Single molecule force spectroscopy of telomeric H3.3 variant nucleosomes*

Understanding how histone protein variants affect the dynamics of nucleosome unwinding is key for developing a mechanistic model of their role in chromatin maintenance and disease. Nucleosomes are the basic units of chromatin, consisting of 146 bp of DNA wrapped around an octamer made up of two of each of the four core histones—H2A, H2B, H3, H4. Nucleosomes containing histone H3.3, a variant of the canonical histone H3.1, are enriched in telomeric regions. Mutations in the pathway responsible for depositing H3.3 nucleosomes on telomeres are associated with the Alternative Lengthening of Telomeres (ALT) pathway. The ALT pathway allows cells to overcome senescence induced by telomere shortening, a key protection against cancer. There is currently no mechanistic understanding of the role of the H3.3 variant in the ALT+ cancer pathway. To address this gap, we measure the kinetic unwinding rates of single H3.3 or H3.1 nucleosomes assembled on either the Widom 601 or human telomeric DNA sequences using single molecule optical tweezers. Our studies quantify the role of the H3.3 variant in telomeric chromatin maintenance.