scholarly journals Systematic functional characterization of antisense eRNA of protocadherin α composite enhancer

2021 ◽  
Vol 35 (19-20) ◽  
pp. 1383-1394
Author(s):  
Yuxiao Zhou ◽  
Siyuan Xu ◽  
Mo Zhang ◽  
Qiang Wu

Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5′ capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA–DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

2021 ◽  
Author(s):  
Yuxiao Zhou ◽  
Siyuan Xu ◽  
Qiang Wu

Enhancers generate bidirectional noncoding enhancer RNAs that may regulate gene expression. At present, mechanisms of eRNA functions are not fully understood. Here, we report an antisense eRNA PEARL that is transcribed from the protocadherin α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA-fragment editing, CRISPRi, and CRISPRa strategies, in conjunction with ChIRP, MeDIP, and DRIP experiments, we find that PEARL regulates Pcdhα expression by forming local R-loop in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancer and target promoters. These findings have important implications regarding mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.


2021 ◽  
Vol 118 (18) ◽  
pp. e2020125118
Author(s):  
Yoshiaki Kita ◽  
Hirozumi Nishibe ◽  
Yan Wang ◽  
Tsutomu Hashikawa ◽  
Satomi S. Kikuchi ◽  
...  

Precise spatiotemporal control of gene expression in the developing brain is critical for neural circuit formation, and comprehensive expression mapping in the developing primate brain is crucial to understand brain function in health and disease. Here, we developed an unbiased, automated, large-scale, cellular-resolution in situ hybridization (ISH)–based gene expression profiling system (GePS) and companion analysis to reveal gene expression patterns in the neonatal New World marmoset cortex, thalamus, and striatum that are distinct from those in mice. Gene-ontology analysis of marmoset-specific genes revealed associations with catalytic activity in the visual cortex and neuropsychiatric disorders in the thalamus. Cortically expressed genes with clear area boundaries were used in a three-dimensional cortical surface mapping algorithm to delineate higher-order cortical areas not evident in two-dimensional ISH data. GePS provides a powerful platform to elucidate the molecular mechanisms underlying primate neurobiology and developmental psychiatric and neurological disorders.


2020 ◽  
Vol 295 (8) ◽  
pp. 2495-2505 ◽  
Author(s):  
Michalina Janiszewska ◽  
Marina Candido Primi ◽  
Tina Izard

Homeostasis in healthy tissues strongly relies on cell-to-cell adhesion and cell-to-extracellular matrix interactions. For instance, normal epithelial cells maintain tissue structure by adhering to each other and to the extracellular matrix. The proteins that mediate these distinct interactions are collectively called cell adhesion molecules and are divided into four major groups: cadherins, integrins, selectins, and immunoglobulins. They not only physically anchor cells, but also critically integrate signaling between the extracellular microenvironment and cells. These signals include biochemical cues, as adhesion proteins can both act as ligand-activated receptors and activate mechanotransduction triggered by changes in the physical environment. Molecular mechanisms related to cell adhesion signaling have been extensively studied, especially because mutations and changes in expression of these proteins, particularly cadherins and integrins, are frequently associated with diseases ranging from developmental intellectual disability to cancer. In fact, two major hallmarks of cancer, loss of cell-to-cell adhesion and anchorage-independent growth, are both dependent on cell adhesion molecules. Despite many studies elucidating the relationships between malignant transformation and metastasis and cellular adhesion processes, several areas still await exploration. Here, we highlight recently discovered roles of adhesion molecules in collective cancer cell migration and discuss the utility of three-dimensional models in studying cell-cell adhesion. We also describe recent therapeutic approaches targeting adhesion molecules.


2017 ◽  
Vol 44 (4) ◽  
pp. 1559-1577 ◽  
Author(s):  
Andrea Gerbino ◽  
Irene Bottillo ◽  
Serena Milano ◽  
Martina Lipari ◽  
Roberta De Zio ◽  
...  

Background/Aims: Truncating LMNA gene mutations occur in many inherited cardiomyopathy cases, but the molecular mechanisms involved in the disease they cause have not yet been systematically investigated. Here, we studied a novel frameshift LMNA variant (p.D243Gfs*4) identified in three members of an Italian family co-segregating with a severe form of cardiomyopathy with conduction defects. Methods: HEK293 cells and HL-1 cardiomyocytes were transiently transfected with either Lamin A or D243Gfs*4 tagged with GFP (or mCherry). D243Gfs*4 expression, cellular localization and its effects on diverse cellular mechanisms were evaluated with western blotting, laser-scanning confocal microscopy and video-imaging analysis in single cells. Results: When expressed in HEK293 cells, GFP- (or mCherry)-tagged LMNA D243Gfs*4 colocalized with calnexin within the ER. ER mislocalization of LMNA D243Gfs*4 did not significantly induce ER stress response, abnormal Ca2+ handling and apoptosis when compared with HEK293 cells expressing another truncated mutant of LMNA (R321X) which similarly accumulates within the ER. Of note, HEK293-LMNA D243Gfs*4 cells showed a significant reduction of connexin 43 (CX43) expression level, which was completely rescued by activation of the WNT/β-catenin signaling pathway. When expressed in HL-1 cardiomyocytes, D243Gfs*4 significantly impaired the spontaneous Ca2+ oscillations recorded in these cells as result of propagation of the depolarizing waves through the gap junctions between non-transfected cells surrounding a cell harboring the mutation. Furthermore, mCh-D243Gfs*4 HL-1 cardiomyocytes showed reduced CX43-dependent Lucifer Yellow (LY) loading and propagation. Of note, activation of β-catenin rescued both LY loading and LMNA D243Gfs*4 -HL-1 cells spontaneous activity propagation. Conclusion: Overall, the present results clearly indicate the involvement of the aberrant CX43 expression/activity as a pathogenic mechanism for the conduction defects associated to this LMNA truncating alteration.


2021 ◽  
Author(s):  
Christine Rempfer ◽  
Gertrud Wiedemann ◽  
Gabriele Schween ◽  
Klaus L. Kerres ◽  
Jan M. Lucht ◽  
...  

Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome, might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant bryophytes. Whereas angiosperms repair DNA double strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in bryophytes homologous recombination (HR) is the main DNA-DSB repair pathway facilitating the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in the moss Physcomitrella using haploid plants and autodiploid plants, generated via an artificial duplication of the whole genome. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the whole genome duplication event. Among the differentially expressed genes involved in DNA-DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p<0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.


2021 ◽  
Vol 12 ◽  
Author(s):  
Karin Rothkegel ◽  
Alonso Espinoza ◽  
Dayan Sanhueza ◽  
Victoria Lillo-Carmona ◽  
Aníbal Riveros ◽  
...  

Peach (Prunus persica) fruits have a fast ripening process and a shelf-life of days, presenting a challenge for long-distance consuming markets. To prolong shelf-life, peach fruits are stored at low temperatures (0 to 7 °C) for at least two weeks, which can lead to the development of mealiness, a physiological disorder that reduces fruit quality and decreases consumer acceptance. Several studies have been made to understand this disorder, however, the molecular mechanisms underlying mealiness are not fully understood. Epigenetic factors, such as DNA methylation, modulate gene expression according to the genetic background and environmental conditions. In this sense, the aim of this work was to identify differentially methylated regions (DMRs) that could affect gene expression in contrasting individuals for mealiness. Peach flesh was studied at harvest time (E1 stage) and after cold storage (E3 stage) for 30 days. The distribution of DNA methylations within the eight chromosomes of P. persica showed higher methylation levels in pericentromeric regions and most differences between mealy and normal fruits were at Chr1, Chr4, and Chr8. Notably, differences in Chr4 co-localized with previous QTLs associated with mealiness. Additionally, the number of DMRs was higher in CHH cytosines of normal and mealy fruits at E3; however, most DMRs were attributed to mealy fruits from E1, increasing at E3. From RNA-Seq data, we observed that differentially expressed genes (DEGs) between normal and mealy fruits were associated with ethylene signaling, cell wall modification, lipid metabolism, oxidative stress and iron homeostasis. When integrating the annotation of DMRs and DEGs, we identified a CYP450 82A and an UDP-ARABINOSE 4 EPIMERASE 1 gene that were downregulated and hypermethylated in mealy fruits, coinciding with the co-localization of a transposable element (TE). Altogether, this study indicates that genetic differences between tolerant and susceptible individuals is predominantly affecting epigenetic regulation over gene expression, which could contribute to a metabolic alteration from earlier stages of development, resulting in mealiness at later stages. Finally, this epigenetic mark should be further studied for the development of new molecular tools in support of breeding programs.


2002 ◽  
Vol 368 (1) ◽  
pp. 159-169 ◽  
Author(s):  
Seema SEHRAWAT ◽  
Narayanaswamy SRINIVASAN ◽  
Karumathil P. GOPINATHAN

Late gene expression factor 4 (LEF4), a multifunctional protein encoded by the Bombyx mori nucleopolyhedrovirus has been bacterially expressed and characterized. Sequence analyses and three-dimensional modelling of B. mori LEF4 showed that the protein is related to mRNA-capping enzymes, which are organized as two modular domains. Most of the acidic side chains in LEF4 were solvent-exposed and spread all along the fold. A region dominated by negatively charged groups, which protrudes from the larger domain was ideally suited for interactions with proteins having positively charged patches at the surface. The purified LEF4 protein exhibited different enzyme activities associated with mRNA-capping enzymes, i.e. GTP-binding, RNA triphosphatase and guanylate transferase activities. In addition, LEF4 also showed NTP-hydrolysing activity. The kinetic analysis of ATP hydrolysis revealed a sigmoidal response with two deduced binding sites for ATP, whereas the guanylate transferase activity showed a typical hyperbolic response to varying concentrations of GTP with a Km of 330±20μM. Analysis of the modelled three-dimensional structure of LEF4 suggested the presence of crucial residues in sequence motifs important for the integrity of the fold. Mutation of one such conserved and buried tyrosine residue to cysteine in the motif IIIa, located close to the interlobe region of the model, resulted in a 44% loss of guanylate transferase activity of LEF4 but had no effect on the ATPase activity.


Author(s):  
Christine Rempfer ◽  
Gertrud Wiedemann ◽  
Gabriele Schween ◽  
Klaus L. Kerres ◽  
Jan M. Lucht ◽  
...  

Abstract Key message In Physcomitrella, whole-genome duplications affected the expression of about 3.7% of the protein-encoding genes, some of them relevant for DNA repair, resulting in a massively reduced gene-targeting frequency. Abstract Qualitative changes in gene expression after an autopolyploidization event, a pure duplication of the whole genome (WGD), might be relevant for a different regulation of molecular mechanisms between angiosperms growing in a life cycle with a dominant diploid sporophytic stage and the haploid-dominant mosses. Whereas angiosperms repair DNA double-strand breaks (DSB) preferentially via non-homologous end joining (NHEJ), in the moss Physcomitrella homologous recombination (HR) is the main DNA–DSB repair pathway. HR facilitates the precise integration of foreign DNA into the genome via gene targeting (GT). Here, we studied the influence of ploidy on gene expression patterns and GT efficiency in Physcomitrella using haploid plants and autodiploid plants, generated via an artificial WGD. Single cells (protoplasts) were transfected with a GT construct and material from different time-points after transfection was analysed by microarrays and SuperSAGE sequencing. In the SuperSAGE data, we detected 3.7% of the Physcomitrella genes as differentially expressed in response to the WGD event. Among the differentially expressed genes involved in DNA–DSB repair was an upregulated gene encoding the X-ray repair cross-complementing protein 4 (XRCC4), a key player in NHEJ. Analysing the GT efficiency, we observed that autodiploid plants were significantly GT suppressed (p < 0.001) attaining only one third of the expected GT rates. Hence, an alteration of global transcript patterns, including genes related to DNA repair, in autodiploid Physcomitrella plants correlated with a drastic suppression of HR.


2020 ◽  
Vol 21 (22) ◽  
pp. 8478
Author(s):  
Alonso Rodríguez-Caparrós ◽  
Jesús Álvarez-Santiago ◽  
María Jesús del Valle-Pastor ◽  
Carlos Suñé ◽  
Jennifer López-Ros ◽  
...  

The adaptive immune response in vertebrates depends on the expression of antigen-specific receptors in lymphocytes. T-cell receptor (TCR) gene expression is exquisitely regulated during thymocyte development to drive the generation of αβ and γδ T lymphocytes. The TCRα, TCRβ, TCRγ, and TCRδ genes exist in two different configurations, unrearranged and rearranged. A correctly rearranged configuration is required for expression of a functional TCR chain. TCRs can take the form of one of three possible heterodimers, pre-TCR, TCRαβ, or TCRγδ which drive thymocyte maturation into αβ or γδ T lymphocytes. To pass from an unrearranged to a rearranged configuration, global and local three dimensional (3D) chromatin changes must occur during thymocyte development to regulate gene segment accessibility for V(D)J recombination. During this process, enhancers play a critical role by modifying the chromatin conformation and triggering noncoding germline transcription that promotes the recruitment of the recombination machinery. The different signaling that thymocytes receive during their development controls enhancer activity. Here, we summarize the dynamics of long-distance interactions established through chromatin regulatory elements that drive transcription and V(D)J recombination and how different signaling pathways are orchestrated to regulate the activity of enhancers to precisely control TCR gene expression during T-cell maturation.


2019 ◽  
Author(s):  
Xi-Ming Sun ◽  
Anthony Bowman ◽  
Miles Priestman ◽  
Francois Bertaux ◽  
Amalia Martinez-Segura ◽  
...  

ABSTRACTCell size varies during the cell cycle and in response to external stimuli. This requires the tight coordination, or “scaling”, of mRNA and protein quantities with the cell volume in order to maintain biomolecules concentrations and cell density. Evidence in cell populations and single cells indicates that scaling relies on the coordination of mRNA transcription rates with cell size. Here we use a combination of single-molecule fluorescence in situ hybridisation (smFISH), time-lapse microscopy and mathematical modelling in single fission yeast cells to uncover the precise molecular mechanisms that control transcription rates scaling with cell size. Linear scaling of mRNA quantities is apparent in single fission yeast cells during a normal cell cycle. Transcription rates of both constitutive and regulated genes scale with cell size without evidence for transcriptional bursting. Modelling and experimental data indicate that scaling relies on the coordination of RNAPII transcription initiation rates with cell size and that RNAPII is a limiting factor. We show using real-time quantitative imaging that size increase is accompanied by a rapid concentration independent recruitment of RNAPII onto chromatin. Finally, we find that in multinucleated cells, scaling is set at the level of single nuclei and not the entire cell, making the nucleus the transcriptional scaling unit. Integrating our observations in a mechanistic model of RNAPII mediated transcription, we propose that scaling of gene expression with cell size is the consequence of competition between genes for limiting RNAPII.


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