Mapping of DNAase I sensitive regions on mitotic chromosomes

Kerem, Batsheva; Goitein, Ruth; Diamond, Gill; Cedar, Howard; Marcus, Menashe

doi:10.1016/0092-8674(84)90504-x

Cited by 134 publications

(53 citation statements)

References 42 publications

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“…It is tempting to speculate that this tagging mechanism may involve aspects of chromatin structure that are independent of transcription factor binding. Evidence supporting this speculation comes from studies showing that DNase I sensitivity patterns characteristic of actively transcribed genes persist on mitotic chromosomes (25). Similarly, mechanisms would need to exist to maintain transcriptional silencing on those genes that are not to be expressed at the end of mitosis.…”

Section: Discussionmentioning

confidence: 94%

Mitotic Repression of RNA Polymerase II Transcription Is Accompanied by Release of Transcription Elongation Complexes

Parsons

Spencer

1997

Molecular and Cellular Biology

119

111

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Nuclear RNA synthesis is repressed during the mitotic phase of each cell cycle. Although total RNA synthesis remains low throughout mitosis, the degree of RNA polymerase II transcription repression on specific genes has not been examined. In addition, it is not known whether mitotic repression of RNA polymerase II transcription is due to polymerase pausing or ejection of transcription elongation complexes from mitotic chromosomes. In this study, we show that RNA polymerase II transcription is repressed in mammalian cells on a number of specific gene regions during mitosis. We also show that the majority of RNA polymerase II transcription elongation complexes are physically excluded from mitotic chromosomes between late prophase and late telophase. Despite generalized transcription repression and stripping of RNA polymerase II complexes from DNA, arrested RNA polymerase II ternary complexes appear to remain on some gene regions during mitosis. The cyclic repression of transcription and ejection of RNA polymerase II transcription elongation complexes may help regulate the transcriptional events that control cell cycle progression and differentiation.In higher eukaryotes, mitosis is accompanied by dramatic and reversible transformations to the structural organization of both cytoplasm and nucleus (reviewed in references 12, 28, 39, and 40). Mitosis is also accompanied by profound biochemical changes, including a decline in nuclear RNA synthesis. Mitotic repression of transcription was first noted over 30 years ago, in studies analyzing incorporation of RNA precursors during the cell cycle (22,29,44,68,69). In these studies, it was observed that incorporation of radioactive precursors into nuclear RNA declines in early to mid-prophase and resumes in late telophase. The precise degree of mitotic transcription repression remains uncertain, with some studies detecting mitotic RNA synthesis at 16 to 24% of interphase levels (22,27,29,80). In addition, it is not clear to what degree transcription by each of the three nuclear RNA polymerases (RNAPs) is repressed during mitosis. As approximately 75 to 80% of RNA synthesis in cycling cells is due to RNAP I activity (32,46,77), it is possible that some RNAP I, II, or III transcription may escape mitotic repression but be undetectable by pulse-labeling. In addition, pulse-labeling assays measure steady-state RNA levels, which arise through a combination of RNA synthesis and degradation. Therefore, it is possible that part of the loss of labeled steady-state RNA in mitotic cells is due to degradation of nascent labeled RNAs.Although mitotic repression of RNAP I and III activity has been examined in some detail in the last few years (16,19,23,51,54,72,73), few studies of RNAP II transcription during mitosis have been reported. The in situ hybridization studies of Shermoen and O'Farrell (59) demonstrate that nascent transcripts from the Drosophila RNAP II-transcribed gene Ubx are aborted and degraded during mitosis. These data suggest that RNAP II transcription complexes may be ...

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Section: Discussionmentioning

confidence: 94%

Mitotic Repression of RNA Polymerase II Transcription Is Accompanied by Release of Transcription Elongation Complexes

Parsons

Spencer

1997

Molecular and Cellular Biology

119

111

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“…The present paper describes experiments in which a modification ofthe techniques used by others (8,9) is used to explore the nuclear location of exposed DNA in normal Chinese hamster ovary fibroblasts, the malignant CHO-K1, and in the latter cell after reverse transformation by cAMP reagents.…”

mentioning

confidence: 99%

The spatial distribution of exposed nuclear DNA in normal, cancer, and reverse-transformed cells.

Krystosek

Puck²

1990

Proc. Natl. Acad. Sci. U.S.A.

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The malignant CHO-K1 cell is reversetransformed by cAMP, regaining the phenotype of a normal fibroblast. During this reaction, much of its DNA re-acquires sensitivity to hydrolysis by DNase I in a way characteristic of the normal fibroblast. Exposed DNA forms a rim about the nucleus in both the normal and reverse-transformed cell but not in the malignant CHO-K1. Reacquisition of the nuclear rim requires an organized cytoskeleton. Sequestered DNA forms families of different degrees of sequestration. In accordance with previous theoretical developments it is proposed that (1) genes specific to a given differentiation state are stored in the nuclear rim, whereas genes specific to other states are sequestered within the nucleus; (il) only exposed genes are active, and their activity is modulated by regulatory molecules in the fluid medium; (iii) exposure and sequestration are regulated by cytoskeletal and nuclear protein structures; (iv) in at least several types of cancer the regulatory defect lies in the genome exposure process so that the specific DNA sequences and their associated growth regulatory loci have been transferred from the exposed to the sequestered condition with consequent loss of the nuclear rim of exposed DNA. The methodology described should be generally applicable to examining the accessibility state of subsets of DNA during various physiological modulations of cell function.Reverse transformation is a reaction in which malignant cells are restored to a normal phenotype in response to particular agonists as in the CHO-K1 cell treated with cAMP derivatives (1). Reverse transformation involves a succession of complex metabolic changes varying from early alterations in calcium ion dynamics occurring during the first minute (2) to changes in membrane and cytoskeletal structures (3, 4) and protein syntheses and phosphorylations (5-7) that extend over >48 hr. Ultimately, the pathway must result in specific changes in nuclear gene activity that restore specific differentiation and growth-control properties to the affected cells. We showed that an increase in DNA sensitivity to hydrolysis, which affects specific nuclear genes and involves a substantial fraction of the genome, occurs in reverse transformation (10,11). This large-scale increase in DNase I sensitivity of chromatin after treatment of cells with specific reversetransformation agents is now termed genome exposure. It was demonstrated that cytoskeletal integrity is required for this reaction and that the degree of increased exposure achieved in reverse transformation resembles that in normal cells (12).Hutchison and Weintraub (9) showed that DNase I-sensitive regions of the genome in mouse L cells are preferentially localized at the nuclear periphery. The present paper describes experiments in which a modification ofthe techniques used by others (8, 9) is used to explore the nuclear location of exposed DNA in normal Chinese hamster ovary fibroblasts, the malignant CHO-K1, and in the latter cell after reverse transformation by cAMP reag...

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“…In order to analyze the occurrence of SSB directly at a level of individual cells, in situ nick translation (ISNT) could be a powerful tool [8,19,29], one which was originally developed to investigate the region of actively transcribed chromatin [14,22] and was performed with radioactive compounds. However, the radioactive ISNT is generally time-consuming and expensive, and it is hard to localize the sites of nicks precisely on the subnuclear basis.…”

mentioning

confidence: 99%

Nonradioactive in Situ Nick Translation: A Useful Molecular Histochemical Tool to Detect Single-Stranded DNA Breaks.

Koji

1996

Acta Histochem. Cytochem

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Mapping of DNAase I sensitive regions on mitotic chromosomes

Cited by 134 publications

References 42 publications

Mitotic Repression of RNA Polymerase II Transcription Is Accompanied by Release of Transcription Elongation Complexes

Mitotic Repression of RNA Polymerase II Transcription Is Accompanied by Release of Transcription Elongation Complexes

The spatial distribution of exposed nuclear DNA in normal, cancer, and reverse-transformed cells.

Nonradioactive in Situ Nick Translation: A Useful Molecular Histochemical Tool to Detect Single-Stranded DNA Breaks.

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