Show and tell: visualizing gene expression in living cells

Rafalska-Metcalf, Ilona U.; Janicki, Susan M.

doi:10.1242/jcs.008664

Cited by 28 publications

(26 citation statements)

References 72 publications

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“…Since many of these factors were discovered using techniques, which disrupt cellular architecture and average effects in cell populations, it is not known how or when critical regulators are selectively recruited to specific genomic sites in intact cells. One of the pivotal advances to imaging chromatin in single cells has been the development of reporter plasmids carrying sequence elements from bacteria and bacteriophage that allow DNA and RNA to be visualized in living cells (Rafalska-Metcalf and Janicki, 2007). The genomic sites into which these plasmids integrate can be visualized when their binding proteins, fused to auto-fluorescent proteins are expressed.…”

Section: Introductionmentioning

confidence: 99%

Single cell analysis of Daxx and ATRX-dependent transcriptional repression

Newhart¹,

Rafalska-Metcalf²,

Yang³

et al. 2012

Journal of Cell Science

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SummaryHistone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0-expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function.

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Section: Introductionmentioning

confidence: 99%

Single cell analysis of Daxx and ATRX-dependent transcriptional repression

Newhart¹,

Rafalska-Metcalf²,

Yang³

et al. 2012

Journal of Cell Science

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“…Such studies have revealed widespread pausing and abortion by engaged Pol II (5)(6)(7)(8)(9)(10). Pol II dynamics have also been studied using fluorescence imaging (11)(12)(13)(14)(15), but it remains difficult to observe both the rapid recycling of Pol II (14) and the unstable nascent transcripts.…”

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confidence: 99%

A wave of nascent transcription on activated human genes

Wada

Ohta

et al. 2009

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

148

201

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Genome-wide studies reveal that transcription by RNA polymerase II (Pol II) is dynamically regulated. To obtain a comprehensive view of a single transcription cycle, we switched on transcription of five long human genes (>100 kbp) with tumor necrosis factor-␣ (TNF␣) and monitored (using microarrays, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation) the appearance of nascent RNA, changes in binding of Pol II and two insulators (the cohesin subunit RAD21 and the CCCTC-binding factor CTCF), and modifications of histone H3. Activation triggers a wave of transcription that sweeps along the genes at Ϸ3.1 kbp/min; splicing occurs cotranscriptionally, a major checkpoint acts several kilobases downstream of the transcription start site to regulate polymerase transit, and Pol II tends to stall at cohesin/CTCF binding sites.endothelial cell ͉ polymerase II ͉ RNA ͉ tumor necrosis factor alpha

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“…However, multi-copy arrays of transgenes containing sequence elements that allow nuclei acids to be visualized in vivo can be easily localized (Rafalska-Metcalf and Janicki, 2007; Yunger et al, 2013a). When RNA pol II transcription units are included in these transgenes, transcriptional dynamics can be directly visualized at these arrays.…”

Section: Presentationmentioning

confidence: 99%

Seeing Is Believing: Visualizing Transcriptional Dynamics in Single Cells

Newhart

Janicki

2013

Journal Cellular Physiology

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For a gene to be expressed, the functions of multiple molecular machines must be coordinated at the site of transcription. To understand the role of nuclear organization in transcription, it is necessary to visualize the dynamic interactions of regulatory factors with chromatin and RNA. It is currently possible to localize individual transcription sites in single living mammalian cells by engineering reporter gene constructs to include sequence elements which permit the visualization of nucleic acids in vivo. Upon stable integration, these transgenes form chromatinized arrays, which can be imaged during activation to obtain high-resolution quantitative information about transcriptional dynamics. Modeling can suggest new hypotheses about gene regulation, which can be tested both in the single-cell imaging system and at endogenous genes. This gene-specific imaging strategy has the potential to reveal regulatory mechanisms, which would be difficult to imagine outside of single living cells.

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Show and tell: visualizing gene expression in living cells

Cited by 28 publications

References 72 publications

Single cell analysis of Daxx and ATRX-dependent transcriptional repression

Single cell analysis of Daxx and ATRX-dependent transcriptional repression

A wave of nascent transcription on activated human genes

Seeing Is Believing: Visualizing Transcriptional Dynamics in Single Cells

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