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Article: Dynamic transcription regulation at the single-molecule level

Wang, Zuhui / Deng, Wulan

Developmental biology. 2022 Feb., v. 482

2022

Abstract: Cell fate changes during development, differentiation, and reprogramming are largely controlled at the transcription level. The DNA-binding transcription factors (TFs) often act in a combinatorial fashion to alter chromatin states and drive cell type- ... ...

Abstract	Cell fate changes during development, differentiation, and reprogramming are largely controlled at the transcription level. The DNA-binding transcription factors (TFs) often act in a combinatorial fashion to alter chromatin states and drive cell type-specific gene expression. Recent advances in fluorescent microscopy technologies have enabled direct visualization of biomolecules involved in the process of transcription and its regulatory events at the single-molecule level in living cells. Remarkably, imaging and tracking individual TF molecules at high temporal and spatial resolution revealed that they are highly dynamic in searching and binding cognate targets, rather than static and binding constantly. In combination with investigation using techniques from biochemistry, structure biology, genetics, and genomics, a more well-rounded view of transcription regulation is emerging. In this review, we briefly cover the technical aspects of live-cell single-molecule imaging and focus on the biological relevance and interpretation of the single-molecule dynamic features of transcription regulatory events observed in the native chromatin environment of living eukaryotic cells. We also discuss how these dynamic features might shed light on mechanistic understanding of transcription regulation.
Keywords	chromatin ; fluorescence microscopy ; gene expression ; genomics ; transcription (genetics)
Language	English
Dates of publication	2022-02
Size	p. 67-81.
Publishing place	Elsevier Inc.
Document type	Article
ZDB-ID	1114-9
ISSN	1095-564X ; 0012-1606
ISSN (online)	1095-564X
ISSN	0012-1606
DOI	10.1016/j.ydbio.2021.11.004
Database	NAL-Catalogue (AGRICOLA)

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Article ; Online: Dynamic transcription regulation at the single-molecule level.

Wang, Zuhui / Deng, Wulan

Developmental biology

2021 Volume 482, Page(s) 67–81

Abstract	Cell fate changes during development, differentiation, and reprogramming are largely controlled at the transcription level. The DNA-binding transcription factors (TFs) often act in a combinatorial fashion to alter chromatin states and drive cell type-specific gene expression. Recent advances in fluorescent microscopy technologies have enabled direct visualization of biomolecules involved in the process of transcription and its regulatory events at the single-molecule level in living cells. Remarkably, imaging and tracking individual TF molecules at high temporal and spatial resolution revealed that they are highly dynamic in searching and binding cognate targets, rather than static and binding constantly. In combination with investigation using techniques from biochemistry, structure biology, genetics, and genomics, a more well-rounded view of transcription regulation is emerging. In this review, we briefly cover the technical aspects of live-cell single-molecule imaging and focus on the biological relevance and interpretation of the single-molecule dynamic features of transcription regulatory events observed in the native chromatin environment of living eukaryotic cells. We also discuss how these dynamic features might shed light on mechanistic understanding of transcription regulation.
MeSH term(s)	Animals ; Chromatin/metabolism ; DNA/biosynthesis ; Gene Expression Regulation/genetics ; Single Molecule Imaging ; Single-Cell Analysis ; Transcription Factors/metabolism ; Transcription, Genetic/genetics
Chemical Substances	Chromatin ; Transcription Factors ; DNA (9007-49-2)
Language	English
Publishing date	2021-12-09
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	1114-9
ISSN	1095-564X ; 0012-1606
ISSN (online)	1095-564X
ISSN	0012-1606
DOI	10.1016/j.ydbio.2021.11.004
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Article ; Online: Detecting Long-Range Enhancer-Promoter Interactions by Quantitative Chromosome Conformation Capture.

Deng, Wulan / Blobel, Gerd A

Methods in molecular biology (Clifton, N.J.)

2017 Volume 1468, Page(s) 51–62

Abstract: Chromosome conformation capture (3C) technology and its derivatives are currently the primary methodologies measuring contacts among genomic elements. In fact, the lion share of what is currently known about chromosome folding is based on 3C-related ... ...

Abstract	Chromosome conformation capture (3C) technology and its derivatives are currently the primary methodologies measuring contacts among genomic elements. In fact, the lion share of what is currently known about chromosome folding is based on 3C-related approaches. For example, distal enhancers are commonly in physically proximity with their target genes, forming chromatin loops. Additional layers of chromatin organization have been described using 3C-based techniques, including topological domains (TADs) and sub-TADs. Finally, inter-chromosomal interactions have been reported although they are much less frequent. 3C is becoming increasingly widespread in its use for understanding genome organization. Here we provide a protocol for quantitative 3C using real-time PCR analysis, along with essential quality controls and normalization methods.
Language	English
Publishing date	2017
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-4035-6_6
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Article ; Online: Nuclear RNA homeostasis promotes systems-level coordination of cell fate and senescence.

Han, Xue / Xing, Linqing / Hong, Yantao / Zhang, Xuechun / Hao, Bo / Lu, J Yuyang / Huang, Mengyuan / Wang, Zuhui / Ma, Shaoqian / Zhan, Ge / Li, Tong / Hao, Xiaowen / Tao, Yibing / Li, Guanwen / Zhou, Shuqin / Zheng, Zheng / Shao, Wen / Zeng, Yitian / Ma, Dacheng /

Zhang, Wenhao / Xie, Zhen / Deng, Haiteng / Yan, Jiangwei / Deng, Wulan / Shen, Xiaohua

Cell stem cell

2024

Abstract: Understanding cellular coordination remains a challenge despite knowledge of individual pathways. The RNA exosome, targeting a wide range of RNA substrates, is often downregulated in cellular senescence. Utilizing an auxin-inducible system, we observed ... ...

Abstract	Understanding cellular coordination remains a challenge despite knowledge of individual pathways. The RNA exosome, targeting a wide range of RNA substrates, is often downregulated in cellular senescence. Utilizing an auxin-inducible system, we observed that RNA exosome depletion in embryonic stem cells significantly affects the transcriptome and proteome, causing pluripotency loss and pre-senescence onset. Mechanistically, exosome depletion triggers acute nuclear RNA aggregation, disrupting nuclear RNA-protein equilibrium. This disturbance limits nuclear protein availability and hinders polymerase initiation and engagement, reducing gene transcription. Concurrently, it promptly disrupts nucleolar transcription, ribosomal processes, and nuclear exporting, resulting in a translational shutdown. Prolonged exosome depletion induces nuclear structural changes resembling senescent cells, including aberrant chromatin compaction, chromocenter disassembly, and intensified heterochromatic foci. These effects suggest that the dynamic turnover of nuclear RNA orchestrates crosstalk between essential processes to optimize cellular function. Disruptions in nuclear RNA homeostasis result in systemic functional decline, altering the cell state and promoting senescence.
Language	English
Publishing date	2024-04-12
Publishing country	United States
Document type	Journal Article
ZDB-ID	2375354-7
ISSN	1875-9777 ; 1934-5909
ISSN (online)	1875-9777
ISSN	1934-5909
DOI	10.1016/j.stem.2024.03.015
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Article ; Online: Manipulating nuclear architecture.

Deng, Wulan / Blobel, Gerd A

Current opinion in genetics & development

2013 Volume 25, Page(s) 1–7

Abstract: The eukaryotic genome is highly organized in the nucleus. Genes can be localized to specific nuclear compartments in a manner reflecting their activity. A plethora of recent reports has described multiple levels of chromosomal folding that can be related ...

Abstract	The eukaryotic genome is highly organized in the nucleus. Genes can be localized to specific nuclear compartments in a manner reflecting their activity. A plethora of recent reports has described multiple levels of chromosomal folding that can be related to gene-specific expression states. Here we discuss studies designed to probe the causal impact of genome organization on gene expression. The picture that emerges is that of a reciprocal relationship in which nuclear organization is not only shaped by gene expression states but also directly influences them.
MeSH term(s)	Animals ; Cell Nucleus/genetics ; Chromatin/chemistry ; Chromatin/genetics ; Gene Expression ; Genome ; Humans
Chemical Substances	Chromatin
Language	English
Publishing date	2013-12-12
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Review
ZDB-ID	1077312-5
ISSN	1879-0380 ; 0959-437X
ISSN (online)	1879-0380
ISSN	0959-437X
DOI	10.1016/j.gde.2013.10.014
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Article ; Online: The transcriptional coactivator RUVBL2 regulates Pol II clustering with diverse transcription factors.

Wang, Hui / Li, Boyuan / Zuo, Linyu / Wang, Bo / Yan, Yan / Tian, Kai / Zhou, Rong / Wang, Chenlu / Chen, Xizi / Jiang, Yongpeng / Zheng, Haonan / Qin, Fangfei / Zhang, Bin / Yu, Yang / Liu, Chao-Pei / Xu, Yanhui / Gao, Juntao / Qi, Zhi / Deng, Wulan /

Ji, Xiong

Nature communications

2022 Volume 13, Issue 1, Page(s) 5703

Abstract: RNA polymerase II (Pol II) apparatuses are compartmentalized into transcriptional clusters. Whether protein factors control these clusters remains unknown. In this study, we find that the ATPase-associated with diverse cellular activities (AAA + ) ATPase ...

Abstract	RNA polymerase II (Pol II) apparatuses are compartmentalized into transcriptional clusters. Whether protein factors control these clusters remains unknown. In this study, we find that the ATPase-associated with diverse cellular activities (AAA + ) ATPase RUVBL2 co-occupies promoters with Pol II and various transcription factors. RUVBL2 interacts with unphosphorylated Pol II in chromatin to promote RPB1 carboxy-terminal domain (CTD) clustering and transcription initiation. Rapid depletion of RUVBL2 leads to a decrease in the number of Pol II clusters and inhibits nascent RNA synthesis, and tethering RUVBL2 to an active promoter enhances Pol II clustering at the promoter. We also identify target genes that are directly linked to the RUVBL2-Pol II axis. Many of these genes are hallmarks of cancers and encode proteins with diverse cellular functions. Our results demonstrate an emerging activity for RUVBL2 in regulating Pol II cluster formation in the nucleus.
MeSH term(s)	Adenosine Triphosphatases/metabolism ; Chromatin/genetics ; Cluster Analysis ; RNA ; RNA Polymerase II/genetics ; RNA Polymerase II/metabolism ; Transcription Factors/metabolism ; Transcription, Genetic
Chemical Substances	Chromatin ; Transcription Factors ; RNA (63231-63-0) ; RNA Polymerase II (EC 2.7.7.-) ; Adenosine Triphosphatases (EC 3.6.1.-)
Language	English
Publishing date	2022-09-28
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2553671-0
ISSN	2041-1723 ; 2041-1723
ISSN (online)	2041-1723
ISSN	2041-1723
DOI	10.1038/s41467-022-33433-3
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Article ; Online: Piperlongumine analogue L50377 induces pyroptosis via ROS mediated NF-κB suppression in non-small-cell lung cancer.

Li, Qian / Chen, Liping / Dong, Zhaojun / Zhao, Ya / Deng, Hui / Wu, Jianzhang / Wu, Xiaoping / Li, Wulan

Chemico-biological interactions

2019 Volume 313, Page(s) 108820

Abstract: Natural products with potent activity and less toxicity provide major sources for development of novel anti-cancer drugs. Herein, we evaluated the effects and the underlying mechanisms of a novel piperlongumine (PL) analogue L50377 on non-small-cell lung ...

Abstract	Natural products with potent activity and less toxicity provide major sources for development of novel anti-cancer drugs. Herein, we evaluated the effects and the underlying mechanisms of a novel piperlongumine (PL) analogue L50377 on non-small-cell lung cancer (NSCLC) cells. The results revealed that L50377 displayed greater potentials of suppressing cell growth than PL. In addition, L50377 promoted cell apoptosis and pyroptosis via stimulating reactive oxygen species (ROS) generation in NSCLC cells. More interestingly, ROS mediated NF-κB suppression might be implicated in the mechanisms of L50377-induced pyroptosis in NSCLC cells. Taken together, our results suggested that L50377 served as a novel chemical agent might have great potentials for NSCLC treatment.
MeSH term(s)	Antineoplastic Agents, Phytogenic/pharmacology ; Carcinoma, Non-Small-Cell Lung/drug therapy ; Carcinoma, Non-Small-Cell Lung/metabolism ; Carcinoma, Non-Small-Cell Lung/pathology ; Cell Line, Tumor ; Dioxolanes/chemistry ; Dioxolanes/pharmacology ; Humans ; Lung Neoplasms/drug therapy ; Lung Neoplasms/metabolism ; Lung Neoplasms/pathology ; NF-kappa B/antagonists & inhibitors ; NF-kappa B/metabolism ; Pyroptosis/drug effects ; Reactive Oxygen Species/metabolism
Chemical Substances	Antineoplastic Agents, Phytogenic ; Dioxolanes ; NF-kappa B ; Reactive Oxygen Species ; piperlongumine (SGD66V4SVJ)
Language	English
Publishing date	2019-09-10
Publishing country	Ireland
Document type	Journal Article
ZDB-ID	218799-1
ISSN	1872-7786 ; 0009-2797
ISSN (online)	1872-7786
ISSN	0009-2797
DOI	10.1016/j.cbi.2019.108820
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Article ; Online: Do chromatin loops provide epigenetic gene expression states?

Deng, Wulan / Blobel, Gerd A

Current opinion in genetics & development

2010 Volume 20, Issue 5, Page(s) 548–554

Abstract: Control of gene expression involves the concerted action of multiple regulatory elements some of which can act over large genomic distances. Physical interaction among these elements can lead to looping of the chromatin fiber. Although posttranslational ... ...

Abstract	Control of gene expression involves the concerted action of multiple regulatory elements some of which can act over large genomic distances. Physical interaction among these elements can lead to looping of the chromatin fiber. Although posttranslational modifications of chromatin are thought to play a role in the conveyance of epigenetic information, it is largely unknown whether higher order chromatin organization such as looping contributes to epigenetic memory. A related unresolved question is whether chromatin loops are the cause or the effect of transcriptional regulation. Recent work on diverse organisms suggests a memory function for long-range chromatin interactions. It is proposed that higher order folding of the chromatin fiber can serve to maintain active and repressed states of gene expression.
MeSH term(s)	Chromatin/ultrastructure ; Enhancer Elements, Genetic ; Epigenomics ; Gene Expression Regulation ; Protein Folding ; Protein Interaction Mapping ; Transcription Factors/genetics ; Transcription Factors/metabolism
Chemical Substances	Chromatin ; Transcription Factors
Language	English
Publishing date	2010-07-01
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Review
ZDB-ID	1077312-5
ISSN	1879-0380 ; 0959-437X
ISSN (online)	1879-0380
ISSN	0959-437X
DOI	10.1016/j.gde.2010.06.007
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Article ; Online: Design and Green Synthesis of Piperlongumine Analogs and Their Antioxidant Activity against Cerebral Ischemia-Reperfusion Injury.

Li, Ge / Zheng, Yuantie / Yao, Jiali / Hu, Linya / Liu, Qunpeng / Ke, Furong / Feng, Weixiao / Zhao, Ya / Yan, Pencheng / He, Wenfei / Deng, Hui / Qiu, Peihong / Li, Wulan / Wu, Jianzhang

ACS chemical neuroscience

2019 Volume 10, Issue 11, Page(s) 4545–4557

Abstract: The supplementation of exogenous antioxidants to scavenge excessive reactive oxygen species (ROS) is an effective treatment for cerebral ischemia-reperfusion injury (CIRI) in stroke. Piperlongumine (PL), a natural alkaloid, has a great potential as a ... ...

Abstract	The supplementation of exogenous antioxidants to scavenge excessive reactive oxygen species (ROS) is an effective treatment for cerebral ischemia-reperfusion injury (CIRI) in stroke. Piperlongumine (PL), a natural alkaloid, has a great potential as a neuroprotective agent, but it also has obvious toxicity. Moreover, its neuroprotective effects remain to be improved. In this study, we designed a series of novel PL analogs by hybridizing the screened low-toxicity diketene skeleton with antioxidant effect and the 3,4,5-trimethoxyphenyl group, which may increase the antioxidant activity of PL. The intermediate was synthesized by a novel green synthesis method, and 34 compounds were obtained. The compounds without obvious cytotoxicity have remarkable antioxidant effects, especially compared with diketene skeletons and PL. The cytoprotection of the active compound decreased significantly by reduction of the carbon-carbon double bonds of the Michael acceptor in the diketene skeleton. More importantly, further study revealed that compound
MeSH term(s)	Animals ; Antioxidants/chemical synthesis ; Antioxidants/pharmacology ; Antioxidants/therapeutic use ; Astrocytes/drug effects ; Astrocytes/metabolism ; Brain Ischemia/drug therapy ; Brain Ischemia/metabolism ; Cell Survival ; Dioxolanes/chemical synthesis ; Dioxolanes/pharmacology ; Dioxolanes/therapeutic use ; Drug Design ; Green Chemistry Technology/methods ; Male ; Oxidative Stress/drug effects ; Oxidative Stress/physiology ; PC12 Cells ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/antagonists & inhibitors ; Reactive Oxygen Species/metabolism ; Reperfusion Injury/drug therapy ; Reperfusion Injury/metabolism
Chemical Substances	Antioxidants ; Dioxolanes ; Reactive Oxygen Species ; piperlongumine (SGD66V4SVJ)
Language	English
Publishing date	2019-10-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1948-7193
ISSN (online)	1948-7193
DOI	10.1021/acschemneuro.9b00402
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Article ; Online: CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells.

Deng, Wulan / Shi, Xinghua / Tjian, Robert / Lionnet, Timothée / Singer, Robert H

Proceedings of the National Academy of Sciences of the United States of America

2015 Volume 112, Issue 38, Page(s) 11870–11875

Abstract: Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving ... ...

Abstract	Direct visualization of genomic loci in the 3D nucleus is important for understanding the spatial organization of the genome and its association with gene expression. Various DNA FISH methods have been developed in the past decades, all involving denaturing dsDNA and hybridizing fluorescent nucleic acid probes. Here we report a novel approach that uses in vitro constituted nuclease-deficient clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated caspase 9 (Cas9) complexes as probes to label sequence-specific genomic loci fluorescently without global DNA denaturation (Cas9-mediated fluorescence in situ hybridization, CASFISH). Using fluorescently labeled nuclease-deficient Cas9 (dCas9) protein assembled with various single-guide RNA (sgRNA), we demonstrated rapid and robust labeling of repetitive DNA elements in pericentromere, centromere, G-rich telomere, and coding gene loci. Assembling dCas9 with an array of sgRNAs tiling arbitrary target loci, we were able to visualize nonrepetitive genomic sequences. The dCas9/sgRNA binary complex is stable and binds its target DNA with high affinity, allowing sequential or simultaneous probing of multiple targets. CASFISH assays using differently colored dCas9/sgRNA complexes allow multicolor labeling of target loci in cells. In addition, the CASFISH assay is remarkably rapid under optimal conditions and is applicable for detection in primary tissue sections. This rapid, robust, less disruptive, and cost-effective technology adds a valuable tool for basic research and genetic diagnosis.
MeSH term(s)	Animals ; Binding Sites ; CRISPR-Associated Proteins/metabolism ; Carbocyanines/metabolism ; Clustered Regularly Interspaced Short Palindromic Repeats/genetics ; Color ; DNA/metabolism ; Electrophoretic Mobility Shift Assay ; Embryo, Mammalian/cytology ; Fibroblasts/metabolism ; Fluorescent Dyes/metabolism ; Genetic Loci ; Humans ; In Situ Hybridization, Fluorescence ; Mice ; RNA, Guide, CRISPR-Cas Systems ; Staining and Labeling
Chemical Substances	CRISPR-Associated Proteins ; Carbocyanines ; Fluorescent Dyes ; RNA, Guide, CRISPR-Cas Systems ; cyanine dye 5 ; DNA (9007-49-2)
Language	English
Publishing date	2015-08-31
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.1515692112
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