Project description:Our previous work on the histone H3.3 chaperone HIRA revealed its importance for maintaining the targeting of H3.3 to its pre-existing sites. In concert with replication fork-coupled deposition of the replicative H3.1 variant, this establishes boundaries of H3.3/H3.1, which define early replication initiation zones, which are disrupted in the absence of HIRA (Gatto et al., 2022). We have also recently shown a role of HIRA for active gene organisation and compartment A interactions by Hi-C. Here, we performed a HIRA rescue experiment in HIRA KO HeLa cells combined with a G1/S synchronisation to assay the recovery of H3.1 and H3.3 distribution (prior to S phase entry) by SNAP capture-seq and the pattern of nascent DNA synthesis in early S phase (2h) by EdU-seq. We also include H3.3 SNAP capture-seq from an asynchronous HIRA rescue we performed to assay recovery of genome organisation by Hi-C (submitted as a separate ArrayExpress entry).
Project description:We performed Hi-C in asynchronous HeLa cells that knock-out (KO) for the H3.3-specific chaperone HIRA and bear an exogenous H3.1-SNAP and H3.3-SNAP gene transfected with HIRA-YFP (HIRA) or YFP only (control) plasmid.
Project description:We performed total RNA-seq of G1/S-synchronised HeLa cells that are wild-type (WT) or knock-out (KO) for the H3.3-specific chaperone HIRA and bear an exogenous H3.1-SNAP and H3.3-SNAP gene.
Project description:We profiled accessibility by ATAC-seq of G1/S-synchronised HeLa cells that are wild-type (WT) or knock-out (KO) for the H3.3-specific chaperone HIRA and bear an exogenous H3.1-SNAP and H3.3-SNAP gene.
Project description:We developed a new sequencing assay to track the de novo deposition of the histone H3 variants H3.1 and H3.3 during S phase. We use cells stably expressing H3.1-SNAP or H3.3-SNAP, and synchronize them in G1/S by double-thymidine block. The SNAP-tag enables to discriminate newly synthesized histones from preexisting ones, via a quench-chase-capture strategy. We applied this strategy to isolate new H3.1 and H3.3 after releasing cells into S phase, and probed their distribution by MNase digestion and sequencing. We could thus characterize H3.1 and H3.3 dynamics from early to mid S phase at genome-wide resolution. We further applied our method to investigate the consequences of perturbations upon deletion of the H3.3 chaperone HIRA. We used HIRA knockout and control cells, and compared H3.1 and H3.3 distribution to early replication patterns by EdU labeling and sequencing of nascent DNA.
Project description:We performed Hi-C in asynchronous HeLa cells that are wild-type (WT) or knock-out (KO) for the H3.3-specific chaperone HIRA and bear an exogenous H3.1-SNAP and H3.3-SNAP gene.
Project description:We profiled the enrichment of active (H3K4me1, H3K4me3, H3K27ac) and inactive (H3K9me3, H3K27me3) histone PTMs in cells that are wild-type (WT) or knock-out (KO) for the H3.3-specific chaperone HIRA. We performed native ChIP-seq following MNase digestion to isolate nucleosomes in H3.1-SNAP and H3.3-SNAP-bearing HeLa cells. We had previously assayed the distribution of the H3.1 and H3.3 histone variants (Gatto et al., 2022) in the same cell lines, so we generated this PTM ChIP-seq data to compare the behaviour of the variants with that of H3 modifications.
Project description:Age-associated functional decline is partly driven by progressive chromatin degeneration. Maintenance of chromatin integrity preserves cell identity and promotes healthy aging, but through different mechanisms in proliferating and non-proliferating cells. However, specific mechanisms of chromatin maintenance and their compensatory capacity in proliferating and non-proliferating cells are undefined. The histone chaperone HIRA deposits the histone variant H3.3 in a DNA replication-independent manner, leading to its accumulation in aging, non-proliferating cells. Here, we show that hepatocyte-specific loss of HIRA causes loss of cell identity, metabolic dysfunction, and accelerated fibrotic pathology with age. Transcriptomic and epigenomic analyses indicate that HIRA-H3.3 preserves chromatin integrity and sustains transcription of highly expressed genes, including cell identity genes. Partial hepatectomy, associated with induced proliferation, restores identity of HIRA knockout livers with compensatory deposition of canonical histones H3.1/2. Together, these results demonstrate that HIRA-mediated H3.3 deposition is essential for safeguarding cell identity and tissue function during aging of non-proliferating cells, but this function can be rescued by tissue regeneration and associated cell proliferation.