Chromosome Compartments on the Inactive X Guide TAD Formation Independently of Transcription during X-Reactivation

AbstractA hallmark of chromosome organization is the partition into transcriptionally active A and repressed B compartments, and into topologically associating domains (TADs). Both structures were regarded to be absent from the inactive mouse X chromosome, but to be re-established with transcriptional reactivation and chromatin opening during X-reactivation. Here, we combine a tailor-made mouse iPSC reprogramming system and high-resolution Hi-C to produce a time course combining gene reactivation, chromatin opening and chromosome topology during X-reactivation. Contrary to previous observations, we observe A/B-like compartments on the inactive X harbouring multiple subcompartments. While partial X-reactivation initiates within a compartment rich in X-inactivation escapees, it then occurs rapidly along the chromosome, concomitant with downregulation of Xist. Importantly, we find that TAD formation precedes transcription and initiates from Xist-poor compartments. Here, we show that TAD formation and transcriptional reactivation are causally independent during X-reactivation while establishing Xist as a common denominator.

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Hemimethylation and hypersensitivity are early events in transcriptional reactivation of human inactive X-linked genes in a hamster x human somatic cell hybrid.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3819 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3819-3826 ◽

Cited By ~ 24

Author(s):

T Sasaki ◽

R S Hansen ◽

S M Gartler

Keyword(s):

Somatic Cell ◽

X Chromosome ◽

Cell Hybrid ◽

Somatic Cell Hybrid ◽

Time Course ◽

Cpg Island ◽

Hprt Gene ◽

Inactive X Chromosome ◽

Growing Cell ◽

Transcriptional Reactivation

Reactivation of the hypoxanthine phosphoribosyltransferase (HPRT) gene on an inactive human X chromosome in a somatic cell hybrid was analyzed following exposure to 5-aza-2'-deoxycytidine. Hemimethylation and chromatin hypersensitivity in the 5' CpG island appeared by 6 h after exposure and continued to increase for 24 h in an exponentially growing cell culture. These results imply that the conformation of inactive chromatin requires a symmetrically methylated 5' G+C-rich promoter region. In addition, quantitative analysis of the time course patterns suggest that chromatin sensitivity changes may depend on strand-specific demethylation. Symmetrically demethylated DNA was first detected at 24 h and continued to increase until 48 h. HPRT mRNA was first detected at 24 h and increased in a biphasic pattern until 48 h. These results suggest that hemimethylation permits nuclease attack but not transcription factor binding, which requires symmetrically demethylated DNA. We also show that in G1-arrested cells, 5-aza-2'-deoxycytidine has no effect on methylation, chromatin conformation, or transcription. We conclude that reactivation of the HPRT gene present on the inactive X chromosome of a somatic cell hybrid involves the initial events of DNA hemimethylation and chromatin hypersensitivity at the 5' CpG island, followed by symmetrical demethylation and transcriptional reactivation.

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Hemimethylation and hypersensitivity are early events in transcriptional reactivation of human inactive X-linked genes in a hamster x human somatic cell hybrid

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3819-3826.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3819-3826

Author(s):

T Sasaki ◽

R S Hansen ◽

S M Gartler

Keyword(s):

Somatic Cell ◽

X Chromosome ◽

Cell Hybrid ◽

Somatic Cell Hybrid ◽

Time Course ◽

Cpg Island ◽

Hprt Gene ◽

Inactive X Chromosome ◽

Growing Cell ◽

Transcriptional Reactivation

Reactivation of the hypoxanthine phosphoribosyltransferase (HPRT) gene on an inactive human X chromosome in a somatic cell hybrid was analyzed following exposure to 5-aza-2'-deoxycytidine. Hemimethylation and chromatin hypersensitivity in the 5' CpG island appeared by 6 h after exposure and continued to increase for 24 h in an exponentially growing cell culture. These results imply that the conformation of inactive chromatin requires a symmetrically methylated 5' G+C-rich promoter region. In addition, quantitative analysis of the time course patterns suggest that chromatin sensitivity changes may depend on strand-specific demethylation. Symmetrically demethylated DNA was first detected at 24 h and continued to increase until 48 h. HPRT mRNA was first detected at 24 h and increased in a biphasic pattern until 48 h. These results suggest that hemimethylation permits nuclease attack but not transcription factor binding, which requires symmetrically demethylated DNA. We also show that in G1-arrested cells, 5-aza-2'-deoxycytidine has no effect on methylation, chromatin conformation, or transcription. We conclude that reactivation of the HPRT gene present on the inactive X chromosome of a somatic cell hybrid involves the initial events of DNA hemimethylation and chromatin hypersensitivity at the 5' CpG island, followed by symmetrical demethylation and transcriptional reactivation.

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A Three-Pool Model Dissecting Readily Releasable Pool Replenishment at the Calyx of Held

Scientific Reports ◽

10.1038/srep09517 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 8

Author(s):

Jun Guo ◽

Jian-long Ge ◽

Mei Hao ◽

Zhi-cheng Sun ◽

Xin-sheng Wu ◽

...

Keyword(s):

Time Course ◽

Current View ◽

Vesicle Recycling ◽

Releasable Pool ◽

Readily Releasable Pool ◽

Central Synapse ◽

Pool Model ◽

Underlying Mechanisms ◽

First Time ◽

Intense Stimulation

Abstract Although vesicle replenishment is critical in maintaining exo-endocytosis recycling, the underlying mechanisms are not well understood. Previous studies have shown that both rapid and slow endocytosis recycle into a very large recycling pool instead of within the readily releasable pool (RRP) and the time course of RRP replenishment is slowed down by more intense stimulation. This finding contradicts the calcium/calmodulin-dependence of RRP replenishment. Here we address this issue and report a three-pool model for RRP replenishment at a central synapse. Both rapid and slow endocytosis provide vesicles to a large reserve pool (RP) ~42.3 times the RRP size. When moving from the RP to the RRP, vesicles entered an intermediate pool (IP) ~2.7 times the RRP size with slow RP-IP kinetics and fast IP-RRP kinetics, which was responsible for the well-established slow and rapid components of RRP replenishment. Depletion of the IP caused the slower RRP replenishment observed after intense stimulation. These results establish, for the first time, a realistic cycling model with all parameters measured, revealing the contribution of each cycling step in synaptic transmission. The results call for modification of the current view of the vesicle recycling steps and their roles.

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Load Signals Assist the Generation of Movement-Dependent Reflex Reversal in the Femur–Tibia Joint of Stick Insects

Journal of Neurophysiology ◽

10.1152/jn.00625.2006 ◽

2006 ◽

Vol 96 (6) ◽

pp. 3532-3537 ◽

Cited By ~ 25

Author(s):

Turgay Akay ◽

Ansgar Büschges

Keyword(s):

Time Course ◽

Stick Insect ◽

Motor Output ◽

Chordotonal Organ ◽

Frequency Of Occurrence ◽

Stick Insects ◽

Extensor Motoneuron ◽

Motoneuron Activity ◽

First Time ◽

Stimulation Of

Reinforcement of movement is an important mechanism by which sensory feedback contributes to motor control for walking. We investigate how sensory signals from movement and load sensors interact in controlling the motor output of the stick insect femur–tibia (FT) joint. In stick insects, flexion signals from the femoral chordotonal organ (fCO) at the FT joint and load signals from the femoral campaniform sensilla (fCS) are known to individually reinforce stance-phase motor output of the FT joint by promoting flexor and inhibiting extensor motoneuron activity. We quantitatively compared the time course of inactivation in extensor tibiae motoneurons in response to selective stimulation of fCS and fCO. Stimulation of either sensor generates extensor activity in a qualitatively similar manner but with a significantly different time course and frequency of occurrence. Inactivation of extensor motoneurons arising from fCS stimulation was more reliable but more than threefold slower compared with the extensor inactivation in response to flexion signals from the fCO. In contrast, simultaneous stimulation of both sense organs produced inactivation in motoneurons with a time course typical for fCO stimulation alone, but with a frequency of occurrence characteristic for fCS stimulation. This increase in probability of occurrence was also accompanied by a delayed reactivation of the extensor motoneurons. Our results indicate for the first time that load signals from the leg affect the processing of movement-related feedback in controlling motor output.

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Induction of the lateral flagellar system of Vibrio shilonii is an early event after inhibition of the sodium ion flux in the polar flagellum

Canadian Journal of Microbiology ◽

10.1139/cjm-2014-0579 ◽

2015 ◽

Vol 61 (3) ◽

pp. 183-191 ◽

Cited By ~ 2

Author(s):

Yael González ◽

Laura Camarena ◽

Georges Dreyfus

Keyword(s):

Sodium Channel ◽

Time Course ◽

Marine Bacterium ◽

Channel Blocker ◽

Morphological Characteristics ◽

Early Event ◽

Basal Bodies ◽

Sodium Channel Blocker ◽

Rt Pcr ◽

First Time

In this study, we show the induction of lateral flagella by the action of the sodium channel blocker phenamil, in the marine bacterium Vibrio shilonii, a coral pathogen that causes bleaching. We analyzed the growth and morphology of cells treated with phenamil. A time course analysis showed that after 30 min of exposure to the sodium channel blocker, lateral flagella were present and could be detected by electron microscopy. Detection of the mRNA of the master regulator (lafK) and lateral flagellin (lafA) by RT–PCR confirmed the expression of lateral flagellar genes. We show the simultaneous isolation of polar and, for the first time, lateral flagellar hook-basal bodies. This allowed us to compare the dimensions and morphological characteristics of the 2 structures.

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PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

ISRN Oncology ◽

10.1155/2013/329063 ◽

2013 ◽

Vol 2013 ◽

pp. 1-9

Author(s):

Tizhi Su ◽

Samuel Straight ◽

Liwei Bao ◽

Xiujie Xie ◽

Caryn L. Lehner ◽

...

Keyword(s):

Protein Kinase C ◽

Cell Membrane ◽

Cell Invasion ◽

Time Course ◽

Time Lapse ◽

Kinase C ◽

Phosphorylation Event ◽

Phosphopeptide Mapping ◽

First Time ◽

Initial Peak

Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA.

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Flow cytometric assessment of distinct physiological stages withinCryptosporidium parvumsporozoites post-excystation

Parasitology ◽

10.1017/s0031182009006519 ◽

2009 ◽

Vol 136 (9) ◽

pp. 953-966 ◽

Cited By ~ 7

Author(s):

B. J. KING ◽

D. HOEFEL ◽

S. P. LIM ◽

B. S. ROBINSON ◽

P. T. MONIS

Keyword(s):

Flow Cytometry ◽

Time Course ◽

Fluorescent Dyes ◽

Cell Attachment ◽

Gastrointestinal Disease ◽

Plasma Membrane Permeability ◽

Flow Cytometric ◽

Vesicular Membrane ◽

Dye Staining ◽

First Time

SUMMARYCryptosporidium parvumare protozoan parasites responsible for outbreaks of gastrointestinal disease worldwide. Within the apical complex of this organism reside numerous vesicular secretory organelles and their discharge has been identified as essential for sporozoite motility, cell attachment and penetration. Traditionally, investigation of apical organelle discharge has relied on microscopic and immunochemical hybridization techniques. In this study we demonstrate for the first time how flow cytometry, in combination with vital dye staining, provides an avenue for discrimination of distinct physiological events occurring withinCryptosporidiumsporozoites post-excystation. Time-course studies of freshly excysted sporozoites were carried out at 37°C in cell-free medium, stained with the fluorescent dyes SYTO9/PI, DiBAC4(3), Fluo-4 AM or FM1-43 and analysed by flow cytometry. Significant decreases in sporozoite plasma membrane permeability and increased membrane depolarization were found to be accompanied by concomitant increases in intracellular calcium. Subsequent to these changes, large increases in exocytosed vesicular membrane were apparent. In addition, by measuring side and forward angle light scatter we were able to assess changes in internal granularity and size of sporozoites post-excystation. These observations were suggestive of rapid mobilization, utilization and discharge of apical organelles within sporozoites, which we relate to changes in sporozoite infectivity, ATP levels and total secreted soluble protein.

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Inflammatory ascites formation induced by macromolecules in mice and rats

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00086.2009 ◽

2009 ◽

Vol 297 (1) ◽

pp. R218-R223 ◽

Cited By ~ 3

Author(s):

Károly Baintner

Keyword(s):

Cell Surface ◽

Ascitic Fluid ◽

Time Course ◽

Transitional Period ◽

Cross Linking ◽

Protein Accumulation ◽

Common Denominator ◽

Fibrinogen Concentration ◽

Peritoneal Permeability ◽

Time Courses

Different macromolecules were administered intraperitoneally to stimulate formation of protein-rich ascitic fluid in rodents. Stimulatory effect of plant lectins depended on the attachment to cell surface carbohydrates, Canavalia ensiformis (ConA) lectin was used in the majority of experiments. The time course of ConA-induced ascites was divided into an early (up to 4 h) and a late (from 6 h on) phase, with a transitional period between the two. Water and protein accumulation showed parallel time courses: volume of the ascitic fluid peaked at around 3 h, and fibrin threads appeared after 6 h. Viscosity of the ascitic fluid and its supernatant increased with time, reaching maximal fibrinogen concentration at around 16 h. Peritoneal permeability, followed by pleural and pericardial effusions, was elicited only by lectins that form soluble complexes with serum glycoproteins, whereas the effect of serum-precipitating lectins was restricted to the peritoneum. Macromolecules with serial positive charges (e.g., polylysine or polyethyleneimine) enhanced peritoneal permeability by ionic interactions with cell surface molecules. Viscosity of the polycation-induced ascitic fluid did not tend to increase with time and corresponded to the early phase of the ConA-induced ascites. Polyglutamate, a polyanionic macromolecule, inhibited the effect of polycations, but not that of ConA. The most efficient stimulatory macromolecules appear to induce ascites by noncovalent cross-linking of cell surface glycoproteins or glycosaminoglycans or both. A similar mechanism may operate in the maintenance of basal secretion to prevent eventual desiccation. Noncovalent cross-linking appears to be a common denominator of both basal and enhanced permeability.

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Disturbance During Biofilm Community Succession Promotes Cooperation and Diversity

10.1101/352914 ◽

2018 ◽

Cited By ~ 1

Author(s):

James P. Stratford ◽

Douglas M. Hodgson ◽

Nelli J. Beecroft ◽

Ann Smith ◽

Julian R. Marchesi ◽

...

Keyword(s):

Time Course ◽

Explanatory Power ◽

16S Rrna Genes ◽

Rrna Genes ◽

Widespread Application ◽

Operational Taxonomic Units ◽

Microbial Biofilms ◽

Macro Scale ◽

First Time ◽

High Diversity

AbstractDiversity-disturbance relationships have found widespread application in ecology, conservation and biodiversity management. In spite of their explanatory power, these conceptual frameworks have yet to be systematically applied to understanding succession in diverse microbial biofilms. Here we investigate community assembly in biofilms formed in replicate microbial bioelectrochemical systems using time-course sequencing of community 16S rRNA genes, corresponding to hundreds of operational taxonomic units (OTUs). For the first time we present a statistical model showing that a simple diversity-disturbance relationship can be used to explain dynamic changes in high diversity biofilm communities. This simple model reveals that succession in these systems is guided towards either a low diversity, generalist-dominated biofilm or a high diversity, cooperative-specialist biofilm, depending on the level of endogenous disturbance measured within the community. The pattern observed shows remarkable symmetry with findings from macro-scale communities such as grasslands, forests and coral reefs.

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