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Transcription at the Ad2 major late promoter with extracts prepared from nuclei extracted at different NaCl concentrations. Extracts were prepared using the standard procedure except that the NaCl concentration for extraction was varied. Extracts were assayed under standard conditions. Lanes 1 and 2 show assays which contained, respectively, 12.5 pl (50 'g protein ) and 25 4l (100 vg protein) of the 0.2 M NaCl extract. Lanes 3 through 6 show, respectively, assays of the 0.3 M NaCl extract (25 p4, 120 u'g protein ), the 0.42 M NaCl extract (25 il, 140 'g protein), and the 0.5 M NaCl extract (25 4l, 150 ig protein). Lanes 7 and 8 show assays which contained, respectively, 25 pl (80 vg protein) and 12.5 p4 (40 pg protein) of the 0.5 M NaCl extract of nuclei previously extracted with 0.2 M NaCl. Lane 9 shows an assay which contained a mixture (12.5 p4 each) of the 0.2 M NaCl extract and the 0.5 M NaCl extract of nuclei previously extracted with 0.2 M NaCl.  

Transcription at the Ad2 major late promoter with extracts prepared from nuclei extracted at different NaCl concentrations. Extracts were prepared using the standard procedure except that the NaCl concentration for extraction was varied. Extracts were assayed under standard conditions. Lanes 1 and 2 show assays which contained, respectively, 12.5 pl (50 'g protein ) and 25 4l (100 vg protein) of the 0.2 M NaCl extract. Lanes 3 through 6 show, respectively, assays of the 0.3 M NaCl extract (25 p4, 120 u'g protein ), the 0.42 M NaCl extract (25 il, 140 'g protein), and the 0.5 M NaCl extract (25 4l, 150 ig protein). Lanes 7 and 8 show assays which contained, respectively, 25 pl (80 vg protein) and 12.5 p4 (40 pg protein) of the 0.5 M NaCl extract of nuclei previously extracted with 0.2 M NaCl. Lane 9 shows an assay which contained a mixture (12.5 p4 each) of the 0.2 M NaCl extract and the 0.5 M NaCl extract of nuclei previously extracted with 0.2 M NaCl.  

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We have developed a procedure for preparing extracts from nuclei of human tissue culture cells that directs accurate transcription initiation in vitro from class II promoters. Conditions of extraction and assay have been optimized for maximum activity using the major late promoter of adenovirus 2. The extract also directs accurate transcription ini...

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... Nuclear extract from MS2-tagged RUS-expressing Neuro2A cells was prepared typically from 8 × 10 7 cells without dialysis, according to Dignam et al (1983). Extract preparation and MS2-affinity purification were carried out at 4°C. Cell pellets were suspended in 5 vol buffer A (10 mM Hepes, pH 7.9 at 4°C, 1.5 mM MgCl 2 , 10 mM KCl, 0.5 mM DTT, and 200 U/ml RNAsin) and incubated for 10 min. ...
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... The supernatant was diluted with an equal volume (300 μl) of 2 × NP-40 lysis buffer. On the other hand, the pellet was suspended in 300 μl of buffer C [20 mM HEPES-KOH (pH 7.9), 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl 2 , 0.2 mM EDTA, 0.5 mM DTT, and protease inhibitor cocktail] 73 and sonicated using Sonifier 450 (Branson Ultrasonics). After centrifugation at 16,000 × g for 10 min, the supernatants were diluted with 1.8 volume (540 μl) of dilution buffer [50 mM Tris-HCl (pH 8.0) and 1.35% NP-40] to generate nuclear extracts containing 150 mM NaCl and 1% NP-40. ...
... HeLa cells were harvested, washed in ice-cold PBS, and resuspended in five packed cell pellet volumes of buffer A [10 mM HEPES-KOH (pH 7.9), 10 mM KCl, 1.5 mM MgCl 2 , 0.5 mM DTT, and protease inhibitor cocktail) 73 . After incubation for 15 min on ice, cells were homogenized using a tight fitting Dounce homogenizer (Weaton type A, 40 strokes), and centrifuged at 500 × g at 4°C for 10 min. ...
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Transcriptional regulation by RNA polymerase II is associated with changes in chromatin structure. Activated and promoter-bound heat shock transcription factor 1 (HSF1) recruits transcriptional co-activators, including histone-modifying enzymes; however, the mechanisms underlying chromatin opening remain unclear. Here, we demonstrate that HSF1 recruits the TRRAP-TIP60 acetyltransferase complex in HSP72 promoter during heat shock in a manner dependent on phosphorylation of HSF1-S419. TRIM33, a bromodomain-containing ubiquitin ligase, is then recruited to the promoter by interactions with HSF1 and a TIP60-mediated acetylation mark, and cooperates with the related factor TRIM24 for mono-ubiquitination of histone H2B on K120. These changes in histone modifications are triggered by phosphorylation of HSF1-S419 via PLK1, and stabilize the HSF1-transcription complex in HSP72 promoter. Furthermore, HSF1-S419 phosphorylation is constitutively enhanced in and promotes proliferation of melanoma cells. Our results provide mechanisms for HSF1 phosphorylation-dependent establishment of an active chromatin status, which is important for tumorigenesis. Here the authors show phosphorylation of heat shock factor 1 (HSF1) at S419 via the chromatin-bound kinase PLK1, promotes HSF1 recruitment of histone acetyltransferases and histone acetylation reader proteins TRIM33 and TRIM24, which actually also execute histone H2BK120 mono-ubiquitination at target genes. Furthermore, HSF1 phosphorylation has an impact on melanoma cell proliferation.
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... One part was homogenized in RIPA buffer (Beyotime, China) containing PMSF to extract total protein content. The second part was used to extract total nuclear protein using the method described by Dignam et al. [29]. Briefly, the skin was homogenized with a tissue homogenizer in an ice water bath, 10 s each time, 30 s apart, 7 Oxidative Medicine and Cellular Longevity and nuclei were separated by centrifugation at 800 × g for 10 min. ...
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Hair follicles (HFs) maintain homeostasis through the hair cycles; therefore, disrupting the hair cycle may lead to hair loss. Our previous study showed that apoptosis-inducing factor (AIF) nuclear translocation and poly [ADP-ribose] polymerase 1 (PARP1) upregulation induced apoptosis in mouse hair follicles during the hair cycle transition from anagen to catagen. However, the mechanism underlying this phenomenon remains unclear. In this study, we found that intrinsic ROS levels increased during the hair follicle cycle transition from anagen to catagen, followed by abrupt DNA breaks and activation of homologous recombinant and nonhomologous end joining DNA repair, along with the enhancement of apoptosis. Mice in different stages of the hair cycle were sacrificed, and the dorsal skins were collected. The results of western blot and histological staining indicated that AIF-PARP1 plays a key role in HF apoptosis, but their role in the regulation of the HF cycle is not clear. Mice were treated with inhibitors from anagen to catagen: treatment with BMN 673, a PARP1 inhibitor, increased DNA breaks and activated the cytochrome c/caspase-3-mediated apoptotic pathway, accelerating HF regression. Ac-DEVD-CHO (Ac), a caspase-3 inhibitor, attenuated HF degeneration by upregulating PARP1 expression, suggesting a seesaw relationship between cytochrome c-caspase-3- and AIF-PARP1-mediated apoptosis, wherein PARP1 may be the fulcrum. In addition, macrophages were involved in regulating the hair cycle, and the rate of M1 macrophages around HFs increased during catagen, while more M2 macrophages were found during anagen and telogen. Our results indicate that intrinsic ROS drive HF cycle progression through DNA damage and repair, followed by apoptosis. Intrinsic ROS drive hair follicle cycle progression by modulating DNA damage and repair, and consecutively, hair follicle apoptosis and macrophage polarization work together to promote the hair follicle cycle.
... The frozen cell pellet was produced by the Cell Culture Company, LLC (Minneapolis, MN), using our specifications for cell growth rate and density. The nuclear extract was prepared using a modified protocol based on the method of Dignam (40 The frozen cells were thawed in a 37 • C water bath while keeping the cells cold. The rest of the procedures were performed at 4 • C or on wet ice. ...
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... Nuclear protein extraction. The nuclear protein extraction protocol was adapted from Dignam JD et al. 47 . ...
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Vacuolar protein sorting 35 (VPS35) is a major component of the retromer complex that regulates endosomal trafficking in eukaryotic cells. Recent studies have shown that VPS35 promotes tumor cell proliferation and affects the nuclear accumulation of its interacting partner. In this study, isobaric tags for relative and absolute quantitation (iTRAQ)-based mass spectrometry were used to measure the changes in nuclear protein abundance in VPS35-depleted HeLa cells. A total of 47 differentially expressed proteins were identified, including 27 downregulated and 20 upregulated proteins. Gene ontology (GO) analysis showed that the downregulated proteins included several minichromosome maintenance (MCM) proteins described as cell proliferation markers, and these proteins were present in the MCM2-7 complex, which is essential for DNA replication. Moreover, we validated that loss of VPS35 reduced the mRNA and protein expression of MCM2-7 genes. Notably, re-expression of VPS35 in VPS35 knockout HeLa cells rescued the expression of these genes. Functionally, we showed that VPS35 contributes to cell proliferation and maintenance of genomic stability of HeLa cells. Therefore, these findings reveal that VPS35 is involved in the regulation of MCM2-7 gene expression and establish a link between VPS35 and cell proliferation.
... In order to save the precious ALN tissue, the experimental approach included the use of HeLa cell culture to confirm the fractionation protocol described in Methods. Historically, HeLa cell fractionation into nuclear and cytosolic fractions had been introduced in molecular biology research about four decades ago [20]. Moreover, previous studies have established that transcription factors including cFOS are nuclear markers of mammalian cells as demonstated in HeLa cell nuclear extracts [21]. ...
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... Purification of RNF219-associated complexes and mass spectrometry. RNF219 complexes were purified from Dignam S100 extracts 36 derived from 2 × 10 9 HeLa S3 cells stably expressing RNF219 fused to a FLAG and an HA tag at the C-terminus (FHA-RNF219) by two-step affinity chromatography, according to a standard method 37 . 5% of FLAG and HA immunoaffinity purified FHA-RNF219 or mock immunoprecipitations (IP) from four liters of culture were resolved on SDS-PAGE and stained with the Silverquest kit (Invitrogen). ...
... We purified RNF219 protein complexes to address its cellular function. TAP-tag experiments 37 were performed using cytoplasmic S100 extract 36 from HeLa S3 cells stably expressing FLAG and HA-tagged RNF219 (FHA-RNF219). Immunoprecipitated material was analyzed by tandem mass spectrometry. ...
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Post-transcriptional regulatory mechanisms play a role in many biological contexts through the control of mRNA degradation, translation and localization. Here, we show that the RING finger protein RNF219 co-purifies with the CCR4-NOT complex, the major mRNA deadenylase in eukaryotes, which mediates translational repression in both a deadenylase activity-dependent and -independent manner. Strikingly, RNF219 both inhibits the deadenylase activity of CCR4-NOT and enhances its capacity to repress translation of a target mRNA. We propose that the interaction of RNF219 with the CCR4-NOT complex directs the translational repressive activity of CCR4-NOT to a deadenylation-independent mechanism.
... The FLAG-DPF2 cell line was selected from HEK293T cells transfected with a FLAG-DPF2-pCAG-IP plasmid. Derived nuclear extracts (Dignam et al., 1983) were incubated with M2 agarose in binding buffer (20 mM Tris-HCl [pH 7.3], 300 mM KCl, 0.2 mM EDTA, 25% glycerol, 1.5 mM MgCl 2 , 10 mM 2-mercaptoethanol. and 0.2 mM PMSF) at 4 °C for 4 hr. ...
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Inositol polyphosphate multikinase (IPMK), a key enzyme in inositol polyphosphate (IP) metabolism, is a pleiotropic signaling factor involved in major biological events, including transcriptional control. In the yeast, IPMK and its IP products promote the activity of the chromatin remodeling complex SWI/SNF, which plays a critical role in gene expression by regulating chromatin accessibility. However, the direct link between IPMK and chromatin remodelers remains unclear, raising the question of how IPMK contributes to transcriptional regulation in mammals. By employing unbiased screening approaches and in vivo/in vitro immunoprecipitation, here we demonstrate that mammalian IPMK physically interacts with the SWI/SNF complex by directly binding to SMARCB1, BRG1, and SMARCC1. Furthermore, we identified the specific domains required for IPMK-SMARCB1 binding. Notably, using CUT&RUN and ATAC-seq assays, we discovered that IPMK co-localizes with BRG1 and regulates BRG1 localization as well as BRG1-mediated chromatin accessibility in a genome-wide manner in mouse embryonic stem cells. Together, these findings show that IPMK regulates the promoter targeting of the SWI/SNF complex, thereby contributing to SWI/SNF-meditated chromatin accessibility, transcription, and differentiation in mouse embryonic stem cells.
... Nuclear extracts were prepared using a protocol described by Dignam and colleagues. 33 EMSAs, competition EMSAs and supershift assays were performed as described previously. 34 Oligonucleotides sequences used for the probes are available upon request. ...
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Background The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2’-deoxycytidine (5-AzadC). Methods We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4⁺ T-cell models and ex vivo cultures of PBMCs from HIV⁺ individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24Gag protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. Findings We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. Interpretation A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV⁺ patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. Funding Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin », the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation »), « Les Amis des Instituts Pasteur à Bruxelles, asbl », the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the “Alsace contre le Cancer” Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.