Density imaging of heterochromatin in live cells using orientation-independent-DIC microscopy

In eukaryotic cells, highly condensed inactive/silenced chromatin has long been called “heterochromatin.” However, recent research suggests that such regions are in fact not fully transcriptionally silent and that there exists only a moderate access barrier to heterochromatin. To further investigate this issue, it is critical to elucidate the physical properties of heterochromatin such as its total density in live cells. Here, using orientation-independent differential interference contrast (OI-DIC) microscopy, which is capable of mapping optical path differences, we investigated the density of the total materials in pericentric foci, a representative heterochromatin model, in live mouse NIH3T3 cells. We demonstrated that the total density of heterochromatin (208 mg/ml) was only 1.53-fold higher than that of the surrounding euchromatic regions (136 mg/ml) while the DNA density of heterochromatin was 5.5- to 7.5-fold higher. We observed similar minor differences in density in typical facultative heterochromatin, the inactive human X chromosomes. This surprisingly small difference may be due to that nonnucleosomal materials (proteins/RNAs) (∼120 mg/ml) are dominant in both chromatin regions. Monte Carlo simulation suggested that nonnucleosomal materials contribute to creating a moderate access barrier to heterochromatin, allowing minimal protein access to functional regions. Our OI-DIC imaging offers new insight into the live cellular environments.

Download Full-text

A Physical Organic Approach to Tuning Reagents for Selective and Stable Methionine Bioconjugation

10.26434/chemrxiv.8176823.v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Alec Christian ◽

Shang Jia ◽

Patricia Zhang ◽

Arismel Tena Meza ◽

Matthew S. Sigman ◽

...

Keyword(s):

Structural Features ◽

Data Driven ◽

Live Cells ◽

Design And Synthesis ◽

Use Of Data ◽

Physical Organic ◽

Driving Stability ◽

Peptide Stapling ◽

Insight Into ◽

Unique Chemical

We report a data-driven, physical organic approach to the development of new methionine-selective bioconjugation reagents with tunable adduct stabilities. Statistical modeling of structural features described by intrinsic physical organic parameters was applied to the development of a predictive model and to gain insight into features driving stability of adducts formed from the chemoselective coupling of oxaziridine and methionine thioether partners through Redox Activated Chemical Tagging (ReACT). From these analyses, a correlation between sulfimide stabilities and sulfimide  (C=O) stretching frequencies was revealed. We ex-ploited the rational gains in adduct stability exposed by this analysis to achieve the design and synthesis of a bis-oxaziridine reagent for peptide stapling. Indeed, we observed that a macrocyclic peptide formed by ReACT stapling at methionine exhibited improved uptake into live cells compared to an unstapled congener, highlighting the potential utility of this unique chemical tool for thioether modification. This work provides a template for the broader use of data-driven approaches to bioconjugation chemistry and other chemical biology applications.

Download Full-text

Telomere repeats induce domains of H3K27 methylation in Neurospora

10.1101/189258 ◽

2017 ◽

Author(s):

Kirsty Jamieson ◽

Kevin J. McNaught ◽

Tereza Ormsby ◽

Neena A. Leggett ◽

Shinji Honda ◽

...

Keyword(s):

Genome Organization ◽

Chromosomal Rearrangements ◽

Histone H3 ◽

Facultative Heterochromatin ◽

Large Domain ◽

Heterochromatin Formation ◽

H3k27 Methylation ◽

And Control ◽

The Relationship ◽

Insight Into

ABSTRACTDevelopment in higher organisms requires selective gene silencing, directed in part by di-/tri-methylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG)17, can induce a large domain (∼225 kb) of H3K27me2/3. This provides an example of a cis-acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.

Download Full-text

Studies on Metatherian Sex Chromosomes III. The Use of Tritiated Uridine-induced Chromosome Aberrations to Distinguish Active and Inactive X Chromosomes

Australian Journal of Biological Sciences ◽

10.1071/bi9770103 ◽

1977 ◽

Vol 30 (2) ◽

pp. 103 ◽

Cited By ~ 6

Author(s):

Jennifer A Donald ◽

DW Cooper

Keyword(s):

X Chromosome ◽

Sex Chromosomes ◽

Chromosome Aberrations ◽

X Inactivation ◽

Facultative Heterochromatin ◽

Hybrid Female ◽

X Chromosomes ◽

Toxicity Effect ◽

Red Kangaroo ◽

Chromosome Regions

The paternal X inactivation system of kangaroos has been investigated in this study by using tritiated uridine-induced chromosome aberrations to distinguish the active from the inactive X. Previous work in eutherian mammals has demonstrated that constitutive heterochromatic chromosome regions are less susceptible to breakage by tritiated uri dine than euchromatic regions. The results of a comparison between the paternal X chromosome of a wallaroo x red kangaroo hybrid female and the two X chromosomes of a red kangaroo female suggested that the facultative heterochromatin of the X is also less susceptible to breakage by this treatment. However there were significantly more breaks of the paternal X in fibroblasts than in lymphocytes of the hybrid female, which agrees with biochemical findings suggesting activation of the paternal X in fibroblasts. Our results strengthen the suggestion of other workers that the reduced number of aberrations in heterochromatin occurs because such breaks occur principally when the DNA and labelled RNA are in apposition during transcription. Some evidence was found of an apparent toxicity effect of the tritiated uridine solution on the cells.

Download Full-text

Segmental pycnotic reversion of facultative heterochromatin during the second male meiosis in tettigonioid species (Orthoptera)

Genome ◽

10.1139/g87-095 ◽

1987 ◽

Vol 29 (4) ◽

pp. 570-577 ◽

Cited By ~ 1

Author(s):

J. Fernández Piqueras ◽

C. Sentís

Keyword(s):

X Chromosome ◽

Male Meiosis ◽

Silver Impregnation ◽

Telomeric Region ◽

Impregnation Method ◽

Facultative Heterochromatin ◽

X Chromosomes ◽

Chromosomal Proteins ◽

Tandem Fusion ◽

Facultative Heterochromatinization

The X chromosome invariably exhibits isopycnosis during the most condensed stages of male meiosis after conventional staining procedures in all studied species of Tettigonioidea (Orthoptera). However, differential negative staining of the X chromosome can be achieved during these stages by a silver impregnation method after extraction of chromosomal proteins with 2 × SSC. This differential response to silver may be indicative of a distinctive chromatin organization in the X chromosome with respect to that of the autosomes. Following 2 × SSC – silver impregnation treatment, pycnotic reversion of part of the X chromosome can be observed from the metaphase II stage on in tettigonioids. Pycnotic reversion is initiated from the telomeric region of the X chromosome in the subfamily Ephyppigerinae, while in the subfamily Pycnogastrinae pycnotic reversion occurs at the centromeric region of this chromosome. These two modes of segmental pycnotic reversion may indicate the onset of a reversion of the facultative heterochromatinized state of the X chromosome (euchromatinization) during male meiosis in tettigonioids. An initiation center may be postulated for this process that is located either at the end distal to or proximal to the centromere of the X chromosome. This hypothesis is supported by the observed pycnotic behaviour of neo-X chromosomes in two different neo-XY systems, one involving centric and the other tandem fusion. Facultative heterochromatinization never involves the autosomally derived segments (XR) of the neo-X chromosomes. Occasional failures in the heterochromatinization process of part of the X chromosome, as well as of the neo-X chromosome, in two species suggests that both facultative heterochromatinization and its reversion are two processes that act segmentally. Key words: male meiosis, X chromosome, facultative heterochromatin, segmental euchromatinization.

Download Full-text

Direct quantification of ligand-induced lipid and protein microdomains with distinctive signaling properties

10.1101/2021.09.28.462247 ◽

2021 ◽

Author(s):

Daniel Wirth ◽

Michael Paul ◽

Elena B Pasquale ◽

Kalina Hristova

Keyword(s):

Plasma Membrane ◽

Lipid Rafts ◽

Receptor Tyrosine Kinase ◽

Mammalian Cells ◽

Dissociation Constants ◽

Live Cells ◽

Downstream Effector ◽

Plasma Membrane Heterogeneity ◽

Signaling Components ◽

Insight Into

Lipid rafts are known as highly ordered lipid domains that are enriched in saturated lipids such as the ganglioside GM1. While lipid rafts are believed to exist in cells and to serve as signaling platforms through their enrichment in signaling components, they have never been directly observed in the plasma membrane without treatments that artificially cluster GM1 into large lattices. Here we report that microscopic GM1-enriched domains can form, without lipid crosslinking, in the plasma membrane of live mammalian cells expressing the EphA2 receptor tyrosine kinase in response to its ligand ephrinA1-Fc. The GM1-enriched microdomains form concomitantly with EphA2-enriched microdomains, but only partially co-localize with them. To gain insight into how plasma membrane heterogeneity controls signaling, we quantify the degree of EphA2 segregation and study initial EphA2 signaling steps in both EphA2-enriched and EphA2-depleted domains. By measuring dissociation constants, we demonstrate that EphA2 oligomerization is the same in EphA2-enriched and -depleted domains. However, EphA2 interacts preferentially with its downstream effector SRC in EphA2-depleted domains. The ability to induce microscopic GM1-enriched domains in live cells using a ligand for a transmembrane receptor will give us unprecedented opportunities to study the biology of lipid rafts.

Download Full-text

Late Replication of the Inactive X Chromosome Is Independent of the Compactness of Chromosome Territory in Human Pluripotent Stem Cells

Acta Naturae ◽

10.32607/20758251-2013-5-2-54-61 ◽

2013 ◽

Vol 5 (2) ◽

pp. 54-61

Author(s):

A. V. Panova ◽

E. D. Nekrasov ◽

M. A. Lagarkova ◽

S. L. Kiselev ◽

A. N. Bogomazova

Keyword(s):

Stem Cells ◽

X Chromosome ◽

Pluripotent Stem Cells ◽

Replication Timing ◽

Chromosome Territory ◽

Late Replication ◽

Facultative Heterochromatin ◽

X Chromosomes ◽

Inactive X Chromosome ◽

The Status

Dosage compensation of the X chromosomes in mammals is performed via the formation of facultative heterochromatin on extra X chromosomes in female somatic cells. Facultative heterochromatin of the inactivated X (Xi), as well as constitutive heterochromatin, replicates late during the S-phase. It is generally accepted that Xi is always more compact in the interphase nucleus. The dense chromosomal folding has been proposed to define the late replication of Xi. In contrast to mouse pluripotent stem cells (PSCs), the status of X chromosome inactivation in human PSCs may vary significantly. Fluorescence in situ hybridization with a whole X-chromosome-specific DNA probe revealed that late-replicating Xi may occupy either compact or dispersed territory in human PSCs. Thus, the late replication of the Xi does not depend on the compactness of chromosome territory in human PSCs. However, the Xi reactivation and the synchronization in the replication timing of X chromosomes upon reprogramming are necessarily accompanied by the expansion of X chromosome territory.

Download Full-text

Monoubiquitylation of H2A.Z Distinguishes Its Association with Euchromatin or Facultative Heterochromatin

Molecular and Cellular Biology ◽

10.1128/mcb.00241-07 ◽

2007 ◽

Vol 27 (18) ◽

pp. 6457-6468 ◽

Cited By ~ 111

Author(s):

Elizabeth Sarcinella ◽

Philip C. Zuzarte ◽

Priscilla N. I. Lau ◽

Ryan Draker ◽

Peter Cheung

Keyword(s):

Mammalian Cells ◽

Regulation Of Gene Expression ◽

Histone H2a ◽

Facultative Heterochromatin ◽

X Chromosomes ◽

Genome Wide ◽

New Findings ◽

Mouse Cells ◽

Heterochromatin Silencing

ABSTRACT H2A.Z is a histone H2A variant that is essential for viability in organisms such as Tetrahymena thermophila, Drosophila melanogaster, and mice. In Saccharomyces cerevisiae, loss of H2A.Z is tolerated, but proper regulation of gene expression is affected. Genetics and genome-wide localization studies show that yeast H2A.Z physically localizes to the promoters of genes and functions in part to protect active genes in euchromatin from being silenced by heterochromatin spreading. To date, the function of H2A.Z in mammalian cells is less clear, and evidence so far suggests that it has a role in chromatin compaction and heterochromatin silencing. In this study, we found that the bulk of H2A.Z is excluded from constitutive heterochromatin in differentiated human and mouse cells. Consistent with this observation, analyses of H2A.Z- or H2A-containing mononucleosomes show that the H3 associated with H2A.Z has lower levels of K9 methylation but higher levels of K4 methylation than those associated with H2A. We also found that a fraction of mammalian H2A.Z is monoubiquitylated and that, on the inactive X chromosomes of female cells, the majority of this histone variant is modified by ubiquitin. Finally, ubiquitylation of H2A.Z is mediated by the RING1b E3 ligase of the human polycomb complex, further supporting a silencing role of ubiquitylated H2A.Z. These new findings suggest that mammalian H2A.Z is associated with both euchromatin and facultative heterochromatin and that monoubiquitylation is a specific mark that distinguishes the H2A.Z associated with these different chromatin states.

Download Full-text

CPVT-associated calmodulin variants N53I and A102V dysregulate calcium signalling via different mechanisms

Journal of Cell Science ◽

10.1242/jcs.258796 ◽

2021 ◽

Author(s):

Ohm Prakash ◽

Marie Held ◽

Liam F. McCormick ◽

Nitika Gupta ◽

Lu-Yun Lian ◽

...

Keyword(s):

Conformational Changes ◽

Molecular Mechanisms ◽

Structural Characteristics ◽

Calcium Signalling ◽

Hek293 Cells ◽

Polymorphic Ventricular Tachycardia ◽

Live Cells ◽

Key Driver ◽

Fatal Cardiac Arrhythmia ◽

Insight Into

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited condition that can cause fatal cardiac arrhythmia. Human mutations in the Ca2+ sensor calmodulin (CaM) have been associated with CPVT susceptibility, suggesting that CaM dysfunction is a key driver of the disease. However, the detailed molecular mechanism remains unclear. Focusing on the interaction with the cardiac ryanodine receptor (RyR2), we determined the effect of CPVT-associated variants N53I and A102V on the structural characteristics of CaM and on Ca2+ fluxes in live cells. We provide novel data showing that binding of both Ca2+/CaM-N53I and Ca2+/CaM-A102V to RyR23583-3603 is decreased. Ca2+/CaM:RyR23583-3603 high-resolution crystal structures highlight subtle conformational changes for the N53I variant, with A102V being similar to wild-type. We show that co-expression of CaM-N53I or CaM-A102V with RyR2 in HEK293 cells significantly increased the duration of Ca2+ events, CaM-A102V exhibited a lower frequency of Ca2+ oscillations. In addition, we show that CaMKIIδ phosphorylation activity is increased for A102V, compared to CaM-WT. This paper provides novel insight into the molecular mechanisms of CPVT-associated CaM variants and will facilitate development of strategies for future therapies.

Download Full-text

A Note on Computing Interval Overlap Statistics

10.1101/517987 ◽

2019 ◽

Author(s):

Shahab Sarmashghi ◽

Vineet Bafna

Keyword(s):

Approximate Method ◽

Finite Length ◽

Line Segment ◽

Null Hypothesis ◽

Statistical Significance ◽

Interval Data ◽

Combinatorial Algorithm ◽

Link Type ◽

Functional Regions ◽

Insight Into

AbstractWe consider the following problem: Let I and If each describe a collection of n and m non-overlapping intervals on a line segment of finite length. Suppose that k of the m intervals of If are intersected by some interval(s) in I. Under the null hypothesis that intervals in I are randomly arranged w.r.t If, what is the significance of this overlap? This is a natural abstraction of statistical questions that are ubiquitous in the post-genomic era. The interval collections represent annotations that reveal structural or functional regions of the genome, and overlap statistics can provide insight into the correlation between different structural and functional regions. However, the statistics of interval overlaps have not been systematically explored. In this manuscript, we formulate a statistical significance problem which considers the length and structure of intervals. We describe a combinatorial algorithm for a constrained interval overlap problem that can accurately compute very small p-values. We also propose a fast approximate method to facilitate problems consisted of very large number of intervals. These methods are all implemented in a tool, iStat. We applied iStat to simulated interval data to obtain precise estimates of low p-values, and characterize the performance of our methods. We also test iStat on real datasets from previous studies, and compare iStat results with the reported p-values using basic permutation or parametric tests. The iStat software is made publicly available on https://github.com/shahab-sarmashghi/ISTAT.git

Download Full-text

Telomere repeats induce domains of H3K27 methylation in Neurospora

eLife ◽

10.7554/elife.31216 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 14

Author(s):

Kirsty Jamieson ◽

Kevin J McNaught ◽

Tereza Ormsby ◽

Neena A Leggett ◽

Shinji Honda ◽

...

Keyword(s):

Genome Organization ◽

Chromosomal Rearrangements ◽

Histone H3 ◽

Facultative Heterochromatin ◽

Heterochromatin Formation ◽

Cis Acting ◽

H3k27 Methylation ◽

And Control ◽

The Relationship ◽

Insight Into

Development in higher organisms requires selective gene silencing, directed in part by di-/trimethylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG)17, can induce a large domain (~225 kb) of H3K27me2/3. This provides an example of a cis-acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.

Download Full-text