Application of Recombination -Induced Tag Exchange (RITE) to study histone dynamics in human cells

Abstract

In eukaryotes, nucleosomes form a barrier to DNA templated reactions and must be dynamically disrupted to provide access to the genome. During nucleosome (re)assembly, histones can be replaced by new histones, erasing post-translational modifications. Measuring histone turnover in mammalian cells has mostly relied on inducible overexpression of histones, which may influence and distort natural histone deposition rates. We have previously used recombination-induced tag exchange (RITE) to study histone dynamics in budding yeast. RITE is a method to follow protein turnover by genetic switching of epitope tags using Cre recombinase and does not rely on inducible overexpression. Here, we applied RITE to study the dynamics of the replicationindependent histone variant H3.3 in human cells. Epitope tag-switching could be readily detected upon induction of Cre-recombinase, enabling the monitoring old and new H3.3 in the same pool of cells. However, the rate of tag-switching was lower than in yeast cells. Analysis of histone H3.3 incorporation by chromatin immunoprecipitation did not recapitulate previously reported aspects of H3.3 dynamics such as high turnover rates in active promoters and enhancers. We hypothesize that asynchronous Cre-mediated DNA recombination in the cell population leads to a low time resolution of the H3.3-RITE system in human cells. We conclude that RITE enables the detection of old and new proteins in human cells and that the timescale of tag-switching prevents the capture of high turnover events in a population of cells. Instead, RITE might be more suited for tracking long-lived histone proteins in human cells.

SETD2 negatively regulates cell size through its catalytic activity and SRI domain

“…We therefore depleted SETD2 in human retinal pigment epithelial cells transduced with the telomerase gene (RPE1-hTERT), which is a non-transformed near diploid human cell line (designated RPE1 from here on). To monitor H3.3 (which differs five amino acids from H3.1 and four amino acids from H3.2), we used RPE1 cells carrying a endogenously V5 epitope-tagged copy of the H3.3 gene H3F3B (Molenaar et al 2020). Despite being frequently inactivated in cancer, SETD2 is an essential gene in several human cell lines (Blomen et al 2015; Wang et al 2015; Bertomeu et al 2018).…”

Anin vivo“turning model” reveals new RanBP9 interactions in lung macrophages

“…The copyright holder for this preprint this version posted May 24, 2024. ; https://doi.org/10.1101/2024.05.22.595416 doi: bioRxiv preprint approach was used also in human cells 72,73 . However, the RanBP9 TurnX model represents the first example of RITE in mouse.…”

“…However, the RanBP9 TurnX model represents the first example of RITE in mouse. Notably, the TurnX allele that we designed does not include selection cassettes that were necessary to generate RITE alleles in human cells and lower organisms (Figure 1, Supplementary Figure 1) [71][72][73] . All considered, the TurnX mouse is relatively easy to engineer by CRISPR targeting and represents a prototype that can be mimicked to create other in vivo models when the addition of small tags does not interfere with the biological functions of the protein of interest.…”

NEDDylated Cullin 3 mediates the adaptive response to topoisomerase 1 inhibitors

“…Last, publicly available RNA-seq data in RPE-1 cells (Gene Expression Omnibus {GEO} accession number GSE146121) were used for determination of gene expression. These data were generated by Molenaar et al ( 120 ); for this purpose, the table of read counts was downloaded from GEO, and Reads Per Kilobase Million values per gene were calculated. Last, by combining the publicly available datasets of Repli-seq, OK-seq, and RNA-seq, replication initiation sites were determined.…”