A Computational Analysis of Alternative Splicing across Mammalian Tissues Reveals Circadian and Ultradian Rhythms in Splicing Events

Mounting evidence points to a role of the circadian clock in the temporal regulation of post-transcriptional processes in mammals, including alternative splicing (AS). In this study, we carried out a computational analysis of circadian and ultradian rhythms on the transcriptome level to characterise the landscape of rhythmic AS events in published datasets covering 76 tissues from mouse and olive baboon. Splicing-related genes with 24-h rhythmic expression patterns showed a bimodal distribution of peak phases across tissues and species, indicating that they might be controlled by the circadian clock. On the output level, we identified putative oscillating AS events in murine microarray data and pairs of differentially rhythmic splice isoforms of the same gene in baboon RNA-seq data that peaked at opposing times of the day and included oncogenes and tumour suppressors. We further explored these findings using a new circadian RNA-seq dataset of human colorectal cancer cell lines. Rhythmic isoform expression patterns differed between the primary tumour and the metastatic cell line and were associated with cancer-related biological processes, indicating a functional role of rhythmic AS that might be implicated in tumour progression. Our data shows that rhythmic AS events are widespread across mammalian tissues and might contribute to a temporal diversification of the proteome

Analysis of the Circadian Regulation of Cancer Hallmarks by a Cross-Platform Study of Colorectal Cancer Time-Series Data Reveals an Association with Genes Involved in Huntington's Disease

Accumulating evidence points to a link between circadian clock dysfunction and the molecular events that drive tumorigenesis. Here, we investigated the connection between the circadian clock and the hallmarks of cancer in an in vitro model of colorectal cancer (CRC). We used a cross-platform data normalization method to concatenate and compare available microarray and RNA-sequencing time series data of CRC cell lines derived from the same patient at different disease stages. Our data analysis suggests differential regulation of molecular pathways between the CRC cells and identifies several of the circadian and likely clock-controlled genes (CCGs) as cancer hallmarks and circadian drug targets. Notably, we found links of the CCGs to Huntington's disease (HD) in the metastasis-derived cells. We then investigated the impact of perturbations of our candidate genes in a cohort of 439 patients with colon adenocarcinoma retrieved from the Cancer Genome Atlas (TCGA). The analysis revealed a correlation of the differential expression levels of the candidate genes with the survival of patients. Thus, our study provides a bioinformatics workflow that allows for a comprehensive analysis of circadian properties at different stages of colorectal cancer, and identifies a new association between cancer and HD

The Interplay between Colon Cancer Cells and Tumour-Associated Stromal Cells Impacts the Biological Clock and Enhances Malignant Phenotypes

Cancer cells interrelate with the bordering host microenvironment that encompasses the extracellular matrix and a nontumour cellular component comprising fibroblasts and immune-competent cells. The tumour microenvironment modulates cancer onset and progression, but the molecular factors managing this interaction are not fully understood. Malignant transformation of a benign tumour is among the first crucial events in colorectal carcinogenesis. The role of tumour stroma fibroblasts is well-described in cancer, but less well-characterized in benign tumours. In the current work we utilized fibroblasts isolated from tubulovillous adenoma, which has high risk for malignant transformation, to study the interaction between benign tumour stroma and the circadian clock machinery. We explored the role of the biological clock in this interplay taking advantage of an experimental model, represented by the co-culture of colon cancer cells with normal fibroblasts or tumour-associated fibroblasts, isolated from human colorectal tumour specimens. When co-cultured with tumour-associated fibroblasts, colon cancer cells showed alterations in their circadian and metabolic parameters, with decreased apoptosis, increased colon cancer cell viability, and increased resistance to chemotherapeutic agents. In conclusion, the interactions among colon cancer cells and tumour-associated fibroblasts affect the molecular clockwork and seem to aggravate malignant cell phenotypes, suggesting a detrimental effect of this interplay on cancer dynamics

Computergestützte Analyse von circadianen Spleißereignissen in menschlichen Krebszelllinien und Säugetiergeweben

The circadian clock regulates physiology and behavior of various organisms in synchrony with daily environmental rhythms. At the cellular level, circadian rhythmicity is driven by the interplay of clock genes and proteins that interact via negative feedback loops, thereby causing oscillations with a period of 24 h in the expression of numerous target genes. The resulting rhythms in the abundance of proteins and other biomolecules are responsible for the temporal organization of diverse biological processes. Accumulating evidence suggests that alternative splicing might be one of these clock-controlled processes. Alternative splicing describes a versatile mechanism of gene regulation that generates several distinct protein isoforms from a single gene via the differential inclusion or exclusion of alternate RNA regions. Both disruptions of the circadian clock and aberrant splicing are associated with carcinogenesis and tumor progression. This dissertation seeks to answer the question whether mammalian alternative splicing is regulated by the circadian clock, and whether the hypothesized regulation differs between cancer cells in different tumor stages. In particular, it tries to elucidate whether changes in circadian regulated splicing events could be responsible for the production of protein isoforms that contribute to the malignant development of cancer cells. The study is based on data from two human colon cancer cell lines, SW480 and SW620, that have been derived from a primary tumor and a metastasis of the same patient and thus serve as an in vitro model of colorectal tumor progression. A computational analysis was conducted to identify 24 h rhythmic genes and alternative splicing events on transcriptome-level based on the time-series data of both cell lines. As a reference, previously published time-series data of numerous healthy tissues from mouse and baboon organs were analyzed. The analysis revealed differences in the circadian phenotype of the two cell lines, with the metastasis-derived cell line SW620 exhibiting a stronger dysregulation of circadian rhythmicity. Furthermore, this work shows that splicing-related genes and putative splicing events display 24 h rhythms that differ between primary tumor- and metastasis-derived cells. Both in healthy tissues and cancer cells, rhythmic splicing was found to affect many genes that are themselves involved in splicing, suggesting a partial autoregulation of the process. Several of the spliced candidate genes encode for protein isoforms that are involved in processes promoting tumor progression, such as migration and angiogenesis. Taken together, the results presented in this dissertation point to a circadian regulation of alternative splicing that plays a role in cancer development.Im Körper zahlreicher Organismen regelt eine innere Uhr den Ablauf physiologischer Prozesse im Einklang mit dem Tagesrhythmus der Umwelt. Auf zellulärer Ebene entsteht die sogenannte circadiane Rhythmik durch ein Zusammenspiel von Uhrgenen und -proteinen, welche durch negative Rückkopplungsschleifen miteinander interagieren und Oszillationen mit einer 24-Stunden-Periodik in der Expression zahlreicher Zielgene auslösen. Die hieraus resultierenden Rhythmen in der Verfügbarkeit von Proteinen und anderen biologischen Molekülen sind wiederum für die zeitliche Organisation einer Vielzahl biologischer Prozesse verantwortlich, darunter möglicherweise alternatives Spleißen. Alternatives Spleißen beschreibt einen Mechanismus der Genregulation, bei dem aus einem einzigen Gen durch variable Kombination von RNA-Teilabschnitten mehrere verschiedene Proteinvarianten mit zum Teil unterschiedlichen Eigenschaften erstellt werden können. Sowohl Störungen der circadianen Uhr als auch anomales Spleißen werden mit der Entstehung und Weiterentwicklung von Krebserkrankungen in Verbindung gebracht. Die vorliegende Dissertation geht der Frage nach, inwieweit eine circadiane Regulation von alternativem Spleißen in Säugerzellen vorliegt und ob diese sich für Krebszellen unterschiedlicher Tumorstadien unterscheiden. Im Besonderen wird die Hypothese untersucht, ob Veränderungen von circadian regulierten Spleiß-Ereignissen zur Produktion von Proteinvarianten führen könnten, die zur bösartigen Entwicklung von Krebszellen beitragen. Die der Arbeit zugrundeliegenden Daten stammen von zwei menschlichen Darmkrebszelllinien, SW480 und SW620, welche ursprünglich von einem Primärtumor und einer Metastase desselben Patienten etabliert wurden und als in vitro-Modell der kolorektalen Tumorprogression dienen. Basierend auf Zeitreihendaten der Genexpression in den beiden Zelllinien wurde eine computergestützte Analyse durchgeführt, bei der 24-Stunden-rhythmische Gene und alternative Spleiß-Ereignisse auf Transkriptomebene identifiziert wurden. Als Vergleichsbasis wurden bereits publizierte Zeitreihendaten zahlreicher gesunder Gewebesorten analysiert, die von Maus- und Pavianorganen stammen. Die Analyse offenbarte Unterschiede im circadianen Phänotyp zwischen den beiden Zelllinien, und eine stärker deregulierte circadiane Rhythmik der Metastasen-Zelllinie. Es konnte zudem gezeigt werden, dass am Spleißvorgang beteiligte Gene sowie mutmaßliche Spleißereignisse 24-Stunden-Rhythmen aufweisen, die sich ebenfalls zwischen den Krebsstadien unterscheiden. Sowohl in gesunden als auch in Krebszellen waren viele jener Gene rhythmisch gespleißt, die selbst am Spleißvorgang beteiligt sind, was auf eine Autoregulierung des Prozesses schließen lässt. Mehrere der gespleißten Kandidatengene codieren zudem für Proteinvarianten, die an Prozessen beteiligt sind, welche eine Weiterentwicklung des Tumors begünstigen, darunter Zellmigration und Angiogenese. Insgesamt legen die Beobachtungen eine circadiane Regulation von alternativem Spleißen in Säugerzellen nah, welche einen Einfluss auf die Krebsentwicklung hat

A Computational Analysis of Alternative Splicing across Mammalian Tissues Reveals Circadian and Ultradian Rhythms in Splicing Events

Mounting evidence points to a role of the circadian clock in the temporal regulation of post-transcriptional processes in mammals, including alternative splicing (AS). In this study, we carried out a computational analysis of circadian and ultradian rhythms on the transcriptome level to characterise the landscape of rhythmic AS events in published datasets covering 76 tissues from mouse and olive baboon. Splicing-related genes with 24-h rhythmic expression patterns showed a bimodal distribution of peak phases across tissues and species, indicating that they might be controlled by the circadian clock. On the output level, we identified putative oscillating AS events in murine microarray data and pairs of differentially rhythmic splice isoforms of the same gene in baboon RNA-seq data that peaked at opposing times of the day and included oncogenes and tumour suppressors. We further explored these findings using a new circadian RNA-seq dataset of human colorectal cancer cell lines. Rhythmic isoform expression patterns differed between the primary tumour and the metastatic cell line and were associated with cancer-related biological processes, indicating a functional role of rhythmic AS that might be implicated in tumour progression. Our data shows that rhythmic AS events are widespread across mammalian tissues and might contribute to a temporal diversification of the proteome

The BAM Data Store: Piloting an openBIS-Based Research Data Infrastructure for Materials Science and Engineering

This presentation was shown as part of the 1st Research Data Infrastructure (CoRDI) – Connecting communities conference of the National Research Data Infrastructure (NFDI) in Karlsruhe organized in cooperation with the Karlsruhe Institute of Technology (KIT) from September 12 to 14, 2023. All conference abstracts have been published by the organizers, including the article on “The BAM Data Store” Vol. 1 (2023): 1st Conference on Research Data Infrastructure (CoRDI) - Connecting Communities | Proceedings of the Conference on Research Data Infrastructure (tib-op.org). This presentation gave a short introduction to the German Federal Institute for Materials Research and Testing (“Bundesanstalt für Materialforschung und -prüfung (BAM)”), followed by an overview of the structure of the BAM Data Store and the underlying framework and open-source software (openBIS) developed by the ETH Zürich. The BAM Data Store project includes work packages to develop the institutional infrastructure for implementing openBIS as the central Electronic Lab Notebook (ELN) and data storage framework at BAM and to address the research data management (RDM) needs of BAM researchers. The presentation also shows the results of the pilot phase of the BAM Data Store in 2021 and presents the advances in RDM made by the nanoPlattform project during the pilot phase. The presentation concludes with a list of lessons learned and an outline of the project prospects with the start of the institute-wide rollout phase in 2023. The authors are interested in an active discussion with colleagues developing RDM infrastructure (and are open to share their gained experiences and best practices), especially on topics related to ELN interoperability and harmonization of research data stored in ELNs, not exclusively for the field of Materials Science and Engineering

The Interplay between Colon Cancer Cells and Tumour-Associated Stromal Cells Impacts the Biological Clock and Enhances Malignant Phenotypes

Cancer cells interrelate with the bordering host microenvironment that encompasses the extracellular matrix and a nontumour cellular component comprising fibroblasts and immune-competent cells. The tumour microenvironment modulates cancer onset and progression, but the molecular factors managing this interaction are not fully understood. Malignant transformation of a benign tumour is among the first crucial events in colorectal carcinogenesis. The role of tumour stroma fibroblasts is well-described in cancer, but less well-characterized in benign tumours. In the current work we utilized fibroblasts isolated from tubulovillous adenoma, which has high risk for malignant transformation, to study the interaction between benign tumour stroma and the circadian clock machinery. We explored the role of the biological clock in this interplay taking advantage of an experimental model, represented by the co-culture of colon cancer cells with normal fibroblasts or tumour-associated fibroblasts, isolated from human colorectal tumour specimens. When co-cultured with tumour-associated fibroblasts, colon cancer cells showed alterations in their circadian and metabolic parameters, with decreased apoptosis, increased colon cancer cell viability, and increased resistance to chemotherapeutic agents. In conclusion, the interactions among colon cancer cells and tumour-associated fibroblasts affect the molecular clockwork and seem to aggravate malignant cell phenotypes, suggesting a detrimental effect of this interplay on cancer dynamics

Diurnal variations in the expression of core-clock genes correlate with resting muscle properties and predict fluctuations in exercise performance across the day

Objectives In this study, we investigated daily fluctuations in molecular (gene expression) and physiological (biomechanical muscle properties) features in human peripheral cells and their correlation with exercise performance. Methods 21 healthy participants (13 men and 8 women) took part in three test series: for the molecular analysis, 15 participants provided hair, blood or saliva time-course sampling for the rhythmicity analysis of core-clock gene expression via RT-PCR. For the exercise tests, 16 participants conducted strength and endurance exercises at different times of the day (9h, 12h, 15h and 18h). Myotonometry was carried out using a digital palpation device (MyotonPRO), five muscles were measured in 11 participants. A computational analysis was performed to relate core-clock gene expression, resting muscle tone and exercise performance. Results Core-clock genes show daily fluctuations in expression in all biological samples tested for all participants. Exercise performance peaks in the late afternoon (15-18 hours for both men and women) and shows variations in performance, depending on the type of exercise (eg, strength vs endurance). Muscle tone varies across the day and higher muscle tone correlates with better performance. Molecular daily profiles correlate with daily variation in exercise performance. Conclusion Training programmes can profit from these findings to increase efficiency and fine-tune timing of training sessions based on the individual molecular data. Our results can benefit both professional athletes, where a fraction of seconds may allow for a gold medal, and rehabilitation in clinical settings to increase therapy efficacy and reduce recovery times