HBO1 complexes. This functional data are the first direct evidence supporting the critical role of ING5 in directing the MOZ/MORF and HBO1 complexes to chromatin, which consequently increases the local HAT activity and stimulates chromatin remodeling.

Reverse Genetics of Nodaviruses

Publisher Summary This chapter focuses on the reverse genetics of Nodaviruses. With the construction of full-length complementary DNA (cDNA) clones of the two genomic RNAs of an alpha nodavirus, it became possible to address all stages of the virus life cycle using reverse genetic methods. Since then, progress has been made in investigating the cis- and trans-acting requirements for RNA replication, its mechanism of regulation, the encapsidation of RNA, the assembly and structures of provirions and virus-like particles, and the efficacy of Nodaviruses as platforms for the antigenic presentation of foreign epitopes. Nodavirus RNA replication has been transplanted to the cells of higher vertebrates and to the yeast Saccharomyces cerevisiae in studies aimed at exploring its potential as the basis of an expression vector. The chapter discusses various contributions of reverse genetics, such as functions of viral nonstructural proteins, mechanism of RNA replication, regulation of RNA replication, virus structure and assembly, and epitope presentation.

Nodamura Virus Nonstructural Protein B2 Can Enhance Viral RNA Accumulation in both Mammalian and Insect Cells

ABSTRACT During infection of both vertebrate and invertebrate cell lines, the alphanodavirus Nodamura virus (NoV) expresses two nonstructural proteins of different lengths from the B2 open reading frame. The functions of these proteins have yet to be determined, but B2 of the related Flock House virus suppresses RNA interference both in Drosophila cells and in transgenic plants. To examine whether the NoV B2 proteins had similar functions, we compared the replication of wild-type NoV RNA with that of mutants unable to make the B2 proteins. We observed a defect in the accumulation of mutant viral RNA that varied in extent from negligible in some cell lines (e.g., baby hamster kidney cells) to severe in others (e.g., human HeLa and Drosophila DL-1 cells). These results are consistent with the notion that the NoV B2 proteins act to circumvent an innate antiviral response such as RNA interference that differs in efficacy among different host cells.

Nodamura Virus RNA Replication in Saccharomyces cerevisiae: Heterologous Gene Expression Allows Replication-Dependent Colony Formation

ABSTRACT Nodamura virus (NoV) and Flock House virus (FHV) are members of the family Nodaviridae. The nodavirus genome is composed of two positive-sense RNA segments: RNA1 encodes the viral RNA-dependent RNA polymerase and RNA2 encodes the capsid protein precursor. A small subgenomic RNA3, which encodes nonstructural proteins B1 and B2, is transcribed from RNA1 during RNA replication. Previously, FHV was shown to replicate both of its genomic RNAs and to transcribe RNA3 in transiently transfected yeast cells. FHV RNAs and their derivatives could also be expressed from plasmids containing RNA polymerase II promoters. Here we show that all of these features can be recapitulated for NoV, the only nodavirus that productively infects mammals. Inducible plasmid-based systems were used to characterize the RNA replication requirements for NoV RNA1 and RNA2 in Saccharomyces cerevisiae. Induced NoV RNA1 replication was robust. Three previously described NoV RNA1 mutants behaved in yeast as they had in mammalian cells. Yeast colonies were selected from cells expressing NoV RNA1, and RNA2 replicons that encoded yeast nutritional markers, from plasmids. Unexpectedly, these NoV RNA replication-dependent yeast colonies were recovered at frequencies 104-fold lower than in the analogous FHV system. Molecular analysis revealed that some of the NoV RNA replication-dependent colonies contained mutations in the NoV B2 open reading frame in the replicating viral RNA. In addition, we found that NoV RNA1 could support limited replication of a deletion derivative of the heterologous FHV RNA2 that expressed the yeast HIS3 selectable marker, resulting in formation of HIS+ colonies.

Unusual features of integrated cDNAs generated by infection with genome-free retroviruses.

We previously demonstrated that when nonretroviral RNAs are encapsidated in retroviral particles they can be reverse transcribed into cDNAs, which are then integrated into the cellular genome. This transfer of genetic information via retroviral infection has been designated retrofection. Further analyses of three genes transferred in this manner (retrogenes) revealed that each was present in a single copy at a different site in the recipient quail cell genome and included a transcriptional promoter encoded by the encapsidated neo RNA. A unique feature of the retrogenes was a common 16-nucleotide sequence at or near a recombination border, which was not present in either recombination partner. The existence of this sequence suggests a common mechanism of retrogene formation and/or integration mediated by retrofection.