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.

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.

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).

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.

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.

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.

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.

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.

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.

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.