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Marise R Heerma van Voss,1,2 Farhad Vesuna,2 Guus M Bol,1,2 Jan Meeldijk,1,3 Ana Raman,4 G Johan Offerhaus,1 Horst Buerger,5 Arvind H Patel,6 Elsken van der Wall,7,8 Paul J van Diest,1,8 Venu Raman1,2,8 1Department of Pathology, University Medical Center Utrecht, Utrecht, the Netherlands; 2Department of Radiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 3Laboratory of Translational Immunology, University Medical Center Utrecht, Utrecht, the Netherlands; 4Department of Pharmacology, Johns Hopkins University School of Medicine, Baltimore, MD, USA; 5Institute of Pathology, Paderborn, Germany; 6Centre for Virus Research, MRC-University of Glasgow, Glasgow, UK; 7Cancer Center, University Medical Center Utrecht, Utrecht, the Netherlands; 8Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA Purpose: DEAD box protein 3 (DDX3) is an RNA helicase with oncogenic properties that shuttles between the cytoplasm and nucleus. The majority of DDX3 is found in the cytoplasm, but a subset of tumors has distinct nuclear DDX3 localization of yet unknown biological significance. This study aimed to evaluate the significance of and mechanisms behind nuclear DDX3 expression in colorectal and breast cancer.Methods: Expression of nuclear DDX3 and the nuclear exporter chromosome region maintenance 1 (CRM1) was evaluated by immunohistochemistry in 304 colorectal and 292 breast cancer patient samples. Correlations between the subcellular localization of DDX3 and CRM1 and the difference in overall survival between patients with and without nuclear DDX3 were studied. In addition, DDX3 mutants were created for in vitro evaluation of the mechanism behind nuclear retention of DDX3.Results: DDX3 was present in the nucleus of 35% of colorectal and 48% of breast cancer patient samples and was particularly strong in the nucleolus. Nuclear DDX3 correlated with worse overall survival in both colorectal (hazard ratio [HR] 2.34, P

The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences among the closely related KDM4 double tudor domains (DTDs). KDM4A/B DTDs bind strongly to H3K23me3, a poorly understood histone modification recently shown to be enriched in meiotic chromatin of ciliates and nematodes. The 2.28 Å co-crystal structure of KDM4A-DTD in complex with H3K23me3 peptide reveals key intermolecular interactions for H3K23me3 recognition. Furthermore, analysis of the 2.56 Å KDM4B-DTD crystal structure pinpoints the underlying residues required for exclusive H3K23me3 specificity, an interaction supported by in vivo co-localization of KDM4B and H3K23me3 at heterochromatin in mammalian meiotic and newly postmeiotic spermatocytes. In vitro demethylation assays suggest H3K23me3 binding by KDM4B stimulates H3K36 demethylation. Together, these results provide a possible mechanism whereby H3K23me3-binding by KDM4B directs localized H3K36 demethylation during meiosis and spermatogenesis., KDM4 histone demethylases target specific chromatin regions by a mechanism that is not fully characterised. Here, the authors identify trimethyl-lysine histone-binding preferences for closely related KDM4 double tudor domains and use structural and biochemical information to examine the molecular details of this interaction.

Homeobox protein HOXA5 functions as a transcriptional factor for genes that are not only involved in segmentation identity but also in cell differentiation. Although HOXA5 has been shown to regulate the expression of the tumor-suppressor protein p53, its role in breast tumorigenesis is not well understood. Using yeast as a model system, we now demonstrate that overexpression of HOXA5 in yeast can be used to identify downstream target genes that are homologous in humans. One such identified gene was that of the mismatch repair pathway component Mutt homolog 1. Analysis of the promoter region of the gene for human Mutt homolog 1 (hMLH1) displayed several putative HOXA5-binding sites. In transient transfection experiments, the overexpression of HOXA5 transactivated the hMLH1 promoter-reporter construct. In addition, chromatin immunoprecipitation assay using a human breast cancer cell line MCF-7 demonstrated that HOXA5 binds to the hMLH1 promoter in vivo. Furthermore, we demonstrate that, in the presence of HOXA5, there is an increase in in vivo repair activity in MCF-7 cells. Taken together, our results indicate that HOXA5 is a transcriptional regulator of hMLH1 in breast cancer cells.

The homeotic gene HOXA5 has been shown to play an important role in breast tumorigenesis. We have shown that loss of p53 correlated with loss of a developmentally regulated transcription factor, HOXA5, in primary breast cancer. Searching for potential protein interacting partners we found that HOXA5 binds to an anti-apoptotic protein, Twist. Furthermore, Twist-overexpressing MCF-7 cells displayed a deregulated p53 response to gamma-radiation and decreased regulation of downstream target genes. Using a p53-promoter-reporter system, we demonstrated that HOXA5 could partially restore the inhibitory effects of Twist on p53 target genes. These effects are likely mediated through both the transcriptional up-regulation of p53 and the protein-protein interaction between HOXA5 and Twist. Thus, the loss of HOXA5 expression could lead to the functional activation of Twist resulting in aberrant cell cycle regulation and promoting breast carcinogenesis.

In yeast, the conserved histone acetyltransferase (HAT) Gcn5 associates with Ada2 and Ada3 to form the catalytic module of the ADA and SAGA transcriptional coactivator complexes. Gcn5 also contains an acetyl-lysine binding bromodomain that has been implicated in regulating nucleosomal acetylation in vitro, as well as at gene promoters in cells. However, the contribution of the Gcn5 bromodomain in regulating site specificity of HAT activity remains unclear. Here, we used a combined acid-urea gel and quantitative mass spectrometry approach to compare the HAT activity of wild-type and Gcn5 bromodomain-mutant ADA subcomplexes (Gcn5-Ada2-Ada3). Wild-type ADA subcomplex acetylated H3 lysines with the following specificity; H3K14 > H3K23 > H3K9 ≈ H3K18 > H3K27 > H3K36. However, when the Gcn5 bromodomain was defective in acetyl-lysine binding, the ADA subcomplex demonstrated altered site-specific acetylation on free and nucleosomal H3, with H3K18ac being the most severely diminished. H3K18ac was also severely diminished on H3K14R, but not H3K23R, substrates in wild-type HAT reactions, further suggesting that Gcn5-catalyzed acetylation of H3K14 and bromodomain binding to H3K14ac are important steps preceding H3K18ac. In sum, this work details a previously uncharacterized cross-talk between the Gcn5 bromodomain "reader" function and enzymatic HAT activity that might ultimately affect gene expression. Future studies of how mutations in bromodomains or other histone post-translational modification readers can affect chromatin-templated enzymatic activities will yield unprecedented insight into a potential "histone/epigenetic code." MS data are available via ProteomeXchange with identifier PXD001167.

Genetically related diploid strains of Saccharomyces cerevisiae that accumulate varied amounts of trehalose during starvation for nitrogen have been constructed. Strains that produced > 5% trehalose (dry cell weight) were more tolerant of thermal, or freeze-thaw stresses than strains that produced < 4% trehalose. Thus trehalose appears to play a role in stress tolerance of yeast. The significance of these results is that, for the first time, a series of related, unmutated strains have been used to test the effect of trehalose on thermotolerance. Previous studies employed either heat shock treatment, or mutated strains to provide trehalose variations, and as such the contribution of the disaccharide to stress tolerance could not necessarily be separated from other factors such as heat shock proteins.

Errors during the process of translating mRNA information into protein products occur infrequently. Frameshift errors occur less frequently than other types of errors, suggesting that the translational machinery has more robust mechanisms for precluding that kind of error. Despite these mechanisms, mRNA sequences have evolved that increase the frequency up to 10,000-fold. These sequences, termed programmed frameshift sites, usually consist of a heptameric nucleotide sequence, at which the change in frames occurs along with additional sequences that stimulate the efficiency of frameshifting. One such stimulatory site derived from the Ty3 retrotransposon of the yeast Saccharomyces cerevisiae (the Ty3 stimulator) comprises a 14 nucleotide sequence with partial complementarity to a Helix 18 of the 18S rRNA, a component of the ribosome's accuracy center. A model for the function of the Ty3 stimulator predicts that it base pairs with Helix 18, reducing the efficiency with which the ribosome rejects erroneous out of frame decoding. We have tested this model by making a saturating set of single-base mutations of the Ty3 stimulator. The phenotypes of these mutations are inconsistent with the Helix 18 base-pairing model. We discuss the phenotypes of these mutations in light of structural data on the path of the mRNA on the ribosome, suggesting that the true target of the Ty3 stimulator may be rRNA and ribosomal protein elements of the ribosomal entry tunnel, as well as unknown constituents of the solvent face of the 40S subunit.

Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stimulates the error probably by interacting with an element of the ribosome to interfere with error correction. One such primary mRNA sequence, the Ty3 stimulator, increases programmed +1 frameshifting 7.5 times in the yeast Saccharomyces cerevisiae. Here we show that this stimulator also increases the usage of non-AUG initiation codons in the bacterium Escherichia coli but not in S. cerevisiae. These data suggest that in E. coli, though not in yeast, an element of the ribosome's elongation accuracy mechanism ensures initiation accuracy.

Programmed translational frameshifting is a ubiquitous but rare mechanism of gene expression in which mRNA sequences cause the translational machinery to shift reading frames with extreme efficiency, up to at least 50%. The mRNA sequences responsible are deceptively simple; the sequence CUU-AGG-C causes about 40% frameshifting when inserted into an mRNA in the yeast Saccharomyces cerevisiae. The high efficiency of this site depends on a set of S. cerevisiae tRNA isoacceptors that perturb the mechanism of translation to cause the programmed translational error. The simplicity of the system might suggest that it could evolve frequently and perhaps be lost as easily. We have investigated the history of programmed +1 frameshifting in fungi. We find that frameshifting has persisted in two structural genes in budding yeasts, ABP140 and EST3 for about 150 million years. Further, the tRNAs that stimulate the event are equally old. Species that diverged from the lineage earlier both do not employ frameshifting and have a different complement of tRNAs predicted to be inimical to frameshifting. The stability of the coevolution of protein coding genes and tRNAs suggests that frameshifting has been selected for during the divergence of these species.