Targeting the Histone Locus: New Therapeutic Options for Cancer Treatment
Introduction
The paradigm of modern cytotoxic chemotherapy aims to impair DNA replication to prevent cell division. A necessary step in DNA replication involves wrapping newly synthesized DNA around histones produced de novo to create the fundamental unit of chromatin, the nucleosome. Despite the more than 50 approved oncological agents that target DNA synthesis, there are no therapeutic strategies approved or in clinical trials that target nucleosome production. Thus, nucleosome assembly represents an untapped target to develop novel, safer, and more efficacious antineoplastic therapies. The vast majority of core histone synthesis (>95%) occurs during S-phase, thereby presenting a therapeutic window in which quiescent, healthy cells would be relatively unaffected by perturbations to histone synthesis. Human histone genes are highly redundant having many codon-identical copies distributed across three major loci. The four core histones are considered some of the most highly evolutionarily conserved proteins in eukaryotes and of these, histone H4 is the most highly conserved. Previous work has identified a highly conserved 17 base pair motif, the “H4-Box”, in the promoter region of all human histone H4 genes that plays a critical role in regulating histone gene transcription.
Methods
CRISPR/Cas9 was used to introduce DNA edits to the H4-Box in human cancer cell lines. RT-qPCR was used to evaluate changes in gene expression in these edited cell lines. A luminescent cell viability assay was performed to quantify proliferative defect and assay rescue attempts. Tumor xenografts were injected into immune-deficient mice to measure proliferative defect in vivo.
Results
Perturbation to the sequence of the H4-Box led to global defects in the transcription of each of the core histones as well as various associated histone chaperones. These defects were not limited to genes neighboring the edited site but involved long-range perturbations. Sequencing confirmed that alterations in gene expression could not be explained by off-target editing. Gene cassettes were constructed to reintroduce histone H4 into edited cell lines using conditional promoters. High-level H4 expression required 3’ elements known to be required for histone mRNA processing. Despite robust H4 re-expression, these edited cell lines exhibited a proliferative defect both in vitro and in vivo that was not rescued by overexpression of H4 alone.
Conclusion
Nucleosome synthesis remains an untargeted pathway for cancer therapeutics. Small Cas9-mediated deletions in one of the 14 human histone H4 gene promoters have been shown to induce dramatic defects in global histone synthesis, cell proliferation, and are impervious to overexpression rescue of the directly affected gene. This work identifies the H4-box as a core gene regulatory element that likely plays a role in coordinated histone gene expression in cancer cells that is required for rapid cell division.