The Nuclear Matrix: Fractionation Techniques and Analysis

Wilson, Rosemary H C; Hesketh, Emma L.; Coverley, Dawn

doi:10.1101/pdb.top074518

Cited by 10 publications

(14 citation statements)

References 52 publications

(45 reference statements)

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“…DNA halo preparations have been used for some time now to study interactions of the genome with the nucleoskeleton (Wilson et al 2016 ). They are prepared from cells grown on glass, permeabilised with detergent and digitonin and extracted with high salt DNA halo extraction buffer.…”

Section: Resultsmentioning

confidence: 99%

Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson–Gilford progeria syndrome fibroblasts

et al. 2018

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Hutchinson–Gilford progeria syndrome (HGPS) is a rare and fatal premature ageing disease in children. HGPS is one of several progeroid syndromes caused by mutations in the LMNA gene encoding the nuclear structural proteins lamins A and C. In classic HGPS the mutation G608G leads to the formation of a toxic lamin A protein called progerin. During post-translational processing progerin remains farnesylated owing to the mutation interfering with a step whereby the farnesyl moiety is removed by the enzyme ZMPSTE24. Permanent farnesylation of progerin is thought to be responsible for the proteins toxicity. Farnesyl is generated through the mevalonate pathway and three drugs that interfere with this pathway and hence the farnesylation of proteins have been administered to HGPS children in clinical trials. These are a farnesyltransferase inhibitor (FTI), statin and a bisphosphonate. Further experimental studies have revealed that other drugs such as N-acetyl cysteine, rapamycin and IGF-1 may be of use in treating HGPS through other pathways. We have shown previously that FTIs restore chromosome positioning in interphase HGPS nuclei. Mis-localisation of chromosomes could affect the cells ability to regulate proper genome function. Using nine different drug treatments representing drug regimes in the clinic we have shown that combinatorial treatments containing FTIs are most effective in restoring specific chromosome positioning towards the nuclear periphery and in tethering telomeres to the nucleoskeleton. On the other hand, rapamycin was found to be detrimental to telomere tethering, it was, nonetheless, the most effective at inducing DNA damage repair, as revealed by COMET analyses.Electronic supplementary materialThe online version of this article (10.1007/s10522-018-9758-4) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson–Gilford progeria syndrome fibroblasts

et al. 2018

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“…No reuse allowed without permission. To confirm and extend our findings on the role of PLD1 and PLD2 in PCa cell nuclei we have now examined the association of PLD1 and PLD2 with the nucleus by treating patientderived PCa cells and PC3 cells in a well-documented fractionation procedure which exposes core nuclear matrix (NM) proteins [35][36][37] Nuclei treated by this fractionation scheme leave a protein-rich matrix which includes actin and fibrillarin 38 and nuclear lamina (NL) proteins 39 .…”

Section: Introductionmentioning

confidence: 72%

Phospholipases D1 and D2 regulate cell cycling in primary prostate cancer cells and are differentially associated with the nuclear matrix

Hogg

Bourgoin

et al. 2021

Preprint

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Phospholipases D1 and D2 (PLD1/2) have been implicated in tumorigenesis. We previously detected higher expression of PLD in the nuclei of patient-derived prostate cancer (PCa) cells and prostate cancer cell lines. Here we have examined whether PLD1 or PLD2 are associated with the nuclear matrix and influence cell cycling. PLD1/PLD2 were detected by qualitative immunofluorescence in cultured PCa cells and extracted with a standardised protocol to reveal nuclear matrix-associated proteins. The effects of isoform-specific inhibition of PLD1or PLD2 on PCa cell cycle progression were analysed by flow cytometry. PLD2 mainly co-localised with the nucleolar marker fibrillarin in PCa cells. However, even after complete extraction, some PLD2 remained associated with the nuclear matrix. Inhibiting PLD2 effectively reduced PCa cell cycling into and through S phase. In contrast, PLD1 inhibition effects were weaker, and a subpopulation of cycling patient-derived PCa cells was unaffected by PLD1 inhibition. When associated with the nuclear matrix PLD2 could generate phosphatidic acid to regulate nuclear mTOR and control downstream transcriptional events. The association of PLD2 with the nucleolus also implies a role in stress regulation. The cell cycling results highlight the importance of PLD2 inhibition as a novel potential prostate cancer therapeutic mechanism by differential regulation of cell proliferation.

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“…05056489001; F Hoffmann-La Roche Ltd, Sigma Aldrich] and 1 mM DTT) with or without 0.1% Triton X-100 [39] for 30 s, then transferred to PBS and fixed for 20 min in 4% paraformaldehyde in PBS. Primary antibodies were incubated for 1.5 h at 37°C and secondary antibodies for 1 h at 37°C in the dark in antibody buffer (PBS, 10 mg/ml BSA, 0.2% SDS, 0.1% Triton X-100).…”

Section: Methodsmentioning

confidence: 99%

“…Cells were fractionated as previously described [39,40]. In brief, coverslips were washed with detergent (0.1% Triton-X-100 in CSK), followed by the same buffer with 0.5 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

CIZ1-F, an alternatively spliced variant of the DNA replication protein CIZ1 with distinct expression and localisation, is overrepresented in early stage common solid tumours

et al. 2018

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CIZ1 promotes cyclin-dependent DNA replication and resides in sub-nuclear foci that are part of the protein nuclear matrix (NM), and in RNA assemblies that are enriched at the inactive X chromosome (Xi) in female cells. It is subjected to alternative splicing, with specific variants implicated in adult and pediatric cancers. CIZ1-F is characterized by a frame shift that results from splicing exons 8–12 leading to inclusion of a short alternative reading frame (ARF), excluding the previously characterized C-terminal NM anchor domain. Here, we apply a set of novel variant-selective molecular tools targeted to the ARF to profile the expression of CIZ1-F at both transcript and protein levels, with focus on its relationship with the RNA-dependent and -independent fractions of the NM. Unlike full-length CIZ1, CIZ1-F does not accumulate at Xi, though like full-length CIZ1 it does resist extraction with DNase. Notably, CIZ1-F is sensitive to RNase identifying it as part of the RNA-fraction of the NM. In quiescent cells CIZ1-F transcript expression is suppressed and CIZ1-F protein is excluded from the nucleus, with re-expression not observed until the second cell cycle after exit from quiescence. Importantly, CIZ1-F is over-expressed in common solid tumors including colon and breast, pronounced in early stage but not highly-proliferative late stage tumors. Moreover, expression was significantly higher in hormone receptor negative breast tumors than receptor positive tumors. Together these data show that CIZ1-F is expressed in proliferating cells in an unusual cell cycle-dependent manner, and suggest that it may have potential as a tumor biomarker.

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The Nuclear Matrix: Fractionation Techniques and Analysis

Abstract: The first descriptions of an insoluble nuclear structure appeared more than 70 years ago, but it is only in recent years that a sophisticated picture of its significance has begun to emerge. Here we introduce multiple methods for the study of the nuclear matrix.

Cited by 10 publications

References 52 publications

Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson–Gilford progeria syndrome fibroblasts

Farnesyltransferase inhibitor and rapamycin correct aberrant genome organisation and decrease DNA damage respectively, in Hutchinson–Gilford progeria syndrome fibroblasts

Phospholipases D1 and D2 regulate cell cycling in primary prostate cancer cells and are differentially associated with the nuclear matrix

CIZ1-F, an alternatively spliced variant of the DNA replication protein CIZ1 with distinct expression and localisation, is overrepresented in early stage common solid tumours

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