Histone H3 Lysine 4 Methylation Disrupts Binding of Nucleosome Remodeling and Deacetylase (NuRD) Repressor Complex

Abstract

Histone N-terminal tails are post-translationally modified in many ways. At lysine residues, histones can be either acetylated or methylated. Both modifications lead to the binding of specific proteins; bromodomain proteins, such as GCN5, bind acetyl lysines and the chromodomain protein, HP1, binds methyl lysine 9 of histone H3. Here we show that the previously characterized transcriptional repressor complex NuRD (nucleosome remodeling and deacetylase) binds to the histone H3 N-terminal tail and that methylation at lysine 4, but not lysine 9, prevents binding. Given that lysine 4 methylation is found at sites of active transcription, these results suggest that a function of lysine 4 methylation is to disrupt the association of histones with a repressor complex.The N-terminal tails of histones are multiply post-translationally modified. Such evolutionarily conserved modifications include phosphorylation, acetylation, and methylation (1, 2). The "histone code hypothesis" predicts that combinations of modifications on the histone N termini will serve as binding sites for different protein modules (1, 3). Furthermore, this differential binding of proteins to histone tails is proposed to affect chromatin structure and transcriptional regulation (4). Indeed bromodomains have been shown to bind to acetylated lysines in histone N-terminal tails (5, 6), and in vivo data suggest that the bromodomain of GCN5 can target transcriptional activators to acetylated promoter regions (7).Although methylation of histone lysines was discovered over 35 years ago (8), only recent studies have elucidated that the transcriptional repressor SUV39H1 can methylate lysine 9 of histone H3 (9). In accordance with the histone code hypothesis, methyl lysine 9 of histone H3 provides a binding site for the chromodomain of HP1 (10, 11). The binding of HP1 is essential for heterochromatic silencing (10,12) and is also involved in the repression of euchromatic genes (13).Consistent with a repressive role for transcription, lysine 9 methylation is found at silent heterochromatic sites (14 -16). In contrast, methylation of histone H3 lysine 4 localizes to sites of active transcription (14,15), suggesting that this modification may be stimulatory for transcription.Here we identify a complex of proteins that specifically binds to H3 tails and the binding of which is abrogated by lysine 4 methylation. Using mass spectrometry, we identify that this complex is the previously characterized repressor complex NuRD (nucleosome remodeling and deacetylase). We postulate that the displacement of NuRD from histone tails may be the activatory mechanism of lysine 4 methylation. EXPERIMENTAL PROCEDURESPeptides-Histone peptides, trimethylated at different lysine residues, were made by G. Bloomberg (Bristol University, UK). The C terminus of the peptides contained a two-glycine spacer followed by a cysteine. Peptides were immobilized onto Sulfolink coupling gel (Pierce) via the C-terminal cysteine at a concentration of 1 mg/ml.Affinity Purification from HeLa Nuclear Ex...

Identifying novel proteins recognizing histone modifications using peptide pull-down assay

“…Although this approach has been successfully used by several laboratories (see for example: [10,11]), in our hands pull-downs with SulfoLink conjugated peptides tend to give much higher unspecific backgrounds compared with pulldowns with avidin-bound peptides.…”

“…However, salt concentration of 250 mM disrupts association of NuRD complex with H3 peptides [10]. Therefore 300 mM salt washes may be too stringent to retain some interactions, and washing with buffer containing lower salt may result in better recovery of the protein of interest.…”

Effect of maternal diet on the epigenome: implications for human metabolic disease

“…The latter binds to methylated cytosines and, in turn, recruits histone modifying complexes to the DNA (63) . Methyl CpG-binding protein 2 recruits both histone deacetylases, which remove acetyl groups from the histones, and histone methyl transferases that methylate Lys9 on His3, resulting in a closed chromatin structure and transcriptional silencing (63)(64)(65) . MicroRNA, which are small non-coding RNA, can also regulate gene expression, they have been shown to modulate gene expression at the post-transcriptional level through the induction of mRNA degradation or translational repression of a target mRNA (66) .…”

Regulation of chromatin by histone modifications

“…They can also function to disrupt an interaction between the histone and a chromatin factor. For instance, H3K4me3 can prevent the NuRD complex from binding to the H3 N-terminal tail [79,80]. This simple mechanism seems to make sense because NuRD is a general transcriptional repressor and H3K4me3 is a mark of active transcription.…”