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Nuclear Protein Sorting

Nuclear Protein Sorting

Nuclear protein sorting is the selective trafficking of histones, polymerases, gene regulatory proteins into the nucleus and exporting RNAs and ribosomes to the cytosol. It is a tightly controlled process that regulates gene expression within a cell.
Proteins targeted to the nucleus carry nuclear localization signals or NLS recognized by import receptors in the cytosol. Similarly, proteins with nuclear export signals are recognized by export receptors. Import and export receptors are...

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Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

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NuclampFISH enables cell sorting based on nuclear RNA expression for chromatin analysis.

Yifang Liu¹, Yuchen Qiu¹, Keqing Nian¹

¹Department of Bioengineering, Northeastern University, Boston, MA, USA.

|July 2, 2025

Summary

We developed nuclear clampFISH (nuclampFISH) to sort cells by transcriptional bursts, overcoming limitations of previous methods. This technique allows for downstream analysis of chromatin and protein interactions.

Keywords:

Chromatin conformation ClampFISH RNA FISH amplification SmFISH Transcription site Transcriptional burst

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Author Spotlight: Nuclei Isolation from Mouse Cardiac Progenitor Cells for Epigenome and Gene Expression Profiling at Single-Cell Resolution

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Area of Science:

Molecular Biology
Cell Biology
Genetics

Background:

Transcriptional bursts, periods of active gene transcription, vary between cells.
Existing fluorescence in situ hybridization (FISH) methods struggle to target transcription sites effectively.
Formaldehyde fixation in traditional methods hinders downstream molecular analysis.

Purpose of the Study:

To develop a method for sorting cells based on transcriptional bursting activity.
To enable downstream biochemical analysis of chromatin and protein interactions.
To overcome limitations of existing FISH-based cell sorting techniques.

Main Methods:

Developed nuclear clampFISH (nuclampFISH), integrating click-amplified FISH with reversible crosslinkers.
Optimized parameters to enable nuclear RNA signal amplification and cell sorting.
Ensured compatibility with downstream chromatin conformation assays.

Main Results:

Successfully sorted cells based on nuclear RNA expression levels.
Demonstrated that transcriptionally active cells exhibit more accessible chromatin.
Validated the compatibility of nuclampFISH with downstream biochemical analyses.

Conclusions:

The new nuclampFISH method enables cell sorting based on transcriptional bursts.
Combines single-cell transcription site detection specificity with flow cytometry throughput.
Facilitates bulk assays like chromatin conformation analysis without specialized equipment.