379 TREATMENT OF 293T CELLS WITH XENOPUS EGG EXTRACT INDUCES NOT-STRESS-RELATED EPIGENETIC REMODELING OF RIBOSOMAL GENES

2010 ◽  
Vol 22 (1) ◽  
pp. 346
Author(s):  
O. Østrup ◽  
P. Hyttel ◽  
D. Klœrke ◽  
P. Collas
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Immunoprecipitation ◽  
Somatic Cells ◽  
Normal Activity ◽  
Ribosomal Genes ◽  
Synthetic Activity ◽  
Relative Level ◽  
Egg Extract ◽  
Epigenetic Remodeling ◽  
H3k9me3 Level

Undefined mechanisms involved in reprogramming of somatic cells by Xenopus leavis egg extract have, to date, prevented clinical applications for cell replacement therapy. The aim of this study was to evaluate the immediate response of somatic cells to exposure to Xenopus leavis egg extract using ribosomal genes (rDNA) as a sensitive marker for stress and/or chromatin remodeling. Human epithelial 293T cells in standard culture (control), starved in glucose-free medium (stress-control), or treated for 1 h with egg extract were fixed at 6 and 24 h after treatment and analyzed for pre-rRNA synthetic activity by quantitative RT-PCR; level of H3K9me3 by immunofluorescence; and for occupancy of rDNA promoter by markers of normal activity (UBF), stress silencing (SIRT1, SUV39H1), and remodeling (SNF2H) using chromatin immunoprecipitation. Relative levels of pre-rRNA decreased in all treated groups to 20 to 35% of control levels. Chromatin immunoprecipitation did not reveal any significant effect of starvation or extract treatment on UBF and SIRT1 binding to the rDNA promoter, presumably because of the pleiotropic property of these proteins. However, starvation specifically led to enhanced SUV39H1 binding 6 h after treatment, indicating early cell response to stress. No differences were found in SUV39H1 binding between extract-treated cells and control. In contrast, binding of SNF2H at 6 h increased in extract-treated cells but decreased in starved cells. The relative level of H3K9me3 increased first after 24 h of starvation equally in all cells, suggesting later involvement of this histone modification in rDNA silencing. Extract treatment, however, led to a decrease in H3K9me3 level at 6 h, and after 24 h, 2 main cell populations were observed: one (85.4% of cells) that retained decreased H3K9me3 and one (12.4% of cells) with significantly enhanced levels. Moreover, H3K9me3 foci in the last group were associated with the periphery of presumptive nucleoli. Thus, extract treatment apparently does not initiate a stress-induced silencing of the rDNA genes; rather, it activates SNF2H-dependent chromatin remodeling, resulting in a long-term decrease in pre-rRNA synthetic activity. The research was supported by Carlsberg Foundation 2008-01-0105.

Remodeling of ribosomal genes in somatic cells by Xenopus egg extract

2011 ◽  
Vol 412 (3) ◽  
pp. 487-493 ◽  
Author(s):  
Olga Østrup ◽  
Poul Hyttel ◽  
Dan A. Klærke ◽  
Philippe Collas
Keyword(s):  
Somatic Cells ◽  
Ribosomal Genes ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Xenopus Egg

scholarly journals Efg1-mediated Recruitment of NuA4 to Promoters Is Required for Hypha-specific Swi/Snf Binding and Activation inCandida albicans

2008 ◽  
Vol 19 (10) ◽  
pp. 4260-4272 ◽  
Author(s):  
Yang Lu ◽  
Chang Su ◽  
Xuming Mao ◽  
Prashna Pala Raniga ◽  
Haoping Liu ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Transcriptional Activation ◽  
Chromatin Immunoprecipitation ◽  
Pathogenic Fungus ◽  
Dependent Manner ◽  
Hyphal Development ◽  
Chromatin Remodeling Complex ◽  
A Subunit ◽  
Human Pathogenic Fungus ◽  
Incandida Albicans

Efg1 is essential for hyphal development and virulence in the human pathogenic fungus Candida albicans. How Efg1 regulates gene expression is unknown. Here, we show that Efg1 interacts with components of the nucleosome acetyltransferase of H4 (NuA4) histone acetyltransferase (HAT) complex in both yeast and hyphal cells. Deleting YNG2, a subunit of the NuA4 HAT module, results in a significant decrease in the acetylation level of nucleosomal H4 and a profound defect in hyphal development, as well as a defect in the expression of hypha-specific genes. Using chromatin immunoprecipitation, Efg1 and the NuA4 complex are found at the UAS regions of hypha-specific genes in both yeast and hyphal cells, and Efg1 is required for the recruitment of NuA4. Nucleosomal H4 acetylation at the promoters peaks during initial hyphal induction in an Efg1-dependent manner. We also find that Efg1 bound to the promoters of hypha-specific genes is critical for recruitment of the Swi/Snf chromatin remodeling complex during hyphal induction. Our data show that the recruitment of the NuA4 complex by Efg1 to the promoters of hypha-specific genes is required for nucleosomal H4 acetylation at the promoters during hyphal induction and for subsequent binding of Swi/Snf and transcriptional activation.

273 SELECTION OF PLURIPOTENT STEM CELLS INDUCED BY XENOPUS EGG EXTRACTS

2009 ◽  
Vol 21 (1) ◽  
pp. 234 ◽  
Author(s):  
C.-Y. Chiang ◽  
P.-C. Tang
Keyword(s):  
Somatic Cells ◽  
Egfp Expression ◽  
3T3 Cells ◽  
Xenopus Egg Extract ◽  
Egg Extract ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Xenopus Egg Extracts ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

It has been reported that Xenopus egg extracts contain molecules that are capable of reprogramming mammalian somatic cells. The reprogrammed somatic cells, which are called extract treated cells (ETC), possess the potential for clinical therapy as embryonic stem (ES) cells do. Therefore, in addition to establishment of an efficient method to reprogram mouse NIH/3T3 cells by Xenopus egg extracts, the aim of this study was to select the ETC cells by the expression of Oct4. In Experiment 1, two methods, electroporation or permeabilization, were conducted to treat mouse NIH/3T3 cells with Xenopus egg extracts. 2 × 105 cells in 200 μL reprogramming mixture containing Xenopus egg extracts were stimulated by a direct current (DC) pulse (80 V mm–1 for 3 msec) three times followed by a pause of incubation at 37°C for 5 min and a single DC pulse (170 V mm–1, for 0.4 msec) subsequently. The electroporated cells were then incubated at 22°C for 1 h. In the other treatment group, NIH/3T3 cells (5 × 105) were permeabilized by streptolysin O (SLO, 500 ng mL–1 in PBS) for 50 min at 37°C before mixed with Xenopus egg extracts at 22°C for 2 h. Cells were cultured in DMEM supplemented with 10% FBS for the first 4 days and then changed to ES medium (DMEM supplemented with 15% FBS, 0.1 mm β-mercaptoethanol, 1000 unit mL–1 mLIF, 0.5% nonessential amino acids, 2 mm L-glutamine) for the last 6 days after Xenopus egg extract treatment. Cell colonies were found in both treatment groups at the end of culture. Examination by immunocytochemical staining, results showed that the extract-treated cell colonies expressed pluripotent marker proteins, such as alkaline phosphatase, Oct4, Nanog and Sox2. In Experiment 2, an enhanced green fluorescent protein (EGFP) expression vector was constructed and EGFP was driven by Oct4 enhancer and promoter (Oct4-EGFP). Mouse NIH/3T3 cells were then transfected with Oct4-EGFP plasmids and selected for stable clone by G418 screening. After 6 passages, the NIH/3T3-Oct4-EGFP cells were treated with egg extracts to induce reprogramming as Experiment 1, and monitored pluripotency based on the expression of EGFP. Results showed that some of the cells or cell colonies expressed green fluorescence driven by Oct4 regulatory element at the 8th day of culture after extract treatment. Our results demonstrated that both methods of electroporation and reversible permeabilization could introduce reprogramming molecules in Xenopus egg extract to the mammalian somatic cells and generate ETCs cells in vitro. Also, with the establishment of NIH/3T3-Oct4-EGFP cell line, the potentially reprogrammed colonies could be easily selected by EGFP expression. The changes of epigenetic modifications in the ETC cells would be investigated in the short future.

scholarly journals BRG1 Is Required for Formation of Senescence-Associated Heterochromatin Foci Induced by Oncogenic RAS or BRCA1 Loss

2013 ◽  
Vol 33 (9) ◽  
pp. 1819-1829 ◽  
Author(s):  
Zhigang Tu ◽  
Xinying Zhuang ◽  
Yong-Gang Yao ◽  
Rugang Zhang
Keyword(s):  
Dna Damage ◽  
Molecular Mechanism ◽  
Chromatin Remodeling ◽  
Cellular Senescence ◽  
Chromatin Immunoprecipitation ◽  
Tumor Suppression ◽  
Gene Promoters ◽  
Independent Manner ◽  
Oncogenic Ras ◽  
Suppression Mechanism

Cellular senescence is an important tumor suppression mechanism. We have previously reported that both oncogene-induced dissociation of BRCA1 from chromatin and BRCA1 knockdown itself drive senescence by promoting formation of s enescence- a ssociated h eterochromatin f oci (SAHF). However, the molecular mechanism by which BRCA1 regulates SAHF formation and senescence is unclear. BRG1 is a chromatin-remodeling factor that interacts with BRCA1 and pRB. Here we show that BRG1 is required for SAHF formation and senescence induced by oncogenic RAS or BRCA1 loss. The interaction between BRG1 and BRCA1 is disrupted during senescence. This correlates with an increased level of chromatin-associated BRG1 in senescent cells. BRG1 knockdown suppresses the formation of SAHF and senescence, while it has no effect on BRCA1 chromatin dissociation induced by oncogenic RAS, indicating that BRG1 functions downstream of BRCA1 chromatin dissociation. Furthermore, BRG1 knockdown inhibits SAHF formation and senescence induced by BRCA1 knockdown. Conversely, BRG1 overexpression drives SAHF formation and senescence in a DNA damage-independent manner. This effect depends upon BRG1's chromatin-remodeling activity as well as the interaction between BRG1 and pRB. Indeed, the interaction between BRG1 and pRB is enhanced during senescence. Chromatin immunoprecipitation analysis revealed that BRG1's association with the human CDKN2A and CDKN1A gene promoters was enhanced during senescence induced by oncogenic RAS or BRCA1 knockdown. Consistently, knockdown of pRB, p21 CIP1 , and p16 INK4a , but not p53, suppressed SAHF formation induced by BRG1. Together, these studies reveal the molecular underpinning by which BRG1 acts downstream of BRCA1 to promote SAHF formation and senescence.

scholarly journals ISWI is a RanGTP-dependent MAP required for chromosome segregation

2009 ◽  
Vol 187 (6) ◽  
pp. 813-829 ◽  
Author(s):  
Hideki Yokoyama ◽  
Sofia Rybina ◽  
Rachel Santarella-Mellwig ◽  
Iain W. Mattaj ◽  
Eric Karsenti
Keyword(s):  
Chromatin Remodeling ◽  
Chromosome Segregation ◽  
Spindle Assembly ◽  
S2 Cells ◽  
Culture Cells ◽  
Egg Extract ◽  
Drosophila S2 Cells ◽  
Egg Extracts ◽  
Localization Signal

Production of RanGTP around chromosomes induces spindle assembly by activating nuclear localization signal (NLS)–containing factors. Here, we show that the NLS protein ISWI, a known chromatin-remodeling ATPase, is a RanGTP-dependent microtubule (MT)-associated protein. Recombinant ISWI induces MT nucleation, stabilization, and bundling in vitro. In Xenopus culture cells and egg extract, ISWI localizes within the nucleus in interphase and on spindles during mitosis. Depletion of ISWI in egg extracts does not affect spindle assembly, but in anaphase spindle MTs disappear and chromosomes do not segregate. We show directly that ISWI is required for the RanGTP-dependent stabilization of MTs during anaphase independently of its effect on chromosomes. ISWI depletion in Drosophila S2 cells induces defects in spindle MTs and chromosome segregation in anaphase, and the cells eventually stop growing. Our results demonstrate that distinctly from its role in spindle assembly, RanGTP maintains spindle MTs in anaphase through the local activation of ISWI and that this is essential for proper chromosome segregation.

scholarly journals Promoter Occupancy Is a Major Determinant of Chromatin Remodeling Enzyme Requirements

2005 ◽  
Vol 25 (7) ◽  
pp. 2698-2707 ◽  
Author(s):  
Archana Dhasarathy ◽  
Michael P. Kladde
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Histone Acetylation ◽  
Binding Site ◽  
Chromatin Remodeling ◽  
Chromatin Immunoprecipitation ◽  
Site Occupancy ◽  
Major Determinant ◽  
Activator Concentration ◽  
Promoter Occupancy

ABSTRACT Chromatin creates transcriptional barriers that are overcome by coactivator activities such as histone acetylation by Gcn5 and ATP-dependent chromatin remodeling by SWI/SNF. Factors defining the differential coactivator requirements in the transactivation of various promoters remain elusive. Induction of the Saccharomyces cerevisiae PHO5 promoter does not require Gcn5 or SWI/SNF under fully inducing conditions of no phosphate. We show that PHO5 activation is highly dependent on both coactivators at intermediate phosphate concentrations, conditions that reduce the nuclear concentration of the Pho4 transactivator and severely diminish its association with PHO5 in the absence of Gcn5 or SWI/SNF. Conversely, physiological increases in Pho4 nuclear concentration and binding at PHO5 suppress the need for both Gcn5 and SWI/SNF, suggesting that coactivator redundancy is established at high Pho4 binding site occupancy. Consistent with this, we demonstrate, using chromatin immunoprecipitation, that Gcn5 and SWI/SNF are directly recruited to PHO5 and other strongly transcribed promoters, including GAL1-10, RPL19B, RPS22B, PYK1, and EFT2, which do not require either coactivator for expression. These results show that activator concentration and binding site occupancy play crucial roles in defining the extent to which transcription requires individual chromatin remodeling enzymes. In addition, Gcn5 and SWI/SNF associate with many more genomic targets than previously appreciated.

scholarly journals Deacetylase Activity Is Required for Recruitment of the Basal Transcription Machinery and Transactivation by STAT5

2003 ◽  
Vol 23 (12) ◽  
pp. 4162-4173 ◽  
Author(s):  
Anne Rascle ◽  
James A. Johnston ◽  
Bruno Amati
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Immunoprecipitation ◽  
Transcription Initiation ◽  
Cellular Response ◽  
Target Genes ◽  
Trichostatin A ◽  
Basal Transcription ◽  
Basal Transcription Machinery ◽  
Transcription Machinery ◽  
Microarray Analyses

ABSTRACT The signal transducer and activator of transcription STAT5 plays a major role in the cellular response to cytokines, but the mechanism by which it activates transcription remains poorly understood. We show here that deacetylase inhibitors (trichostatin A, suberoylanilide hydroxamic acid, and sodium butyrate) prevent induction of endogenous STAT5 target genes, implying that a deacetylase activity is required for that process. Microarray analyses revealed that this requirement is common to all STAT5 target genes. Using chromatin immunoprecipitation, we show that, following STAT5 DNA binding, deacetylase inhibitors block transcription initiation by preventing recruitment of the basal transcription machinery. This inhibition is not due to effects on histone H3 and H4 acetylation or chromatin remodeling within the promoter region. This novel mechanism of transactivation by STAT5 provides a rationale for the use of deacetylase inhibitors for therapeutic intervention in STAT5-associated cancers.

scholarly journals LncRNA NEAT1 remodels chromatin to promote the 5-Fu resistance by maintaining colorectal cancer stemness

2020 ◽  
Vol 11 (11) ◽  
Author(s):  
Yihao Zhu ◽  
Hanqing Hu ◽  
Ziming Yuan ◽  
Qian Zhang ◽  
Huan Xiong ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Chromatin Remodeling ◽  
Chromatin Immunoprecipitation ◽  
Cancer Stemness ◽  
Functional Studies ◽  
Non Coding Rna ◽  
New Strategy ◽  
Lncrna Neat1 ◽  
Long Non Coding Rna

Abstract Resistance of chemotherapy is one of causes of recurrence and poor prognosis in patients with colorectal cancer (CRC). The role of differentially expressed long non-coding RNA (lncRNA) in 5-fluorouracil (5-Fu) resistance has not been fully elucidated. Here we observed that lncRNA NEAT1 was associated with 5-Fu resistance in CRC. Our Functional studies showed that NEAT1 promoted 5-Fu resistance in colorectal cells. In addition, A-TAC sequencing and chromatin immunoprecipitation (ChIP) showed that NEAT1 affected chromatin remodeling, increased the acetylation levels of histones, increased their enrichment at the promoters of ALDH1 and c-Myc, and promoted the expression of ALDH1 and c-Myc. Taken together, our study suggested that NEAT1 promoted 5-Fu resistance and cancer stemness by remodeling chromatin. Our finding provides a novel role of NEAT1 and may provide a new strategy for the treatment of CRC 5-Fu resistance.

scholarly journals Piwi suppresses transcription of Brahma-dependent transposons via Maelstrom in ovarian somatic cells

2020 ◽  
Vol 6 (50) ◽  
pp. eaaz7420
Author(s):  
Ryo Onishi ◽  
Kaoru Sato ◽  
Kensaku Murano ◽  
Lumi Negishi ◽  
Haruhiko Siomi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Chromatin Remodeling ◽  
Adenosine Triphosphatase ◽  
Somatic Cells ◽  
Pol Ii ◽  
Heterochromatin Formation ◽  
Rna Transcripts ◽  
The Core ◽  
Chromatin Remodeling Complex

Drosophila Piwi associates with PIWI-interacting RNAs (piRNAs) and represses transposons transcriptionally through heterochromatinization; however, this process is poorly understood. Here, we identify Brahma (Brm), the core adenosine triphosphatase of the SWI/SNF chromatin remodeling complex, as a new Piwi interactor, and show Brm involvement in activating transcription of Piwi-targeted transposons before silencing. Bioinformatic analyses indicated that Piwi, once bound to target RNAs, reduced the occupancies of SWI/SNF and RNA polymerase II (Pol II) on target loci, abrogating transcription. Artificial piRNA-driven targeting of Piwi to RNA transcripts enhanced repression of Brm-dependent reporters compared with Brm-independent reporters. This was dependent on Piwi cofactors, Gtsf1/Asterix (Gtsf1), Panoramix/Silencio (Panx), and Maelstrom (Mael), but not Eggless/dSetdb (Egg)–mediated H3K9me3 deposition. The λN-box B–mediated tethering of Mael to reporters repressed Brm-dependent genes in the absence of Piwi, Panx, and Gtsf1. We propose that Piwi, via Mael, can rapidly suppress transcription of Brm-dependent genes to facilitate heterochromatin formation.

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