A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling

Abstract

Lysine methylation of histones is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive chromatin domains. Trimethylation of histone H3 lysine 4 (H3K4me3) marks transcription start sites of virtually all active genes. Recently, we reported that the WD40-repeat protein WDR5 is important for global levels of H3K4me3 and control of HOX gene expression. Here we show that a plant homeodomain (PHD) finger of nucleosome remodelling factor (NURF), an ISWI-containing ATP-dependent chromatin-remodelling complex, mediates a direct preferential association with H3K4me3 tails. Depletion of H3K4me3 causes partial release of the NURF subunit, BPTF (bromodomain and PHD finger transcription factor), from chromatin and defective recruitment of the associated ATPase, SNF2L (also known as ISWI and SMARCA1), to the HOXC8 promoter. Loss of BPTF in Xenopus embryos mimics WDR5 loss-of-function phenotypes, and compromises spatial control of Hox gene expression. These results strongly suggest that WDR5 and NURF function in a common biological pathway in vivo, and that NURF-mediated ATP-dependent chromatin remodelling is directly coupled to H3K4 trimethylation to maintain Hox gene expression patterns during development. We also identify a previously unknown function for the PHD finger as a highly specialized methyl-lysine-binding domain.

Mechanisms of ATP dependent chromatin remodeling

“…The presence of such an epitope was found to be essential in generating ATP-dependent regularly spaced nucleosome arrays by RSF [141]. Recent reports have shown that H3K9me3 mark can actively recruit the PHD domain of NURF [142,143]. Similarly, the same mark was also shown to recruit Isw1p ATPase to chromatin [144].…”

MOZ and MORF, two large MYSTic HATs in normal and cancer stem cells

“…PHD fingers may also function as methyl-lysine-binding modules. One PHD finger of NURF recognizes trimethyl lysine 4 of histone H3 to couple ATP-dependent chromatin remodeling to histone methylation Wysocka et al, 2006), and the PHD finger of ING2 binds to the same mark to stimulate methylation-dependent deacetylation (Pena et al, 2006;Shi et al, 2006). Moreover, methylation of histone H3 at lysine 4 promotes association with yeast Yng1, a subunit of the NuA3 acetyltransferase complex (Martin et al, 2006;Taverna et al, 2006).…”

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

“…Confirm specific association of the identified protein with the modified peptide by immunoblotting eluates with specific antibodies, if available, and by performing pull-down assays using purified recombinant protein as input (for examples, see: [8,9]). …”

“…The protocol described here calls for washes with buffer containing 300mM salt, which usually result in relatively low backgrounds in pull-downs and do not disrupt association of HP1, WDR5, BPTF or CBX7 with H3 peptides [8,9,14]. However, salt concentration of 250 mM disrupts association of NuRD complex with H3 peptides [10].…”

“…However, most of these assays are restricted to "candidate" approaches in which a limited, pre-determined set of proteins can be tested for binding to a particular modified or unmodified peptide. One significant advantage of using a peptide pull-down assay from nuclear extracts, in addition to its simplicity, is the fact that it is an unbiased approach, and therefore allows for identification of factors that would otherwise not have been likely candidates (see for example [8,9]). The assay also has its caveats: it is not quantitative and, due to extensive washes needed to minimize background, the association off-rate significantly impacts on observed binding.…”