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Retrospective analysis of enhancer activity and transcriptome history.

ABSTRACT: Cell state changes in development and disease are controlled by gene regulatory networks, the dynamics of which are difficult to track in real time. In this study, we used an inducible DCM-RNA polymerase subunit b fusion protein which labels active genes and enhancers with a bacterial methylation mark that does not affect gene transcription and is propagated in S-phase. This DCM-RNA polymerase fusion protein enables transcribed genes and active enhancers to be tagged and then examined at later stages of development or differentiation. We apply this DCM-time machine (DCM-TM) technology to study intestinal homeostasis, revealing rapid and coordinated activation of enhancers and nearby genes during enterocyte differentiation. We provide new insights in absorptive-secretory lineage decision-making in intestinal stem cell (ISC) differentiation and show that ISCs retain a unique chromatin landscape required to maintain ISC identity and delineate future expression of differentiation-associated genes. DCM-TM has wide applicability in tracking cell states, providing new insights in the regulatory networks underlying cell state changes.

SUBMITTER: Boers R

PROVIDER: S-EPMC10635829 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Retrospective analysis of enhancer activity and transcriptome history.

Boers Ruben R Boers Joachim J Tan Beatrice B van Leeuwen Marieke E ME Wassenaar Evelyne E Sanchez Erlantz Gonzalez EG Sleddens Esther E Tenhagen Yasha Y Mulugeta Eskeatnaf E Laven Joop J Creyghton Menno M Baarends Willy W van IJcken Wilfred F J WFJ Gribnau Joost J

Nature biotechnology 20230223 11

Cell state changes in development and disease are controlled by gene regulatory networks, the dynamics of which are difficult to track in real time. In this study, we used an inducible DCM-RNA polymerase subunit b fusion protein which labels active genes and enhancers with a bacterial methylation mark that does not affect gene transcription and is propagated in S-phase. This DCM-RNA polymerase fusion protein enables transcribed genes and active enhancers to be tagged and then examined at later s ...[more]

PMID: 36823354

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Project description:BackgroundAll learners at the Northern Ontario School of Medicine University complete a longitudinal integrated clerkship experience in their third year, which serves to improve learner experience with community and clinical acute and chronic health needs. Early in the program, Muskoka faculty (two of the 15 LIC sites of NOSM U) became aware that learners never had the occasion to complete a full history and physical exam on a real patient with complex needs. Recognizing this as a critical experience, a program was initiated to provide learners with this opportunity. This manuscript reports on the effectiveness and impact of this novel program and outlines the procedure developed to incorporate a similar program should communities see the relevance.MethodsUsing a mixed method design, feedback was collected from learners and preceptors following the implementation of a novel learning opportunity for clinical clerks. Learners completed a full history and physical exam on volunteer complex patients, with supervision and immediate feedback. Using semi structured surveys, data was collected from each learner and preceptor to determine the program impact and optimize the program. Laurentian University research ethics board, certificate number 6021120.ResultsBoth learners and preceptors agreed this was a valuable experience for learners, a good use of their time and contributed to essential skills including, communication, time management and appropriate data collection. The use of real patients was reported to be very appropriate by learners and faculty and often highlighted gaps in the learner's knowledge that they were then able to address.ConclusionFeedback collected in this study confirms that providing medical learners the opportunity to complete a full history and physical exam with supervision and feedback was significantly beneficial from both a clinical and a skills-based aspect. Requiring learners to complete this task within the established period forced them to manage their time, focus on clinical consideration and remain on task. Enhancing learning opportunities is associated with improved outcomes and understanding in medical learners. Positive community experience is also related to learner retention, which is paramount for attracting new physicians in a time with significantly limited human health resources.

Project description:Preadult determinants of adult fitness and behaviour have been documented in a variety of organisms with complex life cycles, but little is known about expression patterns of genes underlying these adult traits. We explored the effects of differences in egg-to-adult development time on adult transcriptome and cuticular hydrocarbon variation in order to understand the nature of the genetic correlation between preadult development time and premating isolation between populations of Drosophila mojavensis reared in different host cactus environments. Transcriptome variation was analysed separately in flies reared on each host and revealed that hundreds of genes in adults were differentially expressed (FDR P < 0.05) due to development time differences. For flies reared on pitaya agria cactus, longer preadult development times caused increased expression of genes in adults enriched for ribosome production, protein metabolism, chromatin remodelling and regulation of alternate splicing and transcription. Baja California flies reared on organ pipe cactus showed fewer differentially expressed genes in adults due to longer preadult development time, but these were enriched for ATP synthesis and the TCA cycle. Mainland flies reared on organ pipe cactus with shorter development times showed increased transcription of genes enriched for mitochondria and energy production, protein synthesis and glucose metabolism: adults with longer development times had increased expression of genes enriched for adult life span, cuticle proteins and ion binding, although most differentially expressed genes were unannotated. Differences due to population, sex, mating status and their interactions were also assessed. Adult cuticular hydrocarbon profiles also showed shifts due to egg-to-adult development time and were influenced by population and mating status. These results help to explain why preadult life history variation determines subsequent expression of the adult transcriptome along with traits involved with reproductive isolation and revealed previously undocumented connections between genetic and environmental influences over the entire life cycle in this desert insect.

Dataset Information

Retrospective analysis of enhancer activity and transcriptome history.

Publications

Retrospective analysis of enhancer activity and transcriptome history.

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