Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins

Delker, Rebecca K; Munce, Ross H.; Hu, Michelle; Mann, Richard S.

doi:10.1016/j.crmeth.2022.100175

Cited by 6 publications

(13 citation statements)

References 62 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Previous work established ANCHOR as a specific and sensitive method for labelling target ROIs in the genome, without affecting transcriptional regulation in vivo [13,14].…”

Section: Bifor Allows the Revelation Of The Enrichment Of Specific Di...mentioning

confidence: 99%

“…The LacO/LacI system has been used in Drosophila, but it also requires a hundred copies of the LacO cassette to exploit the fluorescent signals emitted by the DNA-bound LacI-FP fusion protein [8]. In addition, the binding of LacI has been shown to interfere with gene transcription and is, therefore, not the most appropriate option for quantifying DNA-bound TFs in the genomic vicinity [14]. In contrast, the ANCHOR system is more neutral in regard to gene transcription [14].…”

Section: Advantages Of Anchor Over Other Dna-labelling Systems For Bi...mentioning

confidence: 99%

“…First applied in yeast [9] and human cell culture [10,11], this system has also been developed to visualize enhancer-promoter interactions [12] or the DNA locus in Drosophila [13]. Moreover, recent analysis showed that the ANCHOR system did not locally perturb transcription in Drosophila (in contrast to the LacI/LacO system: [14]).…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva

Vanderperre,

Merabet

2024

Cells

View full text Add to dashboard Cite

Transcription factors (TFs) regulate gene expression by recognizing specific target enhancers in the genome. The DNA-binding and regulatory activity of TFs depend on the presence of additional protein partners, leading to the formation of versatile and dynamic multimeric protein complexes. Visualizing these protein–protein interactions (PPIs) in the nucleus is key for decrypting the molecular cues underlying TF specificity in vivo. Over the last few years, Bimolecular Fluorescence Complementation (BiFC) has been developed in several model systems and applied in the analysis of different types of PPIs. In particular, BiFC has been applied when analyzing PPIs with hundreds of TFs in the nucleus of live Drosophila embryos. However, the visualization of PPIs at the level of specific target enhancers or genomic regions of interest awaits the advent of DNA-labelling methods that can be coupled with BiFC. Here, we present a novel experimental strategy that we have called BiFOR and that is based on the coupling of BiFC with the bacterial ANCHOR DNA-labelling system. We demonstrate that BiFOR enables the precise quantification of the enrichment of specific dimeric protein complexes on target enhancers in Drosophila salivary gland nuclei. Given its versatility and sensitivity, BiFOR could be applied more widely to other tissues during Drosophila development. Our work sets up the experimental basis for future applications of this strategy.

show abstract

“…Previous work established ANCHOR as a specific and sensitive method for labelling target ROIs in the genome, without affecting transcriptional regulation in vivo [13,14].…”

Section: Bifor Allows the Revelation Of The Enrichment Of Specific Di...mentioning

confidence: 99%

Section: Advantages Of Anchor Over Other Dna-labelling Systems For Bi...mentioning

confidence: 99%

See 1 more Smart Citation

Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva

Vanderperre,

Merabet

2024

Cells

View full text Add to dashboard Cite

show abstract

“…TetO and its variant mut-TetO have been used to track genomic interactions during VDJ recombination in live cells ( 10 ). The requirement of LacO repeat numbers for visualization has decreased to as low as 20 copies in Drosophila, but the signal-to-noise ratio is relatively low ( 11 ). To achieve a high signal-to-noise ratio, the requirement of large-size DNA repeat integration for FROS hampers its widespread applications of genomic DNA imaging.…”

Section: Introductionmentioning

confidence: 99%

Tracking pairwise genomic loci by the ParB–ParS and Noc-NBS systems in living cells

He,

Tan,

Feng

et al. 2024

Nucleic Acids Research

View full text Add to dashboard Cite

The dynamics of genomic loci pairs and their interactions are essential for transcriptional regulation and genome organization. However, a robust method for tracking pairwise genomic loci in living cells is lacking. Here we developed a multicolor DNA labeling system, mParSpot (multicolor ParSpot), to track pairs of genomic loci and their interactions in living cells. The mParSpot system is derived from the ParB/ParS in the parABS system and Noc/NBS in its paralogous nucleoid occlusion system. The insertion of 16 base-pair palindromic ParSs or NBSs into the genomic locus allows the cognate binding protein ParB or Noc to spread kilobases of DNA around ParSs or NBSs for loci-specific visualization. We tracked two loci with a genomic distance of 53 kilobases and measured their spatial distance over time. Using the mParSpot system, we labeled the promoter and terminator of the MSI2 gene span 423 kb and measured their spatial distance. We also tracked the promoter and terminator dynamics of the MUC4 gene in living cells. In sum, the mParSpot is a robust and sensitive DNA labeling system for tracking genomic interactions in space and time under physiological or pathological contexts.

show abstract

“…The methods that can detect genomic sites in live cells include lacO/LacI (Hirakawa et al 2015), tetO/TetR (Tasan et al 2018), ANCHOR (Germier et al 2017, Meschichi et al 2021, the transcription activatorlike effector with a fluorescent protein (TALE-FP) (Miyanari et al 2013, and clustered regularly interspaced short palindromic repeats (CRISPR)-imaging . They have been widely used in recent years for research in mice, human cells, and plants, and even for medical diagnosis (Mine-Hattab and Rothstein 2012, Li et al 2019, Boettiger and Murphy 2020, Meschichi et al 2021, Delker et al 2022. Among these methods, the CRISPR imaging is continuously optimized for its imaging resolution due to its ease of use in specific site recognition and low impact on the local chromatin state.…”

mentioning

confidence: 99%

Various Strategies for Improved Signal-to-Noise Ratio in CRISPR-Based Live Cell Imaging

Matsunaga

2023

CYTOLOGIA

View full text Add to dashboard Cite

The various behaviors of cells are closely related to the temporal and spatial dynamics of chromatin. Live cell imaging techniques for chromatin can provide insight into gene expression at the three dimension of a nucleus and offer the novel knowledge in gene regulation for medical diagnosis, crop improvement, and environmental remediation. However, traditional chromatin imaging techniques that rely on the insertion of a target gene are unable to directly analyze the chromatin dynamics in living cell nuclei. CRISPR-based live imaging, with its high accuracy, speed, and low perturbation, has emerged as a reliable tool for high-resolution imaging of the specific chromatin in the nucleus. This review paper introduces the benefits of CRISPR-based live imaging for chromatin dynamics, classifies and analyzes various successful schemes used to improve the signal-to-noise ratio of this method, and discusses future trends in the field.

show abstract

Fluorescent labeling of genomic loci in Drosophila imaginal discs with heterologous DNA-binding proteins

Cited by 6 publications

References 62 publications

Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva

Visualization of the Association of Dimeric Protein Complexes on Specific Enhancers in the Salivary Gland Nuclei of Drosophila Larva

Tracking pairwise genomic loci by the ParB–ParS and Noc-NBS systems in living cells

Various Strategies for Improved Signal-to-Noise Ratio in CRISPR-Based Live Cell Imaging

Contact Info

Product

Resources

About