Imaging Newly Synthesized and Old Histone Variant Dynamics Dependent on Chaperones Using the SNAP-Tag System

Abstract

Distinct histone variants mark chromatin domains in the nucleus. To understand how these marks are established and maintained, one has to decipher how the dynamic distribution of these variants is orchestrated. These dynamics are associated with all DNA-based processes such as DNA replication, repair, transcription, heterochromatin formation and chromosome segregation. Key factors, known as histone chaperones, have been involved in escorting histones, thereby contributing to the chromatin landscape of given cell types. SNAP-tag-based imaging system enables the distinction between old and newly deposited histones, and has proved to be a powerful method for the visualization of histone variant dynamics on a cell-by-cell basis. This approach enables the tracking of specific variants in vivo and defining their timing and mode of deposition throughout the cell cycle and in different nuclear territories. Here, we provide a detailed protocol to exploit the SNAP-tag technology to assess the dynamics of newly synthesized and old histones. We then show that combining the SNAP-tagging of histones with the knockdown of candidate factors, represents an effective approach to decipher the role of key actors in guiding histone dynamics. Here, we specifically illustrate how this strategy was used to identify the essential role of the chaperone HIRA in deposition of newly synthesized histone variant H3.3.