Epigenetic mechanisms of gene bookmarking during mitosis

The MHCII genes of the major histocompatibily complex play an importantrole in immune response. Their regulation implicates proteins that synergistically bindto the promoter and constitute the MHCII enhanceosome (MCE). Although necessary,MCE is not sufficient to drive transcription. The master coactivator CIITA is requiredfor efficient transcription initiation and elongation. This study approaches theregulation of MHCII genes through the cell cycle and particular during mitosis. Theexistence of gene bookmarking was examined, an important process for the cell inorder to maintain the gene expression profile to the next cell cycle.Immunostaining experiments for components of MCE showed theirassociation on mitotic chromatin in a dynamic manner, although with variable kineticsin different stages of the cell cycle. After the synchronization of a B lymphocyteculture and chromatin immunoprecipitation assays in different cell cycle stages werestudied the kinetics of the recruitment of different regulatory factors on the DRApromoter, a prototypical MHCII gene. The recruitment of the regulatory factors wasalso identified in a better defined mitotic environment.To compare the identified bookmarking mechanism in non-lymphoblastoidcells, epithelial cells, modified to express MHCII genes constitutively, were used.Chromatin immunoprecipitation assays were showed that mitotic occupancy of thefactors on the DRA promoter was diminished, and so does the accessibility, comparedto B lymphocytes. Furthermore transcription was abolished contrary to B lymphocytethat it maintained in low levels and can be fully rescued even by providing CIITAinside mitosis.Also, another regulatory element of the DRA gene, LCR/XL4 was studied.Results showed the mitotic occupancy of the NFY complex on this region and therecruitment of a phosphatase, PP2A, through interaction with NFYA transcriptionfactor. Then, with PP2A knocking down was identified that PP2A bookmarks theDRA gene, through chromatin decondensation, for transcription timing upon entryinto the G1 phase of the cell cycle. Finally, chromatin decondensation could rescuethe DRA expression in epithelial cells during mitosis.Τα MHCII γονίδια του κυρίου συμπλόκου ιστοσυμβατότητας παίζουνσημαντικό ρόλο στην ανοσολογική απόκριση. Η ρύθμιση τους εμπλέκει πρωτεΐνες οιοποίες δρώντας συνεργατικά συγκροτούν το ενισχυόσωμα των MHCII (MCE). ΤοMCE δεν είναι αρκετό για την ενεργοποίηση τους, απαιτείται η παρουσία του CIITA,που αποτελεί τον κύριο ρυθμιστή των MHCII γονιδίων. Η παρούσα εργασίαπροσεγγίζει την ρύθμιση των MHCΙΙ γονιδίων κατά τη διάρκεια του κυτταρικούκύκλου και συγκεκριμένα κατά τη μίτωση. Μελετήθηκε η ύπαρξη γονιδιακήςσήμανσης, μια σημαντική διαδικασία για το κύτταρο προκειμένου να διατηρήσει τοπρότυπο έκφρασης των γονιδίων του στον επόμενο κυτταρικό κύκλο.Mε πειράματα ανοσοφθορισμού για παράγοντες του MCE παρατηρήθηκε οεντοπισμός τους στη μιτωτική χρωματίνη, ενώ η μελέτη της κινητική τους έδειξε ότιη πρόσδεση τους είναι δυναμική. Μετά από συγχρονισμό μιας καλλιέργειας Β-λεμφοκυττάρων και με ανοσοκατακριμνήσεις χρωματίνης σε διάφορες φάσεις τουκυτταρικού κύκλου μελετήθηκε η κινητική της πρόσδεσης διαφόρων ρυθμιστικώνπαραγόντων στον υποκινητή του DRA γονιδίου, ενός πρότυπου MHCII γονιδίου. Ηπρόσδεση των παραγόντων αυτών ταυτοποιήθηκε και σε αμιγώς μιτωτικό πληθυσμό.Θέλοντας να συγκρίνουμε τον μηχανισμό γονιδιακής σήμανσης και σε έναάλλο κυτταρικό περιβάλλον χρησιμοποιήθηκαν επιθηλιακά κύτταρα που εξέφραζαντα MHCII συστατικά. Με ανοσοκατακριμνήσεις χρωματίνης δείχτηκε ότι σταμιτωτικά κύτταρα δεν παρατηρείται πρόσδεση των παραγόντων στον υποκινητή ενώκαι η προσβασιμότητα του ήταν περιορισμένη σε σχέση με των Β-λεμφοκυττάρων.Επίσης, παρατηρήθηκε ότι στα πρώτα η μεταγραφή μηδενιζόταν στη μίτωση σεαντίθεση με τα δεύτερα, που τη διατηρούσαν σε χαμηλά επίπεδα και μετά απόεπαγωγή του CIITA στη μίτωση επανερχόταν στα μεσοφασικά επίπεδα.Στη συνέχεια μελετήθηκε μια άλλη ρυθμιστική περιοχή του DRA γονιδίου, τοLCR/XL-4. Με ανοσοκατακριμνήσεις χρωματίνης ταυτοποιήθηκε η πρόσδεση τουNFY συμπλόκου στην περιοχή αυτή και η στρατολόγηση της φωσφατάσης PP2Aμέσω αλληλεπίδρασης της με τον NFYA. Έπειτα, με αποσιώπηση της PP2Aαποδείχθηκε ότι σημαίνει το DRA γονίδιο, μέσω αποσυμπύκνωσης της χρωματίνης,για την ταχύτερη επανέκφραση του στην επόμενη G1 φάση του κυτταρικού κύκλου.Τέλος, παρατηρήθηκε ότι η αποσυμπύκνωση της χρωματίνης αποκαθιστά τηνέκφραση του DRA γονιδίου στα επιθηλιακά κύτταρα κατά τη μίτωση

Gamma Interferon-Dependent Transcriptional Memory via Relocalization of a Gene Locus to PML Nuclear Bodies▿ †

Memory of past cellular responses is an essential adaptation to repeating environmental stimuli. We addressed the question of whether gamma interferon (IFN-γ)-inducible transcription generates memory that sensitizes cells to a second stimulus. We have found that the major histocompatibility complex class II gene DRA is relocated to promyelocytic leukemia (PML) nuclear bodies upon induction with IFN-γ, and this topology is maintained long after transcription shut off. Concurrent interaction of PML protein with mixed-lineage leukemia generates a prolonged permissive chromatin state on the DRA gene characterized by high promoter histone H3 K4 dimethylation that facilitates rapid expression upon restimulation. We propose that the primary signal-induced transcription generates spatial and epigenetic memory that is maintained through several cell generations and endows the cell with increased responsiveness to future activation signals

Optimal design with the use of pumping flow simulation in the area of Agia in the district of Chania

Περίληψη: Η παρούσα μελέτη έχει στόχο την εύρεση των βέλτιστων παροχών άντλησης των γεωτρήσεων της περιοχής της Αγυιάς του νομού Χανίων. Η εργασία βασίζεται σε μοντέλο προσομοίωσης, το οποίο έχει κατασκευαστεί με χρήση του προγράμματος PTC, για την συγκεκριμένη περιοχή. Για τις ανάγκες της μελέτης ορίζονται δέκα πηγάδια παρατήρησης και τρείς επιπρόσθετες γεωτρήσεις άντλησης στο προαναφερόμενο μοντέλο. Αναζητούνται οι βέλτιστες παροχές άντλησης, οι οποίες καλύπτουν τις ανάγκες της περιοχής, για τρία σενάρια διαφορετικού ελάχιστου υδραυλικού ύψους του εξεταζόμενου υδροφορέα, ένα εκ των οποίων προσομοιάζει την υπάρχουσα κατάσταση του. Η βελτιστοποίηση πραγματοποιείται με χρήση της μεθόδου Simplex, καθώς τα αποτελέσματα των υδραυλικών υψών των πηγαδιών παρατήρησης του μοντέλου προσομοίωσης μειώνονται μη γραμμικά σε συνάρτηση με την παροχή άντλησης. Για τον λόγο αυτόν απαιτείται η διαδοχική εφαρμογή της μεθόδου Simplex ώστε να πραγματοποιηθεί σταδιακή γραμμικοποίηση. Δημιουργείται και εκφράζεται αλγόριθμος από την σύγκληση του οποίου τελικά προκύπτει η βέλτιστη λύση για καθένα από τα τρία εξεταζόμενα σενάρια. Τελικά συγκρίνονται τα αποτελέσματα των τριών σεναρίων με την υπάρχουσα κατάσταση του υδροφορέα της περιοχής της Αγυιάς και εξετάζεται κατά πόσο μπορεί αν αυξηθεί η συνολική παροχή άντλησης χωρίς να επέλθει πτώση του υδροφορέα μεγαλύτερη από το ύψος το οποίο ορίζεται σε κάθε σενάριο.Summarization: The present study aims to find the optimal pumping rates of the Agia area wells, in the district of Chania. The study is based on a simulation model, which is designed via the PTC software. Ten observation wells and three additional pumping wells were considered to the above-mentioned model. The optimal pumping rates which cover the region's needs were examined according to three scenarios of different minimum hydraulic heads of the aquifer, one of which is similar to the existing situation. The optimization is accomplished by using the Simplex method, as the results of the simulation model for the hydraulic heads at the observation wells decrease non-linearly in relation to the flow rate. Due to this decrease of groundwater level, the consecutive application of Simplex method is required, so that the gradual linearization can be achieved. An algorithm is created and, by its convergence, the optimal solution for each scenario separately is developed. Finally, the results of the three scenarios are compared to the data of Agia’s present aquifer state. Also, the possibility of increasing the total pumping rate without the decline of the aquifer level being greater than the level determined in each scenario, is examined

Greening the CAP: what implication of crop diversification

This paper aims to assess the impacts of 2013 CAP reform in Poland. We provide a comprehensive approach to modelling crop diversification options at farm group level. Belonging to the category of mathematical programming tools, AROPAj model is used to determine the number and mix of crops that may be efficiently cultivated. We consider an energy fast-growing plantation, i.e, willow, to enlarge the potential number of eligible crops. In this respect, we explore the impacts of crop diversification when the number crop limit and the payment benefiting areas planted with willow vary

Time-Resolved scRNA-Seq Tracks the Adaptation of a Sensitive MCL Cell Line to Ibrutinib Treatment.

Since the approval of ibrutinib for relapsed/refractory mantle cell lymphoma (MCL), the treatment of this rare mature B-cell neoplasm has taken a great leap forward. Despite promising efficacy of the Bruton tyrosine kinase inhibitor, resistance arises inevitably and the underlying mechanisms remain to be elucidated. Here, we aimed to decipher the response of a sensitive MCL cell line treated with ibrutinib using time-resolved single-cell RNA sequencing. The analysis uncovered five subpopulations and their individual responses to the treatment. The effects on the B cell receptor pathway, cell cycle, surface antigen expression, and metabolism were revealed by the computational analysis and were validated by molecular biological methods. The observed upregulation of B cell receptor signaling, crosstalk with the microenvironment, upregulation of CD52, and metabolic reprogramming towards dependence on oxidative phosphorylation favor resistance to ibrutinib treatment. Targeting these cellular responses provide new therapy options in MCL

Time-Resolved scRNA-Seq Tracks the Adaptation of a Sensitive MCL Cell Line to Ibrutinib Treatment

Since the approval of ibrutinib for relapsed/refractory mantle cell lymphoma (MCL), the treatment of this rare mature B-cell neoplasm has taken a great leap forward. Despite promising efficacy of the Bruton tyrosine kinase inhibitor, resistance arises inevitably and the underlying mechanisms remain to be elucidated. Here, we aimed to decipher the response of a sensitive MCL cell line treated with ibrutinib using time-resolved single-cell RNA sequencing. The analysis uncovered five subpopulations and their individual responses to the treatment. The effects on the B cell receptor pathway, cell cycle, surface antigen expression, and metabolism were revealed by the computational analysis and were validated by molecular biological methods. The observed upregulation of B cell receptor signaling, crosstalk with the microenvironment, upregulation of CD52, and metabolic reprogramming towards dependence on oxidative phosphorylation favor resistance to ibrutinib treatment. Targeting these cellular responses provide new therapy options in MCL

Cellular landscape of adrenocortical carcinoma at single-nuclei resolution

Adrenocortical carcinoma (ACC) is a rare yet devastating tumour of the adrenal gland with a molecular pathology that remains incompletely understood. To gain novel insights into the cellular landscape of ACC, we generated single-nuclei RNA sequencing (snRNA-seq) data sets from twelve ACC tumour samples and analysed these alongside snRNA-seq data sets from normal adrenal glands (NAGs). We find the ACC tumour microenvironment to be relatively devoid of immune cells compared to NAG tissues, consistent with known high tumour purity values for ACC as an immunologically “cold” tumour. Our analysis identifies three separate groups of ACC samples that are characterised by different relative compositions of adrenocortical cell types. These include cell populations that are specifically enriched in the most clinically aggressive and hormonally active tumours, displaying hallmarks of reorganised cell mechanobiology and dysregulated steroidogenesis, respectively. We also identified and validated a population of mitotically active adrenocortical cells that strongly overexpress genes POLQ, DIAPH3 and EZH2 to support tumour expansion alongside an LGR4+ progenitor-like or cell-of-origin candidate for adrenocortical carcinogenesis. Trajectory inference suggests the fate adopted by malignant adrenocortical cells upon differentiation is associated with the copy number or allelic balance state of the imprinted DLK1/MEG3 genomic locus, which we verified by assessing bulk tumour DNA methylation status. In conclusion, our results therefore provide new insights into the clinical and cellular heterogeneity of ACC, revealing how genetic perturbations to healthy adrenocortical renewal and zonation provide a molecular basis for disease pathogenesis.Keyword

scSLAM-seq reveals core features of transcription dynamics in single cells.

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations

Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34(+) stromal cell subsets

Pezoldt J, Wiechers C, Zou M, et al. Postnatal expansion of mesenteric lymph node stromal cells towards reticular and CD34(+) stromal cell subsets. Nature Communications. 2022;13(1): 7227.Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34(+) SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9(+) FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19(+) and Cxcl9(+) FRCs in LNs of Irf3(-/-) mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs. Lymph nodes in various locations of the body differ in their cell composition and gene expression signatures. Here authors show that the rapid postnatal expansion of lymph nodes is governed by CD34 (+) stromal cells and fibroblastic reticular stromal cell progenitors, distinguished by intrinsic, microbiome-independent core epigenetic blueprints

Opposing Wnt signals regulate cervical squamocolumnar homeostasis and emergence of metaplasia.

The transition zones of the squamous and columnar epithelia constitute hotspots for the emergence of cancer, often preceded by metaplasia, in which one epithelial type is replaced by another. It remains unclear how the epithelial spatial organization is maintained and how the transition zone niche is remodelled during metaplasia. Here we used single-cell RNA sequencing to characterize epithelial subpopulations and the underlying stromal compartment of endo- and ectocervix, encompassing the transition zone. Mouse lineage tracing, organoid culture and single-molecule RNA in situ hybridizations revealed that the two epithelia derive from separate cervix-resident lineage-specific stem cell populations regulated by opposing Wnt signals from the stroma. Using a mouse model of cervical metaplasia, we further show that the endocervical stroma undergoes remodelling and increases expression of the Wnt inhibitor Dickkopf-2 (DKK2), promoting the outgrowth of ectocervical stem cells. Our data indicate that homeostasis at the transition zone results from divergent stromal signals, driving the differential proliferation of resident epithelial lineages