Analysis of Phosphorylation-Dependent Protein–Protein Interactions of Histone H3

Multiple
posttranslational modifications (PTMs) of histone proteins
including site-specific phosphorylation of serine and threonine residues
govern the accessibility of chromatin. According to the histone code
theory, PTMs recruit regulatory proteins or block their access to
chromatin. Here, we report a general strategy for simultaneous analysis
of both of these effects based on a SILAC MS scheme. We applied this
approach for studying the biochemical role of phosphorylated S10 of
histone H3. Differential pull-down experiments with H3-tails synthesized
from l- and d-amino acids uncovered that histone
acetyltransferase 1 (HAT1) and retinoblastoma-binding protein 7 (RBBP7)
are part of the protein network, which interacts with the unmodified
H3-tail. An additional H3-derived bait containing the nonhydrolyzable
phospho-serine mimic phosphonomethylen-alanine (Pma) at S10 recruited
several isoforms of the 14-3-3 family and blocked the recruitment
of HAT1 and RBBP7 to the unmodified H3-tail. Our observations provide
new insights into the many functions of H3S10 phosphorylation. In
addition, the outlined methodology is generally applicable for studying
specific binding partners of unmodified histone tails.