The Role of the TSK/TONSL-H3.1 Pathway in Maintaining Genome Stability in Multicellular Eukaryotes

Abstract

Replication-dependent histone H3.1 and replication-independent histone H3.3 are nearly identical proteins in most multicellular eukaryotes. The N-terminal tails of these H3 variants, where the majority of histone post-translational modifications are made, typically differ by only one amino acid. Despite extensive sequence similarity with H3.3, the H3.1 variant has been hypothesized to play unique roles in cells, as it is specifically expressed and inserted into chromatin during DNA replication. However, identifying a function that is unique to H3.1 during replication has remained elusive. In this review, we discuss recent findings regarding the involvement of the H3.1 variant in regulating the TSK/TONSL-mediated resolution of stalled or broken replication forks. Uncovering this new function for the H3.1 variant has been made possible by the identification of the first proteins containing domains that can selectively bind or modify the H3.1 variant. The functional characterization of H3-variant-specific readers and writers reveals another layer of chromatin-based information regulating transcription, DNA replication, and DNA repair.

Keywords: DNA repair; DNA replication; genome stability; histone H3.1 variant; homologous recombination.

Figure 1

The distinct deposition pathways and sequences of histone H3.1 and H3.3 variants: (A) The deposition of H3.1 and H3.3 by their dedicated histone chaperones at different genomic loci. The CAF-1 complex directly interacts with the PCNA clamp and deposits H3.1 at replication forks during DNA replication. In contrast, HIRA inserts H3.3 at active gene regions (i.e., euchromatin) during transcription, while DAXX deposits H3.3 mainly at various heterochromatic loci, including pericentromeric regions and telomeres. Nucleosomes with parental H3.1 or H3.3 histones are shown in gray; nucleosomes with newly synthesized H3 proteins are highlighted in blue (H3.1) or red (H3.3); (B) sequence alignment of histone H3 variants (H3.1/H3.2 and H3.3) from human and Arabidopsis. The sequence folding into the globular domain of H3 is marked by a blue rounded rectangle. Functionally important lysine residues (i.e., K4, K9, K27, and K36) in the N-terminal tails of H3 variants are highlighted in red. Sequence variations between H3.1, H3.2, and H3.3 in humans and Arabidopsis are indicated in pink and green, respectively, with positional information for these variable amino acids indicated above the alignment.

Figure 2

Activity of plant H3K27 methyltransferases on different H3 variants. ATXR5 and ATXR6 catalyze K27 monomethylation on H3.1 variants. PRC2 complexes containing either CLF, MEA, or SWN (catalytic subunits of Arabidopsis PRC2 complexes) trimethylate K27 on H3.1 and H3.3 variants. In contrast, ATXR5, ATXR6, and PRC2 complexes cannot methylate the male-gamete-specific histone variant MGH3, which contributes to reprograming the paternal epigenome in pollen of Arabidopsis.

Figure 3

The TPR domain of TSK/TONSL functions as an H3.1 reader: (A) Domain architecture of human and Arabidopsis TONSL/TSK. TPR: tetratricopeptide repeats, ARD: ankyrin repeat domain, UBL: ubiquitin-like, LRR: leucine-rich repeats. The domains in TSK/TONSL responsible for binding specific histones are marked by dashed rectangles; (B) alignment of TPR domains from multiple animal and plant TSK/TONSL orthologs. NCBI reference sequences: NP_038460.4 (Homo sapiens), NP_898914.3 (Mus musculus), NP_001104618.1 (Danio rerio), Q9VSA4.1 (Drosophila melanogaster), NP_188503.2 (Arabidopsis thaliana), PWZ29356.1 (Zea mays), and GAY58445.1 (Citrus unshiu). Residues in the alignment are colored according to the Clustal X color scheme. The α helices of the animal and plant TPR domains of TONSL/TSK, respectively, shown as pink or violet rectangles above the alignment, were predicted by AlphaFold (animals) or based on the crystal structure of the TPR domain of C. unshiu TSK (plants). Red asterisks indicate the residues of the TPR domains shown (plants) or predicted (animals) to mediate specific binding to H3.1 via recognition of residue A31; (C) top view of the TPR domain of TSK (violet) from C. unshiu co-crystalized with the histone H3.1 N-terminal tail (green) (PDB accession number: 7T7T); (D) (Left panel) structural superposition of the solved TPR domain (violet) of plant TSK (C. unshiu) and the AlphaFold-predicted TPR domain (pink) of TONSL (human), with a focus on the histone H3.1A31-binding pocket. The amino acid residues from the TPR domain of plant TSK or animal TONSL interacting with H3.1A31 (green) are shown. (Right panel) Surface representation of the predicted H3.1A31-binding pocket from the TPR domain of human TONSL.

Figure 4

Comparison of the chromatin-based mechanisms regulating TSK/TONSL activity between mammals and plants: (A) In mammals, TONSL forms a heterodimer with MMS22L and initially interacts with soluble H3.1/H4 using its TPR and ARD domains. After H3.1/H4 incorporation into nascent chromatin, TONSL–MMS22L recruits HR repair proteins to resolve stalled or broken replication forks. Post-replicative chromatin maturation relies on SET8 to monomethylate H4K20, which prevents the binding of TONSL–MMS22L to mature chromatin; (B) plants lack a clear MMS22L homologous protein, and TSK only contains a TPR domain to mediate the interaction with newly synthesized H3.1/H4. In addition, H3.1K27, not H4K20, is monomethylated (via ATXR5/6) to prevent TSK from interacting with mature chromatin. Parental H3/H4 histones are shown in gray; newly synthesized histone proteins are highlighted in blue (H3.1), red (H3.3) or yellow (H4).