Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

Zhen, Chao Yu; Tatavosian, Roubina; Huynh, Thao Ngoc; Duc, Huy Nguyen; Das, Raj Kumar; Kokotovic, Marko; Grimm, Jonathan B.; Lavis, Luke D.; Lee, Jun; Mejia, Frances J; Li, Yang; Yao, Tingting; Ren, Xiaojun

doi:10.7554/elife.17667

Cited by 101 publications

(117 citation statements)

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“…To determine whether the LNA-mediated gene upregulation depended on CBX7 in vivo , we introduced the LNAs into wildtype versus Cbx7−/− ES cells (Cheng et al, 2014; Zhen et al, 2016) (Fig. 6E,S8).…”

Section: Resultsmentioning

confidence: 99%

Denaturing CLIP, dCLIP, Pipeline Identifies Discrete RNA Footprints on Chromatin-Associated Proteins and Reveals that CBX7 Targets 3′ UTRs to Regulate mRNA Expression

et al. 2017

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SUMMARY Interaction networks between chromatin complexes and long noncoding RNAs have become a recurrent theme in epigenetic regulation. However, technical limitations have precluded identification of RNA binding motifs for chromatin-associated proteins. Here we add a denaturation step to UV-crosslink RNA immunoprecipitation (dCLIP), and apply dCLIP to mouse and human chromobox homolog 7 (CBX7), an RNA-binding subunit of Polycomb repressive complex 1 (PRC1). In both species, CBX7 predominantly binds 3’ untranslated regions (UTRs) of messenger RNAs. CBX7 binds with a median RNA “footprint” of 171–183 nucleotides, the small size of which facilitates motif identification by bioinformatics. We find four families of consensus RNA motifs in mouse, and independent analysis of human CBX7 dCLIP data identifies similar motifs. Their mutation abolishes CBX7 binding in vitro. Pharmacological intervention with antisense oligonucleotides paradoxically increases CBX7 binding and enhances gene expression. These data support the utility of dCLIP and reveal an unexpected functional interaction between CBX7 and the 3’UTRs of mRNA.

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Section: Resultsmentioning

confidence: 99%

Denaturing CLIP, dCLIP, Pipeline Identifies Discrete RNA Footprints on Chromatin-Associated Proteins and Reveals that CBX7 Targets 3′ UTRs to Regulate mRNA Expression

et al. 2017

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“…Inspired by computational experiments, the N , N -dimethyl groups in TMR were replaced with four-membered azetidine rings, which increased both the quantum yield (Φ = 0.88) and the photostability [25]. This new compound, named Janelia Fluor 549 (JF 549 ) has proven more suitable for single-molecule tracking in live cells [12, 20, 21, 29]. This azetidine strategy is general and can be used to improve red-shifted rhodamine analogs, such as the silicon-containing JF 646 , allowing the possibility of multi-color imaging [5].…”

Section: Single-molecule Tracking For Extracting Binding Charactermentioning

confidence: 99%

“…In all cases, the goal is to decrease the background signal by eliminating the excitation of out-of-focus molecules. The most common method used to study TF dynamics is HILO [11, 14, 15, 19–21, 34–37], most likely due to its easy implementation. In principle, any microscope capable of a TIRF configuration can do HILO illumination.…”

Section: Single-molecule Tracking For Extracting Binding Charactermentioning

confidence: 99%

“…However, those that perform fitting on the PDF generally report binding events that contain a single component [10, 18], whereas those fitting the CDF always obtain two distinct populations that consist of specific and nonspecific binding [9, 11, 19–21]. We therefore used simulated decay curves of bi-exponentials to objectively compare results obtained from fitting the two types of distributions.…”

Section: Single-molecule Tracking For Extracting Binding Charactermentioning

confidence: 99%

“…These technological advances now provide the means for the visualization and the measurement of the in vivo behavior of TF-binding events at chromatin targets such as enhancers and core promoters [6]. Several studies have now measured TF-binding events in both prokaryotes [7, 8] and eukaryotes [7, 9–21]. These experiments have shown that eukaryotic TFs spend most of their time freely diffusing, while only a small portion is specifically bound to chromatin at any given time [9–11, 13, 20].…”

Section: Introductionmentioning

confidence: 99%

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Quantifying transcription factor binding dynamics at the single-molecule level in live cells

Presman

Ball

Paakinaho

et al. 2017

Methods

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Progressive, technological achievements in the quantitative fluorescence microscopy field are allowing researches from many different areas to start unraveling the dynamic intricacies of biological processes inside living cells. From super-resolution microscopy techniques to tracking of individual proteins, fluorescence microscopy is changing our perspective on how the cell works. Fortunately, a growing number of research groups are exploring single-molecule studies in living cells. However, no clear consensus exists on several key aspects of the technique such as image acquisition conditions, or analysis of the obtained data. Here, we describe a detailed approach to perform single-molecule tracking (SMT) of transcription factors in living cells to obtain key binding characteristics, namely their residence time and bound fractions. We discuss different types of fluorophores, labeling density, microscope, cameras, data acquisition, and data analysis. Using the glucocorticoid receptor as a model transcription factor, we compared alternate tags (GFP, mEOS, HaloTag, SNAP-tag, CLIP-tag) for potential multicolor applications. We also examine different methods to extract the dissociation rates and compare them with simulated data. Finally, we discuss several challenges that this exciting technique still faces.

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CBX8 promotes lung adenocarcinoma growth and metastasis through transcriptional repression of CDKN2C and SCEL

Chen,

Su,

Yang

et al. 2023

Journal Cellular Physiology

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Dysregulation of polycomb group (PcG) proteins that mediate epigenetic gene silencing contributes to tumorigenesis. As core components of the polycomb repressive complex 1 (PRC1), chromobox (CBX) proteins recognize H3K27me3 to recruit PRC1 to maintain a repressive transcriptional state. However, the individual biological functions of these CBX proteins in tumorigenesis warrant in‐depth investigation. In this study, we analyzed the mRNA expression of CBX family genes across multiple cancers using The Cancer Genome Atlas data and found different expression patterns of the five CBX genes in different types of cancer. This analyses together with the result of immunohistochemistry indicated that CBX8 expression was significantly higher in lung adenocarcinoma (LUAD) tissues compared to adjacent nontumor tissues. Overexpression approaches demonstrated that CBX8 facilitated LUAD cell proliferation and migration in vitro. Consistently, CBX8 knockdown reduced LUAD cell proliferation and migration in both cell culture and mouse models. RNA sequencing combined with real‐time RT‐PCR assays revealed CDKN2C and SCEL as target genes of CBX8. Furthermore, chromatin immunoprecipitation assays indicated that CBX8 directly bound to the promoters of CDKN2C and SCEL to establish H2AK119ub. CBX8 depletion reduced the enrichment of H2AK119ub on CDKN2C and SCEL promoters. Moreover, depletion of CDKN2C and SCEL restored the repressed growth and invasion ability of LUAD cells caused by CBX8 knockdown. These findings demonstrate that CBX8 promotes LUAD growth and metastasis through the transcriptional repression of CDKN2C and SCEL. Our study uncovers the oncogenic role of CBX8 in LUAD progression and provides a new target for the diagnosis and therapy of LUAD.

show abstract

Live-cell single-molecule tracking reveals co-recognition of H3K27me3 and DNA targets polycomb Cbx7-PRC1 to chromatin

Cited by 101 publications

References 105 publications

Denaturing CLIP, dCLIP, Pipeline Identifies Discrete RNA Footprints on Chromatin-Associated Proteins and Reveals that CBX7 Targets 3′ UTRs to Regulate mRNA Expression

Denaturing CLIP, dCLIP, Pipeline Identifies Discrete RNA Footprints on Chromatin-Associated Proteins and Reveals that CBX7 Targets 3′ UTRs to Regulate mRNA Expression

Quantifying transcription factor binding dynamics at the single-molecule level in live cells

CBX8 promotes lung adenocarcinoma growth and metastasis through transcriptional repression of CDKN2C and SCEL

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