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 replication-independent 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 time-scale 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.

Keywords: Chromatin; H3; H3.3; epigenetics; exchange; histone; turnover.

Figure 1.

Strategy to knock-in a RITE cassette at H3F3B and Cre-ER^T2 at the AAVS1 safe-harbour locus. (a) A RITE cassette (V5→FLAG) is integrated at the C-terminus of H3.3 (H3F3B gene) using CRISPR-Cas9 and a repair plasmid for homologous recombination. Note that the gRNA is targeted to the last intron of H3F3B to prevent off-target cleavage at homologous histone H3 genes. (b) Genotyping PCR to confirm heterozygous integration of the RITE cassette at H3F3B in RPE1 and K562 cells. The asterisk denotes non-specific PCR amplicons. (c) A PGK1-promoter driven Cre-ER^T2 is integrated at the AAVS1 safe-harbour locus using a HDR (homology-directed repair) plasmid donor derived from Mali et al. [65]. (d) Genotyping PCR to confirm homozygous integration of Cre-ER^T2 is at AAVS1 in RPE1 and K562 cells. The asterisk denotes non-specific PCR amplicons. HA, homology arm; PuroR-2A-EGFP, puromycin resistance gene followed by self-cleaving peptide P2A from porcine teschovirus-1 polyprotein and green fluorescent protein; IRES, internal ribosome entry site; NeoR, neomycin resistance gene.

Figure 2.

RITE allows for simultaneous monitoring of old and new H3.3 histones in human cells. Western blot of old (V5) and new H3.3 (FLAG) after Cre-ER^T2 activation by 4OHT in (a) K562 and (b) RPE1 cells. An antibody against the LoxP-encoded spacer peptide recognizes both old and new H3.3. Note that the antibody directed against the C-terminus of H3 does not seem to recognize C-terminally tagged H3.3.

Figure 3.

Efficiency of Cre-ER^T2 mediated H3.3 tag-switching at the DNA level. (a) Overview of H3F3B-RITE locus before and after Cre-LoxP recombination (tag-switching). (b and c) qPCR on genomic DNA to determine tag-switching efficiency in (b) K562 and (c) RPE1 cells. Unswitched and switched qPCR signals were normalized to a control region (MYC exon 3) to normalize for input gDNA. For the unswitched fraction, the qPCR signal at 0 h was set to 1, while for the switched fraction the qPCR signal at 168 h was set to 1, and a sigmoidal curve was fitted to the data points.

Figure 4.

Loss of old H3.3 and gain of new H3.3 can be detected by ChIP-qPCR at different loci. (a) Experimental overview. Switching of the H3F3B-RITE cassette (H3.3-V5 to H3.3-FLAG) was induced in K562 cells by treatment with 4OHT and chromatin was harvested at different time points. (b) Overview of the loci analysed by ChIP-qPCR. Chr7 PCH stands for a pericentric heterochromatin locus on chromosome 7. Features are based on ENCODE data for K562. (c) ChIP-qPCR (IP/input) for old (V5) and new H3.3 (FLAG) at selected loci in K562 cells. (d) Turnover index [51] at selected loci. A higher index means more turnover.

Figure 5.

ChIP-seq of old and new H3.3 in RPE1 cells. (a) Distribution profiles of steady-state (old) H3.3-V5, and new H3.3-FLAG 8 h and 24 h after switch induction by 4OHT. Gene bodies are scaled to 6kb. (b) Examples of old and new H3.3 enrichment in a highly expressed gene (GAPDH), a mediumly expressed gene (PGK1) and a silent gene (KRT6A).