Biology subjects

Cvetesic, N.

Publications and source records attributed to Cvetesic, N..

10 recordsLinked to original sources

bioRxiv2024

Liver kinase B1 (LKB1) regulates the epigenetic landscape of mouse pancreatic beta cells

Liver kinase B1 (LKB1/STK11) is an important regulator of pancreatic {beta}-cell identity and function. Elimination of Lkb1 from the {beta}-cell results in improved glucose-stimulated insulin secretion and is accompanied by profound changes in gene expression, including the upregulation of several neuronal genes. The mechanisms through which LKB1 controls gene expression are, at present, poorly understood. Here, we explore the impact of {beta} cell- selective deletion of Lkb1 on chromatin accessibility in mouse pancreatic islets. To characterize the role of LKB1 in the regulation of gene expression at the transcriptional level, we combine these data with a map of islet active transcription start sites and histone marks. We demonstrate that LKB1 elimination from {beta}-cells results in widespread changes in chromatin accessibility, correlating with changes in transcript levels. Changes occurred in hundreds of promoter and enhancer regions, many of which were close to neuronal genes. We reveal that dysregulated enhancers are enriched in binding motifs for transcription factors important for {beta}-cell identity, such as FOXA, MAFA or RFX6 and we identify microRNAs (miRNAs) that are regulated by LKB1 at the transcriptional level. Overall, our study provides important new insights into the epigenetic mechanisms by which LKB1 regulates {beta}-cell identity and function.

cell biology↗

bioRxiv2021

Negative catalysis by the editing domain of class I aminoacyl-tRNA synthetases

Aminoacyl-tRNA synthetases (AARS) translate the genetic code by loading tRNAs with the cognate amino acids. The errors in amino acid recognition are cleared at the AARS editing domain through hydrolysis of misaminoacyl-tRNAs. This ensures faithful protein synthesis and cellular fitness. Using Escherichia coli isoleucyl-tRNA synthetase (IleRS) as a model enzyme, we demonstrated that the class I editing domain clears the non-cognate amino acids well-discriminated at the synthetic site with the same rates as the weakly-discriminated fidelity threats. This unveiled low selectivity suggests that evolutionary pressure to optimize the rates against the amino acids that jeopardize translational fidelity did not shape the editing site. Instead, we propose that editing was shaped to safeguard cognate aminoacyl-tRNAs against hydrolysis. Misediting is prevented by the residues that promote negative catalysis through destabilisation of the transition state comprising cognate amino acid. Such powerful design allows broad substrate acceptance of the editing domain along with its exquisite specificity in the cognate aminoacyl-tRNA rejection. Editing proceeds by direct substrate delivery to the editing domain (in cis pathway). However, we found that class I IleRS also releases misaminoacyl-tRNAIle and edits it in trans. This minor editing pathway was up to now recognized only for class II AARSs.

biochemistry↗

bioRxiv2020

Global regulatory transitions at core promoters demarcate the mammalian germline cycle

Core promoters integrate regulatory inputs of genes1-3. Global dynamics of promoter usage can reveal systemic changes in how genomic sequence is interpreted by the cell4 Here we report the first analysis of promoter dynamics and code switching in the mammalian germ line, characterising the full cycle of transitions from embryonic stem cells through germline, oogenesis, and zygotic genome activation. Using Super Low Input Carrier-CAGE5,6 (SLIC-CAGE) we show that mouse germline development starts with the somatic promoter code, followed by a prominent switch to the maternal code during follicular oogenesis. The sequence features underlying the shift from somatic to maternal code are conserved across vertebrates, despite large differences in promoter nucleotide compositions. In addition, we show that, prior to this major shift, the promoters of gonadal germ cells diverge from the canonical somatic transcription initiation. This divergence is distinct from the promoter code used later by developing oocytes and reveals genome-wide promoter remodelling associated with alternative nucleosome positioning during early female and male germline development. Collectively, our findings establish promoter-level regulatory transitions as a central, conserved feature of the vertebrate life cycle.

developmental biology↗

bioRxiv2020

The long non-coding RNA Pax6os1/PAX6-AS1 modulates pancreatic β-cell identity and function.

Aim/HypothesisLong non-coding RNAs (lncRNAs) are emerging as crucial regulators of beta cell development and function. Here, we investigate roles for an antisense lncRNA expressed from the Pax6 locus (annotated as Pax6os1 in mice and PAX6-AS1 in humans) in beta cell identity and functionality. MethodsPax6os1 expression was silenced in MIN6 cells using siRNAs and changes in gene expression were determined by RNA sequencing or qRT-PCR. Mice inactivated for Pax6os1 and human PAX6-AS1-null EndoC-{beta}H1 cells, were generated using CRISPR/Cas9 technology. Human islets were infected with lentiviral vectors bearing a targeted shRNA or PAX6-AS1, which were used to silence or overexpress, respectively, the lncRNA. RNA sequencing or RT-qPCR were used to measure transcriptomic changes and RNA pulldown in mice and human cells followed by mass spectrometry/western blot were performed to explore RNA protein interactions. ResultsPax6os1/PAX6-AS1 expression was upregulated at high glucose concentrations in derived beta cell lines as well as in mouse and human islets, and in pancreatic islets isolated from mice fed a high fat diet (n=6, p=0.003) and patients with type 2 diabetes (n=11-5, p<0.01). Silencing or deletion of Pax6os1/PAX6-AS1 in MIN6 or EndoC-{beta}H1cells increased the expression of several {beta}-cell signature genes, including PDX1 and INS. Female, but not male, Pax6os1 null mice fed a high fat diet showed slightly enhanced glucose clearance. ShRNA-mediated silencing of PAX6-AS1 in human islets robustly increased INS mRNA, enhanced glucose-stimulated insulin secretion and calcium dynamics, while overexpression of the lncRNA exerted opposing effects. Pax6os1/AS-1 interacted with histones H3 and H4 in mouse and human cells, indicating a possible role for this lncRNA in histone modifications in both species. ConclusionsIncreased expression of PAX6-AS1 at high glucose levels may impair beta cell functionality and thus contribute to the development of type 2 diabetes. Thus, targeting PAX6-AS1 may provide a promising strategy to enhance insulin secretion and improve glucose homeostasis in this disease. Research in contextO_ST_ABSWhat is already known about the subject?C_ST_ABSLong non-coding RNAs (lncRNAs) are crucial components of the pancreatic islet regulome, whose misexpression may contribute to the development of diabetes. What is the key question?Is the lncRNA Pax6os1/PAX6-AS1 involved in beta cell functionality and type 2 diabetes? What are the new findings?The expression of Pax6os1/PAX6-AS1 is upregulated in mice fed a high fat diet and in pancreatic islets from type 2 diabetes donors. Overexpression of PAX6-AS1 in human pancreatic islets reduces insulin expression, glucose stimulated secretion and intracellular calcium dynamics. Silencing PAX6-AS1 in human pancreatic islets upregulates insulin expression, enhances glucose stimulated insulin secretion and increases intracellular calcium dynamics. How may this impact the clinic in the foreseeable future?Understanding the genetic factors induced by high glucose/obesity involved in beta cell dysfunction is crucial for the development of new therapies to treat T2D.

cell biology↗

bioRxiv2020

TBPL2/TFIIA complex overhauls oocyte transcriptome during oocyte growth

During oocyte growth, transcription is required to create RNA and protein reserves to achieve maternal competence. During this period, the general transcription factor TATA binding protein (TBP) is replaced by its paralogue, TBPL2 (TBP2 or TRF3), which is essential for RNA polymerase II transcription. We show that in oocytes TBPL2 does not assemble into a canonical TFIID complex. Our transcript analyses demonstrate that TBPL2 mediates transcription of oocyte-expressed genes, including mRNA survey genes, as well as specific endogenous retroviral elements. Transcription start site (TSS) mapping indicates that TBPL2 has a strong preference for TATA-like motif in core promoters driving sharp TSS selection, in contrast with canonical TBP/TFIID-driven TATA-less promoters that have broader TSS architecture. Thus, we show a role for the TBPL2/TFIIA complex in the establishment of the oocyte transcriptome by using a specific TSS recognition code.

molecular biology↗

bioRxiv2020

Partial rescue of neuronal genes deregulated in Cornelia de Lange Syndrome by cohesin

Cornelia de Lange Syndrome (CdLS) is a human developmental disorder caused by mutations that compromise the function of cohesin, a major regulator of 3D genome organization. Cognitive impairment is a universal and as yet unexplained feature of CdLS. We characterized the transcriptional profile of cortical neurons from CdLS patients and found deregulation of hundreds of genes enriched for neuronal functions related to synaptic transmission, signalling processes, learning and behaviour. Inducible proteolytic cleavage of cohesin disrupted 3-D genome organization and transcriptional control in post-mitotic cortical mouse neurons. The genes affected belonged to similar gene ontology classes and showed significant numerical overlap with those deregulated in CdLS. Interestingly, gene expression was largely rescued by subsequent reconstitution of cohesin function. These experiments show that cohesin is continuously required for neuronal gene expression and provide a tractable approach for addressing mechanisms of neuronal dysfunction in CdLS.

cell biology↗

bioRxiv2020

Conservative route to extreme genome compaction in a miniature annelid

The causes and consequences of genome reduction in animals are unclear, because our understanding of this process mostly relies on lineages with often exceptionally high rates of evolution. Here, we decode the compact 73.8 Mb genome of Dimorphilus gyrociliatus, a meiobenthic segmented worm. The D. gyrociliatus genome retains traits classically associated with larger and slower-evolving genomes, such as an ordered, intact Hox cluster, a generally conserved developmental toolkit, and traces of ancestral bilaterian linkage. Unlike some other animals with small genomes, the analysis of the D. gyrociliatus epigenome revealed canonical features of genome regulation, excluding the presence of operons and trans-splicing. Instead, the gene dense D. gyrociliatus genome presents a divergent Myc pathway, a key physiological regulator of growth, proliferation, and genome stability in animals. Altogether, our results uncover a conservative route to genome compaction in annelids, reminiscent of that observed in the vertebrate Takifugu rubripes.

genomics↗

bioRxiv2019

Zebrafish embryonic tissue differentiation is marked by concurrent cell cycle dynamic and gene promoter regulatory changes

The core promoter, a stretch of DNA surrounding the transcription start site (TSS) is a major integration point for regulatory signals controlling gene transcription. The process of cell differentiation is accompanied by a marked divergence in transcriptional repertoire between cells of different fates, accompanied by changes in cellular behaviour, in particular their proliferative activity. Investigation of divergent core promoter architectures suggest distinct regulatory networks act on the core promoter, modulating cell behavior through transcriptional profile changes, which ultimately drives key transitions in cellular behaviour during embryonic development. The role that promoter-associated gene regulatory networks play in development associated transitions in cell cycle dynamics (e.g. during differentiation) however, is poorly understood. In this study we demonstrate in a developing in vivo model, how core promoter variations play a key role in defining transcriptional output in cells transitioning from a proliferative to cell-lineage specifying phenotype. The FUCCI transgenic system, differentially marks cells in G1 and S/G2/M phases of the cell cycle and can therefore be used to separate rapidly and slowly cycling cells in vivo, by virtue of the cell cycle stage they primarily inhabit. Longitudinal assessment of cell proliferation rate during zebrafish embryo development, using this system, revealed a spatial and lineage-specific separation in cell cycling behaviour across post-gastrulation embryos. In order to investigate the role differential promoter usage plays in this process, cap analysis of gene expression (CAGE) was performed on fluorescent associated cell sorted (FACS) FUCCI zebrafish embryos going through somitogenesis, separating cells in accordance with the rate of their cell cycling. This analysis revealed a dramatic increase in lineage and tissue-specific gene expression, concurrent with a slowing of their cell cycling. Core promoters associated with rapidly cycling cells, showed broad distribution of transcription start site usage, featuring positionally constrained CCAAT-box, while slowly cycling cells favoured sharp TSS usage coupled with canonical TATA-box utilisation and enrichment of Sp1 binding sites. These results demonstrate the regulatory role of core promoters in cell cycle-dependent transcription regulation, during somitogenesis stages of embryo development.

developmental biology↗

bioRxiv2018

Saccharomyces cerevisiae displays a stable transcription start site landscape in multiple conditions

One of the fundamental processes that determine cellular fate is regulation of gene transcription. Understanding these regulatory processes is therefore essential for understanding cellular responses to changes in environmental conditions. At the core promoter, the regulatory region containing the transcription start site (TSS), all inputs regulating transcription are integrated. Here, we used Cap Analysis of Gene Expression (CAGE) to analyze the pattern of transcription start sites at four different environmental conditions (limited in ethanol, limited in nitrogen, limited in glucose and limited in glucose under anaerobic conditions) using the Saccharomyces cerevisiae strain CEN.PK113-7D. With this experimental setup we were able to show that the TSS landscape in yeast is stable at different metabolic states of the cell. We also show that the shape index, a characteristic feature of each TSS describing the spatial distribution of transcription initiation events, has a surprisingly strong negative correlation with the measured expression levels. Our analysis supplies a set of high quality TSS annotations useful for metabolic engineering and synthetic biology approaches in the industrially relevant laboratory strain CEN.PK113-7D, and provides novel insights into yeast TSS dynamics and gene regulation.

systems biology↗

bioRxiv2018

SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA

Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of rules of transcription initiation, led to discovery of new core promoter sequence features and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 micrograms), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types. Here, we present SLIC-CAGE, a Super-Low Input Carrier-CAGE approach to capture 5ends of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity is achieved by specially designed, selectively degradable carrier RNA. We demonstrate the ability of SLIC-CAGE to generate data for genome-wide promoterome with 1000-fold less material than required by existing CAGE methods by generating a complex, high quality library from mouse embryonic day (E) 11.5 primordial germ cells.

genomics↗