scholarly journals Confined migration induces heterochromatin formation and alters chromatin accessibility

2021 ◽  
Author(s):  
Chieh-Ren Hsia ◽  
Jawuanna McAllister ◽  
Ovais Hasan ◽  
Julius Judd ◽  
Seoyeon Lee ◽  
...  
Keyword(s):  
Chromatin Accessibility ◽  
Cellular Functions ◽  
Promoter Regions ◽  
Collagen Matrices ◽  
Heterochromatin Formation ◽  
Damage Response ◽  
Intergenic Regions ◽  
And Function ◽  
Extensive Deformation

During migration, cells often squeeze through small constrictions, requiring extensive deformation. We hypothesized that the nuclear deformation associated with such confined migration could alter chromatin organization and function. Studying cells migrating through collagen matrices and microfluidic devices that mimic interstitial spaces in vivo, we found that confined migration results in increased H3K9me3 and H3K27me3 heterochromatin marks that persist for several days. This "confined migration-induced heterochromatin" (CMiH) was distinct from heterochromatin formation during migration initiation. CMiH predominantly decreased chromatin accessibility at intergenic regions near centromeres and telomeres, suggesting heterochromatin spreading from existing heterochromatin sites. Consistent with the overall decrease in chromatin accessibility, global transcription was decreased during confined migration. Intriguingly, we also identified increased accessibility at promoter regions of genes linked to chromatin silencing, tumor invasion, and DNA damage response. Inhibiting CMiH reduced migration speed, suggesting that CMiH promotes confined migration. Together, our findings indicate that confined migration induces chromatin changes that regulate cell migration and other cellular functions.

scholarly journals Protein manipulation using single copies of short peptide tags in cultured cells and in Drosophila melanogaster

2020 ◽  
Author(s):  
M. Alessandra Vigano ◽  
Clara-Maria Ell ◽  
Manuela MM Kustermann ◽  
Gustavo Aguilar ◽  
Shinya Matsuda ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Single Copy ◽  
Specific Protein ◽  
Cellular Functions ◽  
Cellular Processes ◽  
Peptide Tags ◽  
Genetic Technologies ◽  
Protein Binders ◽  
And Function

AbstractCellular development and specialized cellular functions are regulated processes which rely on highly dynamic molecular interactions among proteins, distributed in all cell compartments. Analysis of these interactions and their mechanisms of action has been one of the main topics in cellular and developmental research over the last fifty years. Studying and understanding the functions of proteins of interest (POIs) has been mostly achieved by their alteration at the genetic level and the analysis of the phenotypic changes generated by these alterations. Although genetic and reverse genetic technologies contributed to the vast majority of information and knowledge we have gathered so far, targeting specific interactions of POIs in a time- and space-controlled manner or analyzing the role of POIs in dynamic cellular processes such as cell migration or cell division would require more direct approaches. The recent development of specific protein binders, which can be expressed and function intracellularly, together with several improvements in synthetic biology techniques, have contributed to the creation of a new toolbox for direct protein manipulations. We selected a number of short tag epitopes for which protein binders from different scaffolds have been developed and tested whether these tags can be bound by the corresponding protein binders in living cells when they are inserted in a single copy in a POI. We indeed find that in all cases, a single copy of a short tag allows protein binding and manipulation. Using Drosophila, we also find that single short tags can be recognized and allow degradation and relocalization of POIs in vivo.

scholarly journals Epigenetic Changes Induced by Bacteroides fragilis Toxin

2019 ◽  
Vol 87 (6) ◽  
Author(s):  
Jawara Allen ◽  
Stephanie Hao ◽  
Cynthia L. Sears ◽  
Winston Timp
Keyword(s):  
Bacteroides Fragilis ◽  
Distal Colon ◽  
Chromatin Accessibility ◽  
Tumor Formation ◽  
Content Type ◽  
Intestinal Neoplasia ◽  
And Function ◽  
Gut Microbial Community ◽  
The Impact

ABSTRACT Enterotoxigenic Bacteroides fragilis (ETBF) is a Gram-negative, obligate anaerobe member of the gut microbial community in up to 40% of healthy individuals. This bacterium is found more frequently in people with colorectal cancer (CRC) and causes tumor formation in the distal colon of multiple intestinal neoplasia (Apcmin/+) mice; tumor formation is dependent on ETBF-secreted Bacteroides fragilis toxin (BFT). Because of the extensive data connecting alterations in the epigenome with tumor formation, initial experiments attempting to connect BFT-induced tumor formation with methylation in colon epithelial cells (CECs) have been performed, but the effect of BFT on other epigenetic processes, such as chromatin structure, remains unexplored. Here, the changes in gene expression (transcriptome sequencing [RNA-seq]) and chromatin accessibility (assay for transposase-accessible chromatin using sequencing) induced by treatment of HT29/C1 cells with BFT for 24 and 48 h were examined. Our data show that several genes are differentially expressed after BFT treatment and that these changes relate to the interaction between bacteria and CECs. Further, sites of increased chromatin accessibility are associated with the location of enhancers in CECs and the binding sites of transcription factors in the AP-1/ATF family; they are also enriched for common differentially methylated regions (DMRs) in CRC. These data provide insight into the mechanisms by which BFT induces tumor formation and lay the groundwork for future in vivo studies to explore the impact of BFT on nuclear structure and function.

scholarly journals Aggregation state determines the localization and function of M1– and M23–aquaporin-4 in astrocytes

2014 ◽  
Vol 204 (4) ◽  
pp. 559-573 ◽  
Author(s):  
Alex J. Smith ◽  
Byung-Ju Jin ◽  
Julien Ratelade ◽  
Alan S. Verkman
Keyword(s):  
Plasma Membrane ◽  
Protein Complexes ◽  
Water Channel ◽  
Aquaporin 4 ◽  
Cellular Functions ◽  
Aggregation State ◽  
Functional Roles ◽  
State Dependent ◽  
And Function

The astrocyte water channel aquaporin-4 (AQP4) is expressed as heterotetramers of M1 and M23 isoforms in which the presence of M23–AQP4 promotes formation of large macromolecular aggregates termed orthogonal arrays. Here, we demonstrate that the AQP4 aggregation state determines its subcellular localization and cellular functions. Individually expressed M1–AQP4 was freely mobile in the plasma membrane and could diffuse into rapidly extending lamellipodial regions to support cell migration. In contrast, M23–AQP4 formed large arrays that did not diffuse rapidly enough to enter lamellipodia and instead stably bound adhesion complexes and polarized to astrocyte end-feet in vivo. Co-expressed M1– and M23–AQP4 formed aggregates of variable size that segregated due to diffusional sieving of small, mobile M1–AQP4-enriched arrays into lamellipodia and preferential interaction of large, M23–AQP4-enriched arrays with the extracellular matrix. Our results therefore demonstrate an aggregation state–dependent mechanism for segregation of plasma membrane protein complexes that confers specific functional roles to M1– and M23–AQP4.

scholarly journals Comparative Analysis of Super-enhancers Across Mammals Reveals Their High Function Conservation

2020 ◽  
Author(s):  
Yanling Peng ◽  
Yubo Zhang
Keyword(s):  
Cell Types ◽  
Regulatory Elements ◽  
Biological Processes ◽  
Orthologous Sequence ◽  
Orthologous Genes ◽  
Promoter Regions ◽  
Cell Tissue ◽  
Intergenic Regions ◽  
Regulation Function ◽  
And Function

Abstract BackgroundSuper-enhancers (SEs) are key positive regulatory elements in defining cells/tissues identity in mammals, yet their similarities and differences of sequence and function across mammals have been poor studied. To allow sequence and function comparison across mammalian SEs, we employ H3K27ac ChIP-seq data to six cell types/tissues across human, pig, and mouse, which represent different lineages of mammals in the evolutionary tree.ResultsOverall, a median of 848 human SEs, 888 pig SEs and 503 mouse SEs are identified across cells/tissues. These SEs are largely distributed in promoter regions for human (91.9% in median) and mouse (63.4% in median), while mostly in distal intergenic regions for pig (66.1% in median). Extremely higher unique orthologous SEs frequency (91.6%~92.1%) has been detected for the same cell/tissue across species. Consistently, their overlapping rates are very low for the same cell/tissue across species (0.1%~0.5%). For the SE-associated orthologous genes, they also show high unique frequency for species (63.3%~83.9%) and low overlapping rates (0.8%~1.3%) at inter-species comparison. However, orthologous SEs function comparisons across species have shown similar biological processes related to cells/tissues identity in the top 15 significant enriched terms for the same cell/tissue. Meanwhile, common core transcription factors that determine cells/tissues identity are determined for the same cell/tissue across mammals.ConclusionsThis study highlights the differences of SEs genomic distribution across mammals. It reveals low orthologous sequence overlapping but high function conservation of SEs across mammals. It would improve our understanding of regulation function cis-regulatory elements in mammals.

scholarly journals Regulation of the p53 Family Proteins by the Ubiquitin Proteasomal Pathway

2019 ◽  
Vol 21 (1) ◽  
pp. 261 ◽  
Author(s):  
Scott Bang ◽  
Sandeep Kaur ◽  
Manabu Kurokawa
Keyword(s):  
Genetic Defect ◽  
Tumor Development ◽  
Tumor Suppressor P53 ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
P53 Family ◽  
E3 Ligases ◽  
Damage Response

The tumor suppressor p53 and its homologues, p63 and p73, play a pivotal role in the regulation of the DNA damage response, cellular homeostasis, development, aging, and metabolism. A number of mouse studies have shown that a genetic defect in the p53 family could lead to spontaneous tumor development, embryonic lethality, or severe tissue abnormality, indicating that the activity of the p53 family must be tightly regulated to maintain normal cellular functions. While the p53 family members are regulated at the level of gene expression as well as post-translational modification, they are also controlled at the level of protein stability through the ubiquitin proteasomal pathway. Over the last 20 years, many ubiquitin E3 ligases have been discovered that directly promote protein degradation of p53, p63, and p73 in vitro and in vivo. Here, we provide an overview of such E3 ligases and discuss their roles and functions.

scholarly journals Signaling roles of phosphoinositides in the retina

2020 ◽  
pp. jlr.TR120000806 ◽  
Author(s):  
Raju V. S. Rajala
Keyword(s):  
Cell Types ◽  
Cellular Functions ◽  
Parent Molecule ◽  
Phosphoinositide Signaling ◽  
Wide Range ◽  
The Many ◽  
And Function ◽  
Different Cell Types

The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (PIs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of PI kinases and PI phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule phosphatidylinositol. PI signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane binding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIs in general, in this review, we discuss recent studies and advances in PI lipid signaling in the retina. We specifically focus on PI lipids from vertebrate (e.g. bovine, rat, mice, toad, and zebrafish) and invertebrate (e.g. drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIs revealed from animal models and human diseases, and methods to study PI levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PI-modifying enzymes/phosphatases and further unravel PI regulation and function in the different cell types of the retina.

scholarly journals The nuclear envelope protein Net39 is essential for muscle nuclear integrity and chromatin organization

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Andres Ramirez-Martinez ◽  
Yichi Zhang ◽  
Kenian Chen ◽  
Jiwoong Kim ◽  
Bercin K. Cenik ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nuclear Envelope ◽  
Envelope Protein ◽  
Transmembrane Protein ◽  
Chromatin Accessibility ◽  
Chromatin Organization ◽  
Potential Contribution ◽  
Molecular Etiology ◽  
And Function

AbstractLamins and transmembrane proteins within the nuclear envelope regulate nuclear structure and chromatin organization. Nuclear envelope transmembrane protein 39 (Net39) is a muscle nuclear envelope protein whose functions in vivo have not been explored. We show that mice lacking Net39 succumb to severe myopathy and juvenile lethality, with concomitant disruption in nuclear integrity, chromatin accessibility, gene expression, and metabolism. These abnormalities resemble those of Emery–Dreifuss muscular dystrophy (EDMD), caused by mutations in A-type lamins (LMNA) and other genes, like Emerin (EMD). We observe that Net39 is downregulated in EDMD patients, implicating Net39 in the pathogenesis of this disorder. Our findings highlight the role of Net39 at the nuclear envelope in maintaining muscle chromatin organization, gene expression and function, and its potential contribution to the molecular etiology of EDMD.

scholarly journals Characterizing RNA structures in vitro and in vivo with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

2015 ◽  
Author(s):  
Kyle E Watters ◽  
Angela M Yu ◽  
Eric J Strobel ◽  
Alex H Settle ◽  
Julius Lucks
Keyword(s):  
Primer Extension ◽  
Computational Techniques ◽  
Rna Structures ◽  
Cellular Functions ◽  
Rna Molecules ◽  
Cellular Defense ◽  
Nucleotide Resolution ◽  
And Function

RNA molecules adopt a wide variety of structures that perform many cellular functions, including catalysis, small molecule sensing, and cellular defense, among others. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.

scholarly journals Coordinated genomic control of ciliogenesis and cell movement by RFX2

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Mei-I Chung ◽  
Taejoon Kwon ◽  
Fan Tu ◽  
Eric R Brooks ◽  
Rakhi Gupta ◽  
...  
Keyword(s):  
Gene Expression ◽  
Control Cell ◽  
Cell Movement ◽  
Genomic Analysis ◽  
Apical Surface ◽  
Biological Processes ◽  
Cellular Functions ◽  
Genomic Control ◽  
And Function

The mechanisms linking systems-level programs of gene expression to discrete cell biological processes in vivo remain poorly understood. In this study, we have defined such a program for multi-ciliated epithelial cells (MCCs), a cell type critical for proper development and homeostasis of the airway, brain and reproductive tracts. Starting from genomic analysis of the cilia-associated transcription factor Rfx2, we used bioinformatics and in vivo cell biological approaches to gain insights into the molecular basis of cilia assembly and function. Moreover, we discovered a previously un-recognized role for an Rfx factor in cell movement, finding that Rfx2 cell-autonomously controls apical surface expansion in nascent MCCs. Thus, Rfx2 coordinates multiple, distinct gene expression programs in MCCs, regulating genes that control cell movement, ciliogenesis, and cilia function. As such, the work serves as a paradigm for understanding genomic control of cell biological processes that span from early cell morphogenetic events to terminally differentiated cellular functions.

scholarly journals Autophosphorylation of the CK1 kinase domain regulates enzyme activity and function

2019 ◽  
Author(s):  
Sierra N. Cullati ◽  
Jun-Song Chen ◽  
Kathleen L. Gould
Keyword(s):  
Active Site ◽  
Binding Pocket ◽  
Kinase Domain ◽  
Cellular Functions ◽  
Sleep Phase ◽  
Substrate Phosphorylation ◽  
High Level ◽  
And Function ◽  
Autophosphorylation Site

AbstractCK1 enzymes are conserved, acidophilic serine/threonine kinases with a variety of critical cellular functions; misregulation of CK1 contributes to cancer, neurodegenerative diseases, and sleep phase disorders. Despite this, little is known about how CK1 activity is controlled. Here, we describe a new mechanism of CK1 autoregulation that is conserved in CK1 enzymes from yeast to human – the autophosphorylation of a threonine in the mobile L-EF loop proximal to the active site. Phosphorylation at this site inhibits kinase activity, in contrast to well-characterized T-loop autophosphorylation in other kinase families. Consequently, yeast and human enzymes with phosphoablating mutations at this site are hyperactive. In S. pombe, hyperactive CK1 causes defects in cell growth and morphology at a high level but protection from heat shock at a low level, highlighting the necessity of regulated CK1 function. We propose that phosphorylation on the L-EF loop prevents substrate docking with the kinase domain by shielding the positively charged binding pocket and/or sterically hindering the active site. Due to the strong sequence conservation of this autophosphorylation site and the functional importance of the L-EF loop, which is unique to the CK1 family of kinases, this mechanism is likely to regulate the majority of CK1 enzymes in vivo.Significance StatementKinases in the CK1 family are important signaling enzymes, and they function in multiple pathways within the same cell. Misregulation of CK1 activity contributes to human disease, including cancer, neurodegenerative disease, and sleep phase disorders, yet the mechanisms that control CK1 activity are not well understood. We have identified a conserved autophosphorylation site in the CK1 kinase domain that inhibits substrate phosphorylation. We hypothesize that by using kinase domain autophosphorylation in combination with other regulatory mechanisms, CK1 enzymes can coordinate the phosphorylation of their substrates in different pathways.

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