PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3.

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

Developmental role for ACF1-containing nucleosome remodellers in chromatin organisation.

The nucleosome remodelling complexes CHRAC and ACF of Drosophila are thought to play global roles in chromatin assembly and nucleosome dynamics. Disruption of the gene encoding the common ACF1 subunit compromises fly viability. Survivors show defects in chromatin assembly and chromatin-mediated gene repression at all developmental stages. We now show that ACF1 expression is under strict developmental control. The expression is strongly diminished during embryonic development and persists at high levels only in undifferentiated cells, including the germ cell precursors and larval neuroblasts. Constitutive expression of ACF1 is lethal. Cell-specific ectopic expression perturbs chromatin organisation and nuclear programmes. By monitoring heterochromatin formation during development, we have found that ACF1-containing factors are involved in the initial establishment of diversified chromatin structures, such as heterochromatin. Altering the levels of ACF1 leads to global and variegated deviations from normal chromatin organisation with pleiotropic defects.

Acetylation increases access of remodelling complexes to their nucleosome targets to enhance initiation of V(D)J recombination.

Targeted chromatin remodelling is essential for many nuclear processes, including the regulation of V(D)J recombination. ATP-dependent nucleosome remodelling complexes are important players in this process whose activity must be tightly regulated. We show here that histone acetylation regulates nucleosome remodelling complex activity to boost RAG cutting during the initiation of V(D)J recombination. RAG cutting requires nucleosome mobilization from recombination signal sequences. Histone acetylation does not stimulate nucleosome mobilization per se by CHRAC, ACF or their catalytic subunit, ISWI. Instead, we find the more open structure of acetylated chromatin regulates the ability of nucleosome remodelling complexes to access their nucleosome templates. We also find that bromodomain/acetylated histone tail interactions can contribute to this targeting at limited concentrations of remodelling complex. We therefore propose that the changes in higher order chromatin structure associated with histone acetylation contribute to the correct targeting of nucleosome remodelling complexes and this is a novel way in which histone acetylation can modulate remodelling complex activity.

Site-specific acetylation of ISWI by GCN5.

BACKGROUND: The tight organisation of eukaryotic genomes as chromatin hinders the interaction of many DNA-binding regulators. The local accessibility of DNA is regulated by many chromatin modifying enzymes, among them the nucleosome remodelling factors. These enzymes couple the hydrolysis of ATP to disruption of histone-DNA interactions, which may lead to partial or complete disassembly of nucleosomes or their sliding on DNA. The diversity of nucleosome remodelling factors is reflected by a multitude of ATPase complexes with distinct subunit composition. RESULTS: We found further diversification of remodelling factors by posttranslational modification. The histone acetyltransferase GCN5 can acetylate the Drosophila remodelling ATPase ISWI at a single, conserved lysine, K753, in vivo and in vitro. The target sequence is strikingly similar to the N-terminus of histone H3, where the corresponding lysine, H3K14, can also be acetylated by GCN5. The acetylated form of ISWI represents a minor species presumably associated with the nucleosome remodelling factor NURF. CONCLUSION: Acetylation of histone H3 and ISWI by GCN5 is explained by the sequence similarity between the histone and ISWI around the acetylation site. The common motif RKT/SxGx(Kac)xPR/K differs from the previously suggested GCN5/PCAF recognition motif GKxxP. This raises the possibility of co-regulation of a nucleosome remodelling factor and its nucleosome substrate through acetylation of related epitopes and suggests a direct crosstalk between two distinct nucleosome modification principles.

HP1 binding to chromatin methylated at H3K9 is enhanced by auxiliary factors.

A large portion of the eukaryotic genome is packaged into transcriptionally silent heterochromatin. Several factors that play important roles during the establishment and maintenance of this condensed form have been identified. Methylation of lysine 9 within histone H3 and the subsequent binding of the chromodomain protein heterochromatin protein 1 (HP1) are thought to initiate heterochromatin formation in vivo and to propagate a heterochromatic state lasting through several cell divisions. For the present study we analyzed the binding of HP1 to methylated chromatin in a fully reconstituted system. In contrast to its strong binding to methylated peptides, HP1 binds only weakly to methylated chromatin. However, the addition of recombinant SU(VAR) protein, such as ACF1 or SU(VAR)3-9, facilitates HP1 binding to chromatin methylated at lysine 9 within the H3 N terminus (H3K9). We propose that HP1 has multiple target sites that contribute to its recognition of chromatin, only one of them being methylated at H3K9. These findings have implications for the mechanisms of recognition of specific chromatin modifications in vivo.

The histone fold subunits of Drosophila CHRAC facilitate nucleosome sliding through dynamic DNA interactions.

The chromatin accessibility complex (CHRAC) is an abundant, evolutionarily conserved nucleosome remodeling machinery able to catalyze histone octamer sliding on DNA. CHRAC differs from the related ACF complex by the presence of two subunits with molecular masses of 14 and 16 kDa, whose structure and function were not known. We determined the structure of Drosophila melanogaster CHRAC14-CHRAC16 by X-ray crystallography at 2.4-angstroms resolution and found that they dimerize via a variant histone fold in a typical handshake structure. In further analogy to histones, CHRAC14-16 contain unstructured N- and C-terminal tail domains that protrude from the handshake structure. A dimer of CHRAC14-16 can associate with the N terminus of ACF1, thereby completing CHRAC. Low-affinity interactions of CHRAC14-16 with DNA significantly improve the efficiency of nucleosome mobilization by limiting amounts of ACF. Deletion of the negatively charged C terminus of CHRAC16 enhances DNA binding 25-fold but leads to inhibition of nucleosome sliding, in striking analogy to the effect of the DNA chaperone HMGB1 on nucleosome sliding. The presence of a surface compatible with DNA interaction and the geometry of an H2A-H2B heterodimer may provide a transient acceptor site for DNA dislocated from the histone surface and therefore facilitate the nucleosome remodeling process.

Dynamic chromatin: concerted nucleosome remodelling and acetylation.

The flexibility of chromatin that enables translation of environmental cues into changes in genome utilisation, relies on a battery of enzymes able to modulate chromatin structure in a highly targeted and regulated manner. The most dynamic structural changes are brought about by two kinds of enzymes with different functional principles. Changes in the acetylation status of histones modulate the folding of the nucleosomal fibre. The histone-DNA interactions that define the nucleosome itself can be disrupted by ATP-dependent remodelling factors. This review focuses on recent developments that illustrate various strategies for integrating these disparate activities into complex regulatory schemes. Synergies may be brought about by consecutive or parallel action during the stepwise process of chromatin opening or closing. Tight co-ordination may be achieved by direct interaction of (de-)acetylation enzymes and remodelling ATPases or even permanent residence within the same multi-enzyme complex. The fact that remodelling ATPases can be acetylated by histone acetyltransferases themselves suggests exciting possibilities for the co-ordinate modulation of chromatin structure and remodelling enzymes.

ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts.

The nucleosome remodelling ATPase ISWI resides in several distinct protein complexes whose subunit composition reflects their functional specialization. Association of ISWI with ACF1, the largest subunit of CHRAC and ACF complexes, improves the efficiency of ISWI-induced nucleosome mobilization by an order of magnitude and also modulates the reaction qualitatively. In order to understand the principle by which ACF1 improves the efficiency of ISWI, we mapped their mutual interaction requirements and generated a series of ACF complexes lacking conserved ACF1 domains. Deletion of the C-terminal PHD finger modules of ACF1 or their disruption by zinc chelation profoundly affected the nucleosome mobilization capability of associated ISWI in trans. Interactions of the PHD fingers with the central domains of core histones contribute significantly to the binding of ACF to the nucleosome substrate, suggesting a novel role for PHD modules as nucleosome interaction determinants. Connecting ACF to histones may be prerequisite for efficient conversion of ATP-dependent conformational changes of ISWI into translocation of DNA relative to the histones during nucleosome mobilization.

Nucleosome binding by the bromodomain and PHD finger of the transcriptional cofactor p300.

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.

Crystal structure and functional analysis of a nucleosome recognition module of the remodeling factor ISWI.

Energy-dependent nucleosome remodeling emerges as a key process endowing chromatin with dynamic properties. However, the principles by which remodeling ATPases interact with their nucleosome substrate to alter histone-DNA interactions are only poorly understood. We have identified a substrate recognition domain in the C-terminal half of the remodeling ATPase ISWI and determined its structure by X-ray crystallography. The structure comprises three domains, a four-helix domain with a novel fold and two alpha-helical domains related to the modules of c-Myb, SANT and SLIDE, which are linked by a long helix. An integrated structural and functional analysis of these domains provides insight into how ISWI interacts with the nucleosomal substrate.

Histone acetylation: a switch between repressive and permissive chromatin. Second in review series on chromatin dynamics.

The organization of eukaryotic chromatin has a major impact on all nuclear processes involving DNA substrates. Gene expression is affected by the positioning of individual nucleosomes relative to regulatory sequence elements, by the folding of the nucleosomal fiber into higher-order structures and by the compartmentalization of functional domains within the nucleus. Because site-specific acetylation of nucleosomal histones influences all three aspects of chromatin organization, it is central to the switch between permissive and repressive chromatin structure. The targeting of enzymes that modulate the histone acetylation status of chromatin, in synergy with the effects mediated by other chromatin remodeling factors, is central to gene regulation.