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William C Earnshaw - One of the best experts on this subject based on the ideXlab platform.
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cad dff40 nuclease is dispensable for high molecular weight dna cleavage and stage i Chromatin Condensation in apoptosis
Journal of Biological Chemistry, 2001Co-Authors: Kumiko Samejima, Shigenobu Tone, William C EarnshawAbstract:DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear Chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic Chromatin Condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of Chromatin Condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic Chromatin Condensation.
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CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I Chromatin Condensation in apoptosis.
The Journal of biological chemistry, 2001Co-Authors: K Samejima, Shigenobu Tone, William C EarnshawAbstract:DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear Chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic Chromatin Condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of Chromatin Condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic Chromatin Condensation.
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DNA topoisomerase IIα interacts with CAD nuclease and is involved in Chromatin Condensation during apoptotic execution
Current biology : CB, 2000Co-Authors: F. Durrieu, Kumiko Samejima, John M. Fortune, Stefanie Kandels-lewis, Neil Osheroff, William C EarnshawAbstract:Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution [2-4] and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 [4,7,8] (CAD) can induce apoptotic Chromatin Condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors [6,8]. As Chromatin Condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIalpha (Topo IIalpha), which is essential for both Condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIalpha and caspases completely abolishes apoptotic Chromatin Condensation. In addition, we show that CAD binds to Topo IIalpha, and that their association enhances the decatenation activity of Topo IIalpha in vitro.
Harvey F. Lodish - One of the best experts on this subject based on the ideXlab platform.
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Exportin 7 (RanBP16) Plays An Essential Role in Terminal Erythroid Chromatin Condensation and Enucleation
Blood, 2011Co-Authors: Shilpa M. Hattangadi, Karly Burke, Jennifer C. Eng, Jeffrey D. Cooney, Junxia Wang, Maki Murata-hori, Barry H. Paw, Harvey F. LodishAbstract:Abstract 178 In contrast to compacting Chromatin into highly condensed mitotic chromosomes, the quite distinct process of global Chromatin Condensation culminating in enucleation that occurs during terminal erythroid development is still poorly understood. By examining the protein composition of the erythroid nucleus from early erythroblast to ultimate extrusion, I observed that extruded nuclei are largely depleted of all nuclear proteins. Given my previous observations that the highly-regulated but reportedly nonspecific nuclear export protein, Xpo7 or RanBP16, is highly induced during erythropoiesis and highly erythroid-specific, I hypothesized that its role may be to remove almost all nuclear proteins in order to allow the terminal erythroid Chromatin to condense. Knockdown of Xpo7 using shRNA in primary fetal liver erythroid progenitors resulted in severe inhibition of Chromatin Condensation and enucleation but had little effect on hemoglobin accumulation or erythroid cell surface marker expression. As expected based on my hypothesis, proteomic analysis of nuclei from Xpo7-knockdown cells revealed largely all nuclear proteins, some of which may be responsible for the process of histone redistribution during Chromatin Condensation. Xpo7 is also highly regulated: besides its promoter being bound directly by the erythroid master regulators GATA1 and Klf1 (unpublished data), it is also the target of a miRNA whose level decreases during erythropoiesis, miR-181, and whose overexpression has been shown to result in the inhibition of terminal enucleation. Because Chromatin Condensation occurs in lower vertebrates without subsequent enucleation, I have also explored the localization and function of Xpo7 in zebrafish using in situ hybridization and morpholinos, respectively, and found that this export function is specific to mammalian Chromatin Condensation, providing evidence that Condensation and enucleation are inextricably linked processes in mammals. Disclosures: No relevant conflicts of interest to declare.
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formation of mammalian erythrocytes Chromatin Condensation and enucleation
Trends in Cell Biology, 2011Co-Authors: Maki Muratahori, Harvey F. LodishAbstract:In all vertebrates, the cell nucleus becomes highly condensed and transcriptionally inactive during the final stages of red cell biogenesis. Enucleation, the process by which the nucleus is extruded by budding off from the erythroblast, is unique to mammals. Enucleation has critical physiological and evolutionary significance in that it allows an elevation of hemoglobin levels in the blood and also gives red cells their flexible biconcave shape. Recent experiments reveal that enucleation involves multiple molecular and cellular pathways that include histone deacetylation, actin polymerization, cytokinesis, cell–matrix interactions, specific microRNAs and vesicle trafficking; many evolutionarily conserved proteins and genes have been recruited to participate in this uniquely mammalian process. In this review, we discuss recent advances in mammalian erythroblast Chromatin Condensation and enucleation, and conclude with our perspectives on future studies.
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Histone deacetylase 2 is required for Chromatin Condensation and subsequent enucleation of cultured mouse fetal erythroblasts
Haematologica, 2010Co-Authors: Victor Yeh, Maki Murata-hori, Tzutzuy Ramirez, Harvey F. LodishAbstract:Background During the final stages of differentiation of mammalian erythroid cells, the Chromatin is condensed and enucleated. We previously reported that Rac GTPases and their downstream target, mammalian homolog of Drosophila diaphanous 2 (mDia2), are required for enucleation of in vitro cultured mouse fetal liver erythroblasts. However, it is not clear how Chromatin Condensation is achieved and whether it is required for enucleation. Design and Methods Mouse fetal liver erythroblasts were purified from embryonic day 14.5 pregnant mice and cultured in erythropoietin-containing medium. Enucleation was determined by flow-cytometry based analysis after treatment with histone deacetylase inhibitors or infection with lentiviral short harirpin RNA. Results We showed that histone deacetylases play critical roles in Chromatin Condensation and enucleation in cultured mouse fetal liver erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocked Chromatin Condensation, contractile actin ring formation and enucleation. We further demonstrated that histone deacetylases 1, 2, 3 and 5 are highly expressed in mouse fetal erythroblasts. Short hairpin RNA down-regulation of histone deacetylase 2, but not of the other histone deacetylases, phenotypically mimicked the effect of trichostatin A or valproic acid treatment, causing significant inhibition of Chromatin Condensation and enucleation. Importantly, knock-down of histone deacetylase 2 did not affect erythroblast proliferation, differentiation, or apoptosis. Conclusions These results identify histone deacetylase 2 as an important regulator, mediating Chromatin Condensation and enucleation in the final stages of mammalian erythropoiesis.
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Histone Deacetylases Are Essential for Chromatin Condensation and Enucleation in Mammalian Erythroblasts
Blood, 2008Co-Authors: Francisco J. Sánchez-rivera, Harvey F. LodishAbstract:In the last steps of differentiation mammalian erythroid cells undergo Chromatin Condensation and enucleation; the latter process does not occur in other vertebrates. Enucleation was critical for the evolution of mammals, as it permits an enhanced hemoglobin concentration – and thus oxygen- carrying capacity – in mammalian red blood cells. We previously reported that Rac GTPases and their downstream forming target mDia2 are required for mouse fetal erythroblast enucleation. We also found that the nucleus undergoes a gradual ~10- fold decrease in volume during erythropoiesis. Since histone deacetylases (HDACs) play important roles in Chromatin Condensation, we hypothesized that HDACs are involved in mammalian erythroblast enucleation. To test this hypothesis, we purified E13.5 mouse fetal TER119 negative erythroblasts and cultured them in fibronectin-coated plates. Cells were then treated with Trichostatin A (TSA), a pan-HDAC inhibitor, at different times during erythropoiesis. TSA completely blocked enucleation and kinetic studies showed this inhibitory effect occurred earlier than the step catalyzed by the Rac-mDia2 pathway. This indicates that Chromatin Condensation is required for the final extrusion of the nucleus. We further investigated the activity ofHDAC6 specifically, since HDAC6 is known to interact with mDia2. We found that mDia2is acetylated in vitro and in vivo and that HDAC6 interacts with and deacetylates mDia2. Treatment of purified TER119 negative mouse fetal erythroblasts with a specific HDAC6inhibitor partially blocked enucleation. We conclude that histone deacetylase activities are essential for mammalian erythroblasts to undergo enucleation and that HDACs act both on Chromatin Condensation and mDia2 deacetylation to promote enucleation.
Kumiko Samejima - One of the best experts on this subject based on the ideXlab platform.
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cad dff40 nuclease is dispensable for high molecular weight dna cleavage and stage i Chromatin Condensation in apoptosis
Journal of Biological Chemistry, 2001Co-Authors: Kumiko Samejima, Shigenobu Tone, William C EarnshawAbstract:DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear Chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic Chromatin Condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of Chromatin Condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic Chromatin Condensation.
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DNA topoisomerase IIα interacts with CAD nuclease and is involved in Chromatin Condensation during apoptotic execution
Current biology : CB, 2000Co-Authors: F. Durrieu, Kumiko Samejima, John M. Fortune, Stefanie Kandels-lewis, Neil Osheroff, William C EarnshawAbstract:Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution [2-4] and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 [4,7,8] (CAD) can induce apoptotic Chromatin Condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors [6,8]. As Chromatin Condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIalpha (Topo IIalpha), which is essential for both Condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIalpha and caspases completely abolishes apoptotic Chromatin Condensation. In addition, we show that CAD binds to Topo IIalpha, and that their association enhances the decatenation activity of Topo IIalpha in vitro.
David A. Lee - One of the best experts on this subject based on the ideXlab platform.
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Mechanically Induced Chromatin Condensation Requires Cellular Contractility in Mesenchymal Stem Cells
Biophysical journal, 2016Co-Authors: Su Jin Heo, David A. Lee, Woojin M. Han, Spencer E. Szczesny, Brian D. Cosgrove, Dawn M. Elliott, Randall L. Duncan, Robert L. MauckAbstract:Abstract Mechanical cues play important roles in directing the lineage commitment of mesenchymal stem cells (MSCs). In this study, we explored the molecular mechanisms by which dynamic tensile loading (DL) regulates Chromatin organization in this cell type. Our previous findings indicated that the application of DL elicited a rapid increase in Chromatin Condensation through purinergic signaling mediated by ATP. Here, we show that the rate and degree of Condensation depends on the frequency and duration of mechanical loading, and that ATP release requires actomyosin-based cellular contractility. Increases in baseline cellular contractility via the addition of an activator of G-protein coupled receptors (lysophosphatidic acid) induced rapid ATP release, resulting in Chromatin Condensation independent of loading. Conversely, inhibition of contractility through pretreatment with either a RhoA/Rock inhibitor (Y27632) or MLCK inhibitor (ML7) abrogated ATP release in response to DL, blocking load-induced Chromatin Condensation. With loading, ATP release occurred very rapidly (within the first 10–20 s), whereas changes in Chromatin occurred at a later time point (∼10 min), suggesting a downstream biochemical pathway mediating this process. When cells were pretreated with blockers of the transforming growth factor (TGF) superfamily, purinergic signaling in response to DL was also eliminated. Further analysis showed that this pretreatment decreased contractility, implicating activity in the TGF pathway in the establishment of the baseline contractile state of MSCs (in the absence of exogenous ligands). These data indicate that Chromatin Condensation in response to DL is regulated through the interplay between purinergic and RhoA/Rock signaling, and that ligandless activity in the TGF/bone morphogenetic proteins signaling pathway contributes to the establishment of baseline contractility in MSCs.
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Quantification of Chromatin Condensation level by image processing.
Medical engineering & physics, 2013Co-Authors: Jerome Irianto, David A. Lee, Martin M. KnightAbstract:The level of Chromatin Condensation is related to the silencing/activation of chromosomal territories and therefore impacts on gene expression. Chromatin Condensation changes during cell cycle, progression and differentiation, and is influenced by various physicochemical and epigenetic factors. This study describes a validated experimental technique to quantify Chromatin Condensation. A novel image processing procedure is developed using Sobel edge detection to quantify the level of Chromatin Condensation from nuclei images taken by confocal microscopy. The algorithm was developed in MATLAB and used to quantify different levels of Chromatin Condensation in chondrocyte nuclei achieved through alteration in osmotic pressure. The resulting Chromatin Condensation parameter (CCP) is in good agreement with independent multi-observer qualitative visual assessment. This image processing technique thereby provides a validated unbiased parameter for rapid and highly reproducible quantification of the level of Chromatin Condensation.
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Osmotic Challenge Drives Rapid and Reversible Chromatin Condensation in Chondrocytes
Biophysical journal, 2013Co-Authors: Jerome Irianto, Martin M. Knight, Joe Swift, Rui Pires Martins, Graham D. Mcphail, Dennis E. Discher, David A. LeeAbstract:Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce Chromatin Condensation, while hypoosmotic conditions (100 mOsm/kg) cause deCondensation. Large density changes (p < 0.001) occur over a very narrow range of physiological osmolalities, which suggests that chondrocytes likely experience Chromatin Condensation and deCondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p < 0.01) and Chromatin Condensation (p < 0.001) also differed between chondrocytes in monolayer culture and three-dimensional agarose, suggesting a role for cell adhesion. The relationship between Condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the Chromatin Condensation (
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osmotic challenge drives rapid and reversible Chromatin Condensation in chondrocytes
Biophysical Journal, 2013Co-Authors: Jerome Irianto, Martin M. Knight, Joe Swift, Rui Pires Martins, Graham D. Mcphail, Dennis E. Discher, David A. LeeAbstract:Changes in extracellular osmolality have been shown to alter gene expression patterns and metabolic activity of various cell types, including chondrocytes. However, mechanisms by which physiological or pathological changes in osmolality impact chondrocyte function remain unclear. Here we use quantitative image analysis, electron microscopy, and a DNase I assay to show that hyperosmotic conditions (>400 mOsm/kg) induce Chromatin Condensation, while hypoosmotic conditions (100 mOsm/kg) cause deCondensation. Large density changes (p < 0.001) occur over a very narrow range of physiological osmolalities, which suggests that chondrocytes likely experience Chromatin Condensation and deCondensation during a daily loading cycle. The effect of changes in osmolality on nuclear morphology (p < 0.01) and Chromatin Condensation (p < 0.001) also differed between chondrocytes in monolayer culture and three-dimensional agarose, suggesting a role for cell adhesion. The relationship between Condensation and osmolality was accurately modeled by a polymer gel model which, along with the rapid nature of the Chromatin Condensation (<20 s), reveals the basic physicochemical nature of the process. Alterations in Chromatin structure are expected to influence gene expression and thereby regulate chondrocyte activity in response to osmotic changes.
Shigenobu Tone - One of the best experts on this subject based on the ideXlab platform.
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cad dff40 nuclease is dispensable for high molecular weight dna cleavage and stage i Chromatin Condensation in apoptosis
Journal of Biological Chemistry, 2001Co-Authors: Kumiko Samejima, Shigenobu Tone, William C EarnshawAbstract:DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear Chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic Chromatin Condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of Chromatin Condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic Chromatin Condensation.
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CAD/DFF40 nuclease is dispensable for high molecular weight DNA cleavage and stage I Chromatin Condensation in apoptosis.
The Journal of biological chemistry, 2001Co-Authors: K Samejima, Shigenobu Tone, William C EarnshawAbstract:DNA degradation during apoptotic execution generally occurs at two levels: early as high molecular weight (HMW) fragments and later on as oligonucleosomal fragments. Two nucleases, CAD/CPAN/DFF40 and endonuclease G, can digest nuclear Chromatin to produce the oligonucleosomal fragments, and it has been suggested that CAD might be responsible for HMW DNA cleavage. To more clearly define the role of CAD in nuclear disassembly, we have generated CAD(-/-) sublines of chicken DT40 cells in which the entire CAD open reading frame has been deleted. These cells grow normally and undergo apoptosis with kinetics essentially identical to wild type cells. However, they fail to undergo detectable oligonucleosomal fragmentation, proving that CAD is essential for this stage of DNA cleavage, at least in DT40 cells. Other aspects of nuclear disassembly, including HMW DNA cleavage and early stage apoptotic Chromatin Condensation against the nuclear periphery proceed normally in the absence of CAD. However, the final stages of Chromatin Condensation and nuclear fragmentation do not occur. Our results demonstrate that CAD is required for complete disassembly of the nucleus during apoptosis and reveal the existence of one or more as yet unidentified second factors responsible for HMW DNA cleavage and the early stages of apoptotic Chromatin Condensation.