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.

Chromosome conformation signatures define predictive markers of inadequate response to methotrexate in early rheumatoid arthritis.

BACKGROUND: There is a pressing need in rheumatoid arthritis (RA) to identify patients who will not respond to first-line disease-modifying anti-rheumatic drugs (DMARD). We explored whether differences in genomic architecture represented by a chromosome conformation signature (CCS) in blood taken from early RA patients before methotrexate (MTX) treatment could assist in identifying non-response to DMARD and, whether there is an association between such a signature and RA specific expression quantitative trait loci (eQTL). METHODS: We looked for the presence of a CCS in blood from early RA patients commencing MTX using chromosome conformation capture by EpiSwitch™. Using blood samples from MTX responders, non-responders and healthy controls, a custom designed biomarker discovery array was refined to a 5-marker CCS that could discriminate between responders and non-responders to MTX. We cross-validated the predictive power of the CCS by generating 150 randomized groups of 59 early RA patients (30 responders and 29 non-responders) before MTX treatment. The CCS was validated using a blinded, independent cohort of 19 early RA patients (9 responders and 10 non-responders). Last, the loci of the CCS markers were mapped to RA-specific eQTL. RESULTS: We identified a 5-marker CCS that could identify, at baseline, responders and non-responders to MTX. The CCS consisted of binary chromosome conformations in the genomic regions of IFNAR1, IL-21R, IL-23, CXCL13 and IL-17A. When tested on a cohort of 59 RA patients, the CCS provided a negative predictive value of 90.0% for MTX response. When tested on a blinded independent validation cohort of 19 early RA patients, the signature demonstrated a true negative response rate of 86 and a 90% sensitivity for detection of non-responders to MTX. Only conformations in responders mapped to RA-specific eQTL. CONCLUSIONS: Here we demonstrate that detection of a CCS in blood in early RA is able to predict inadequate response to MTX with a high degree of accuracy. Our results provide a proof of principle that a priori stratification of response to MTX is possible, offering a mechanism to provide alternative treatments for non-responders to MTX earlier in the course of the disease.

Repression of the human dihydrofolate reductase gene by a non-coding interfering transcript.

Alternative promoters within the same gene are a general phenomenon in gene expression. Mechanisms of their selective regulation vary from one gene to another and are still poorly understood. Here we show that in quiescent cells the mechanism of transcriptional repression of the major promoter of the gene encoding dihydrofolate reductase depends on a non-coding transcript initiated from the upstream minor promoter and involves both the direct interaction of the RNA and promoter-specific interference. The specificity and efficiency of repression is ensured by the formation of a stable complex between non-coding RNA and the major promoter, direct interaction of the non-coding RNA with the general transcription factor IIB and dissociation of the preinitiation complex from the major promoter. By using in vivo and in vitro assays such as inducible and reconstituted transcription, RNA bandshifts, RNA interference, chromatin immunoprecipitation and RNA immunoprecipitation, we show that the regulatory transcript produced from the minor promoter has a critical function in an epigenetic mechanism of promoter-specific transcriptional repression.

Circulating Chromosome Conformation Signatures Significantly Enhance PSA Positive Predicting Value and Overall Accuracy for Prostate Cancer Detection.

BACKGROUND: Prostate cancer (PCa) has a high lifetime prevalence (one out of six men), but currently there is no widely accepted screening programme. Widely used prostate specific antigen (PSA) test at cut-off of 3.0 ng/mL does not have sufficient accuracy for detection of any prostate cancer, resulting in numerous unnecessary prostate biopsies in men with benign disease and false reassurance in some men with PCa. We have recently identified circulating chromosome conformation signatures (CCSs, Episwitch® PCa test) allowing PCa detection and risk stratification in line with standards of clinical PCa staging. The purpose of this study was to determine whether combining the Episwitch PCa test with the PSA test will increase its diagnostic accuracy. METHODS: n = 109 whole blood samples of men enrolled in the PROSTAGRAM screening pilot study and n = 38 samples of patients with established PCa diagnosis and cancer-negative controls from Imperial College NHS Trust were used. Samples were tested for PSA, and the presence of CCSs in the loci encoding for of DAPK1, HSD3B2, SRD5A3, MMP1, and miRNA98 associated with high-risk PCa identified in our previous work. RESULTS: PSA > 3 ng/mL alone showed a low positive predicted value (PPV) of 0.14 and a high negative predicted value (NPV) of 0.93. EpiSwitch alone showed a PPV of 0.91 and a NPV of 0.32. Combining PSA and Episwitch tests has significantly increased the PPV to 0.81 although reducing the NPV to 0.78. Furthermore, integrating PSA, as a continuous variable (rather than a dichotomised 3 ng/mL cut-off), with EpiSwitch in a new multivariant stratification model, Prostate Screening EpiSwitch (PSE) test, has yielded a remarkable combined PPV of 0.92 and NPV of 0.94 when tested on the independent prospective cohort. CONCLUSIONS: Our results demonstrate that combining the standard PSA readout with circulating chromosome conformations (PSE test) allows for significantly enhanced PSA PPV and overall accuracy for PCa detection. The PSE test is accurate, rapid, minimally invasive, and inexpensive, suggesting significant screening diagnostic potential to minimise unnecessary referrals for expensive and invasive MRI and/or biopsy testing. Further extended prospective blinded validation of the new combined signature in a screening cohort with low cancer prevalence would be the recommended step for PSE adoption in PCa screening.

Development and Validation of Blood-Based Predictive Biomarkers for Response to PD-1/PD-L1 Checkpoint Inhibitors: Evidence of a Universal Systemic Core of 3D Immunogenetic Profiling across Multiple Oncological Indications.

BACKGROUND: Unprecedented advantages in cancer treatment with immune checkpoint inhibitors (ICIs) remain limited to only a subset of patients. Systemic analyses of the regulatory 3D genome architecture linked to individual epigenetic and immunogenetic controls associated with tumour immune evasion mechanisms and immune checkpoint pathways reveal a highly prevalent molecular profile predictive of response to PD-1/PD-L1 ICIs. A clinical blood test based on a set of eight (8) 3D genomic biomarkers has been developed and validated on the basis of an observational trial to predict response to ICI therapy. METHODS: The predictive eight biomarker set is derived from prospective observational clinical trials, representing 280 treatments with Pembrolizumab, Atezolizumab, Durvalumab, Nivolumab, and Avelumab in a broad range of indications: melanoma, lung, hepatocellular, renal, breast, bladder, colon, head and neck, bone, brain, lymphoma, prostate, vulvar, and cervical cancers. RESULTS: The 3D genomic eight biomarker panel for response to immune checkpoint therapy achieved a high accuracy of 85%, sensitivity of 93%, and specificity of 82%. CONCLUSIONS: This study demonstrates that a 3D genomic approach can be used to develop a predictive clinical assay for response to PD-1/PD-L1 checkpoint inhibition in cancer patients.

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.

Mass spectrometry of RNA.

A complex population of non-coding RNAs is present in higher organisms. These RNAs have a multitude of functions and execute control over gene expression through various, often poorly understood, mechanisms. At present, the identification and analysis of functional regulatory RNAs and disparate ribonucleoprotein complexes remain an experimental challenge for biologists. They require specially designed approaches and techniques in genomics and RNA biochemistry. Developments in technologies based on mass spectrometry could offer sensitive and efficient solutions to analysis of the sequence, structure, modification and composition of RNA.

Autocatalytic RNA cleavage in the human beta-globin pre-mRNA promotes transcription termination.

New evidence indicates that termination of transcription is an important regulatory step, closely related to transcriptional interference and even transcriptional initiation. However, how this occurs is poorly understood. Recently, in vivo analysis of transcriptional termination for the human beta-globin gene revealed a new phenomenon--co-transcriptional cleavage (CoTC). This primary cleavage event within beta-globin pre-messenger RNA, downstream of the poly(A) site, is critical for efficient transcriptional termination by RNA polymerase II. Here we show that the CoTC process in the human beta-globin gene involves an RNA self-cleaving activity. We characterize the autocatalytic core of the CoTC ribozyme and show its functional role in efficient termination in vivo. The identified core CoTC is highly conserved in the 3' flanking regions of other primate beta-globin genes. Functionally, it resembles the 3' processive, self-cleaving ribozymes described for the protein-encoding genes from the myxomycetes Didymium iridis and Physarum polycephalum, indicating evolutionary conservation of this molecular process. We predict that regulated autocatalytic cleavage elements within pre-mRNAs may be a general phenomenon and that functionally it may provide the entry point for exonucleases involved in mRNA maturation, turnover and, in particular, transcriptional termination.

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.

Epigenetic chromatin conformation changes in peripheral blood can detect thyroid cancer.

BACKGROUND: Fine needle aspiration has been the traditional method for diagnosing thyroid cancer. Epigenetic chromatin conformation changes offer an alternative method of diagnosing cancer. The purpose of this study is to evaluate an EpiSwitch assay of epigenetic markers that can be used to diagnose thyroid cancer in blood samples. METHODS: From 2014 to 2016, adult patients with thyroid nodules having thyroidectomy were recruited and grouped based on benign, malignant, and atypia of undetermined significance or follicular lesions of undetermined significance fine needle aspiration cytology. Blood samples were collected before surgery. Final pathologic diagnosis was made from the thyroid specimens. Patients' blood samples were analyzed using the EpiSwitch assay, (Oxford Biodynamics, Oxford, UK), and the results were compared with surgical pathology to determine assay performance. RESULTS: In total, 58 patients were recruited: 20 benign, 20 malignant, and 18 atypia or follicular lesions of undetermined significance. An analysis of the malignant and benign fine needle aspiration groups found 6 epigenetic markers for thyroid. A total of 28 (48%) patients had thyroid cancer. The assay was able to correctly identify 25 of the 28 malignant nodules, showing sensitivity of 89.3% and specificity of 66.7%. The positive predictive value for the assay was 71.4%, whereas the negative predictive value was 87.0%. CONCLUSION: An epigenetic assay of peripheral blood shows high sensitivity in detecting thyroid cancer and provides an additional method for its diagnosis.

Initial Identification of a Blood-Based Chromosome Conformation Signature for Aiding in the Diagnosis of Amyotrophic Lateral Sclerosis.

BACKGROUND: The identification of blood-based biomarkers specific to the diagnosis of amyotrophic lateral sclerosis (ALS) is an active field of academic and clinical research. While inheritance studies have advanced the field, a majority of patients do not have a known genetic link to the disease, making direct sequence-based genetic testing for ALS difficult. The ability to detect biofluid-based epigenetic changes in ALS would expand the relevance of using genomic information for disease diagnosis. METHODS: Assessing differences in chromosomal conformations (i.e. how they are positioned in 3-dimensions) represents one approach for assessing epigenetic changes. In this study, we used an industrial platform, EpiSwitch™, to compare the genomic architecture of healthy and diseased patient samples (blood and tissue) to discover a chromosomal conformation signature (CCS) with diagnostic potential in ALS. A three-step biomarker selection process yielded a distinct CCS for ALS, comprised of conformation changes in eight genomic loci and detectable in blood. FINDINGS: We applied the ALS CCS to determine a diagnosis for 74 unblinded patient samples and subsequently conducted a blinded diagnostic study of 16 samples. Sensitivity and specificity for ALS detection in the 74 unblinded patient samples were 83∙33% (CI 51∙59 to 97∙91%) and 76∙92% (46∙19 to 94∙96%), respectively. In the blinded cohort, sensitivity reached 87∙50% (CI 47∙35 to 99∙68%) and specificity was 75∙0% (34∙91 to 96∙81%). INTERPRETATIONS: The sensitivity and specificity values achieved using the ALS CCS identified and validated in this study provide an indication that the detection of chromosome conformation signatures is a promising approach to disease diagnosis and can potentially augment current strategies for diagnosing ALS. FUND: This research was funded by Oxford BioDynamics and Innovate UK. Work in the Oxford MND Care and Research Centre is supported by grants from the Motor Neurone Disease Association and the Medical Research Council. Additional support was provided by the Northeast ALS Consortium (NEALS).

What is so special about oskar wild?

The amazing world of regulatory noncoding RNA has been at the center of biologists' attention in many different fields, from structural biology to transcriptional regulation and cell signaling. The latest example comes from developmental biology. A mutation in the Drosophila gene Oskar reveals a novel developmental function for the 3' untranslated region (UTR) of the oscar mRNA. This study further suggests that, when transcribed, the noncoding parts of the genome may well carry essential regulatory functions fundamental for the coordinated gene expression and development of multicellular organisms.

Super-Enhancers and Broad H3K4me3 Domains Form Complex Gene Regulatory Circuits Involving Chromatin Interactions.

Stretched histone regions, such as super-enhancers and broad H3K4me3 domains, are associated with maintenance of cell identity and cancer. We connected super-enhancers and broad H3K4me3 domains in the K562 chronic myelogenous leukemia cell line as well as the MCF-7 breast cancer cell line with chromatin interactions. Super-enhancers and broad H3K4me3 domains showed higher association with chromatin interactions than their typical counterparts. Interestingly, we identified a subset of super-enhancers that overlap with broad H3K4me3 domains and show high association with cancer-associated genes including tumor suppressor genes. Besides cell lines, we could observe chromatin interactions by a Chromosome Conformation Capture (3C)-based method, in primary human samples. Several chromatin interactions involving super-enhancers and broad H3K4me3 domains are constitutive and can be found in both cancer and normal samples. Taken together, these results reveal a new layer of complexity in gene regulation by super-enhancers and broad H3K4me3 domains.

Non-coding RNA in transcription initiation.

Diverse classes of non-coding RNAs, including snRNAs (small nuclear RNAs), play fundamental regulatory roles in gene expression. For example, 7SK RNA and the components of the splicing apparatus U1-U6 snRNAs are implicated in the regulation of transcriptional elongation. The first evidence for the involvement of RNA in the regulation of transcriptional initiation is now emerging. TFIIH (transcription factor IIH), a general transcription initiation factor, appears to associate specifically with U1 snRNA, a core splicing component. Reconstituted transcription in vitro demonstrates an increase in the rate of formation of the first phosphodiester bond by RNA polymerase II in presence of U1 snRNA. Reconstituted re-initiation is also stimulated by U1 snRNA. These results suggest that U1 snRNA functions in the regulation of transcription by RNA polymerase II in addition to its role in RNA processing. The implications of these data extend to the development of new technologies that will allow the identification and analysis of diverse RNA species present as regulatory components in transcription-related ribonucleoprotein complexes.

A pilot study of chromosomal aberrations and epigenetic changes in peripheral blood samples to identify patients with melanoma.

Prognosis is markedly improved when melanoma is diagnosed early. Improved methods are needed for earlier detection and screening. We hypothesized that epigenetic analysis of blood samples could discriminate patients with melanoma from patients with other cutaneous lesions and from healthy volunteers. After institutional review board approval and consent, whole blood was obtained from 59 patients with melanoma, 20 patients with other skin cancers, 20 patients with benign skin conditions, and 20 healthy volunteers. Fifteen conformation biomarkers from five gene loci were analyzed on chromatin with the EpiSwitch technology using a modified chromatin conformation capture assay. Differentiation between patients with melanoma and those with nonmelanoma skin cancers was correct 85% of the time, resulting in a sensitivity of 88% and a specificity of 82%. Differentiation of patients with melanoma from healthy controls was correct 80% of the time, resulting in a sensitivity of 85% and a specificity of 75%. The noninvasive test was more accurate in early-stage melanoma (1/10 and 1/16 stage I and stage II patients were misclassified, respectively) and became less accurate with more advanced disease (3/14 and 4/19 stage III and IV patients were misclassified, respectively). We report the results of a noninvasive test using chromosomal aberrations and epigenetic changes identified in peripheral blood that, in this pilot study, distinguished patients with early-stage melanoma from other cohorts.

Formation of distinct chromatin conformation signatures epigenetically regulate macrophage activation.

Microbial-lipopolysacharide (LPS), interleukin 4 (IL-4) and interferon gamma (IFN-γ) polarise macrophages into "innate", "alternative" and "classical", activation states by selective gene regulation. Expression of MARCO, CD200, CD200R1 (innate), MRC1 (alternative) and H2-Eb1 (classical) selectively marks these distinct activation states. Epigenetic events drive such activation upon stimuli and here we study one such mechanism, chromatin conformation signatures implicated in long-range chromatin interactions that regulate transcriptional switch and gene expression. The EpiSwitch™ technology was used to identify and analyse potential markers bordering such conformational signatures for these genes and juxtaposition of markers was compared between resting and activated macrophages. LPS, IL-4 and IFN-γ selectively altered chromatin conformations of their responsive genes in wild type, but not in MyD88(-/-), IL-4R(-/-) and IFN-γR(-/-) macrophages. In addition, two distinct conformations were observed in CD200R1 after LPS and IFN-γ stimulation. In summary, signal-specific alterations in chromatin conformation provide biomarkers that identify and determine distinct gene expression programmes during macrophage activation.

Chromatin barcodes as biomarkers for melanoma.

The major barrier to effective cancer therapy is the presence of genetic and phenotypic heterogeneity within cancer cell populations that provides a reservoir of therapeutically resistant cells. As the degree of heterogeneity present within tumours will be proportional to tumour burden, the development of rapid, robust, accurate and sensitive biomarkers for cancer progression that could detect clinically occult disease before substantial heterogeneity develops would provide a major therapeutic benefit. Here, we explore the application of chromatin conformation capture technology to generate a diagnostic epigenetic barcode for melanoma. The results indicate that binary states from chromatin conformations at 15 loci within five genes can be used to provide rapid, non-invasive multivariate test for the presence of melanoma using as little as 200 µl of patient blood.

Host cell proteins interacting with the 3' end of TGEV coronavirus genome influence virus replication.

Coronavirus RNA synthesis is performed by a multienzymatic replicase complex together with cellular factors. This process requires the specific recognition of RNA cis-acting signals located at the ends of the viral genome. To identify cellular proteins involved in coronavirus RNA synthesis, transmissible gastroenteritis coronavirus (TGEV) genome ends, harboring essential cis-acting signals for replication, were used as baits for RNA affinity protein purification. Ten proteins were preferentially pulled down with either the 5' or 3' ends of the genome and identified by proteomic analysis. Nine of them, including members of the heterogeneous ribonucleoprotein family of proteins (hnRNPs), the poly(A)-binding protein (PABP), the p100 transcriptional co-activator protein and two aminoacyl-tRNA synthetases, showed a preferential binding to the 3' end of the genome, whereas only the polypyrimidine tract-binding protein (PTB) was preferentially pulled down with the 5' end of the genome. The potential function of the 3' end-interacting proteins in virus replication was studied by analyzing the effect of their silencing using a TGEV-derived replicon and the infectious virus. Gene silencing of PABP, hnRNP Q, and glutamyl-prolyl-tRNA synthetase (EPRS) caused a significant 2 to 3-fold reduction of viral RNA synthesis. Interestingly, the silencing of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), initially used as a control gene, caused a 2 to 3-fold increase in viral RNA synthesis in both systems. These data suggest that PABP, hnRNP Q, and EPRS play a positive role in virus infection that could be mediated through their interaction with the viral 3' end, and that GAPDH has a negative effect on viral infection.

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.