Nucleosomal Histone H2A Research Articles

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a ‘pincer-like’ conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5–nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C.

The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a 'pincer-like' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.

Conservation of transcriptional regulation by BRCA1 and BARD1 in Caenorhabditis elegans.

The tumor-suppressor proteins BRCA1 and BARD1 function as an E3 ubiquitin ligase to facilitate transcriptional repression and DNA damage repair. This is mediated in-part through its ability to mono-ubiquitylate histone H2A in nucleosomes. Studies in Caenorhabditis elegans have been used to elucidate numerous functions of BRCA1 and BARD1; however, it has not been established that the C. elegans orthologs, BRC-1 and BRD-1, retain all the functions of their human counterparts. Here we explore the conservation of enzymatic activity toward nucleosomes which leads to repression of estrogen-metabolizing cytochrome P450 (cyp) genes in humans. Biochemical assays establish that BRC-1 and BRD-1 contribute to ubiquitylation of histone H2A in the nucleosome. Mutational analysis shows that while BRC-1 likely binds the nucleosome using a conserved interface, BRD-1 and BARD1 have evolved different modes of binding, resulting in a difference in the placement of ubiquitin on H2A. Gene expression analysis reveals that in spite of this difference, BRC-1 and BRD-1 also contribute to cyp gene repression in C. elegans. Establishing conservation of these functions in C. elegans allows for use of this powerful model organism to address remaining questions regarding regulation of gene expression by BRCA1 and BARD1.

Coordinated DNA and histone dynamics drive accurate histone H2A.Z exchange.

Nucleosomal histone H2A is exchanged for its variant H2A.Z by the SWR1 chromatin remodeler, but the mechanism and timing of histone exchange remain unclear. Here, we quantify DNA and histone dynamics during histone exchange in real time using a three-color single-molecule FRET assay. We show that SWR1 operates with timed precision to unwrap DNA with large displacement from one face of the nucleosome, remove H2A-H2B from the same face, and rewrap DNA, all within 2.3 s. This productive DNA unwrapping requires full SWR1 activation and differs from unproductive, smaller-scale DNA unwrapping caused by SWR1 binding alone. On an asymmetrically positioned nucleosome, SWR1 intrinsically senses long-linker DNA to preferentially exchange H2A.Z on the distal face as observed in vivo. The displaced H2A-H2B dimer remains briefly associated with the SWR1-nucleosome complex and is dissociated by histone chaperones. These findings reveal how SWR1 coordinates DNA unwrapping with histone dynamics to rapidly and accurately place H2A.Z at physiological sites on chromatin.

Prognostic Value and Immunological Role of MORF4-Related Gene-Binding Protein in Human Cancers.

MORF4-related gene-binding protein (MRGBP) is the subunit of the NuA4 histone acetyltransferase complex which is involved in transcriptional activation of select genes principally by acetylation of nucleosomal histones H4 and H2A. Much of the research indicated an oncogenic role of MRGBP in the development of cancers. However, it is still unknown the role MRGBP plays in human cancers, which deserves further exploration. In this research, the expression profile, prognostic value of MRGBP, and the relationship between MRGBP and immune infiltration were explored in 33 types of cancer. The differences in MRGBP expression in tumor and normal tissues were explored using data from The Cancer Genome Atlas, Gene Expression Omnibus and ONCOMINE. Analysis of the association between MRGBP and prognosis using Kaplan-Meier survival curve and COX analysis. The data of Tumor mutational burden (TMB), microsatellite instability (MSI) from TCGA. The relationship Between MRGBP expression and immunity was analyzed using the ESTIMATE algorithm and CIBERSORT. Furthermore, we explored MRGBP expression and the relationship between MRGBP expression and macrophage infiltration using immunohistochemical analysis in lower grade glioma (LGG). Our results revealed that MRGBP was highly expressed in most cancer tissues compared with normal tissues. Tumors with increased MRGBP expression had a high clinicopathologic stage and poor prognosis. The expression of MRGBP was closely related to the TMB, MSI. We also found a significant negative correlation between MRGBP expression and stromal scores and immune scores in various types of cancer. Furthermore, MRGBP expression was associated with a variety of immune cells including B cells, NK cells, T cells, and macrophages. LGG and LIHC was selected as representative cancer types for further study, the results of immunohistochemistry indicated that the protein levels of MRGBP were significantly elevated in tumor tissues. Moreover, our LIHC data analysis showed that patients with high MRGBP expression were associated with short survival rates and MRGBP was a risk factor to determine OS. Immunohistochemistry also confirmed that M0 macrophage infiltration in the MRGBP-high group significantly increased. In conclusion, these results reveal that MRGBP can serve as a potential prognostic biomarker and it plays an important role in tumor immune infiltration in various tumors, especially in LGG and LIHC.

Structural Basis for the Activation of the Deubiquitinase Calypso by the Polycomb Protein ASX

Ubiquitin C-terminal hydrolase deubiquitinase BAP1 is an essential tumor suppressor involved in cell growth control, DNA damage response, and transcriptional regulation. As part of the Polycomb repression machinery, BAP1 is activated by the deubiquitinase adaptor domain of ASXL1 mediating gene repression by cleaving ubiquitin (Ub) from histone H2A in nucleosomes. The molecular mechanism of BAP1 activation by ASXL1 remains elusive, as no structures are available for either BAP1 or ASXL1. Here, we present the crystal structure of the BAP1 ortholog from Drosophila melanogaster, named Calypso, bound to its activator, ASX, homolog of ASXL1. Based on comparative structural andfunctional analysis, we propose a model for Ub binding by Calypso/ASX, uncover decisive structural elements responsible for ASX-mediated Calypso activation, and characterize the interaction with ubiquitinated nucleosomes. Our results give molecular insight into Calypso function and its regulation by ASX and provide the opportunity for the rational design of mechanism-based therapeutics to treat human BAP1/ASXL1-related tumors.

De novo mutation in RING1 with epigenetic effects on neurodevelopment

RING1 is an E3-ubiquitin ligase that is involved in epigenetic control of transcription during development. It is a component of the polycomb repressive complex 1, and its role in that complex is to ubiquitylate histone H2A. In a 13-year-old girl with syndromic neurodevelopmental disabilities, we identified a de novo mutation, RING1 p.R95Q, which alters a conserved arginine residue in the catalytic RING domain. In vitro assays demonstrated that the mutant RING1 retains capacity to catalyze ubiquitin chain formation, but is defective in its ability to ubiquitylate histone H2A in nucleosomes. Consistent with this in vitro effect, cells of the patient showed decreased monoubiquitylation of histone H2A. We modeled the mutant RING1 in Caenorhabditis elegans by editing the comparable amino acid change into spat-3, the suggested RING1 ortholog. Animals with either the missense mutation or complete knockout of spat-3 were defective in monoubiquitylation of histone H2A and had defects in neuronal migration and axon guidance. Relevant to our patient, animals heterozygous for either the missense or knockout allele also showed neuronal defects. Our results support three conclusions: mutation of RING1 is the likely cause of a human neurodevelopmental syndrome, mutation of RING1 can disrupt histone H2A ubiquitylation without disrupting RING1 catalytic activity, and the comparable mutation in C. elegans spat-3 both recapitulates the effects on histone H2A ubiquitylation and leads to neurodevelopmental abnormalities. This role for RING1 adds to our understanding of the importance of aberrant epigenetic effects as causes of human neurodevelopmental disorders.

BARD1 is necessary for ubiquitylation of nucleosomal histone H2A and for transcriptional regulation of estrogen metabolism genes

Missense mutations that disrupt the RING domain of the tumor suppressor gene BRCA1 lead to increased risk of breast and ovarian cancer. The BRCA1 RING domain is a ubiquitin ligase, whose structure and function rely critically on forming a heterodimer with BARD1, which also harbors a RING domain. The function of the BARD1 RING domain is unknown. In families severely affected with breast cancer, we identified inherited BARD1 missense mutations Cys53Trp, Cys71Tyr, and Cys83Arg that alter three zinc-binding residues of the BARD1 RING domain. Each of these mutant BARD1 proteins retained the ability to form heterodimeric complexes with BRCA1 to make an active ubiquitin ligase, but the mutant BRCA1/BARD1 complexes were deficient in binding to nucleosomes and in ubiquitylating histone H2A. The BARD1 mutations also caused loss of transcriptional repression of BRCA1-regulated estrogen metabolism genes CYP1A1 and CYP3A4; breast epithelial cells edited to create heterozygous loss of BARD1 showed significantly higher expression of CYP1A1 and CYP3A4 Reintroduction of wild-type BARD1 into these cells restored CYP1A1 and CYP3A4 transcription to normal levels, but introduction of the cancer-predisposing BARD1 RING mutants failed to do so. These results indicate that an intact BARD1 RING domain is critical to BRCA1/BARD1 binding to nucleosomes and hence to ubiquitylation of histone H2A and also critical to transcriptional repression of BRCA1-regulated genes active in estrogen metabolism.

Skin vaccination using microneedles coated with a plasmid DNA cocktail encoding nucleosomal histones of Leishmania spp.

Vaccine delivery using microneedles (MNs) represents a safe, easily disposable and painless alternative to traditional needle immunizations. The MN delivery of DNA vaccines to the dermis may result in a superior immune response and/or an equivalent immune response at a lower vaccine dose (dose-sparing). This could be of special interest for immunization programs against neglected tropical diseases such as leishmaniasis. In this work, we loaded a MN device with 60μg of a plasmid DNA cocktail encoding the Leishmania infantum nucleosomal histones H2A, H2B, H3 and H4 and compared its immunogenicity and protective capacity against conventional s.c. or i.d. injection of the plasmid. Mice immunized with MNs showed increased ratios of IFN-γ/IL-10, IFN-γ/IL-13, IFN-γ/IL-4, and IFN-γ/TGF-β in the spleens and lymph nodes compared with mice immunized by s.c. and i.d. routes. Furthermore, CCXCL9, CXCL10 and CCL2 levels were also higher. These data suggest that the nucleic acid immunization using MNs produced a better bias towards a Th1 response. However, none of the immunizations strategies were able to control Leishmania major infection in BALB/c mice, as illustrated by an increase in lesion size and parasite burden.

H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast.

The histone variant H2A.Z is a universal mark of gene promoters, enhancers, and regulatory elements in eukaryotic chromatin. The chromatin remodeler SWR1 mediates site-specific incorporation of H2A.Z by a multi-step histone replacement reaction, evicting histone H2A-H2B from the canonical nucleosome and depositing the H2A.Z-H2B dimer. Binding of both substrates, the canonical nucleosome and the H2A.Z-H2B dimer, is essential for activation of SWR1. We found that SWR1 primarily recognizes key residues within the α2 helix in the histone-fold of nucleosomal histone H2A, a region not previously known to influence remodeler activity. Moreover, SWR1 interacts preferentially with nucleosomal DNA at superhelix location 2 on the nucleosome face distal to its linker-binding site. Our findings provide new molecular insights on recognition of the canonical nucleosome by a chromatin remodeler and have implications for ATP-driven mechanisms of histone eviction and deposition.

Crystal structure of the PRC1 ubiquitylation module bound to the nucleosome.

The Polycomb group of epigenetic enzymes represses expression of developmentally regulated genes in higher eukaryotes. This group includes the Polycomb repressive complex 1 (PRC1), which ubiquitylates nucleosomal histone H2A Lys119 using its E3 ubiquitin ligase subunits, Ring1B and Bmi1, together with an E2 ubiquitin-conjugating enzyme, UbcH5c. However, the molecular mechanism of nucleosome substrate recognition by PRC1 or other chromatin enzymes is unclear. Here we present the crystal structure of the Ring1B/Bmi1/UbcH5c E3-E2 complex (the PRC1 ubiquitylation module) bound to its nucleosome core particle substrate. The structure shows how a chromatin enzyme achieves substrate specificity by interacting with multiple nucleosome surfaces spatially distinct from the site of catalysis. Our structure further reveals an unexpected role for the ubiquitin E2 enzyme in substrate recognition, and provides insight into how the related histone H2A E3 ligase, BRCA1, interacts with and ubiquitylates the nucleosome.

The Catalytic Subunit of the SWR1 Remodeler Is a Histone Chaperone for the H2A.Z-H2B Dimer

Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multisubunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. How SWR1 catalyzes the replacement reaction is largely unknown. Here, we determined the crystal structure of the N-terminal region (599-627) of the catalytic subunit Swr1, termed Swr1-Z domain, in complex with the H2A.Z-H2B dimer at 1.78 Å resolution. The Swr1-Z domain forms a 310 helix and an irregular chain. A conserved LxxLF motif in the Swr1-Z 310 helix specifically recognizes the αC helix of H2A.Z. Our results show that the Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3-H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism.

The Catalytic Subunit of the SWR1 Remodeler has a Histone Chaperone Role for the H2A.Z-H2B Dimer

Histone variant H2A.Z-containing nucleosomes exist at most eukaryotic promoters and play important roles in gene transcription and genome stability. The multi-subunit nucleosome-remodeling enzyme complex SWR1, conserved from yeast to mammals, catalyzes the ATP-dependent replacement of histone H2A in canonical nucleosomes with H2A.Z. The over-incorporation of H2A.Z in chromatin, however, is associated with breast cancer, and therefore H2A.Z and p400/SRCAP are targets for cancer therapy. How SWR1 catalyzes the replacement reaction is largely unknown. Here we determined the crystal structure of the region (599-627) of the catalytic subunit Swr1, termed Swr1-Z domain, N-terminal to the ATPase domain, in complex with the H2A.Z-H2B dimer at 1.78 A resolution. The Swr1-Z domain forms a 3(10) helix and an irregular chain, separated by an 8-residue disordered loop. Swr1-Z forms hydrophobic interactions with the a3 and aC helices of H2A.Z, and also with the a1 and a2 helices of H2B. Importantly, a conserved LxxLF motif in the Swr1-Z specifically recognizes the αC helix of H2A.Z, which leads to an extension of two helical turns of the aC helix in H2A.Z. Additionally, the Asp-rich region in Swr1-Z forms electrostatic interactions with Arg85 in the L2 loop, and with Arg89 in the a3 helix of H2A.Z, and with Lys60 in the a2 helix of H2B, which makes the complex more stable. Furthermore, in vitro studies show that Swr1-Z domain can deliver the H2A.Z-H2B dimer to the DNA-(H3-H4)2 tetrasome to form the nucleosome by a histone chaperone mechanism, and in vivo studies indicate that Swr1-Z has an important functional role. Our results provide the first structural insights into how Swr1 recognizes H2AZ-H2B, and help identify similar binding regions between human H2AZ-H2B and p400/SRCAP, paving the road for structure-based cancer drug design.

Phospho-dependent recruitment of NuA4 by MRX at DNA breaks regulates RPA dynamics during resection

Background The NuA4 histone acetyltransferase is a highly conserved multisubunit complex responsible for acetylation of nucleosomal histone H4 and H2A. Mutations in different NuA4 subunits create cell cycle arrest or delay in G2/M. The NuA4 complex is important for gene expression but also the efficient repair of DNA double strand breaks (DSBs) in vivo. NuA4 is rapidly recruited to chromatin surrounding a DSB in vivo at the same time histone H2A (X) is phosphorylated in the neighboring region (g-H2AX). While we have shown that NuA4 can interact with phosphorylated H2A(X), we speculated that another interaction was required for the initial recruitment of NuA4 at the break. Based on the presence of an ATM-related factor within NuA4, the subunitTra1 (TRRAP), it was tempting to postulate that DSB sensing factors known to recruit ATM family PIKK factors could be responsible for NuA4 recruitment.

In vitro molecular magnetic resonance imaging detection and measurement of apoptosis using superparamagnetic iron oxide + antibody as ligands for nucleosomes

Recent research in cell biology as well as oncology research has focused on apoptosis or programmed cell death as a means of quantifying the induced effects of treatment. A hallmark of late-stage apoptosis is nuclear fragmentation in which DNA is degraded to release nucleosomes with their associated histones. In this work, a method was developed for detecting and measuring nucleosome concentration in vitro with magnetic resonance imaging (MRI). The indirect procedure used a commercially available secondary antibody-superparamagnetic iron oxide (SPIO) particle complex as a contrast agent that bound to primary antibodies against nucleosomal histones H4, H2A and H2B. Using a multiple-echo spin-echo sequence on a 1.5 T clinical MRI scanner, significant T2 relaxation enhancement as a function of in vitro nucleosomal concentration was measured. In addition, clustering or aggregation of the contrast agent was demonstrated with its associated enhancement in T2 effects. The T2 clustering enhancement showed a complex dependence on relative concentrations of nucleosomes, primary antibody and secondary antibody + SPIO. The technique supports the feasibility of using MRI measurements of nucleosome concentration in blood as a diagnostic, prognostic and predictive tool in the management of cancer.

Stepwise Histone Replacement by SWR1 Requires Dual Activation with Histone H2A.Z and Canonical Nucleosome

Histone variant H2A.Z-containing nucleosomes are incorporated at most eukaryotic promoters. This incorporation is mediated by the conserved SWR1 complex, which replaces histone H2A in canonical nucleosomes with H2A.Z in an ATP-dependent manner. Here, we show that promoter-proximal nucleosomes are highly heterogeneous for H2A.Z in Saccharomyces cerevisiae, with substantial representation of nucleosomes containing one, two, or zero H2A.Z molecules. SWR1-catalyzed H2A.Z replacement in vitro occurs in a stepwise and unidirectional fashion, one H2A.Z-H2B dimer at a time, producing heterotypic nucleosomes as intermediates and homotypic H2A.Z nucleosomes as end products. The ATPase activity of SWR1 is specifically stimulated by H2A-containing nucleosomes without ensuing histone H2A eviction. Remarkably, further addition of free H2A.Z-H2B dimer leads to hyperstimulation of ATPase activity, eviction of nucleosomal H2A-H2B, and deposition of H2A.Z-H2B. These results suggest that the combination of H2A-containing nucleosome and free H2A.Z-H2B dimer acting as both effector and substrate for SWR1 governs the specificity and outcome of the replacement reaction.

NuA4-dependent Acetylation of Nucleosomal Histones H4 and H2A Directly Stimulates Incorporation of H2A.Z by the SWR1 Complex

Structural and functional analyses of nucleosomes containing histone variant H2A.Z have drawn a lot of interest over the past few years. Important work in budding yeast has shown that H2A.Z (Htz1)-containing nucleosomes are specifically located on the promoter regions of genes, creating a specific chromatin structure that is poised for disassembly during transcription activation. The SWR1 complex is responsible for incorporation of Htz1 into nucleosomes through ATP-dependent exchange of canonical H2A-H2B dimers for Htz1-H2B dimers. Interestingly, the yeast SWR1 complex is functionally linked to the NuA4 acetyltransferase complex in vivo. NuA4 and SWR1 are physically associated in higher eukaryotes as they are homologous to the TIP60/p400 complex, which encompasses both histone acetyltransferase (Tip60) and histone exchange (p400/Domino) activities. Here we present work investigating the impact of NuA4-dependent acetylation on SWR1-driven incorporation of H2A.Z into chromatin. Using in vitro histone exchange assays with native chromatin, we demonstrate that prior chromatin acetylation by NuA4 greatly stimulates the exchange of H2A for H2A.Z. Interestingly, we find that acetylation of H2A or H4 N-terminal tails by NuA4 can independently stimulate SWR1 activity. Accordingly, we demonstrate that mutations of H4 or H2A N-terminal lysine residues have similar effects on H2A.Z incorporation in vivo, and cells carrying mutations in both tails are nonviable. Finally, depletion experiments indicate that the bromodomain-containing protein Bdf1 is important for NuA4-dependent stimulation of SWR1. These results provide important mechanistic insight into the functional cross-talk between chromatin acetylation and ATP-dependent exchange of histone H2A variants.

Regulation of the polycomb protein Ring1B by self-ubiquitination or by E6-AP may have implications to the pathogenesis of Angelman syndrome

The polycomb repressive complex (PRC) 1 protein Ring1B is an ubiquitin ligase that modifies nucleosomal histone H2A, a modification which plays a critical role in regulation of gene expression. We have shown that self-ubiquitination of Ring1B generates multiply branched, "noncanonical" polyubiquitin chains that do not target the ligase for degradation, but rather stimulate its activity toward histone H2A. This finding implies that Ring1B is targeted by a heterologous E3. In this study, we identified E6-AP (E6-associated protein) as a ligase that targets Ring1B for "canonical" ubiquitination and subsequent degradation. We further demonstrated that both the self-ubiquitination of Ring1B and its modification by E6-AP target the same lysines, suggesting that the fate of Ring1B is tightly regulated (e.g., activation vs. degradation) by the type of chains and the ligase that catalyzes their formation. As expected, inactivation of E6-AP affects downstream effectors: Ring1B and ubiquitinated H2A levels are increased accompanied by repressed expression of HoxB9, a PRC1 target gene. Consistent with these findings, E6-AP knockout mice display an elevated level of Ring1B and ubiquitinated histone H2A in various tissues, including cerebellar Purkinje neurons, which may have implications to the pathogenesis of Angelman syndrome, a neurodevelopmental disorder caused by deficiency of E6-AP in the brain.

N Terminus of Swr1 Binds to Histone H2AZ and Provides a Platform for Subunit Assembly in the Chromatin Remodeling Complex

Variant histone H2AZ-containing nucleosomes are involved in the regulation of gene expression. In Saccharomyces cerevisiae, chromatin deposition of histone H2AZ is mediated by the fourteen-subunit SWR1 complex, which catalyzes ATP-dependent exchange of nucleosomal histone H2A for H2AZ. Previous work defined the role of seven SWR1 subunits (Swr1 ATPase, Swc2, Swc3, Arp6, Swc5, Yaf9, and Swc6) in maintaining complex integrity and H2AZ histone replacement activity. Here we examined the function of three additional SWR1 subunits, bromodomain containing Bdf1, actin-related protein Arp4 and Swc7, by analyzing affinity-purified mutant SWR1 complexes. We observed that depletion of Arp4 (arp4-td) substantially impaired the association of Bdf1, Yaf9, and Swc4. In contrast, loss of either Bdf1 or Swc7 had minimal effects on overall complex integrity. Furthermore, the basic H2AZ histone replacement activity of SWR1 in vitro required Arp4, but not Bdf1 or Swc7. Thus, three out of fourteen SWR1 subunits, Bdf1, Swc7, and previously noted Swc3, appear to have roles auxiliary to the basic histone replacement activity. The N-terminal region of the Swr1 ATPase subunit is necessary and sufficient to direct association of Bdf1 and Swc7, as well as Arp4, Act1, Yaf9 and Swc4. This same region contains an additional H2AZ-H2B specific binding site, distinct from the previously identified Swc2 subunit. These findings suggest that one SWR1 enzyme might be capable of binding two H2AZ-H2B dimers, and provide further insight on the hierarchy and interdependency of molecular interactions within the SWR1 complex.

The essential function of Swc4p - a protein shared by two chromatin-modifying complexes of the yeast Saccharomyces cerevisiae - resides within its N-terminal part.

The Swc4p protein, encoded by an essential gene, is shared by two chromatin-remodeling complexes in Saccharomyces cerevisiae cells: NuA4 (nucleosome acetyltransferase of H4) and SWR1. The SWR1 complex catalyzes ATP-dependent exchange of the nucleosomal histone H2A for H2AZ (Htz1p). The activity of NuA4 is responsible mainly for the acetylation of the H4 histone but also for the acetylation of H2A and H2AZ. In this work we investigated the role of the Swc4p protein. Using random mutagenesis we isolated a collection of swc4 mutants and showed that the essential function of Swc4p resides in its N-terminal part, within the first 269 amino acids of the 476-amino acid-long protein. We also demonstrated that Swc4p is able to accommodate numerous mutations without losing its functionality under standard growth conditions. However, when swc4 mutants were exposed to methyl methanesulfonate (MMS), hydroxyurea or benomyl, severe growth deficiencies appeared, pointing to an involvement of Swc4p in many chromatin-based processes. The mutants' phenotypes did not result from an impairment of histone acetylation, as in the mutant which bears the shortest isolated variant of truncated Swc4p, the level of overall H4 acetylation was unchanged.