Binding of the MLL PHD3 finger to histone H3K4me3 is required for MLL-dependent gene transcription - PubMed (original) (raw)

Binding of the MLL PHD3 finger to histone H3K4me3 is required for MLL-dependent gene transcription

Pei-Yun Chang et al. J Mol Biol. 2010.

Abstract

The MLL (mixed-lineage leukemia) proto-oncogene encodes a histone methyltransferase that creates the methylated histone H3K4 epigenetic marks, commonly associated with actively transcribed genes. In addition to its canonical histone methyltransferase SET domain, the MLL protein contains three plant homeodomain (PHD) fingers that are well conserved between species but whose potential roles and requirements for MLL function are unknown. Here, we demonstrate that the third PHD domain of MLL (PHD3) binds histone H3 trimethylated at lysine 4 (H3K4me3) with high affinity and specificity and H3K4me2 with 8-fold lower affinity. Biochemical and structural analyses using NMR and fluorescence spectroscopy identified key amino acids essential for the interaction with H3K4me3. Site-directed mutations of the residues involved in recognition of H3K4me3 compromised in vitro H3K4me3 binding but not in vivo localization of full-length MLL to chromatin sites in target promoters of MEIS1 and HOXA genes. Whereas intact PHD3 finger was necessary for MLL occupancy at these promoters, H3K4me3 binding was critical for MLL transcriptional activity. These results demonstrate that MLL occupancy and target gene activation can be functionally separated. Furthermore, these findings reveal that MLL not only "writes" the H3K4me3 mark but also binds the mark, and this binding is required for the transcriptional maintenance functions of MLL.

Copyright (c) 2010 Elsevier Ltd. All rights reserved.

PubMed Disclaimer

Figures

Figure 1

Figure 1. The MLL PHD3 finger binds histone H3K4me3

(A, B) Immuno-detection (anti-GST antibody) shows binding of GST-PHD3 to the indicated biotinylated histone peptides in a peptide pull-down assay (A) and peptide microarray (B). (C) Western blot using antibodies to the indicated epitopes shows binding of GST-PHD3 to a calf thymus histone mixture. (D, F) Shown are superimposed 1H, 15N HSQC spectra of 0.2 mM PHD3, collected while the indicated histone peptides were titrated in. The spectra are color-coded according to the peptide concentrations (inset). (E) The histogram displays normalized 1H, 15N chemical shift changes observed in backbone amides (or the side chain of W1594) in the corresponding (D) spectra of the PHD3 finger. Colored bars indicate significant change being greater than an average plus one-half standard deviation. Asterisk indicates loss of M1585 resonance.

Figure 2

Figure 2. Binding affinities of the MLL PHD3 finger

(A) Alignment of PHD domain sequences: zinc-coordinating and absolutely and moderately conserved residues are colored brown, blue and green, respectively. Residues of the MLL PHD3 finger that were mutated are marked with a black dot above the sequences and labeled. (B) Immuno-detection (anti-GST) shows binding of wt or mutant GST-PHD3 proteins indicated on the left to biotinylated histone peptides in a peptide pull-down assay. (C) Peptide microarray results are shown for binding of wt or mutant GST-PHD3 to the indicated biotinylated histone peptides. (D) The Kd values for interaction of wild type or mutant MLL PHD3 fingers with the indicated histone peptides were measured by tryptophan fluorescence (a) or NMR (b). (E) A representative binding curve used to determine Kds for wild type PHD3 by tryptophan fluorescence.

Figure 3

Figure 3. MLL PHD3-to-histone H3K4me3 association is not required for MLL occupancy on select gene loci

(A) Quantitative real-time PCR was used to analyze chromatin immunoprecipitation results in 293T cells expressing wild type or mutant MLL proteins. (B) MLL protein levels were analyzed by western blot analysis.

Figure 4

Figure 4. MLL PHD3-to-histone H3K4me3 association is critical for MLL dependent target gene expression

(A) Quantitative real-time RT-PCR was used to analyze gene expression levels in 293T cells expressing wild-type or mutant MLL proteins. (B) Model for MLL PHD3-mediated functions.

Similar articles

Cited by

References

    1. David Allis C, Danny Reinberg TJ. Marie-Laure Caparros. Epigenetics
    1. Slany RK. When epigenetics kills: MLL fusion proteins in leukemia. Hematol Oncol. 2005;23:1–9. - PubMed
    1. Slany RK. Chromatin control of gene expression: mixed-lineage leukemia methyltransferase SETs the stage for transcription. Proc Natl Acad Sci U S A. 2005;102:14481–2. - PMC - PubMed
    1. Fair K, Anderson M, Bulanova E, Mi H, Tropschug M, Diaz MO. Protein interactions of the MLL PHD fingers modulate MLL target gene regulation in human cells. Mol Cell Biol. 2001;21:3589–97. - PMC - PubMed
    1. Sutherland HG, Mumford GK, Newton K, Ford LV, Farrall R, Dellaire G, Caceres JF, Bickmore WA. Large-scale identification of mammalian proteins localized to nuclear sub-compartments. Hum Mol Genet. 2001;10:1995–2011. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources