A dynamic mode of mitotic bookmarking by transcription factors

Teves, Sheila S; An, Luye; Hansen, Anders S.; Xie, Liangqi; Darzacq, Xavier; Tjian, Robert

doi:10.7554/elife.22280

Cited by 242 publications

(379 citation statements)

References 55 publications

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“…2Y,Z). We thus conclude that detection of RNAP2 in mitotic chromatin is both antibody dependent and that cases of exclusion are likely due to fixation artifacts, as previously reported for transcription factors (Lerner et al 2016;Teves et al 2016). Importantly, the presence of an active form of RNAP2 in mitosis has been reported at the centromere (Liu et al 2015), but our observation agrees with various hints throughout the literature that it is also present at low levels throughout mitotic chromatin.…”

Section: Rna Pol II During Mitosissupporting

confidence: 91%

“…Despite the physical condensation of mitotic chromosomes, global ATAC-seq profiles are highly similar between asynchronous and mitotic cells (Teves et al 2016), indicating an overall retention in accessibility at the molecular level. At a finer level, it has been shown that promoter accessibility is maintained during mitosis (Martinez-Balbas et al 1995;Hsiung et al 2015), although enhancer accessibility is lost (Hsiung et al 2015).…”

Section: Enhancer Usagementioning

confidence: 98%

“…These results suggest that low levels of transcribing RNAP2 are present, and that only when elongation is blocked and there is a backup of RNAP2 at the promoter is the signal detected. In addition, recent studies have shown that formaldehyde cross-linking artifactually removes proteins from mitotic chromatin (Lerner et al 2016;Teves et al 2016). Even though transcription factors were visibly excluded from mitotic chromatin by immunofluorescence (IF), fluorescently labeled fusions of the same proteins colocalized with mitotic chromatin in live cells.…”

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confidence: 99%

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Low-Level, Global Transcription during Mitosis and Dynamic Gene Reactivation during Mitotic Exit

Palozola

Liu

Nicetto

et al. 2017

Cold Spring Harb Symp Quant Biol

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Mitosis is thought to be a period of transcriptional silence due to the compact nature of mitotic chromosomes and the apparent exclusion of RNA Pol II and many transcription factors from mitotic chromatin. Yet accurate reactivation of a cell's specific gene expression program is needed to reestablish functional cell identity after mitosis. The majority of studies on protein regulation and localization during mitosis have relied extensively on antibodies and cross-linking-based approaches that are known to artifactually exclude proteins from mitotic chromatin. Here we show that RNA Pol II localization in mitosis is antibody-and fixation-dependent, and that direct assessment of transcription by pulse-labeling nascent RNA reveals global, low-level mitotic transcription. We also find a hierarchy of gene reactivation as the cells transition from mitosis to their interphase amplitude of gene expression. Resetting of gene transcription during mitotic exit is coincident with enhancer transcription. Our work thus shifts focus from assessing mitotic exit as a binary transcription switch to a more nuanced concert of transcription amplitude and enhancer usage. We suggest that understanding how gene expression patterns are conserved during mitosis rests upon deciphering how transcription is maintained by promoters.During development, lineage specification and differentiation are performed by the successive expression of key transcription factors. Once a cell's functional identity has been established, it is imperative that its identity be maintained by the inheritance of the cell type-specific gene expression profile through cell division. Mitosis is the period of the cell cycle during which the recently duplicated sister chromatids are divided into two separate nuclei. At the onset of mitosis, chromosomes condense to help ensure separation of the sister chromatids. It has been thought that this condensation excludes transcriptional machinery (Martinez-Balbas et al. 1995;Prasanth et al. 2003) resulting in the termination of transcription (Prescott and Bender 1962;Parsons and Spencer 1997), and others showed a global repression of RNA synthesis by nucleotide incorporation (Prescott and Bender 1962;Konrad 1963;Johnson and Holland 1965;Parsons and Spencer 1997). However, there are caveats to each of these studies surrounding the approaches used and conclusions drawn that may have misinterpreted the transcriptional state of mitotic cells.The carboxy-terminal domain (CTD) of Rpb1, the largest and enzymatic subunit of RNAP2, is composed of multiple, conserved heptapeptide repeats of Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Many studies have described the role of CTD posttranslational modifications (PTMs) in the transcription regulatory cycle of recruitment, pausing, initiation, elongation, and termination (Heidemann et al. 2013). In general, phospho-serine 5 RNAP2 (Ser5-P) is found at the transcription start site and diminishes toward the 3′ end of the transcribed region of a gene, whereas phospho-serine 2 RNAP2 (Ser2-P) is low near th...

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Section: Rna Pol II During Mitosissupporting

confidence: 91%

Section: Enhancer Usagementioning

confidence: 98%

mentioning

confidence: 99%

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Low-Level, Global Transcription during Mitosis and Dynamic Gene Reactivation during Mitotic Exit

Palozola

Liu

Nicetto

et al. 2017

Cold Spring Harb Symp Quant Biol

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“…It has been known for ~ 60 years that mitotic chromosome condensation is correlated with transcriptional repression 39,40 . Studies in the past two decades have since shown that this repression is not absolute because transcription factors can still access the interior of mitotic chromosomes [41][42][43] and because lowlevel transcription is detectable in mitotic mammalian cells [44][45][46] . In S. pombe G0 cells, we also observed a correlation between smaller nucleus size (a proxy for chromatin condensation) and transcriptional repression, in line with previous studies 8, 10 .…”

Section: Discussionmentioning

confidence: 99%

Macromolecular and cytological changes in fission yeast G0 nuclei

Cai

Nie

et al. 2020

Preprint

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Cells change their cytology in response to environmental cues and stress. Notably, large changes in nuclear architecture are accompanied by transcriptional reprogramming. When starved of nitrogen, Schizosaccharomyces pombe cells become rounder and they enter a quiescent "G0" state. These cells have smaller nuclei and undergo near-global transcriptional repression. Here we use electron cryotomography (cryo-ET) and cell-biology approaches to investigate the structural and biochemical changes of G0 S. pombe nuclei. We find that G0 cells have a denser nucleoplasm and fewer chromatin-associated multi-megadalton globular complexes (megacomplexes) than proliferating cells. These structural changes are correlated with mild histone deacetylation. Induced histone hyperacetylation in G0 results in cells that have larger nuclei and less condensed chromatin. However, these histone-hyperacetylated G0 cells still have repressed transcription, few megacomplexes, and a dense nucleoplasm. Like in budding yeast, S. pombe G0 nuclear phenotypes are controlled by multiple biochemical factors.

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“…29 Moreover, chemical fixation of cells has been demonstrated to cause artefacts in the dynamic detection of transcription factors. 30 Live-cell imaging can visualise the movement of select proteins dynamically, but is limited to transgenic cell lines, which limits multiplexing (number of targets measured concurrently).…”

Section: Introductionmentioning

confidence: 99%

Electrophoretic cytometry of adherent cells

Herr

2017

Lab Chip

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Cell-matrix and cell-cell interactions influence intracellular signalling and play an important role in physiologic and pathologic processes. Detachment of cells from the surrounding microenvironment alters intracellular signalling. Here, we demonstrate and characterise an integrated microfluidic device to culture single and clustered cells in tuneable microenvironments and then directly analyse the lysate of each cell in situ, thereby eliminating the need to detach cells prior to analysis. First, we utilise microcontact printing to pattern cells in confined geometries. We then utilise a microscale isoelectric focusing (IEF) module to separate, detect, and analyse lamin A/C from substrate-adhered cells seeded and cultured at varying (500, 2000, and 9000 cells per cm2) densities. We report separation performance (minimum resolvable pI difference of 0.11) that is on par with capillary IEF and independent of cell density. Moreover, we map lamin A/C and β-tubulin protein expression to morphometric information (cell area, circumference, eccentricity, form factor, and cell area factor) of single cells and observe poor correlation with each of these parameters. By eliminating the need for cell detachment from substrates, we enhance detection of cell receptor proteins (CD44 and β-integrin) and dynamic phosphorylation events (pMLCS19) that are rendered undetectable or disrupted by enzymatic treatments. Finally, we optimise protein solubilisation and separation performance by tuning lysis and electrofocusing (EF) durations. We observe enhanced separation performance (decreased peak width) with longer EF durations by 25.1% and improved protein solubilisation with longer lysis durations. Overall, the combination of morphometric analyses of substrate-adhered cells, with minimised handling, will yield important insights into our understanding of adhesion-mediated signalling processes.

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A dynamic mode of mitotic bookmarking by transcription factors

Cited by 242 publications

References 55 publications

Low-Level, Global Transcription during Mitosis and Dynamic Gene Reactivation during Mitotic Exit

Low-Level, Global Transcription during Mitosis and Dynamic Gene Reactivation during Mitotic Exit

Macromolecular and cytological changes in fission yeast G0 nuclei

Electrophoretic cytometry of adherent cells

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