Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization

Clements, Craig Steven; Bikkul, Ural; Ahmed, Mai Hassan; Foster, Helen A.; Godwin, Lauren S.; Bridger, Joanna M.

doi:10.1007/978-1-4939-3530-7_24

Cited by 8 publications

(21 citation statements)

References 51 publications

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“…Error bars represent SE of the mean ( SEM ). F displays the nuclear position of the telomeres in NB1, NB1, T06, and T08 cells after erosion analysis measuring the percentage of the cy3 telomere signal (%), normalized by the percentage of DAPI signal, over five concentric shells of equal area from the nuclear periphery to interior. The x ‐axis displays the shells from 1 to 5 (left to right), with 1 being the most peripheral shell and 5 being the most internal shell.…”

Section: Resultsmentioning

confidence: 99%

“…Aged slides were transferred into a denaturing solution (70% (vol/vol) formamide, 2X saline sodium citrate (SSC) at 70°C for 2 minutes. The slides were plunged into ice‐cold 70% ethanol and then passed through the ethanol row …”

Section: Methodsmentioning

confidence: 99%

“…At least 50 images per slide were captured by Smart Capture 3.00 software and signal position analyzed with an erosion analysis script using IPLab Spectrum software. The erosion analysis script (used with kind permission of Prof. Wendy Bickmore and Dr. Paul Perry, MRC Human Genetics Unit) was devised to divide each captured nucleus into five concentric shells of equal area, the first shell being at the periphery of the nuclei ending in the interior of the nuclei (fifth shell) . The erosion script measures the pixel signal intensity of DAPI and the chromosome probe.…”

Section: Methodsmentioning

confidence: 99%

“…Wendy Bickmore and Dr. Paul Perry, MRC Human Genetics Unit) was devised to divide each captured nucleus into five concentric shells of equal area, the first shell being at the periphery of the nuclei ending in the interior of the nuclei (fifth shell). 33,77 The erosion script measures the pixel signal intensity of DAPI and the chromosome probe. The percentage chromosome signal intensity measurement per shell was divided by the percentage DNA signal intensity measurement of the same shell in order to normalize the data.…”

Section: Metaphase Chromosome Preparationsmentioning

confidence: 99%

“…Aged slides were transferred into a denaturing solution (70% (vol/vol) formamide, 2X saline sodium citrate (SSC) at 70 C for 2 minutes. The slides were plunged into ice-cold 70% ethanol and then passed through the ethanol row 77. The chromosome templates 18 and X were made in-house byamplifying flow-sorted chromosome arms (a kind gift from Dr Michael Bittner) by degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR).…”

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confidence: 99%

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Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson‐Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform

Bikkul

Faragher

Worthington

et al. 2019

Genes Chromosomes & Cancer

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Immortalizing primary cells with human telomerase reverse transcriptase (hTERT) has been common practice to enable primary cells to be of extended use in the laboratory because they avoid replicative senescence. Studying exogenously expressed hTERT in cells also affords scientists models of early carcinogenesis and telomere behavior. Control and the premature ageing disease—Hutchinson‐Gilford progeria syndrome (HGPS) primary dermal fibroblasts, with and without the classical G608G mutation have been immortalized with exogenous hTERT. However, hTERT immortalization surprisingly elicits genome reorganization not only in disease cells but also in the normal control cells, such that whole chromosome territories normally located at the nuclear periphery in proliferating fibroblasts become mislocalized in the nuclear interior. This includes chromosome 18 in the control fibroblasts and both chromosomes 18 and X in HGPS cells, which physically express an isoform of the LINC complex protein SUN1 that has previously only been theoretical. Additionally, this HGPS cell line has also become genomically unstable and has a tetraploid karyotype, which could be due to the novel SUN1 isoform. Long‐term treatment with the hTERT inhibitor BIBR1532 enabled the reduction of telomere length in the immortalized cells and resulted that these mislocalized internal chromosomes to be located at the nuclear periphery, as assessed in actively proliferating cells. Taken together, these findings reveal that elongated telomeres lead to dramatic chromosome mislocalization, which can be restored with a drug treatment that results in telomere reshortening and that a novel SUN1 isoform combined with elongated telomeres leads to genomic instability. Thus, care should be taken when interpreting data from genomic studies in hTERT‐immortalized cell lines.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Metaphase Chromosome Preparationsmentioning

confidence: 99%

mentioning

confidence: 99%

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Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson‐Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform

Bikkul

Faragher

Worthington

et al. 2019

Genes Chromosomes & Cancer

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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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PIWI silencing mechanism involving the retrotransposonnimbusorchestrates resistance to infection withSchistosoma mansoniin the snail vector,Biomphalaria glabrata

Smith

Yadav

Akinyele

et al. 2021

Preprint

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BackgroundSchistosomiasis remains widespread in many regions despite efforts at its elimination. By examining changes in the transcriptome at the host-pathogen interface in the snail Biomphalaria glabrata and the blood fluke Schistosoma mansoni, we previously demonstrated that an early stress response in juvenile snails, manifested by induction of heat shock protein 70 (Hsp 70) and Hsp 90 and of the reverse transcriptase (RT) domain of the B. glabrata non-LTR-retrotransposon, nimbus, were critical for B. glabrata susceptibility to S. mansoni. Subsequently, juvenile B. glabrata BS90 snails, resistant to S. mansoni at 25°C become susceptible by the F2 generation when maintained at 32°C, indicating an epigenetic response.Methodology/Principal FindingsTo better understand this plasticity in susceptibility of the BS90 snail, mRNA sequences were examined from S. mansoni exposed juvenile BS90 snails cultured either at 25°C (permissive temperature) or 32°C (non-permissive). Comparative analysis of transcriptomes from snails cultured at the non-permissive and permissive temperatures revealed that whereas stress related transcripts dominated the transcriptome of susceptible BS90 juvenile snails at 32°C, transcripts encoding proteins with a role in epigenetics, such as PIWI (BgPiwi), chromobox protein homolog 1 (BgCBx1), histone acetyl transferase histone deacetylase (HDAC) and metallotransferase (MT) were highly expressed in those cultured at 25°C. To further determine a role for BgPiwi in B. glabrata susceptibility to S. mansoni, siRNA corresponding to the BgPiwi encoding transcript was utilized to suppress expression of BgPiwi, rendering the resistant BS90 juvenile snail susceptible to infection at 25°C. Given transposon silencing activity of PIWI as a facet of its role as guardian of the integrity of the genome, we examined the expression of the nimbus RT encoding transcript at 120 min after infection of resistant BS90 piwi-siRNA treated snails. We observed that nimbus RT was upregulated, indicating that modulation of the transcription of the nimbus RT was associated with susceptibility to S. mansoni in BgPiwi- siRNA treated BS90 snails. Furthermore, treatment of susceptible snails with the RT inhibitor lamivudine, before exposure to S. mansoni, blocked S. mansoni infection concurrent with downregulation of the nimbus RT transcript and upregulation of the BgPiwi encoding transcript in the lamivudine-treated, schistosome-exposed susceptible snails.Conclusions and SignificanceThese findings support a role for the interplay of BgPiwi and nimbus in the epigenetic modulation of plasticity of resistance/susceptibility in the snail-schistosome relationship.

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Visualizing the Spatial Relationship of the Genome with the Nuclear Envelope Using Fluorescence In Situ Hybridization

Cited by 8 publications

References 51 publications

Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson‐Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform

Telomere elongation through hTERT immortalization leads to chromosome repositioning in control cells and genomic instability in Hutchinson‐Gilford progeria syndrome fibroblasts, expressing a novel SUN1 isoform

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

PIWI silencing mechanism involving the retrotransposonnimbusorchestrates resistance to infection withSchistosoma mansoniin the snail vector,Biomphalaria glabrata

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