Abstract
The decoding of histone post-translational modifications by chromatin-binding modules ("readers") constitutes one major mechanism of epigenetic regulation. Nuclear antigen Sp100 (SPECKLED, 100 kDa), a constitutive component of the promyelocytic leukemia nuclear bodies, plays key roles in intrinsic immunity and transcriptional repression. Sp100C, a splicing isoform specifically up-regulated upon interferon stimulation, harbors a unique tandem plant homeodomain (PHD) finger and bromodomain at its C terminus. Combining structural, quantitative binding, and cellular co-localization studies, we characterized Sp100C PHD finger as an unmethylated histone H3 Lys(4) (H3K4me0) reader that tolerates histone H3 Thr(3) phosphorylation (H3T3ph), histone H3 Lys(9) trimethylation (H3K9me3), and histone H3 Ser(10) phosphorylation (H3S10ph), hallmarks associated with the mitotic chromosome. In contrast, whereas H3K4me0 reader activity is conserved in Sp140, an Sp100C paralog, the multivalent tolerance of H3T3ph, H3K9me3, and H3S10ph was lost for Sp140. The complex structure determined at 2.1 Å revealed a highly coordinated lysine ϵ-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 Sp100C. An additional complex structure solved at 2.7 Å revealed that H3T3ph is recognized by the arginine residue, Arg(713), that is unique to the PHD finger of Sp100C. Consistent with a restrictive cellular role of Sp100C, these results establish a direct chromatin targeting function of Sp100C that may regulate transcriptional gene silencing and promyelocytic leukemia nuclear body-mediated intrinsic immunity in response to interferon stimulation.
Highlights
In eukaryotic organisms, epigenetic mechanisms are commonly employed to establish particular chromatin states in response to intrinsic or extrinsic signals [1]
Sp100CPB Is an Unmethylated H3K4 Reader, an Activity Conserved in Sp140PB—Human Sp100C and its paralog Sp140 share similar domain architectures, with a characteristic PHDBromo cassette located at the C terminus (Fig. 1A)
Despite the fact that H3 binding activity was not observed in Sp140 in a previous study using the plant homeodomain (PHD) finger alone [13], sequence alignment with TRIM24 and TRIM33 suggests that the PHD fingers in Sp100C and Sp140 are unmethylated histone H3K4 readers, given the presence of candidate H3K4me0-binding acidic residues near the N terminus (Fig. 1B, triangle)
Summary
Molecular Cloning and Recombinant Protein Preparation— The full-length gene of human Sp100C was PCR-amplified from human cDNA and cloned into the pEGFP-C1 vector (Clontech) for the transfection assay. The PHDBromo domains encompassing residues 696 – 878 or 701– 878 of Sp100C and 684 – 867 or 687– 867 of Sp140 were subcloned into a pSUMOH10 vector (modified based on pET28b) containing an N-terminal His10-SUMO tag. Bound proteins were eluted with a 20 –1000 mM imidazole gradient, and the resultant peak was pooled and subjected to SUMO protease ULP1 digestion. The His10-SUMO tag was removed by reloading onto the HisTrap column. The Sp100CPB[696–878] sample flow-through was collected, concentrated, and further purified by size exclusion chromatography on a Superdex G75 column (GE Healthcare) in elution buffer containing 20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 1 mM DTT. All other constructs of the wild type and mutant Sp100CPB and Sp140PB were purified essentially using the same procedure as described above for Sp100CPB(696 – 878)
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