Isolation of Extrachromosomal Elements by Histone Immunoprecipitation

Kuschak, Theodore I.; Kuschak, Brenda; Smith, G.M.; Wright, Jim A.; Mai, Sabine

doi:10.2144/01305rr05

Cited by 5 publications

(2 citation statements)

References 23 publications

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“…The supernatant was phenol-chloroform extracted three times and the Hirt DNA was ethanol precipitated. To check for any genomic DNA contamination before phenol-chloroform extraction, the Hirt supernatant was subjected to methanol-acetic acid fixation on slides and examined by DAPI staining 48 . The precipitated Hirt DNA was also examined by standard electrophoresis agarose gel and ethidium bromide staining.…”

Section: Hirt Ecc Dna Isolation and Detectionmentioning

confidence: 99%

H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability

2006

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Investigations aimed at identifying regulators of nuclear architecture in Drosophila demonstrated that cells lacking H3K9 methylation and RNA interference (RNAi) pathway components displayed disorganized nucleoli, ribosomal DNA (rDNA) and satellite DNAs. The levels of H3K9 dimethylation (H3K9me2) in chromatin associated with repeated DNAs decreased dramatically in Su(var)3-9 and dcr-2 (dicer-2) mutant tissues compared with wild type. We also observed a substantial increase in extrachromosomal circular (ecc) repeated DNAs in mutant tissues. The disorganized nucleolus phenotype depends on the presence of Ligase 4 and ecc DNA formation is not induced by removal of cohesin. We conclude that the structural integrity and organization of repeated DNAs and nucleoli are regulated by the H3K9 methylation and RNAi pathways, and other regulators of heterochromatin-mediated silencing. In addition, repeated DNA stability involves suppression of non-homologous end joining (NHEJ) or other recombination pathways. These results suggest a mechanism for how local chromatin structure can regulate genome stability, and the organization of chromosomal elements and nuclear organelles.Nuclei and chromosomes maintain specific and dynamic architectures that are required for many essential functions 1 . Nuclear bodies are involved in diverse biological processes and exhibit dynamic mobility, and individual chromosomes occupy distinct domains within interphase nuclei2. Chromosomes in the metazoan interphase nucleus are comprised of two types of cytologically and functionally distinct chromatin, euchromatin and heterochromatin3. Patterns of posttranslational histone modifications associated with these domains are strongly correlated with functions such as gene regulation, chromosome inheritance and replication timing4. For example, regions that display heterochromatinmediated gene silencing are rich in histone H3K9 methylation and lack many histone acetylations, whereas histones in transcriptionally active euchromatic regions are highly acetylated and methylated at H3K4 (ref. 5).The first indication that chromosome organization can affect gene expression stems from the discovery of position effect variegation (PEV) in Drosophila 6 . PEV describes the epigenetic inactivation or silencing of a euchromatic gene that has been positioned close to or within ©2007 Nature Publishing Group 3 Correspondence should be addressed to G.H.K. (karpen@fruitfly.org). AUTHOR CONTRIBUTIONS I.P. performed all the experiments, and J.P. and G.K. collaborated on data analysis and project planning.Note: Supplementary Information is available on the Nature Cell Biology website. COMPETING FINANCIAL INTERESTSThe authors declare that they have no competing financial interests. The nucleolus, the site of ribosome assembly, is an example of an essential nuclear organelle. The nucleolus organizer region (NOR) contains tandemly repeated ribosomal DNAs (rDNAs), and is embedded in heterochromatin in most eukaryotes. Single rDNA genes inserted into euchromatin are able...

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Section: Hirt Ecc Dna Isolation and Detectionmentioning

confidence: 99%

H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability

2006

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“…Previously it has been shown that plasmids containing matrix attachment regions (MARs) are maintained in double minute extrachromosomal elements in HeLa cells [65]. These are autonomously replicating extrachromosomal elements that are up to a few Mb in size [66], have been known to be associated with active histones [67] and can be purified by histone immunoprecipitation [68]. MARs and scaffold attachment regions (SARs), together named as S/MARs, are regions on the DNA which attach to nuclear matrix and have been associated with functions such as anchoring of DNA, maintenance of nuclear architecture, regulation of transcription and replication.…”

Section: Discussionmentioning

confidence: 99%

BAC-based cellular model for screening regulators of BDNF gene transcription

et al. 2014

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BackgroundBrain derived neurotrophic factor (BDNF) belongs to a family of structurally related proteins called neurotrophins that have been shown to regulate survival and growth of neurons in the developing central and peripheral nervous system and also to take part in synaptic plasticity related processes in adulthood. Since BDNF is associated with several nervous system disorders it would be beneficial to have cellular reporter system for studying its expression regulation.MethodsUsing modified bacterial artificial chromosome (BAC), we generated several transgenic cell lines expressing humanised Renilla luciferase (hRluc)-EGFP fusion reporter gene under the control of rat BDNF gene regulatory sequences (rBDNF-hRluc-EGFP) in HeLa background. To see if the hRluc-EGFP reporter was regulated in response to known regulators of BDNF expression we treated cell lines with substances known to regulate BDNF and also overexpressed transcription factors known to regulate BDNF gene in established cell lines.ResultsrBDNF-hRluc-EGFP cell lines had high transgene copy numbers when assayed with qPCR and FISH analysis showed that transgene was maintained episomally in all cell lines. Luciferase activity in transgenic cell lines was induced in response to ionomycin-mediated rise of intracellular calcium levels, treatment with HDAC inhibitors and by over-expression of transcription factors known to increase BDNF expression, indicating that transcription of the transgenic reporter is regulated similarly to the endogenous BDNF gene.ConclusionsGenerated rBDNF-hRluc-EGFP BAC cell lines respond to known modulators of BDNF expression and could be used for screening of compounds/small molecules or transcription factors altering BDNF expression.

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Formation of non-random extrachromosomal elements during development, differentiation and oncogenesis

Kuttler

Mai

2007

Seminars in Cancer Biology

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Isolation of Extrachromosomal Elements by Histone Immunoprecipitation

Cited by 5 publications

References 23 publications

H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability

H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability

BAC-based cellular model for screening regulators of BDNF gene transcription

Formation of non-random extrachromosomal elements during development, differentiation and oncogenesis

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