Nucleosome Binding by the Bromodomain and PHD Finger of the Transcriptional Cofactor p300

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Abstract

The PHD finger and the bromodomain are small protein domains that occur in many proteins associated with phenomena related to chromatin. The bromodomain has been shown to bind acetylated lysine residues on histone tails. Lysine acetylation is one of several histone modifications that have been proposed to form the basis for a mechanism for recording epigenetically stable marks in chromatin, known as the histone code. The bromodomain is therefore thought to read a part of the histone code. Since PHD fingers often occur in proteins next to bromodomains, we have tested the hypothesis that the PHD finger can also interact with nucleosomes. Using two different in vitro assays, we found that the bromodomain/PHD finger region of the transcriptional cofactor p300 can bind to nucleosomes that have a high degree of histone acetylation. In a nucleosome retention assay, both domains were required for binding. Replacement of the p300 PHD finger with other PHD fingers resulted in loss of nucleosome binding. In an electrophoretic mobility shift assay, each domain alone showed, however, nucleosome-binding activity. The binding of the isolated PHD finger to nucleosomes was independent of the histone acetylation levels. Our data are consistent with a model where the two domains cooperate in nucleosome binding. In this model, both the bromodomain and the PHD finger contact the nucleosome while simultaneously interacting with each other.

Introduction

Chromatin plays an active role in gene regulation. A gene can be packaged in chromatin such that it becomes inaccessible to the transcription apparatus. If inactive chromatin structures persist through cell generations, this becomes, in effect, a mechanism for propagating stable patterns of gene expression. It is similarly thought that active chromatin configurations can be made mitotically stable. Two classes of enzymes have been identified that are responsible for modulation of chromatin structure. These are (i) the enzymes that post-translationally modify the N-terminal histone tails by acetylation, methylation, phosphorylation and ubiquitinylation; and (ii) those that remodel nucleosome structure in an ATP-dependent manner. The patterns of some of the histone modifications can, apparently, persist through cell generations. Furthermore, since each histone tail can be modified on several residues by different mechanisms, it has been proposed that chromatin carries epigenetically stable marks in the form of a “histone code”.1., 2., 3. While histone acetyltransferases (HATs), deacetylases (HDACs) and methyltransferases (HMTases) serve to “write” the histone code, other mechanisms must exist for “reading” the histone code.

Two protein domains have recently been found to have properties expected for readers of the histone code: (i) the bromodomain, which has been shown to bind to acetylated lysine residues on histone tails4., 5., 6. and (ii) the chromodomain of HP1, which has been demonstrated to bind histone H3 tails tri-methylated on lysine 9 (H3K9me3).7., 8., 9.

Bromo- and chromodomains often occur together with PHD fingers.10 PHD fingers are small protein domains built around two zinc ions coordinated by cysteine residues (C) and a histidine (H) in a C4HC3 motif.11., 12. It was first related to epigenetic regulation when it was discovered in Drosophila Trithorax (Trx), the prototype trithorax group (trxG) protein and the polycomb group (PcG) protein Polycomblike (Pcl).10 Intriguingly, the three trxG proteins Ash1,13 Ash214 and Lid15 have also been found to carry PHD fingers. Other PHD finger proteins include several classes of transcriptional cofactors including p300/CBP,16 the TIF1-family,17 the Mi-2 protein,18., 19. a component of the NuRD histone deacetylase complex20 and the ACF1/WSTF family, subunits of chromatin remodelling complexes.21 A further connection between PHD fingers and chromatin comes from the identification of SET domains as histone methyltransferases. Both Trx and Ash1 contain PHD fingers and a SET domain. Furthermore, two SET domain protein complexes in yeast contain PHD finger proteins.22., 23. Taken together, these observations strongly suggest that the PHD finger has a function related to chromatin. This correlation is underscored by the general underrepresentation of PHD fingers in sequence-specific DNA-binding transcription factors and proteins involved in other nuclear processes such as splicing, replication, recombination and repair. Despite this strong correlation with chromatin, PHD fingers are very diverse in sequence, suggesting that the molecular function related to chromatin is also diverse.

We have suggested three possible functions for PHD fingers: (i) as many other zinc fingers, they may bind to DNA or RNA; (ii) like the double zinc finger LIM domain, they may mediate protein–protein interactions or (iii) they may recognise the differentially modified histone tails.10 A few reports have provided evidence that PHD fingers may be protein–protein interaction domains: first, the PHD finger/bromodomain region of human KAP-1 (alias TIF1β) has been shown to interact with the KID domain of Mi-2α;24 second, the region containing the two PHD fingers of Drosophila Pcl has been implicated in the interaction of Pcl with Enhancer of zeste (E(z)).25 While the recently described complexes containing E(z) and Extra sex combs (Esc) did not contain Pcl,26., 27., 28. it has been shown that Pcl resides in a larger 1-MDa complex with E(z), Esc, Rpd3 and several other proteins.29 These authors further showed that a region containing the two PHD fingers of Pcl was sufficient to bind to Rpd3 in vitro.29 Third, the PHD finger region of MLL, a human homologue of Trx, has been shown to interact with the cyclophilin Cyp33.30 Cyp33 was, however, not among the 28 polypetides in the recently described human MLL supercomplex.31

Another type of function for the PHD finger was recently suggested by the requirement for the PHD finger for the histone acetyltransferase (HAT) activity of CBP.32 The PHD finger of the closely related p300 protein was, however, dispensable for its HAT activity. More recently, the PHD finger in MEKK1 kinase was proposed to be a ubiquitin ligase.33 This finger has later been shown, however, to be a RING finger, a domain known to have such activity.34 Furthermore, the PHD fingers of ING2 and several other proteins were reported to function as phosphoinositide receptors,35 much like the related FYVE fingers.36 The significance of this finding is not yet clear. Finally, the PHD fingers in AIRE, encoded by a gene mutated in autoimmune disease, have recently been found to be required for subnuclear targeting of this protein in dot-like structures in the nucleus.37., 38., 39., 40., 41.

While it is apparent that PHD fingers could have several functions, we have investigated the hypothesis that PHD fingers can interact directly with nucleosomes.

Section snippets

The bromodomain–PHD finger region of p300 binds to nucleosomes in vitro

Here, we describe experiments with the PHD finger of the human transcriptional coactivator p300.16 In this protein, the PHD finger is placed next to a bromodomain centrally in the protein (Figure 1(a)). We expressed the p300 bromodomain/PHD finger region (p300BP) and derivatives in bacteria as fusion proteins with glutathione-S-transferase (GST) and purified them by affinity chromatography (Figure 1(b)). The proteins were assayed in nucleosome retention assays and nucleosome mobility shift

Discussion

The occurrence of PHD fingers in a diverse set of proteins implicated in chromatin-based gene regulation led us to investigate the possibility that this domain can bind nucleosomes in vitro. Since many PHD fingers occur next to bromo- and chromodomains, we selected the PHD finger of p300 as a representative. In this protein, the PHD finger is situated next to a bromodomain which is expected to bind to histone tails carrying acetylated lysine residues.

Using two different assays, we have shown

Construction of protein expression vectors

The starting point for all vectors for bacterial expression of p300-derived protein fragments (see Figure 1(a)) was pSXG-p300BP, which carries a fragment of a human p300 cDNA cloned in our laboratory by RT-PCR using primers B5 (5′ ggc cat atg gaattc CCG GCT CCA GGA CAG TCA AA) and P3 (5′ cgg ctg cag gtcgac TTC TTT CCT AGT TCG TGC AC; for all primers, p300 sequences are shown with capital letters and the relevant reading frame is indicated by triplets; relevant restriction sites are underlined,

Acknowledgements

H.V. and R.A. were supported by the Norwegian Cancer Society and A.R., S.E. and A.M.Ø. by the Norwegian Research Council. Part of this work was supported by a grant to R.A. from the L. Meltzer Foundation at the University of Bergen. We also appreciate a short term FEBS fellowship to A.R. We thank Javad Azadehdel, Synne Winterthun, and Christine Gjerdrum for technical assistance, Karim Bouazone, Axel Imhof and Alex Brehm for the generous gift of acetylated recombinant histones, Pål Puntervoll,

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    Supplementary data associated with this article can be found at doi=10.1016/j.jmb.2004.01.051

    Present addresses: H. Valvatne, Department of Microbiology and Immunology, University of Bergen, Norway; V. Årskog, Department of Biology, University of Oslo, Norway; A. M. Øyan, Center for Research in Virology, Department of Microbiology and Immunology, University of Bergen, Norway.

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