Multiple roles for PARP1 in ALC1-dependent nucleosome remodeling

The SNF2 family ATPase Amplified in Liver Cancer 1 (ALC1) is the only chromatin remodeling enzyme with a poly(ADP-ribose) (PAR) binding macrodomain. ALC1 functions together with poly(ADP-ribose) polymerase PARP1 to remodel nucleosomes. Activation of ALC1 cryptic ATPase activity and the subsequent nucleosome remodeling requires binding of its macrodomain to PAR chains synthesized by PARP1 and NAD⁺. A key question is whether PARP1 has a role(s) in ALC1-dependent nucleosome remodeling beyond simply synthesizing the PAR chains needed to activate the ALC1 ATPase. Here, we identify PARP1 separation-of-function mutants that activate ALC1 ATPase but do not support nucleosome remodeling by ALC1. Investigation of these mutants has revealed multiple functions for PARP1 in ALC1-dependent nucleosome remodeling and provides insights into its multifaceted role in chromatin remodeling.

The human ALC1 (Amplified in Liver Cancer 1) protein (also referred to as CHD1L or Chromodomain-Helicase-DNA-binding protein 1-Like) is a SNF2 family chromatin remodeling enzyme that functions together with the poly(ADP-ribose) polymerase PARP1 to catalyze ATP- and NAD⁺-dependent nucleosome remodeling. The ALC1 gene is amplified in a subset of hepatocellular carcinomas, and overexpression of the ALC1 protein leads to transformation of cultured cells and appearance of spontaneous tumors in mice (1, 2). Although the precise mechanism(s) by which ALC1 overexpression contributes to tumorigenesis remains unknown, ALC1 has been implicated in multiple DNA damage repair pathways (3–6). Several recent studies have shown that ALC1 overexpression confers resistance to PARP inhibitors used in treatment of DNA repair–deficient tumors, while loss or reduction of ALC1 expression renders cells exquisitely sensitive to these drugs (7–10). Hence, understanding the functional relationships between ALC1 and PARP1 is of considerable interest.We and others initially demonstrated that ALC1 has cryptic DNA-dependent ATPase and ATP-dependent nucleosome sliding activities that are strongly activated in the presence of PARP1 and NAD⁺ (3, 11), which PARP1 and other PARPs use as substrate for synthesis of poly(ADP-ribose) (PAR) (12). ALC1 is unique among SNF2 family members in containing a macrodomain. The macrodomain, located at the enzyme’s C terminus, binds PAR chains containing three or more ADP-ribose residues (3, 11, 13–15). ALC1 macrodomain mutations that abolish PAR binding block ALC1 ATPase and nucleosome remodeling, indicating that PAR binding by the macrodomain is important for ALC1 activation. Recent studies have led to a working model for how binding of PAR to ALC1 macrodomain contributes to nucleosome remodeling. According to this model, ALC1 SNF2 ATPase domain interacts with and is held in an inactive state by the macrodomain. Upon binding of PAR to the macrodomain, this interaction is released, leading to structural changes in the ALC1 ATPase domain that relieve autoinhibition (13, 16). In subsequent steps, nucleosome binding by ALC1 stabilizes the catalytically active conformation of the ATPase, and a linker region between the ATPase and macrodomains contacts an acidic patch on nucleosomes to couple ATP hydrolysis to nucleosome sliding (17).Although it is well established that one key role of PARP1 in ALC1-dependent nucleosome remodeling is to produce PAR, it is less clear whether it makes additional contributions. Recent findings indicating that free tri-ADP ribose is sufficient to activate ALC1 ATPase activity in the absence of PARP1 (13) suggest that the role of PARP1 might be limited merely to synthesizing PAR chains. In this case, PARP1 might act simply as a bystander in ALC1-dependent nucleosome remodeling. On the other hand, PARP1 possesses both nucleosome binding and histone chaperone activities (18). This, together with our previous evidence that ALC1 and PARP1 bind cooperatively to nucleosomes to form an ALC1–PARP1–nucleosome intermediate prior to remodeling (14), makes it tempting to speculate that PARP1 might play a more active role.In the course of experiments investigating the mechanism(s) by which PARP1 contributes to ALC1-dependent nucleosome remodeling, we identified PARP1 mutants capable of activating ALC1 ATPase, but defective in supporting ALC1-catalyzed nucleosome remodeling. By investigating the properties of these and additional PARP1 mutants, we show that both the PARP1 C-terminal ADP-ribosyl transferase domain and its N-terminal region, which contains nucleosome binding activity, play important roles in ALC1-dependent nucleosome remodeling. We report these findings, which bring to light a role for PARP1 in chromatin remodeling.     

Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly

Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.

The precise inheritance of genetic information relies on the accurate segregation of chromosomes in mitosis and meiosis. Kinetochores are large protein complexes assembled on centromeres and play a crucial role in chromosome segregation. The kinetochore links the chromosome to microtubule polymers, drives the movement of chromosomes, and ensures correct microtubule–kinetochores attachment (1–3). The kinetochore assembly is thus tightly regulated. Yet, the mechanism by which kinetochores are precisely assembled through the cell cycle remains poorly understood.The kinetochore comprises an outer region and an inner region. The outer kinetochore interacts with microtubules and is assembled on the platform of the inner kinetochore. The inner kinetochore consists of a complex of 14 to 16 subunits known as the constitutive centromere–associated network (CCAN) that is directly built on centromeric chromatin (4–6). In centromeres, the histone H3 variant, CENP-A, replaces the canonical histone H3 to form CENP-A–containing nucleosomes (7–9). Most eukaryotes contain large complex regional centromeres where CENP-A–containing nucleosomes are interspersed with canonical H3–containing nucleosomes (10–12). Regional centromeres are epigenetically specified by CENP-A (12–14). But how CENP-A– and histone H3–containing nucleosomes are balanced in centromeres remains unclear.CENP-T, an integral component of CCAN, is also a histone fold–containing protein. CENP-T provides a platform for the assembly of the Ndc80 complex (Ndc80C), an essential outer kinetochore component, during mitosis (5, 15–18). Ndc80C acts as the interface between microtubules and kinetochores and mediates the microtubule attachments (19, 20). The long N terminus of CENP-T contains a conserved Ndc80 receptor motif. The motif forms an alpha-helix that directly interacts with the Spc24-Spc25 heterodimer in Ndc80C (15, 16). The motif can be phosphorylated by cyclin-dependent kinase 1 (CDK1) to stabilize the interaction between CENP-T and Ndc80C (16, 21–24). However, how CENP-T is regulated through the cell cycle to mediate the assembly of Ndc80C is still not well understood.CENP-T has been shown to interact with three other histone fold–containing proteins, CENP-W, CENP-S, and CENP-X, to form the heterotetrameric nucleosome-like structure in vitro (25, 26). The CENP-T-W-S-X complex directly associates with centromeric DNA. The DNA binding activity of the complex is important for kinetochore formation (5, 25). Interestingly, the complex also directly associates with histone H3, not with CENP-A (5, 27), suggesting that CENP-T particles and the CENP-A nucleosome occupy different positions in centromeres. How the spatial relationship between the CENP-A nucleosome and CENP-T particles in centromeres is regulated remains unclear.The fission yeast Schizosaccharomyces pombe contains large regional centromeres and is considered to be a model system for centromere study. The CENP-A homolog, Cnp1, is enriched in centromere cores, which are surrounded by pericentromeric heterochromatin (28–30). CENP-A^Cnp1 nucleosomes nucleate kinetochore assembly. Mislocalization of CENP-A^Cnp1 results in severe chromosome segregation defects in fission yeast (28, 31–34). Fission yeast also contains the CENP-T homolog, Cnp20, which associates with centromeres throughout the cell cycle. The same as in higher eukaryotes, CENP-T^Cnp20 in S. pombe is essential for viability (35).Recently, Ccp1, a nucleosome assembly protein (NAP) family protein, has been shown to play an important role in antagonizing the loading of CENP-A in fission yeast (36). Ccp1 forms a homodimer and is enriched at centromeres. Ccp1 acts as a key player in balancing CENP-A and histone H3 levels in the region (36). How Ccp1 regulates the CENP-A level in centromeres remains elusive. Interestingly, its centromere localization is cell cycle regulated. Ccp1 is dissociated from centromeres at the onset of mitosis and reassociates with centromeres at the end of mitosis (36, 37). The biological importance of the cell cycle–dependent interaction between Ccp1 and centromeres is unknown.Here using mass spectrometry, we found that Ccp1 interacts directly with CENP-T^Cnp20 in fission yeast. We further identified a conserved Ccp1-interaction motif (CIM) at the N terminus of CENP-T^Cnp20, which is adjacent to the Ndc80 receptor motif. We demonstrated that CIM is important for Ccp1 localization. Furthermore, our data suggested that CDK1-mediated phosphorylation of the CIM motif at the onset of mitosis dissociates Ccp1 from CENP-T^Cnp20, allowing proper positioning of Ndc80C. Ccp1 associates with centromeres during mitosis in the phospho-null mutant of the CIM domain, leading to mislocalization of Ndc80C and severe chromosome segregation defects. Our study uncovers a previously unrecognized mechanism regulating kinetochore organization in regional centromeres.     

Generation of double Holliday junction DNAs and their dissolution/resolution within a chromatin context

Four-way DNA intermediates, also known as Holliday junctions (HJs), are formed during homologous recombination and DNA repair, and their resolution is necessary for proper chromosome segregation. To facilitate the biochemical analysis of HJ processing, we developed a method involving DNAzyme self-cleavage to generate 1.8-kb DNA molecules containing either single (sHJ) or double Holliday junctions (dHJs). We show that dHJ DNAs (referred to as HoJo DNAs) are dissolved by the human BLM–TopIIIα–RMI1–RMI2 complex to form two noncrossover products. However, structure-selective endonucleases (human GEN1 and SMX complex) resolve DNA containing single or double HJs to yield a mixture of crossover and noncrossover products. Finally, we demonstrate that chromatin inhibits the resolution of the double HJ by GEN or SMX while allowing BTRR-mediated dissolution.

Homologous recombination (HR) provides an important mechanism for the repair of DNA double-stranded breaks and the restoration of broken replication forks (1). Recombination in mitotic cells generally occurs between sister chromatids and can lead to the formation of DNA intermediates in which the sisters are covalently linked by four-way DNA junctions, known as Holliday junctions (HJs) (2). Failure to process these DNA intermediates leads to improper chromosome segregation and cell death (3, 4). Recombination also plays an important role in meiotic cells, when interactions occur between homologous chromosomes, and is responsible for the generation of genetic diversity.In mitotic cells, HJs are primarily processed by “dissolution,” in which two adjacent HJs (double Holliday junctions [dHJs]) converge in an adenosine triphosphate (ATP)-hydrolysis–dependent reaction to form a hemicatenane that is subsequently decatenated by topoisomerase action. In human cells, this two-step process involves the BLM–topoisomerase IIIα–RMI1–RMI2 (BTRR) complex (5–7). In yeast, similar reactions are driven by the Sgs1–Top3–Rmi1 (STR) complex (8, 9). Dissolution yields exclusively noncrossover products, which help to maintain the heterozygous state, as a loss of heterozygosity can cause cancer development (10). Mutations in the BLM gene are linked to a human inherited disorder known as Bloom syndrome, which is characterized by short stature, sensitivity to sunlight, and a greatly increased risk of a broad range of cancers (11, 12). In the clinic, patients with Bloom syndrome are diagnosed by a cytogenetic test that detects elevated levels of sister chromatid exchanges. Pathogenic mutations in the TOP3A and RMI1 genes also cause a Bloom syndrome–like disorder, consistent with the fact that these genes participate in the same molecular pathway (12).Persistent HJs that escape processing by BTRR, and single Holliday junctions (sHJs), are resolved by structure-selective endonucleases (SSEs), which specifically recognize and cleave HJs by mediating a nucleolytic attack on two opposing strands at the junction point (2). In humans, these endonucleases include GEN1 and the SMX trinuclease, comprising SLX1–SLX4–MUS81–EME1–XPF–ERCC1 (3, 13–15). Unlike dissolution, resolution gives rise to both crossover and noncrossover products, thereby elevating the frequency of sister chromatid exchanges and increasing the potential for loss of heterozygosity.In contrast to BTRR-mediated dHJ dissolution, which is active throughout the cell cycle, the actions of SMX and GEN1 are tightly regulated. Firstly, SMX complex formation is restricted to prometaphase, as it is dependent upon the phosphorylation of EME1 by CDK1 and PLK1, which stimulates its association with the SLX4 scaffold (15, 16). Secondly, GEN1 is mainly sequestered from the cell nucleus and gains access to DNA after the breakdown of the nuclear envelope during cell division (17).Despite the importance of the BTRR complex in maintaining genetic stability, a detailed picture of dissolution is lacking. Mechanistic studies, using protein complexes from various organisms, led to a model in which two HJs are converged by the branch migration activity of the BLM helicase (18–20). Convergent migration generates positive supercoiling that is relaxed by topoisomerase IIIα and generates a hemicatenane that is processed by topoisomerase IIIα with the aid of RMI1–RMI2 (6–9, 21).Studies of dHJ dissolution have utilized two model systems: 1) a small dHJ prepared by annealing two synthetic oligos (5–7), and 2) a larger plasmid-sized molecule in which two HJs are separated by 165 bp (20, 22). However, the small size of the synthetic DNA substrate eliminates any possibility for branch migration as the two HJs are separated by only 14 bp, raising concerns as to whether these substrates recapitulate the physiological aspects of dissolution (19). The plasmid-sized substrate has been utilized for the dissolution of dHJs by Saccharomyces cerevisiae STR, Drosophila melanogaster BTR, and more recently human BTRR complex (8, 20, 23). However, there is the significant drawback that generation of this substrate is laborious (taking several weeks), requires purified Cre recombinase and reverse gyrase, and leads to low yields of product.To facilitate mechanistic analysis of dissolution and resolution, we developed a rapid and scalable methodology to prepare a 1.8-kb DNA containing single or double HJs. In the dHJ molecules, the two HJs are separated by a maximum of 746 bp of homologous sequence, allowing the two HJs to migrate within the region of homology. We demonstrate that these dHJ molecules are efficiently dissolved by the human BTRR complex to generate noncrossover products. We also show that GEN1 or SMX resolves the single or double HJs to yield the expected mixture of crossover and noncrossover products. Finally, we find that GEN1/SMX are unable to resolve HJs on chromatinized templates, whereas BTRR-mediated dissolution events are unaffected by nucleosome assembly, potentially indicative of an additional level of regulatory control that favors dissolution over resolution.     

Structural insights into the histone H1-nucleosome complex

Linker H1 histones facilitate formation of higher-order chromatin structures and play important roles in various cell functions. Despite several decades of effort, the structural basis of how H1 interacts with the nucleosome remains elusive. Here, we investigated Drosophila H1 in complex with the nucleosome, using solution nuclear magnetic resonance spectroscopy and other biophysical methods. We found that the globular domain of H1 bridges the nucleosome core and one 10-base pair linker DNA asymmetrically, with its α3 helix facing the nucleosomal DNA near the dyad axis. Two short regions in the C-terminal tail of H1 and the C-terminal tail of one of the two H2A histones are also involved in the formation of the H1–nucleosome complex. Our results lead to a residue-specific structural model for the globular domain of the Drosophila H1 in complex with the nucleosome, which is different from all previous experiment-based models and has implications for chromatin dynamics in vivo.Eukaryotic genomic DNA is packaged into chromatin through association with positively charged histones to form the nucleosome, the structural unit of chromatin (1–3). The nucleosome core consists of an octamer of histones with two copies of H2A, H2B, H3, and H4, around which ∼146 bp of DNA winds in ∼1.65 left-handed superhelical turns (4). At this level of the DNA packaging, chromatin resembles a beads-on-a-string structure, with the nucleosome core as the beads and the linker DNA between them as the strings (5). At the next level of DNA packaging, H1 histones bind to the linker DNA and the nucleosome to further condense the chromatin structure (6, 7). H1-mediated chromatin condensation plays important roles in cellular functions such as mitotic chromosome architecture and segregation (8), muscle differentiation (9), and regulation of gene expression (10, 11).Linker H1 histones typically are ∼200 amino acid residues in length, with a short N-terminal region, followed by a ∼70–80-amino acid structured globular domain (gH1) and a ∼100-amino acid unstructured C-terminal domain that is highly enriched in Lys residues. H1 stabilizes the nucleosome and facilitates folding of nucleosome arrays into higher-order structures (12–15). gH1 alone confers the same protection from micrococcal nuclease digestion to the nucleosome as the full-length H1 does (16). The N-terminal region of H1 is not important for nucleosome binding (16, 17), whereas the C terminus is required for H1 binding to chromatin in vivo (18, 19) and for the formation of a stem structure of linker DNA in vitro (17, 20, 21).The globular domain structures of avian H5 (22) and budding yeast Hho1 (23), which are both H1 homologs, have been determined at atomic resolution and show similar structures. In addition, numerous studies have indicated that gH1/gH5 binds around the dyad region of the nucleosome (14, 24), leading to many conflicting structural models for how the globular domain of H1/H5 binds to the nucleosome (SI Appendix, Fig. S1) (24–26). These models are divided into two major classes, symmetric and asymmetric, on the basis of the location of gH1/gH5 in the nucleosome. In the symmetric class, gH1/gH5 binds to the nucleosomal DNA at the dyad and interacts with both linker DNAs (16, 17, 27, 28). In the asymmetric class, gH1/gH5 binds to the nucleosomal DNA in the vicinity of the dyad axis and to 10 bp (27, 29–32) or 20 bp (19, 29, 33, 34) of one linker DNA, or is located inside the DNA gyres, where it interacts with histone H2A (35). In addition, Zhou and colleagues also characterized the orientation of gH5 in the gH5-nucleosome complex (29). The use of nonuniquely positioned nucleosomes and indirect methods may have contributed to the differences in these models (SI Appendix, Fig. S1).Multidimensional nuclear magnetic resonance (NMR), and in particular methyl-based NMR, provides a direct approach to the structural characterization of macromolecular complexes (36, 37). We have previously assigned chemical shifts of the methyl groups of the side chains of residues Ile, Leu, and Val in the core histones (38) and the backbone amides in the disordered histone tails (39), which provide the fingerprints for investigating the interactions between H1 and the nucleosome. Here, we used NMR, along with several other methods, to determine the location and orientation of the globular domain of a stable mutant of Drosophila H1 on a well-positioned nucleosome.     

Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair

The repair of DNA double-strand breaks (DSBs) requires open, flexible chromatin domains. The NuA4–Tip60 complex creates these flexible chromatin structures by exchanging histone H2A.Z onto nucleosomes and promoting acetylation of histone H4. Here, we demonstrate that the accumulation of H2A.Z on nucleosomes at DSBs is transient, and that rapid eviction of H2A.Z is required for DSB repair. Anp32e, an H2A.Z chaperone that interacts with the C-terminal docking domain of H2A.Z, is rapidly recruited to DSBs. Anp32e functions to remove H2A.Z from nucleosomes, so that H2A.Z levels return to basal within 10 min of DNA damage. Further, H2A.Z removal by Anp32e disrupts inhibitory interactions between the histone H4 tail and the nucleosome surface, facilitating increased acetylation of histone H4 following DNA damage. When H2A.Z removal by Anp32e is blocked, nucleosomes at DSBs retain elevated levels of H2A.Z, and assume a more stable, hypoacetylated conformation. Further, loss of Anp32e leads to increased CtIP-dependent end resection, accumulation of single-stranded DNA, and an increase in repair by the alternative nonhomologous end joining pathway. Exchange of H2A.Z onto the chromatin and subsequent rapid removal by Anp32e are therefore critical for creating open, acetylated nucleosome structures and for controlling end resection by CtIP. Dynamic modulation of H2A.Z exchange and removal by Anp32e reveals the importance of the nucleosome surface and nucleosome dynamics in processing the damaged chromatin template during DSB repair.The repair of DNA double-strand breaks (DSBs), which cleave the DNA backbone, requires remodeling of the local chromatin architecture. This reorganization of the chromatin is important for promoting access to the site of damage, for creating a template for the repair machinery, and for repackaging the chromatin and resetting the epigenetic landscape following repair. Chromatin remodeling at DSBs is linked to changes in posttranslational modification of histones. DSBs activate the ataxia-telangiectasia mutated (ATM) and DNA–PKcs kinases, which phosphorylate multiple DNA repair proteins, including histone H2AX. Phosphorylated H2AX (γH2AX) provides a binding site for mdc1, which promotes spreading of γH2AX for hundreds of kilobases either side of the break (1–3). DSBs also promote complex patterns of chromatin ubiquitination, including ubiquitination of H2A/H2AX by the RNF8/RNF168 ubiquitin ligases, which, in turn, creates binding sites for repair proteins such as 53BP1 and brca1 (4–7). DSBs also lead to increased methylation of histone H3 on lysine 36, which can regulate DNA repair pathway choice (8, 9) and methylation of H3 on lysine 9 (10), which drives activation of the Tip60 acetyltransferase and the ATM kinase (11, 12). Further, the NuA4–Tip60 complex (5, 13–15) promotes acetylation of histone H4 at DSBs and drives the formation of open, flexible chromatin domains at DSBs (5, 13, 14). The repair of DSBs is therefore fundamentally a chromatin-driven process requiring dynamic changes in histone modification and chromatin reorganization, which directly promote recruitment of DSB repair proteins (16).The NuA4–Tip60 remodeling complex plays a central role in nucleosome reorganization at DSBs (16). NuA4–Tip60 is a 16 subunit complex containing 2 key subunits—the p400 SWI/SNF ATPase and the Tip60 acetyltransferase. The p400 ATPase promotes exchange of H2A for the histone variant H2A.Z (13, 17). This increase in H2A.Z at DSBs then promotes acetylation of histone H4 by the Tip60, creating open, flexible chromatin at sites of DNA damage (5, 11, 14). Inactivation of NuA4–Tip60 blocks both H2A.Z exchange and H4 acetylation, leading to a reduction in chromatin mobility at DSBs. Consequently, loss of H2A.Z exchange leads to defective DSB repair, increased sensitivity to DNA damage, and genomic instability (13, 18, 19).Here, we demonstrate that H2A.Z exchange at DSBs is dynamic, with H2A.Z accumulating at DSBs within minutes of damage, followed by rapid H2A.Z eviction. Further, we show that Anp32e, an H2A.Z-specific histone chaperone, binds specifically to the docking domain of H2A.Z and is required to remove H2A.Z from the damaged chromatin template. Failure to remove H2A.Z leads to defects in DSB repair, including a loss of H4 acetylation, defects in nonhomologous end joining (NHEJ), and increased end resection of DSBs.