The ability of MLL PHD3 to bind modified histone peptides was examined by pull-down experiments and peptide microarray analyses (). The GST-PHD3 fusion was incubated with biotinylated histone H3 peptides (either mono-, di- or tri-methylated at Lys 4, Lys 9, Lys 27 or Lys 36) in the presence of streptavidin sepharose beads, and the peptide-bound protein was detected using anti-GST antibodies. As shown in , GST-PHD3 recognized H3K4me3 peptide and, to a lesser degree, H3K4me2. Minimal or no interaction was observed with peptides methylated at other lysine residues (). Strong preference of the PHD3 finger for trimethylated Lys 4 was also seen in a microarray assay, in which GST-PHD3 and biotinylated peptides immobilized on a streptavidin coated chip were used ( and Suppl. Fig. 1
). The MLL PHD3 finger bound to H3K4me3/me2 but did not associate with H3 and H4 peptides containing other post-translational histone modifications, including acetylation and phosphorylation marks, or methylated p53 peptides. Mono-methylation and asymmetrical di-methylation at Arg2 disrupted the MLL PHD3-H3K4me3 interaction whereas symmetrical di-methylation of Arg2 did not significantly alter the interaction. To determine whether the specificity of the PHD3 finger was preserved within intact histones, it was tested in a pull down assay using calf thymus histones. GST-PHD3 associated with intact histone H3 containing methylated marks on Lys 4 (). Thus, the PHD3 finger of MLL selectively binds to the histone H3 tail poly-methylated at Lys 4.
The MLL PHD3 finger binds histone H3K4me3
The molecular basis of H3K4me3 recognition by MLL PHD3 was investigated by NMR spectroscopy. The sequence-specific 1
C and 15
N resonance assignments of PHD3 were obtained using a set of triple-resonance NMR experiments. Subsequently, histone peptide binding was characterized by collecting 1
N HSQC (heteronuclear single quantum coherence) spectra of the 15
N-labeled protein. Substantial chemical shift changes were observed in the MLL PHD3 finger when H3K4me3 peptide was titrated in (). Addition of a small amount of peptide caused line broadening of many NMR resonances revealing an intermediate-to-fast exchange regime on the NMR time scale and a high-affinity interaction. A large number of the amide resonances were significantly perturbed, including that of N1567, F1568, C1569, Y1576, D1578, S1583, M1585, M1586, Q1587, W1594, N1601, L1602, D1604, Y1607, L1613, and V1617, suggesting an extensive binding site () as was also seen in the case of the ING2 PHD finger 9
. We note that some PHD3 residues were in conformational exchange that precluded observation of their amide resonances in NMR spectra.
Alignment of the MLL PHD3 amino acid sequence with the sequences of other H3K4me3-binding PHD fingers revealed a number of conserved residues () To determine the role of the most conserved and perturbed F1568, Y1576, M1585 and W1594 residues in binding to H3K4me3, each of these residues was substituted by alanine, and the corresponding mutant proteins were assessed by pull-down experiments, peptide microarrays and NMR spectroscopy. An additional mutant, in which the acidic residue D1592 was replaced with Ala, was generated. Among the five mutants, F1568A and D1592A retained the ability to associate with the histone H3 tail in a methylation dependent manner in peptide binding assays () and peptide microarray () although slightly weaker than the wild type protein, indicating that these residues are not essential for binding (). The remaining mutants (Y1576A, M1585A, W1594A) were more severely compromised in their abilities to recognize H3K4me3. Although the Y1576A and M1585A mutants remained structured (Suppl. Fig. 2
), small to negligible changes in their 1
N HSQC spectra, induced by the H3K4me3 peptide, indicated that binding was essentially abolished (data not shown). In contrast, the Ala substitution of W1594 caused unfolding of PHD3, impeding the ability to bind H3K4me3 and suggesting the critical role of this residue for structural stability (Suppl. Fig. 2
). Taken together, these data indicate that the aromatic residues Y1576 and W1594, and the hydrophobic residue M1585 are required for high affinity interaction of the MLL PHD3 finger with H3K4me3.
Binding affinities of the MLL PHD3 finger
H3K4me2 and H3K4me1 peptides induced a similar pattern of chemical shift perturbations in 1H,15N HSQC spectra of the MLL PHD3 finger, suggesting that these peptides were bound in the same binding site as H3K4me3, albeit much more weakly (). The affinities of the PHD3 finger for di- and mono- methylated H3K4 were eight- and fifty three-fold lower (), corroborating the data obtained by pull-down experiments. Addition of histone H3 peptides methylated at Lys 9, Lys 27 or Lys 36 to the PHD3 NMR sample caused no significant chemical shift changes, demonstrating again that MLL PHD3 is unable to recognize these methylation marks and displays a clear preference for H3K4me3 ().
The dissociation constant (Kd) for the interaction of MLL PHD3 with H3K4me3 was determined to be 19 μM as measured by tryptophan fluorescence (). Comparable affinities, in the low μM range, are exhibited by other histone-binding modules including bromodomains, chromodomains, Tudor and MBT, which have been shown to be functionally relevant, suggesting that similarly the MLL PHD3-H3K4me3 interaction might be functionally significant.
To determine the potential role of MLL PHD3 recognition of H3K4me3 for MLL function, point mutations (F1568A, M1585A, D1592A, W1594A and H1596A) were engineered into full-length MLL. Following transfection into 293T cells, MLL targeting was assessed by chromatin immunoprecipitation (ChIP). Occupancy of exogenous wild type MLL was substantially enriched at the MEIS1 promoter, a physiologic MLL target, compared to the MEIS1 3’ genomic () and coding (data not shown) regions (occupancy of 12 versus 4). MLL mutants F1568A and D1592A that maintained their ability to bind H3K4me3 in vitro also occupied the MEIS1 promoter. In contrast, MLL proteins harboring either the W1594A (shown to be unstructured in vitro) or H1596A (zinc coordinating His residue, mutation of which should completely disrupt the structure of PHD3) substitutions displayed virtually no binding to the MEIS1 genomic sites (occupancy of ~2-4). Furthermore, MLL containing the “loss-of-function” M1585A mutation, which essentially eliminated H3K4me3 binding in vitro, also occupied the MEIS1 promoter. A similar ChIP analysis of four distinct regions within the HOXA9 promoter showed that MLL proteins harboring the “loss-of-structure” W1594A or H1596A mutations consistently displayed no or minimal localization at the HOXA9 promoter sites in contrast to M1585A, F1568A and D1592A mutants, which occupied most HOXA9 sites. Reduced occupancy observed for the W1594A mutant likely reflected at least in part its reduced stability in vivo (). These observations indicate that high affinity binding of PHD3 to H3K4me3 is not necessary for MLL association with promoter chromatin, which is likely mediated by alternative motifs (e.g. CXXC, AT hooks) implicated in MLL DNA binding, however the structural integrity of PHD3 is essential.
MLL PHD3-to-histone H3K4me3 association is not required for MLL occupancy on select gene loci
The consequences of PHD3 mutation were assessed for expression of MLL target genes using real-time PCR (). Exogenous wild type MLL increased expression of HOXA9, HOXA10, and MEIS1, whereas MLL proteins containing the “loss-of-structure” mutations H1596A and W1594A were unable to promote target gene expression, consistent with the effects of these mutations on chromatin localization and in vitro binding to H3K4me3. MLL containing the F1568A and D1592A mutations that bind H3K4me3 in vitro induced target gene expression similar to wild type MLL, which is consistent with their abilities to associate with chromatin in target genes. Conversely, the ability of the “loss-of-function” M1585A mutant to induce HOXA9, HOXA10 or MEIS1 expression was substantially impaired despite an ability to associate with promoter chromatin, but nevertheless consistent with an inability to bind H3K4me3 in vitro. Thus, binding of H3K4me3 by PHD3 is dispensable for localization at target chromatin sites, however it is necessary for the transcription-promoting effects of MLL.
MLL PHD3-to-histone H3K4me3 association is critical for MLL dependent target gene expression
Our studies demonstrate that MLL not only “writes” the H3K4me3 histone code, but also “reads” it as well, which contributes to its transcriptional function. Although recognition of H3K4me3 by PHD3 is required, its specific role in the cascade of molecular events underlying transcriptional maintenance by MLL remains unclear. One possibility is that processive reading of the newly deposited H3K4me3 mark may contribute to sliding of the MLL complex along the gene to establish a broad, methylated chromatin domain. Alternatively, PHD3 may facilitate presentation or protection of the mark for competitive binding by heterologous proteins that facilitate translation of the histone code into a transcriptional output. Future studies will focus on distinguishing among these possibilities.