Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Recognition of Trimethylated K4 of Histone H3 by the TFIID Subunit TAF3
Post-translational modifications of residues in the N-terminal tails of the histone proteins play an important role in the regulation of gene expression by enabling or disabling interaction with chromatin regulatory proteins.
Methylation of lysine 4 of histone H3 (H3K4Me3) is a hallmark of active genes. Recently, it was discovered that trimethylated K4 is specifically recognized by PHD finger domains (2,3). Interestingly, a direct link between the basal transcription factor TFIID and H3K4me3 has been established (4). The PHD finger of the TFIID-subunit TAF3 specifically binds H4K4Me3, which might potentiate the recruitment of the RNA polymerase II complex to active genes.
Here, we investigate the molecular basis of the TAF3-H3K4me3 interaction using NMR spectroscopy, mutational analysis and affinity measurements. We present the solution structure of the PHD finger of the TAF3 subunit in its free state and when bound to the histone tail of histone H3 trimethylated at lysine 4(1) (Figure 1A). The structures of the free and bound form are nearly identical, suggesting that the predefined interaction surface has an important role as a ?folding template? for the H3 tail.
We will discuss the importance of the cation-pi interaction between K4me3 and the PHD domain as the main determinant of affinity and specificity. The K4me3-binding pocket of TAF3A contains a unique local structure rearrangement due to a conserved sequence insertion to allow the presence of two tryptophan residues close to the trimethylated amino group of K4. Detailed analysis of several trimethylated lysine complexes reveals that two aromatic residues are required to bind Kme3, one of which is a tryptophan in a parallel orientation to the lysine side chain.
The TAF3 PHD domain has a high affinity for the H3K4me3 peptide (0.3 µM). This affinity likely results from the combination of: i) two tryptophans in the binding pocket that can generate strong cation-pi interaction; ii) deep burial of the N-terminus and A1 and iii) a large network of electrostatic interactions. The TAF3 PHD domain binds specifically to trimethylated K4, although the discrimination against dimethylated K4 is limited and seems to be conferred solely by alterations in the cation-pi strength. Interestingly, the potential hydrogen bond acceptor D887 in the K4 pocket is too remote to influence this specificity by hydrogen bonding to the dimethylated amino group.
Finally, we show that the H3K4me3 interaction is sensitive to crosstalk by other histone modifications (Figure 1B). Both chemical shift and mutation data suggest that the methylated R2 is too bulky to fit in its pocket on the TAF3 surface. Interference by asymmetric dimethylation of arginine 2 suggests that a H3R2/K4 ??methyl-methyl?? switch in the histone ?code? dynamically regulates TFIID-promoter association.
Figure 1.(A) Solution structure of TAF3-PHD ? H3K4me3 complex, showing residues 1-6 of the H3K4me3 peptide in stick representation and the interaction surface of the PHD domain. The K4, T3 and R2 interaction pockets are shown in cyan, brown and orange, respectively. (B) Overlay of NMR spectra of free (black) TAF3, bound to H3K4me3 (red) and bound to H3R2me2aK4me3 (green), showing significant chemical shift perturbation for R2 pocket residues.
References and Footnotes
H. van Ingen1,2
1Bijvoet Centre for Biomolecular Research, Utrecht University