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

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.

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Dynamic changes in chromatin accessibility, altered adipogenic gene expression, and total versus de novo fatty acid synthesis in subcutaneous adipose stem cells of normal-weight polycystic ovary syndrome (PCOS) women during adipogenesis: evidence of cellular programming

Clinical Epigenetics ◽

10.1186/s13148-020-00970-x ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Karen L. Leung ◽

Smriti Sanchita ◽

Catherine T. Pham ◽

Brett A. Davis ◽

Mariam Okhovat ◽

...

Keyword(s):

Gene Expression ◽

Fatty Acid ◽

De Novo ◽

Polycystic Ovary ◽

Total Content ◽

Chromatin Accessibility ◽

Normal Weight ◽

Triacylglycerol Synthesis ◽

And Function

Abstract Background Normal-weight polycystic ovary syndrome (PCOS) women exhibit adipose resistance in vivo accompanied by enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with accelerated lipid accumulation per cell in vitro. The present study examines chromatin accessibility, RNA expression and fatty acid (FA) synthesis during SC abdominal ASC differentiation into adipocytes in vitro of normal-weight PCOS versus age- and body mass index-matched normoandrogenic ovulatory (control) women to study epigenetic/genetic characteristics as well as functional alterations of PCOS and control ASCs during adipogenesis. Results SC abdominal ASCs from PCOS women versus controls exhibited dynamic chromatin accessibility during adipogenesis, from significantly less chromatin accessibility at day 0 to greater chromatin accessibility by day 12, with enrichment of binding motifs for transcription factors (TFs) of the AP-1 subfamily at days 0, 3, and 12. In PCOS versus control cells, expression of genes governing adipocyte differentiation (PPARγ, CEBPα, AGPAT2) and function (ADIPOQ, FABP4, LPL, PLIN1, SLC2A4) was increased two–sixfold at days 3, 7, and 12, while that involving Wnt signaling (FZD1, SFRP1, and WNT10B) was decreased. Differential gene expression in PCOS cells at these time points involved triacylglycerol synthesis, lipid oxidation, free fatty acid beta-oxidation, and oxidative phosphorylation of the TCA cycle, with TGFB1 as a significant upstream regulator. There was a broad correspondence between increased chromatin accessibility and increased RNA expression of those 12 genes involved in adipocyte differentiation and function, Wnt signaling, as well as genes involved in the triacylglycerol synthesis functional group at day 12 of adipogenesis. Total content and de novo synthesis of myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1), and oleic (C18:1) acid increased from day 7 to day 12 in all cells, with total content and de novo synthesis of FAs significantly greater in PCOS than controls cells at day 12. Conclusions In normal-weight PCOS women, dynamic chromatin remodeling of SC abdominal ASCs during adipogenesis may enhance adipogenic gene expression as a programmed mechanism to promote greater fat storage.

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Nuclear Organization in Stress and Aging

Cells ◽

10.3390/cells8070664 ◽

2019 ◽

Vol 8 (7) ◽

pp. 664 ◽

Cited By ~ 11

Author(s):

Romero-Bueno ◽

de la Cruz Ruiz ◽

Artal-Sanz ◽

Askjaer ◽

Dobrzynska

Keyword(s):

Gene Expression ◽

Nuclear Envelope ◽

Nuclear Organization ◽

3 Dimensional ◽

Cellular Processes ◽

Nuclear Lamins ◽

Nuclear Composition ◽

Histone Modifiers ◽

And Function ◽

Gene Expression Alterations

The eukaryotic nucleus controls most cellular processes. It is isolated from the cytoplasm by the nuclear envelope, which plays a prominent role in the structural organization of the cell, including nucleocytoplasmic communication, chromatin positioning, and gene expression. Alterations in nuclear composition and function are eminently pronounced upon stress and during premature and physiological aging. These alterations are often accompanied by epigenetic changes in histone modifications. We review, here, the role of nuclear envelope proteins and histone modifiers in the 3-dimensional organization of the genome and the implications for gene expression. In particular, we focus on the nuclear lamins and the chromatin-associated protein BAF, which are linked to Hutchinson–Gilford and Nestor–Guillermo progeria syndromes, respectively. We also discuss alterations in nuclear organization and the epigenetic landscapes during normal aging and various stress conditions, ranging from yeast to humans.

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Functions of the nuclear envelope and lamina in development and disease

Biochemical Society Transactions ◽

10.1042/bst0361329 ◽

2008 ◽

Vol 36 (6) ◽

pp. 1329-1334 ◽

Cited By ~ 26

Author(s):

Tatiana V. Cohen ◽

Lidia Hernandez ◽

Colin L. Stewart

Keyword(s):

Transcriptional Regulation ◽

Nuclear Envelope ◽

Chromatin Organization ◽

Signalling Pathways ◽

Mechanical Integrity ◽

Inherited Diseases ◽

And Function

Recent findings that some 24 inherited diseases and anomalies are caused by defects in proteins of the NE (nuclear envelope) and lamina have resulted in a fundamental reassessment of the functions of the NE and underlying lamina. Instead of just regarding the NE and lamina as a molecular filtering device, regulating the transfer of macromolecules between the cytoplasm and nucleus, we now envisage the NE/lamina functioning as a key cellular ‘hub’ in integrating critical functions that include chromatin organization, transcriptional regulation, mechanical integrity of the cell and signalling pathways, as well as acting as a key component in the organization and function of the cytoskeleton.

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P0658MATRIX AND FLOW MODULATE GENE EXPRESSION IN A 3D VASCULARISED TUBULE MODEL

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0658 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Julie Williams ◽

Sanlin Robinson ◽

Babak Alaei ◽

Kimberly Homan ◽

Maryam Clausen ◽

...

Keyword(s):

Gene Expression ◽

Extracellular Matrix ◽

Impact Analysis ◽

Cell Types ◽

Drug Uptake ◽

Shear Stresses ◽

The Matrix ◽

And Function

Abstract Background and Aims Questions abound regarding the translation of in vitro 2D cell culture systems to the human setting. This is especially true of the kidney in which there is a complex hierarchical structure and a multitude of cell types. While it is well accepted that extracellular matrix plays a large part in directing cellular physiology emerging research has highlighted the importance of shear stresses and flow rates too. To fully recapitulate the normal gene expression and function of a particular renal cell type how important is it to completely reconstitute their in vivo surroundings? Method To answer this question, we have cultured proximal tubular (PT) epithelial cells in a 3-dimensional channel embedded within an engineered extracellular matrix (ECM) under physiological flow that is colocalised with an adjacent channel lined with renal microvascular endothelial cells that mimic a peritubular capillary. Modifications to the system were made to allow up to 12 chips to be run in parallel in an easily handleable form. After a period of maturation under continuous flow, both cell types were harvested for RNAseq analyses. RNA expression data was compared with cells cultured under static 2-dimensional conditions on plastic or the engineered ECM. Additionally, the perfusion of glucose through this 3D vascularised PT model has been investigated in the presence and absence of known diabetes modulating agents. Results PCA of RNAseq data showed that a) static non-coated, b) static matrix-coated and c) flow matrix-coated conditions separated into 3 distinct groups, while cell co-culture had less impact. Analysis of transcriptomic signatures showed that many genes were modulated by the matrix with additional genes influenced under flow conditions. Several of these genes, classified as transporters, are of particular importance when using this model to assess drug uptake and safety implications. Co-culture regulated some interesting genes, but fewer than anticipated. Preliminary experiments are underway to monitor glucose uptake and transport between tubules under different conditions. Conclusion We have developed a medium throughput system in which matrix and flow modulate gene expression. This system can be used to study the physiology of molecular cross-talk between cells. Ongoing analysis will further consider relevance to human physiology.

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Chromatin potential identified by shared single cell profiling of RNA and chromatin

10.1101/2020.06.17.156943 ◽

2020 ◽

Cited By ~ 6

Author(s):

Sai Ma ◽

Bing Zhang ◽

Lindsay LaFave ◽

Zachary Chiang ◽

Yan Hu ◽

...

Keyword(s):

Gene Expression ◽

Single Cell ◽

Cell Fate ◽

Mouse Skin ◽

Chromatin Accessibility ◽

Lineage Commitment ◽

Single Cell Profiling ◽

Lineage Priming ◽

And Function ◽

Computational Strategy

SummaryCell differentiation and function are regulated across multiple layers of gene regulation, including the modulation of gene expression by changes in chromatin accessibility. However, differentiation is an asynchronous process precluding a temporal understanding of the regulatory events leading to cell fate commitment. Here, we developed SHARE-seq, a highly scalable approach for measurement of chromatin accessibility and gene expression within the same single cell. Using 34,774 joint profiles from mouse skin, we develop a computational strategy to identify cis-regulatory interactions and define Domains of Regulatory Chromatin (DORCs), which significantly overlap with super-enhancers. We show that during lineage commitment, chromatin accessibility at DORCs precedes gene expression, suggesting changes in chromatin accessibility may prime cells for lineage commitment. We therefore develop a computational strategy (chromatin potential) to quantify chromatin lineage-priming and predict cell fate outcomes. Together, SHARE-seq provides an extensible platform to study regulatory circuitry across diverse cells within tissues.

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Apical restriction of the planar cell polarity component VANGL in pancreatic ducts is required to maintain epithelial integrity

10.1101/778332 ◽

2019 ◽

Author(s):

Lydie Flasse ◽

Siham Yennek ◽

Cédric Cortijo ◽

Irene Seijo Barandiaran ◽

Marine R-C Kraus ◽

...

Keyword(s):

Cell Polarity ◽

Planar Cell Polarity ◽

Transmembrane Protein ◽

Pancreatic Ducts ◽

Apical Surface ◽

Epithelial Integrity ◽

Ductal Cells ◽

And Function ◽

Rock Activity

ABSTRACTCell polarity is essential for the architecture and function of numerous epithelial tissues. Here we show how planar cell polarity (PCP), so far studied principally in flat epithelia, is deployed during the morphogenesis of a tubular organ. Using the mammalian pancreas as a model, we report that components of the core PCP pathway such as the transmembrane protein Van Gogh-like (VANGL), are progressively apically-restricted. VANGL expression becomes asymmetrically localized at the apical surface of ductal cells, revealing a planar polarization of the pancreatic duct. We further show that restricting VANGL to these discrete sites of expression is crucial for epithelial integrity. Expansion of expression on basolateral membranes of the progenitors leads to their death and extrusion from the epithelium, as previously observed for perturbations of apico-basal polarity. Using organoids and in vivo analyses, we show that cell elimination is induced by a decrease of Rock activity via Dishevelled.

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TRAF6 directs Foxp3 localization and facilitates Treg function through K63-type ubiquitination

10.1101/316000 ◽

2018 ◽

Author(s):

Xuhao Ni ◽

Jinhui Tao ◽

Jian Gu ◽

Benjamin V. Park ◽

Zuojia Chen ◽

...

Keyword(s):

Gene Expression ◽

T Cells ◽

Transcription Factor ◽

Transcriptional Regulation ◽

Tumor Immunity ◽

Immune Control ◽

Treg Function ◽

And Function ◽

Expression And Activity

AbstractRegulatory T cells (Treg) are crucial mediators of immune control. The characteristic gene expression and suppressive function of Treg depend considerably on the stable expression and activity of the transcription factor Foxp3. While transcriptional regulation of the Foxp3 gene has been studied in depth, both the expression and function of Foxp3 are also modulated at the protein level. However, the molecular players involved in posttranslational Foxp3 regulation are just beginning to be elucidated. Here we found TRAF6-deficient Tregs were dysfunctional in vivo; mice with Treg-restricted deletion of TRAF6 were resistant to B16 melanomas and displayed enhanced anti-tumor immunity. We further determined that Foxp3 undergoes lysine-63 chain (K63) ubiquitination at lysine 262 mediated by the E3 ligase TRAF6. When deprived of TRAF6 activity or rendered insensitive to K63 ubiquitination, Foxp3 displayed aberrant, perinuclear accumulation, disrupted function. Thus, Foxp3 ubiquitination by TRAF6 ensures proper localization of Foxp3 and facilitates Foxp3’s gene-regulating activity in Tregs. These results implicate TRAF6 as a key posttranslational, Treg-stabilizing force that may be targeted in novel tolerance-breaking therapies.

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Tendon-derived biomimetic surface topographies induce phenotypic maintenance of tenocytes in vitro

10.1101/2020.07.23.217224 ◽

2020 ◽

Author(s):

Aysegul Dede Eren ◽

Aliaksei Vasilevich ◽

E. Deniz Eren ◽

Phanikrishna Sudarsanam ◽

Urandelger Tuvshindorj ◽

...

Keyword(s):

Gene Expression ◽

Cell Shape ◽

Flat Surface ◽

Flat Surfaces ◽

Tissue Culture Polystyrene ◽

Surface Topographies ◽

And Function ◽

Shape Changes

AbstractThe tenocyte niche contains biochemical and biophysical signals that are needed for tendon homeostasis. The tenocyte phenotype is correlated with cell shape in vivo and in vitro, and shape-modifying cues are needed for tenocyte phenotypical maintenance. Indeed, cell shape changes from elongated to spread when cultured on a flat surface, and rat tenocytes lose the expression of phenotypical markers throughout five passages. We hypothesized that tendon gene expression can be preserved by culturing cells in the native tendon shape. To this end, we reproduced the tendon topographical landscape into tissue culture polystyrene, using imprinting technology. We confirmed that the imprints forced the cells into a more elongated shape, which correlated with the level of Scleraxis expression. When we cultured the tenocytes for seven days on flat surfaces and tendon imprints, we observed a decline in tenogenic marker expression on flat but not on imprints. This research demonstrates that native tendon topography is an important factor contributing to the tenocyte phenotype. Tendon imprints therefore provide a powerful platform to explore the effect of instructive cues originating from native tendon topography on guiding cell shape, phenotype and function of tendon-related cells.

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Profiling of chromatin accessibility identifies transcription factor binding sites across the genome of Aspergillus species

BMC Biology ◽

10.1186/s12915-021-01114-0 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Lianggang Huang ◽

Xuejie Li ◽

Liangbo Dong ◽

Bin Wang ◽

Li Pan

Keyword(s):

Gene Expression ◽

Transcription Factor ◽

Filamentous Fungi ◽

Binding Sites ◽

Transcription Factor Binding ◽

Chromatin Accessibility ◽

Culture Conditions ◽

Factor Binding ◽

Tn5 Transposase

Abstract Background The identification of open chromatin regions and transcription factor binding sites (TFBs) is an important step in understanding the regulation of gene expression in diverse species. ATAC-seq is a technique used for such purpose by providing high-resolution measurements of chromatin accessibility revealed through integration of Tn5 transposase. However, the existence of cell walls in filamentous fungi and associated difficulty in purifying nuclei have precluded the routine application of this technique, leading to a lack of experimentally determined and computationally inferred data on the identity of genome-wide cis-regulatory elements (CREs) and TFBs. In this study, we constructed an ATAC-seq platform suitable for filamentous fungi and generated ATAC-seq libraries of Aspergillus niger and Aspergillus oryzae grown under a variety of conditions. Results We applied the ATAC-seq assay for filamentous fungi to delineate the syntenic orthologue and differentially changed chromatin accessibility regions among different Aspergillus species, during different culture conditions, and among specific TF-deleted strains. The syntenic orthologues of accessible regions were responsible for the conservative functions across Aspergillus species, while regions differentially changed between culture conditions and TFs mutants drove differential gene expression programs. Importantly, we suggest criteria to determine TFBs through the analysis of unbalanced cleavage of distinct TF-bound DNA strands by Tn5 transposase. Based on this criterion, we constructed data libraries of the in vivo genomic footprint of A. niger under distinct conditions, and generated a database of novel transcription factor binding motifs through comparison of footprints in TF-deleted strains. Furthermore, we validated the novel TFBs in vivo through an artificial synthetic minimal promoter system. Conclusions We characterized the chromatin accessibility regions of filamentous fungi species, and identified a complete TFBs map by ATAC-seq, which provides valuable data for future analyses of transcriptional regulation in filamentous fungi.

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Epigenetic Changes Induced by Bacteroides fragilis Toxin

Infection and Immunity ◽

10.1128/iai.00447-18 ◽

2019 ◽

Vol 87 (6) ◽

Cited By ~ 12

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.

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