Abstract
The incorporation of histone variant H3.3 into the genome plays a critical role in regulating gene transcription, genomic stability, and mitosis progression. However, the precise mechanisms underlying the influence of H3.3 on nucleosome stability and dynamics remain poorly understood. In this study, we demonstrate that while the incorporation of H3.3 into nucleosomes does not significantly alter their stability, it enhances the maintenance of nucleosome integrity. Notably, H3.3 recruits the FACT complexes more efficiently than the canonical H3, counteracting FACT's destabilizing effect on nucleosomes. The binding of FACT to H3.3-nucleosome further stabilizes the nucleosome structure, which can be reversed by phosphorylation at Serine 31 (H3.3S31ph). Through genome-wide analyses, we show that the deposition of H3.3 and its phosphorylation at Ser31 dynamically modulate the nucleosome states, influencing FACT binding and regulating the transcriptional responses in macrophages upon stimulation. The selective phosphorylation at H3.3S31 functions as a pivotal switch, transforming the H3.3-nucleosome from a stable, maintenance-oriented state to a more dynamic, active configuration. This molecular switch enables a rapid response to environmental stimuli, thereby facilitating transcriptional activation. Our findings provide new mechanistic insights into how H3.3 and its Ser31 phosphorylation modulate nucleosome dynamics and transcriptional response, with significant implications for immune response pathways in macrophages.