Structural And Functional Studies On The Histone Reader Sp100C And Tubulin Acetyltransferase ?TAT1

The decoding of histone post-translational modifications(PTMs)by chromatin-binding modules(“readers”)constitutes one major mechanism of epigenetic regulation.Nuclear antigen Sp100(speckled protein 100 kDa),a constitutive component of the promyelocytic leukemia nuclear bodies(PML-NBs),plays key roles in intrinsic immunity and transcriptional repression.Sp100 C,a splicing isoform specifically up-regulated upon interferon stimulation,harbors a unique tandem plant homeodomain finger and bromodomain(PHD-Bromo)at its C-terminus.Combining structural,quantitative binding and cellular co-localization studies,we characterized Sp100 C PHD finger as an unmethylated histone H3K4(H3K4me0)reader that tolerates H3T3 phosphorylation(H3T3ph),H3K9 trimethylation(H3K9me3)and H3S10 phosphorylation(H3S10ph)–hallmarks associated with mitotic chromosomes.In contrast,while H3K4me0 reader activity is conserved in Sp140,an Sp100 C paralog,the multivalent tolerance of H3T3 ph,H3K9me3 and H3S10 ph was lost for Sp140.The complex structure determined at 2.1 ? revealed a highly coordinated lysine ?-amine recognition sphere formed by an extended N-terminal motif for H3K4me0 readout.Interestingly,reader pocket rigidification by disulfide bond formation enhanced H3K4me0 binding by Sp100 C.An additional complex structure solved at 2.7 ? revealed that H3T3 ph is recognized by the arginine residue R713 that is unique to Sp100 C.Consistent with the restrictive function of Sp100 C in transcription and cell cycle regulation,these results establish a direct chromatin targeting function of Sp100 C that may regulate PML-NB-mediated intrinsic immunity and gene silencing in response to interferon stimulation.Similarly,there is a “tubulin code” on microtubules in the cytoplasm.A highly conserved protein,?-tubulin acetyltransferase 1(?TAT1)mediates acetylation of ?-tubulin on lysine 40(K40ac),the only PTM identified hitherto in the luminal surface of microtubule.Although ?TAT1 has been extensively studied about its role in microtubule stability and tumor invasion regulation,the molecular mechanism for catalysis remains unknown.Here,we determined the structure of mouse ?TAT1 catalytic domain bound to its cofactor acetyl-coenzyme A(AcCoA)at 1.2 ?.The complex structure reveals that ?TAT1 adopts a canonical GNAT acetyltransferase superfamily core fold but a rather tight Ac CoA binding site,with its adenosine moiety sandwiched in between.Additionally,substrate recognition involves a special ?4/?5 insertion and positive patches in proximity to the putative catalytic site as well as a hydrophobic N-terminal region.Peptide titration and enzymatic assays indicate that the acetyltransferase activity of ?TAT1 depends on the K40 loop conformation and the lateral interaction of microtubule protofilaments.Furthermore,electron microscopy experiments show that in addition to the K40 site,?TAT1 may bind to the outer surface of microtubule.In conclusion,the unique features of ?TAT1 revealed by our structural and biochemical studies provide a molecular basis for understanding its substrate recognition mode and the regulatory role of ?TAT1 in microtubule-related processes.