Interactions with multiple inner kinetochore proteins determine mitotic localization of FACT

Area of Science:

• Cell Biology
• Molecular Biology
• Epigenetics

Background:

• The FAcilitates Chromatin Transcription (FACT) complex is a histone chaperone involved in DNA-templated processes.
• FACT interacts with centromeric nucleosomes containing CENP-A and CCAN proteins, but the functional significance is unclear.

Purpose of the Study:

• To elucidate the functional significance of FACT interactions with the constitutive centromere-associated network (CCAN).
• To investigate the regulation and role of FACT-CCAN interactions in centromere function.

Main Methods:

• Biochemical assays to identify FACT binding sites on CCAN.
• Phosphorylation studies using casein kinase II.
• Mitotic localization studies using microscopy.
• DNA displacement assays.

Main Results:

• FACT binds to multiple sites on CCAN, and this interaction is enhanced by FACT phosphorylation.
• FACT's kinetochore localization depends on specific CCAN subcomplexes.
• CENP-TW requires FACT for stable localization.
• DNA can displace FACT from CCAN, suggesting recruitment via a non-chromatin-integrated pool.

Conclusions:

• FACT plays a role in chaperoning CCAN during transcription.
• FACT may stabilize CCAN at the centromere during the cell cycle.
• FACT-CCAN interactions are regulated by phosphorylation and DNA binding, impacting centromere integrity.