Recognition of histone H3 methylation states by the PHD1 domain of histone demethylase KDM5A

Abstract

PHD reader domains are chromatin binding modules often responsible for the recruitment of large protein complexes that contain histone modifying enzymes, chromatin remodelers and DNA repair machinery. PHD domains recognize N-terminal residues of histone H3 and are sensitive to the methylation state of Lys4 in histone H3 (H3K4). Histone demethylase KDM5A, an epigenetic eraser enzyme that contains three PHD domains, is often overexpressed in various cancers and its demethylation activity is allosterically enhanced when its PHD1 domain is bound to the H3 tail. The allosteric regulatory function of PHD1 expands roles of reader domains, suggesting unique features of this chromatin interacting module. Our previous studies determined the H3 binding site of PHD1, although it remains unclear how the H3 tail interacts with the N-terminal residues of PHD1 and how PHD1 discriminates against H3 tails with varying degrees of H3K4 methylation. Here we show that the N-terminal region of PHD1 is initially unstructured, but becomes ordered upon binding of the high affinity H3 peptide ligands. We also observe differential interactions of conserved binding residues with differently methylated H3K4 peptides (me0, me1, me2 or me3), providing a rational for this PHD1 domain's preference for lower methylation states of H3K4.We further assessed the contributions of various H3 interacting residues in the PHD1 domain to the binding of H3 peptides using mutagenesis. The insights into the H3 binding site could provide useful information to aid development of allosteric small molecule modulators of KDM5A.

Exploring the Ligand Preferences of the PHD1 Domain of Histone Demethylase KDM5A Reveals Tolerance for Modifications of the Q5 Residue of Histone 3

“…This finding is consistent with the observation of hydrogen bonding between the hydroxyl group of H3T3 and the amide of H3T6, which contributes to the H3 peptide's adoption of a stabilized helical conformation when bound to PHD1, and hydrophobic interactions between the methyl group of H3T3 and the L309 and V330 residues of PHD1. 42 The FP results for the T3 single mutant ligands largely agree with the PI-SAMDI array data, which showed relatively low affinity for each of these ligands except T3Y, which displayed a higher fold enhancement (Figure 4B). Additionally, we observed that PHD1 displays an affinity similar to the wild-type ligand for every Q5 single mutant ligand tested in the FP assay, suggesting that Q5 is dispensable for binding (Table 1, entries 8−14; Figure S4).…”

“…This may be due to charge−charge interactions between the positively charged Q5R residue and the negatively charged E305 and D306 residues in PHD1. 42 We did observe several inconsistencies between the PI-SAMDI and FP results and therefore sought to better understand the sources of this discrepancy. We selected three ligands with which to compare the two assay formats: wild-type, a well-characterized, high-affinity ligand that showed only moderate fold enhancement in the PI-SAMDI array; Q5E, which displayed low fold enhancement in the PI-SAMDI array but showed high affinity in the FP assay; and Q5Y, which showed high affinity in both assays.…”

Missense Variants Reveal Functional Insights Into the Human ARID Family of Gene Regulators

“…In particular, residues D292/Y294/L308 in JARID1A and W1502/W512 in JARID1B, which are required for histone peptide binding, 54. , 55. are affected by neutral missense variants ( Figure 4 (C)–(D)).…”

“…Conversely, residues responsible for histone peptide binding in PHD3 of JARID1A ( Figure 4 (C)) 53. , 55. and PHD1 of JARID1B ( Figure 4 (D)) 52.…”

Joining the PARty: PARP Regulation of KDM5A during DNA Repair (and Transcription?)

“…[ 51 ] Interestingly, these PHD domains are also involved in KDM5A DNA damage functions where the PHD1 domain is required to localize KDM5A to DSBs, while the PHD3 domain, which is dispensable for break recruitment, is necessary for KDM5A DSB repair functions downstream of break recruitment (Figure 1A). [ 34,52 ] KDM5A can promote DNA repair and transcriptional silencing through the recruitment of additional effector complexes, including ZMYND8 and the NuRD complex. [ 34 ] At DSBs, ZMYND8 requires the histone acetyltransferase (HAT) TIP60 and its bromodomain (BRD) to localize to breaks, suggesting an important, yet poorly understood, requirement for acetylation in this pathway.…”