Chapter 22
Using RNA Tags for Multicolor Live Imaging of Chromatin
Loci and Transcription in Drosophila Embryos
Hongtao Chen and Thomas Gregor
Abstract
Elucidating the biological implications of higher order chromatin architectures in animal development
requires simultaneous, quantitative measurements of chromatin dynamics and transcriptional activity in
living specimen. Here we describe a multicolor labeling and live imaging approach in embryos of the fruit fly
Drosophila melanogaster. The method allows simultaneous measurement of movements of specific loci and
their transcriptional activity for developmental genes, enabling new approaches to probe the interaction
between 3D chromatin architecture and regulation of gene expression in development.
Key words Quantitative live imaging, Drosophila embryos, Transcription, 3D genome architecture
1 Introduction
There is growing evidence for a regulatory role of the 3D
architecture of chromatin for the control of gene expression during
development and disease processes [1–3]. Ligation or thin
sectioning-based genomic approaches provide snapshots of the
global DNA connectivity map [4, 5]. However, the dynamics of
the 3D chromatin architecture at the single-cell level has been
difficult to assess due to fixed and pooled samples applied in such
genomic assays. Here we present a multicolor labeling and live
imaging method to visualize the movement of specific DNA loci
in fly embryos. In particular, we combine molecular systems based
on nascent RNA visualization and systems based on direct tagging
of DNA loci to simultaneously measure the transcriptional activity
of key developmental genes and their cis-regulatory elements using
fluorescence microscopy. This method allows measurement and
analysis of 3D chromatin dynamics and gene activity in real time
and with single live-cell resolution.
Using CRISPR-Cas9 technology, endogenous genes are edited
with RNA stem-loop cassettes, which were pioneered in singlecelled organisms to study RNA transcription, splicing, and
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_22, © Springer Science+Business Media, LLC, part of Springer Nature 2020
373
Using RNA Tags for Multicolor Live Imaging of Chromatin
Loci and Transcription in Drosophila Embryos
Hongtao Chen and Thomas Gregor
Abstract
Elucidating the biological implications of higher order chromatin architectures in animal development
requires simultaneous, quantitative measurements of chromatin dynamics and transcriptional activity in
living specimen. Here we describe a multicolor labeling and live imaging approach in embryos of the fruit fly
Drosophila melanogaster. The method allows simultaneous measurement of movements of specific loci and
their transcriptional activity for developmental genes, enabling new approaches to probe the interaction
between 3D chromatin architecture and regulation of gene expression in development.
Key words Quantitative live imaging, Drosophila embryos, Transcription, 3D genome architecture
1 Introduction
There is growing evidence for a regulatory role of the 3D
architecture of chromatin for the control of gene expression during
development and disease processes [1–3]. Ligation or thin
sectioning-based genomic approaches provide snapshots of the
global DNA connectivity map [4, 5]. However, the dynamics of
the 3D chromatin architecture at the single-cell level has been
difficult to assess due to fixed and pooled samples applied in such
genomic assays. Here we present a multicolor labeling and live
imaging method to visualize the movement of specific DNA loci
in fly embryos. In particular, we combine molecular systems based
on nascent RNA visualization and systems based on direct tagging
of DNA loci to simultaneously measure the transcriptional activity
of key developmental genes and their cis-regulatory elements using
fluorescence microscopy. This method allows measurement and
analysis of 3D chromatin dynamics and gene activity in real time
and with single live-cell resolution.
Using CRISPR-Cas9 technology, endogenous genes are edited
with RNA stem-loop cassettes, which were pioneered in singlecelled organisms to study RNA transcription, splicing, and
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_22, © Springer Science+Business Media, LLC, part of Springer Nature 2020
373