Surprising complexity of the Asf1 histone chaperone-Rad53 kinase interaction.

The histone chaperone Asf1 and the checkpoint kinase Rad53 are found in a complex in budding yeast cells in the absence of genotoxic stress. Our data suggest that this complex involves at least three interaction sites. One site involves the H3-binding surface of Asf11 with an as yet undefined surface of Rad53. A second site is formed by the Rad53-FHA1 domain binding to Asf1-T(270) phosphorylated by casein kinase II. The third site involves the C-terminal 21 amino acids of Rad53 bound to the conserved Asf1 N-terminal domain. The structure of this site showed that the Rad53 C-terminus binds Asf1 in a remarkably similar manner to peptides derived from the histone cochaperones HirA and CAF-I. We call this binding motif, (R/K)R(I/A/V) (L/P), the AIP box for Asf1-Interacting Protein box. Furthermore, C-terminal Rad53-F(820) binds the same pocket of Asf1 as does histone H4-F(100). Thus Rad53 competes with histones H3-H4 and cochaperones HirA/CAF-I for binding to Asf1. Rad53 is phosphorylated and activated upon genotoxic stress. The Asf1-Rad53 complex dissociated when cells were treated with hydroxyurea but not methyl-methane-sulfonate, suggesting a regulation of the complex as a function of the stress. We identified a rad53 mutation that destabilized the Asf1-Rad53 complex and increased the viability of rad9 and rad24 mutants in conditions of genotoxic stress, suggesting that complex stability impacts the DNA damage response.

An ENU-induced point mutation in the mouse Btaf1 gene causes post-gastrulation embryonic lethality and protein instability.

The mouse Btaf1 gene, an ortholog of yeast MOT1, encodes a highly conserved general transcription factor. The function of this SNF2-like ATPase has been studied mainly in yeast and human cells, which has revealed that it binds directly to TBP, forming the B-TFIID complex. This complex binds to core promoters of RNA polymerase II-transcribed genes and, of crucial importance, BTAF1-TBP interactions have been shown to affect the kinetics of TBP-promoter interactions. Here we report the isolation of a mouse line carrying a Btaf1 allele containing an ENU-induced point mutation that causes a substitution mutation in the BTAF1 ATPase domain. Embryos homozygous for this loss-of-function mutation appear to be morphologically normal until early somite stages, but die between embryonic days 9 and 10.5 displaying growth arrest and edema. Analyses in vitro suggest that the altered protein is less stable and, independent from this, functionally impaired in releasing of TBP from chromatin, but not in binding to TBP.

Chromatin interaction of TATA-binding protein is dynamically regulated in human cells.

Gene transcription in mammalian cells is a dynamic process involving regulated assembly of transcription complexes on chromatin in which the TATA-binding protein (TBP) plays a central role. Here, we investigate the dynamic behaviour of TBP by a combination of fluorescence recovery after photobleaching (FRAP) and biochemical assays using human cell lines of different origin. The majority of nucleoplasmic TBP and other TFIID subunits associate with chromatin in a highly dynamic manner. TBP dynamics are regulated by the joint action of the SNF2-related BTAF1 protein and the NC2 complex. Strikingly, both BTAF1 and NC2 predominantly affect TBP dissociation rates, leaving the association rate unchanged. Chromatin immunoprecipitation shows that BTAF1 negatively regulates TBP and NC2 binding to active promoters. Our results support a model for a BTAF1-mediated release of TBP-NC2 complexes from chromatin.

Comparing genome-wide chromatin profiles using ChIP-chip or ChIP-seq.

MOTIVATION: ChIP-chip and ChIP-seq technologies provide genome-wide measurements of various types of chromatin marks at an unprecedented resolution. With ChIP samples collected from different tissue types and/or individuals, we can now begin to characterize stochastic or systematic changes in epigenetic patterns during development (intra-individual) or at the population level (inter-individual). This requires statistical methods that permit a simultaneous comparison of multiple ChIP samples on a global as well as locus-specific scale. Current analytical approaches are mainly geared toward single sample investigations, and therefore have limited applicability in this comparative setting. This shortcoming presents a bottleneck in biological interpretations of multiple sample data. RESULTS: To address this limitation, we introduce a parametric classification approach for the simultaneous analysis of two (or more) ChIP samples. We consider several competing models that reflect alternative biological assumptions about the global distribution of the data. Inferences about locus-specific and genome-wide chromatin differences are reached through the estimation of multivariate mixtures. Parameter estimates are obtained using an incremental version of the Expectation-Maximization algorithm (IEM). We demonstrate efficient scalability and application to three very diverse ChIP-chip and ChIP-seq experiments. The proposed approach is evaluated against several published ChIP-chip and ChIP-seq software packages. We recommend its use as a first-pass algorithm to identify candidate regions in the epigenome, possibly followed by some type of second-pass algorithm to fine-tune detected peaks in accordance with biological or technological criteria. AVAILABILITY: R source code is available at http://gbic.biol.rug.nl/supplementary/2009/ChromatinProfiles/. Access to Chip-seq data: GEO repository GSE17937.

Quantitative proteomics reveals regulation of dynamic components within TATA-binding protein (TBP) transcription complexes.

Affinity purification in combination with isotope labeling of proteins has proven to be a powerful method to discriminate specific from nonspecific interactors. However, in the standard SILAC (stable isotope labeling by amino acids in cell culture) approach dynamic components may easily be assigned as nonspecific. We compared two affinity purification protocols, which in combination revealed information on the dynamics of protein complexes. We focused on the central component in eukaryotic transcription, the human TATA-binding protein, which is involved in different complexes. All known TATA-binding protein-associated factors (TAFs) were detected as specific interactors. Interestingly one of them, BTAF1, exchanged significantly in cell extracts during the affinity purification. The other TAFs did not display this behavior. Cell cycle synchronization showed that BTAF1 exchange was regulated during mitosis. The combination of the two affinity purification protocols allows a quantitative approach to identify transient components in any protein complex.

Modulation of Ubc4p/Ubc5p-mediated stress responses by the RING-finger-dependent ubiquitin-protein ligase Not4p in Saccharomyces cerevisiae.

The Ccr4-Not complex consists of nine subunits and acts as a regulator of mRNA biogenesis in Saccharomyces cerevisiae. The human ortholog of yeast NOT4, CNOT4, displays UbcH5B-dependent ubiquitin-protein ligase (E3 ligase) activity in a reconstituted in vitro system. However, an in vivo role for this enzymatic activity has not been identified. Site-directed mutagenesis of the RING finger of yeast Not4p identified residues required for interaction with Ubc4p and Ubc5p, the yeast orthologs of UbcH5B. Subsequent in vitro assays with purified Ccr4-Not complexes showed Not4p-mediated E3 ligase activity, which was dependent on the interaction with Ubc4p. To investigate the in vivo relevance of this activity, we performed synthetic genetic array (SGA) analyses using not4Delta and not4L35A alleles. This indicates involvement of the RING finger of Not4p in transcription, ubiquitylation, and DNA damage responses. In addition, we found a phenotypic overlap between deletions of UBC4 and mutants encoding single-amino-acid substitutions of the RING finger of Not4p. Together, our results show that Not4p functions as an E3 ligase by modulating Ubc4p/Ubc5p-mediated stress responses in vivo.

The histone chaperone Asf1 at the crossroads of chromatin and DNA checkpoint pathways.

Nucleosome assembly involves deposition of a heterotetramer of histones H3/H4 onto DNA followed by two heterodimers of histones H2A/H2B. Cycles of nucleosome assembly and disassembly are essential to cellular events such as replication, transcription, and DNA repair. After synthesis in the cytoplasm, histones are shuttled into the nucleus where they are associated with chaperone proteins. Chaperones of histones H3/H4 include CAF-I, the Hir proteins, and Asf1. CAF-I and the Hir proteins function as replication-coupled and replication-independent deposition factors for H3/H4, respectively, whereas Asf1 may play a role in both pathways. In addition to acting as assembly factors, histone chaperones assist nucleosome dissociation from DNA and they may recruit other proteins to chromatin. The past few years have witnessed a notable accumulation of genetic, biochemical, and structural data on Asf1, which motivated this review. We discuss the sequence and structural features of Asf1 before considering its roles in nucleosome assembly/disassembly, the cellular response to DNA damage, and the regulation of gene expression. We emphasize the key role of Asf1 as a central node in a network of partners that place it at the crossroads of chromatin and DNA checkpoint pathways.

Single-spanning membrane protein insertion in membrane mimetic systems: role and localization of aromatic residues.

Membrane protein insertion in the lipid bilayer is determining for their activity and is governed by various factors such as specific sequence motifs or key amino-acids. A detailed fluorescence study of such factors is exemplified with PMP1, a small (38 residues) single-membrane span protein that regulates the plasma membrane H(+)-ATPase in yeast and specifically interacts with phosphatidylserines. Such interactions may stabilize raft domains that have been shown to contain H(+)-ATPase. Previous NMR studies of various fragments have focused on the critical role of interfacial residues in the PMP1 structure and intermolecular interactions. The C-terminal domain contains a terminal Phe (F38), a single Trp (W28) and a single Tyr (Y25) that may act together to anchor the protein in the membrane. In order to describe the location and dynamics of W28 and the influence of Y25 on protein insertion within membrane, we carried out a detailed steady-state and time-resolved fluorescence study of the synthetic G13-F38 fragment and its Tyr-less mutant, Y25L in various membrane mimetic systems. Detergent micelles are conveniently used for this purpose. We used dodecylphosphocholine (DPC) in order to compare with and complement previous NMR results. In addition, dodecylmaltoside (DM) was used so that we could apply our recently described new quenching method by two brominated analogs of DM (de Foresta et al. 2002, Eur. Biophys. J. 31:185-97). In both systems, and in the presence and absence of Y25, W28 was shown to be located below but close to the polar headgroup region, as shown by its maximum emission wavelengths (lambda(max)), curves for the quenching of Trp by the brominated analogs of DM and bimolecular constants for quenching (k(q)) by acrylamide. Results were interpreted by comparison with calibration data obtained with fluorescent model peptides. Time-resolved anisotropy measurements were consistent with PMP1 fragment immobilization within peptide-detergent complexes. We tentatively assigned the two major Trp lifetimes to the Trp (chi(1)=60 degrees and 180 degrees ) rotamers, based on the recent lifetime-rotamer correlation proposed for model cyclic peptides (Pan and Barkley 2004, Biophys J 86:3828-35). We also analyzed the role of the hydrophobic anchor, by comparing the micelle binding of fragments of various lengths including the synthesized full-length protein and detected peculiar differences for protein interaction with the polar headgroups of DM or DPC.

Structural basis for the interaction of Asf1 with histone H3 and its functional implications.

Asf1 is a conserved histone chaperone implicated in nucleosome assembly, transcriptional silencing, and the cellular response to DNA damage. We solved the NMR solution structure of the N-terminal functional domain of the human Asf1a isoform, and we identified by NMR chemical shift mapping a surface of Asf1a that binds the C-terminal helix of histone H3. This binding surface forms a highly conserved hydrophobic groove surrounded by charged residues. Mutations within this binding site decreased the affinity of Asf1a for the histone H3/H4 complex in vitro, and the same mutations in the homologous yeast protein led to transcriptional silencing defects, DNA damage sensitivity, and thermosensitive growth. We have thus obtained direct experimental evidence of the mode of binding between a histone and one of its chaperones and genetic data suggesting that this interaction is important in both the DNA damage response and transcriptional silencing.

Deciphering the role of individual acyl chains in the interaction network between phosphatidylserines and a single-spanning membrane protein.

PMP1 is a small single-spanning membrane protein functioning as a regulatory subunit of the yeast plasma membrane H(+)-ATPase. This protein forms a unique helix and exhibits a positively charged cytoplasmic domain that is able to specifically segregate phosphatidylserines (PSs). A marked groove formed at the helix surface is thought to play a major role in the related lipid-protein interaction network. Mutational analysis and (1)H NMR experiments were therefore performed on a synthetic PMP1 fragment using DPC-d(38) micelles as a membrane-like environment, in the presence of small amounts of POPS. A mutation designed for altering the helix groove was shown to disfavor the POPS binding specificity as much as that affecting the electrostatic interaction network. From POPS titration experiments monitored by a full set of one- and two-dimensional NOESY spectra, the association between the phospholipids and the PMP1 peptide has been followed. Our data reveal that the clustering of POPS molecules is promoted from a stabilized framework obtained by coupling the PMP1 helix groove to a POPS sn-2 chain. To our knowledge, the NOE-based titration plots displayed in this report constitute the first NMR data that directly distinguish the role of the sn-1 and sn-2 acyl chains in a lipid-protein interaction. The results are discussed while taking into account our accurate knowledge of the yeast plasma membrane composition and its ability to form functional lipid rafts.