| 1. |
- Dass, Randall A., et al.
(författare)
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Wnt5a Signals through DVL1 to Repress Ribosomal DNA Transcription by RNA Polymerase I
- 2016
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Ingår i: PLoS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 12:8
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Tidskriftsartikel (refereegranskat)abstract
- Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in cancer. Accordingly, numerous oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast cancer, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we show that Wnt5a rapidly represses rDNA gene transcription in breast cancer cells and generates a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer regions (NORs) and binds to rDNA regions of the chromosome. Upon DVL1 binding, the Pol I transcription activator and deacetylase Sirtuin 7 (SIRT7) releases from rDNA loci, concomitant with disassembly of Pol I transcription machinery at the rDNA promoter. These findings reveal that Wnt5a signals through DVL1 to suppress rRNA transcription. This provides a novel mechanism for how Wnt5a exerts tumor suppressive effects and why disruption of Wnt5a signaling enhances mammary tumor growth in vivo.
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| 2. |
- Almuzzaini, Bader, et al.
(författare)
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In beta-actin knockouts, epigenetic reprogramming and rDNA transcription inactivation lead to growth and proliferation defects
- 2016
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Ingår i: The FASEB Journal. - 0892-6638 .- 1530-6860. ; 30:8, s. 2860-2873
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Tidskriftsartikel (refereegranskat)abstract
- Actin and nuclear myosin 1 (NM1) are regulators of transcription and chromatin organization. Using a genome-wide approach, we report here that beta-actin binds intergenic and genic regions across the mammalian genome, associated with both protein-coding and rRNA genes. Within the rDNA, the distribution of beta-actin correlated with NM1 and the other subunits of the B-WICH complex, WSTF and SNF2h. In beta-actin(-/-) mouse embryonic fibroblasts (MEFs), we found that rRNA synthesis levels decreased concomitantly with drops in RNA polymerase I (Pol I) and NM1 occupancies across the rRNA gene. Reintroduction of wild-type beta-actin, in contrast to mutated forms with polymerization defects, efficiently rescued rRNA synthesis underscoring the direct role for a polymerization-competent form of beta-actin in Pol I transcription. The rRNA synthesis defects in the beta-actin(-/-) MEFs are a consequence of epigenetic reprogramming with up-regulation of the repressive mark H3K4me1 (mono-methylation of lys4 on histone H3) and enhanced chromatin compaction at promoter-proximal enhancer (T0 sequence), which disturb binding of the transcription factor TTF1. We propose a novel genome-wide mechanism where the polymerase-associated beta-actin synergizes with NM1 to coordinate permissive chromatin with Pol I transcription, cell growth, and proliferation.
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| 3. |
- Almuzzaini, Bader, et al.
(författare)
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Nuclear myosin 1 contributes to a chromatin landscape compatible with RNA polymerase II transcription activation
- 2015
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Ingår i: BMC Biology. - : Springer Science and Business Media LLC. - 1741-7007. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- Background: Nuclear myosin 1c (NM1) is emerging as a regulator of transcription and chromatin organization. Results: Using chromatin immunoprecipitation and deep sequencing (ChIP-Seq) in combination with molecular analyses, we investigated the global association of NM1 with the mammalian genome. Analysis of the ChIP-Seq data demonstrates that NM1 binds across the entire mammalian genome with occupancy peaks correlating with distributions of RNA Polymerase II (Pol II) and active epigenetic marks at class II gene promoters. In mouse embryonic fibroblasts subjected to RNAi mediated NM1 gene silencing, we show that NM1 synergizes with polymerase-associated actin to maintain active Pol II at the promoter. NM1 also co-localizes with the nucleosome remodeler SNF2h at class II promoters where they assemble together with WSTF as part of the B-WICH complex. A high resolution micrococcal nuclease (MNase) assay and quantitative real time PCR shows that this mechanism is required for local chromatin remodeling. Following B-WICH assembly, NM1 mediates physical recruitment of the histone acetyl transferase PCAF and the histone methyl transferase Set1/Ash2 to maintain and preserve H3K9acetylation and H3K4trimethylation for active transcription. Conclusions: We propose a novel genome-wide mechanism where myosin synergizes with Pol II-associated actin to link the polymerase machinery with permissive chromatin for transcription activation.
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| 4. |
- Huynh, Hang Thuy, et al.
(författare)
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Biochemical Separation of Cytoplasmic and Nuclear Fraction for Downstream Molecular Analysis
- 2024
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Ingår i: CURRENT PROTOCOLS. - 2691-1299. ; 4:5
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Tidskriftsartikel (refereegranskat)abstract
- Biochemical fractionation is a technique used to isolate and separate distinct cellular compartments, critical for dissecting cellular mechanisms and molecular pathways. Herein we outline a biochemical fraction methodology for isolation of ultra-pure nuclei and cytoplasm. This protocol utilizes hypotonic lysis buffer to suspend cells, coupled with a calibrated centrifugation strategy, for enhanced separation of cytoplasm from the nuclear fraction. Subsequent purification steps ensure the integrity of the isolated nuclear fraction. Overall, this method facilitates accurate protein localization, essential for functional studies, demonstrating its efficacy in separating cellular compartments. (c) 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol: Biochemical fractionationSupport Protocol 1: Protein quantification using Bradford assaySupport Protocol 2: SDS/PAGE and Western blotting
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| 5. |
- Nowak, Iwona, et al.
(författare)
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Argonaute Proteins Take Center Stage in Cancers
- 2021
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Ingår i: Cancers. - : MDPI AG. - 2072-6694. ; 13:4
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Tidskriftsartikel (refereegranskat)abstract
- Simple Summary The dysregulation of RNA interference (RNAi) has often been observed in cancers, where the main focus of research has been on the small RNA molecules directing RNAi. In this review, we focus on the activity of Argonaute proteins, central components of RNAi, in tumorigenesis, and also highlight their potential applications in grading tumors and anti-cancer therapies. Argonaute proteins (AGOs) play crucial roles in RNA-induced silencing complex (RISC) formation and activity. AGOs loaded with small RNA molecules (miRNA or siRNA) either catalyze endoribonucleolytic cleavage of target RNAs or recruit factors responsible for translational silencing and target destabilization. miRNAs are well characterized and broadly studied in tumorigenesis; nevertheless, the functions of the AGOs in cancers have lagged behind. Here, we discuss the current state of knowledge on the role of AGOs in tumorigenesis, highlighting canonical and non-canonical functions of AGOs in cancer cells, as well as the biomarker potential of AGO expression in different of tumor types. Furthermore, we point to the possible application of the AGOs in development of novel therapeutic approaches.
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| 6. |
- Rosen, R., et al.
(författare)
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Identification, Location, and Emergence of the Cercaria of Leuceruthrus micropteri (Trematoda: Azygiidae) Recovered from the Snail Pleurocera semicarinata (Gastropoda: Pleuroceridae) at North Elkhorn Creek, Kentucky
- 2019
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Ingår i: Comparative Parasitology. - : Helminthological Society. - 1525-2647. ; 86:2, s. 142-148
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Tidskriftsartikel (refereegranskat)abstract
- We have encountered azygiid cercariae shed from the snail, Pleurocera semicarinata, at North Elkhorn Creek, Scott County, Kentucky, since 2004. Samples of these cercariae were frozen, and their DNA analyzed. The resulting alignment total length of 394 base pairs from partial 5.8S and 28S genes and the complete ribosomal internal transcribed spacer 2 (ITS2) confirmed our identification of the North Elkhorn Creek cercaria as Leuceruthrus micropteri. There was a 100% match to adult L. micropteri obtained from largemouth bass at Wheeler Reservoir, Alabama. Prevalence of infections by L. micropteri in snails was low at North Elkhorn Creek during 2004 (1.8% [7/400]) and 2017 (0.9% [4/432]). Quantitative data were obtained regarding cercarial emergence (daily and long-term patterns), site of infection of rediae, and functional morphology of the distome and tail stem. Most (96.1%) cercariae were released during the 12-hr light phase of a 12-hr light:12-hr dark cycle, and the average number of cercariae released per 7 snails per day over 21 d ranged between 0 and 2.3. Individual snails most frequently shed between 0 and 2 (range 0-6) cercariae per day. Snails shed cercariae for an average of 12.6 +/- 3.1 d of the 21-d sampling period. Histology revealed rediae in the bottom whorl of the snail within the perintestinal sinus separated from the mantle cavity and gills by a thin mantle membrane. Envelopment of the distome body by the tail stem in vitro required less than 2 min. The lip of the tail chamber moved forward over the distome body until the latter was completely enclosed.
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| 7. |
- Sarshad, Aishe A., et al.
(författare)
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Glycogen Synthase Kinase (GSK) 3 beta Phosphorylates and Protects Nuclear Myosin 1c from Proteasome-Mediated Degradation to Activate rDNA Transcription in Early G1 Cells
- 2014
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Ingår i: PLOS Genetics. - : Public Library of Science (PLoS). - 1553-7390 .- 1553-7404. ; 10:6, s. e1004390-
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Tidskriftsartikel (refereegranskat)abstract
- Nuclear myosin 1c (NM1) mediates RNA polymerase I (pol I) transcription activation and cell cycle progression by facilitating PCAF-mediated H3K9 acetylation, but the molecular mechanism by which NM1 is regulated remains unclear. Here, we report that at early G1 the glycogen synthase kinase (GSK) 3 beta phosphorylates and stabilizes NM1, allowing for NM1 association with the chromatin. Genomic analysis by ChIP-Seq showed that this mechanism occurs on the rDNA as active GSK3 beta selectively occupies the gene. ChIP assays and transmission electron microscopy in GSK3 beta(-/-) mouse embryonic fibroblasts indicated that at G1 rRNA synthesis is suppressed due to decreased H3K9 acetylation leading to a chromatin state incompatible with transcription. We found that GSK3 beta directly phosphorylates the endogenous NM1 on a single serine residue (Ser-1020) located within the NM1 C-terminus. In G1 this phosphorylation event stabilizes NM1 and prevents NM1 polyubiquitination by the E3 ligase UBR5 and proteasome-mediated degradation. We conclude that GSK3 beta-mediated phosphorylation of NM1 is required for pol I transcription activation.
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