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Abstract <p><b>Objective:</b> The study of histone post-translational modifications (PTMs) is a rapidly developing field, yet the tools available for detecting and interpreting these modifications are limited. Histone modifications, such as methylation, acetylation, and phosphorylation, play a crucial role in regulating chromatin dynamics and gene expression. Specific binding of histone modification “reader” domains (HMRDs) is central to this regulation, allowing for the recruitment of proteins that facilitate chromatin remodeling. This research aims to develop genetically encoded sensors based on HMRDs to study histone modifications in live cells, offering a more efficient and flexible method for studying epigenetic changes. <b>Methods:</b> We designed genetically encoded sensors that utilize HMRDs and splitFAST to bind specifically to different histone modifications. These sensors were incorporated into cells to track the dynamic changes in histone modifications. The performance of these sensors was evaluated through live-cell imaging, using fluorescent microscopy to monitor histone modifications. <b>Results and Discussion:</b> The genetically encoded sensors demonstrated high specificity and sensitivity to various histone modifications. Sensors based on SplitFAST and HMRDs MPP8 and AF9 exhibited specific distributions for H3K9me3 and H3K9ac. Moreover, the combination of these two domains with different parts of SplitFAST showed spatial proximity between H3K9me3 and H3K9ac. These findings suggest that the integration of HMRD-based sensors, MPP8 and AF9, with SplitFAST could provide valuable tools for live-cell monitoring of histone modifications and their roles in gene regulation and cellular response mechanisms. <b>Conclusions:</b> The development of genetically encoded sensors for histone modifications based on HMRDs provides a powerful new tool for studying chromatin dynamics in live cells. These sensors offer a more direct and real-time approach to understanding the complex mechanisms of histone modification and their impact on gene expression.</p>

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Live-Cell Visualization of Histone Modification Using Bimolecular Complementation

  • 期刊论文

  • 发表日期：

    2025年2月12日

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  • A. I. Stepanov,
  • L. V. Putlyaeva,
  • A. A. Shuvaeva,
  • M. A. Andrushkin,
  • M. S. Baranov,
  • N. G. Gurskaya,
  • K. A. Lukyanov

摘要

Abstract

Objective: The study of histone post-translational modifications (PTMs) is a rapidly developing field, yet the tools available for detecting and interpreting these modifications are limited. Histone modifications, such as methylation, acetylation, and phosphorylation, play a crucial role in regulating chromatin dynamics and gene expression. Specific binding of histone modification “reader” domains (HMRDs) is central to this regulation, allowing for the recruitment of proteins that facilitate chromatin remodeling. This research aims to develop genetically encoded sensors based on HMRDs to study histone modifications in live cells, offering a more efficient and flexible method for studying epigenetic changes. Methods: We designed genetically encoded sensors that utilize HMRDs and splitFAST to bind specifically to different histone modifications. These sensors were incorporated into cells to track the dynamic changes in histone modifications. The performance of these sensors was evaluated through live-cell imaging, using fluorescent microscopy to monitor histone modifications. Results and Discussion: The genetically encoded sensors demonstrated high specificity and sensitivity to various histone modifications. Sensors based on SplitFAST and HMRDs MPP8 and AF9 exhibited specific distributions for H3K9me3 and H3K9ac. Moreover, the combination of these two domains with different parts of SplitFAST showed spatial proximity between H3K9me3 and H3K9ac. These findings suggest that the integration of HMRD-based sensors, MPP8 and AF9, with SplitFAST could provide valuable tools for live-cell monitoring of histone modifications and their roles in gene regulation and cellular response mechanisms. Conclusions: The development of genetically encoded sensors for histone modifications based on HMRDs provides a powerful new tool for studying chromatin dynamics in live cells. These sensors offer a more direct and real-time approach to understanding the complex mechanisms of histone modification and their impact on gene expression.