A genetic code alteration generates a proteome of high diversity in the human pathogen Candida albicans

Background - Genetic code alterations have been reported in mitochondrial, prokaryotic, and eukaryotic cytoplasmic translation systems, but their evolution and how organisms cope and survive such dramatic genetic events are not understood. Results - Here we used an unusual decoding of leucine CUG codons as serine in the main human fungal pathogen Candida albicans to elucidate the global impact of genetic code alterations on the proteome. We show that C. albicans decodes CUG codons ambiguously and tolerates partial reversion of their identity from serine back to leucine on a genome-wide scale. Conclusion - Such codon ambiguity expands the proteome of this human pathogen exponentially and is used to generate important phenotypic diversity. This study highlights novel features of C. albicans biology and unanticipated roles for codon ambiguity in the evolution of the genetic code.publishe

Monocytes acquire prostate cancer specific chromatin conformations upon indirect co-culture with prostate cancer cells

Background: Three-dimensional chromosome loop conformations are powerful regulators of gene expression. These chromosome conformations can be detected both in tumour and in circulating cells and have significant disease biomarker potential. We have recently detected specific chromosome conformations in circulating cells of patients with prostate cancer (PCa) which were similar to ones found in their primary tumours, however, the possibility of horizontal transfer of chromosome conformations was not studied previously. Methods: Human monocytes (U937) were co-cultured in Boyden chambers through 0.4 uM membrane with or without PC-3 human PCa cells or their conditioned media and a custom DNA microarray for 900,000 chromosomal loops covering all coding loci and non-coding RNA genes was performed on each part of the co-culture system. Results: We have detected 684 PC-3 cell-specific chromosome conformations across the whole genome that were absent in naïve monocytes but appeared in monocytes co-cultured with PC-3 cells or with PC-3-conditioned media. Comparing PC3-specific conformations to the ones we have previously detected in systemic circulation of high-risk PCa patients revealed 9 positive loops present in both settings. Conclusions: Our results demonstrate for the first time a proof of concept for horizontal transfer of chromosome conformations without direct cell-cell contact. This carries high clinical relevance as we have previously observed chromatin conformations in circulating cells of patients with melanoma and PCa similar to ones in their primary tumours. These changes can be used as highly specific biomarkers for diagnosis and prognosis. Further studies are required to elucidate the specific mechanism of chromosome conformations transfer and its clinical significance in particular diseases

Chromatin conformation changes in peripheral blood can detect prostate cancer and stratify disease risk groups

Background Current diagnostic blood tests for prostate cancer (PCa) are unreliable for the early stage disease, resulting in numerous unnecessary prostate biopsies in men with benign disease and false reassurance of negative biopsies in men with PCa. Predicting the risk of PCa is pivotal for making an informed decision on treatment options as the 5-year survival rate in the low-risk group is more than 95% and most men would benefit from surveillance rather than active treatment. Three-dimensional genome architecture and chromosome structures undergo early changes during tumourigenesis both in tumour and in circulating cells and can serve as a disease biomarker. Methods In this prospective study we screened whole blood of newly diagnosed, treatment naïve PCa patients (n = 140) and cancer-free controls (n = 96) for the presence of 14,241 chromosomal loops in the loci of 425 genes. Results We have detected specific chromosome conformation changes in the loci of ETS1, MAP3K14, SLC22A3 and CASP2 genes in peripheral blood from PCa patients yielding PCa detection with 80% sensitivity and 80% specificity. Further analysis between PCa risk groups yielded prognostic validation sets consisting of HSD3B2, VEGFC, APAF1, BMP6, ERG, MSR1, MUC1, ACAT1 and DAPK1 genes that achieved 80% sensitivity and 93% specificity stratifying high-risk category 3 vs low risk category 1 and 84% sensitivity and 89% specificity stratifying high risk category 3 vs intermediate risk category 2 disease. Conclusions Our results demonstrate specific chromosome conformations in the blood of PCa patients that allow PCa diagnosis and risk stratification with high sensitivity and specificity

TFIIH: a key component in multiple DNA transactions

The transcription factor TFIIH is a versatile, multi-functional protein complex with multiple engagements. Apart from its role in basal transcription, TFIIH is intimately implicated in DNA repair and (probably) in cell cycle control (both of which are required to prevent carcinogenesis) as well as having possible roles in other processes. Thus, it is a striking example of the efficient use of one component for many purposes. Ingeniously, the incorporation of this essential factor into important, but non-essential, mechanisms, such as DNA repair, protects against cancer. The critical role of TFIIH in transcription function renders inactivating TFIIH mutations lethal to cells. Without this transcription connection, such mutations would lead to genetic instability and oncogenesis

Both XPD alleles contribute to the phenotype of compound heterozygote xeroderma pigmentosum patients

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH result in the rare recessive genetic disorder xeroderma pigmentosum (XP). Many XP patients are compound heterozygotes with a “causative” XPD point mutation R683W and different second mutant alleles, considered “null alleles.” However, there is marked clinical heterogeneity (including presence or absence of skin cancers or neurological degeneration) in these XPD/R683W patients, thus suggesting a contribution of the second allele. Here, we report XP patients carrying XPD/R683W and a second XPD allele either XPD/Q452X, /I455del, or /199insPP. We performed a systematic study of the effect of these XPD mutations on several enzymatic functions of TFIIH and found that each mutation exhibited unique biochemical properties. Although all the mutations inhibited the nucleotide excision repair (NER) by disturbing the XPD helicase function, each of them disrupted specific molecular steps during transcription: XPD/Q452X hindered the transactivation process, XPD/I455del disturbed RNA polymerase II phosphorylation, and XPD/199insPP inhibited kinase activity of the cdk7 subunit of TFIIH. The broad range and severity of clinical features in XP patients arise from a broad set of deficiencies in NER and transcription that result from the combination of mutations found on both XPD alleles

The Prospective Study of Epigenetic Regulatory Profiles in Sport and Exercise Monitored Through Chromosome Conformation Signatures

The integration of genetic and environmental factors that regulate the gene expression patterns associated with exercise adaptation is mediated by epigenetic mechanisms. The organisation of the human genome within three-dimensional space, known as chromosome conformation, has recently been shown as a dynamic epigenetic regulator of gene expression, facilitating the interaction of distal genomic regions due to tight and regulated packaging of chromosomes in the cell nucleus. Technological advances in the study of chromosome conformation mean a new class of biomarker—the chromosome conformation signature (CCS)—can identify chromosomal interactions across several genomic loci as a collective marker of an epigenomic state. Investigative use of CCSs in biological and medical research shows promise in identifying the likelihood that a disease state is present or absent, as well as an ability to prospectively stratify individuals according to their likely response to medical intervention. The association of CCSs with gene expression patterns suggests that there are likely to be CCSs that respond, or regulate the response, to exercise and related stimuli. The present review provides a contextual background to CCS research and a theoretical framework discussing the potential uses of this novel epigenomic biomarker within sport and exercise science and medicine

Interaction of Rio1 Kinase with Toyocamycin Reveals a Conformational Switch That Controls Oligomeric State and Catalytic Activity

Rio1 kinase is an essential ribosome-processing factor required for proper maturation of 40 S ribosomal subunit. Although its structure is known, several questions regarding its functional remain to be addressed. We report that both Archaeoglobus fulgidus and human Rio1 bind more tightly to an adenosine analog, toyocamycin, than to ATP. Toyocamycin has antibiotic, antiviral and cytotoxic properties, and is known to inhibit ribosome biogenesis, specifically the maturation of 40 S. We determined the X-ray crystal structure of toyocamycin bound to Rio1 at 2.0 Å and demonstrated that toyocamycin binds in the ATP binding pocket of the protein. Despite this, measured steady state kinetics were inconsistent with strict competitive inhibition by toyocamycin. In analyzing this interaction, we discovered that Rio1 is capable of accessing multiple distinct oligomeric states and that toyocamycin may inhibit Rio1 by stabilizing a less catalytically active oligomer. We also present evidence of substrate inhibition by high concentrations of ATP for both archaeal and human Rio1. Oligomeric state studies show both proteins access a higher order oligomeric state in the presence of ATP. The study revealed that autophosphorylation by Rio1 reduces oligomer formation and promotes monomerization, resulting in the most active species. Taken together, these results suggest the activity of Rio1 may be modulated by regulating its oligomerization properties in a conserved mechanism, identifies the first ribosome processing target of toyocamycin and presents the first small molecule inhibitor of Rio1 kinase activity

Requirement of TFIIH kinase subunit Mat1 for RNA Pol II C-terminal domain Ser5 phosphorylation, transcription and mRNA turnover

The relevance of serine 5 phosphorylation of RNA polymerase II carboxy-terminal domain during initiation has been difficult to determine in mammalian cells as no general in vivo Ser5 kinase has been identified. Here, we demonstrate that deletion of the TFIIH kinase subunit Mat1 in mouse fibroblasts leads to dramatically reduced Pol II Ser5 phosphorylation. This is associated with defective capping and reduced Ser2 phosphorylation, decreased Pol II progression into elongation and severely attenuated transcription detected through analysis of nascent mRNAs, establishing a general requirement for mammalian Mat1 in transcription. Surprisingly, the general defect in Pol II transcription in Mat1−/− fibroblasts is not reflected in the majority of steady-state mRNAs. This indicates widespread stabilization of mRNAs and points to the existence of a regulatory mechanism to stabilize mRNAs following transcriptional attenuation, thus revealing a potential caveat in similar studies limited to analysis of steady-state mRNAs