Human FACT subunits coordinate to catalyze both disassembly and reassembly of nucleosomes.

The histone chaperone FACT (facilitates chromatin transcription) enhances transcription in eukaryotic cells, targeting DNA-protein interactions. FACT, a heterodimer in humans, comprises SPT16 and SSRP1 subunits. We measure nucleosome stability and dynamics in the presence of FACT and critical component domains. Optical tweezers quantify FACT/subdomain binding to nucleosomes, displacing the outer wrap of DNA, disrupting direct DNA-histone (core site) interactions, altering the energy landscape of unwrapping, and increasing the kinetics of DNA-histone disruption. Atomic force microscopy reveals nucleosome remodeling, while single-molecule fluorescence quantifies kinetics of histone loss for disrupted nucleosomes, a process accelerated by FACT. Furthermore, two isolated domains exhibit contradictory functions; while the SSRP1 HMGB domain displaces DNA, SPT16 MD/CTD stabilizes DNA-H2A/H2B dimer interactions. However, only intact FACT tethers disrupted DNA to the histones and supports rapid nucleosome reformation over several cycles of force disruption/release. These results demonstrate that key FACT domains combine to catalyze both nucleosome disassembly and reassembly.

The BRCT domain of PARP1 binds intact DNA and mediates intrastrand transfer.

PARP1 is a key player in the response to DNA damage and is the target of clinical inhibitors for the treatment of cancers. Binding of PARP1 to damaged DNA leads to activation wherein PARP1 uses NAD+ to add chains of poly(ADP-ribose) onto itself and other nuclear proteins. PARP1 also binds abundantly to intact DNA and chromatin, where it remains enzymatically inactive. We show that intact DNA makes contacts with the PARP1 BRCT domain, which was not previously recognized as a DNA-binding domain. This binding mode does not result in the concomitant reorganization and activation of the catalytic domain. We visualize the BRCT domain bound to nucleosomal DNA by cryogenic electron microscopy and identify a key motif conserved from ancestral BRCT domains for binding phosphates on DNA and phospho-peptides. Finally, we demonstrate that the DNA-binding properties of the BRCT domain contribute to the "monkey-bar mechanism" that mediates DNA transfer of PARP1.

Solution structure(s) of trinucleosomes from contrast variation SAXS.

Nucleosomes in all eukaryotic cells are organized into higher order structures that facilitate genome compaction. Visualizing these organized structures is an important step in understanding how genomic DNA is efficiently stored yet remains accessible to information-processing machinery. Arrays of linked nucleosomes serve as useful models for understanding how the properties of both DNA and protein partners affect their arrangement. A number of important questions are also associated with understanding how the spacings between nucleosomes are affected by the histone proteins, chromatin remodelers, or other chromatin-associated protein partners. Contrast variation small angle X-ray scattering (CVSAXS) reports the DNA conformation within protein-DNA complexes and here is applied to measure the conformation(s) of trinucleosomes in solution, with specific sensitivity to the distance between and relative orientation of linked nucleosomes. These data are interpreted in conjunction with DNA models that account for its sequence dependent mechanical properties, and Monte-Carlo techniques that generate realistic structures for comparison with measured scattering profiles. In solution, trinucleosomes segregate into two dominant populations, with the flanking nucleosomes stacked or nearly equilaterally separated, e.g. with roughly equal distance between all pairs of nucleosomes. These populations are consistent with previously observed magnesium-dependent structures of trinucleosomes with shorter linkers.

HPF1 and nucleosomes mediate a dramatic switch in activity of PARP1 from polymerase to hydrolase.

Poly(ADP-ribose) polymerase 1 (PARP1) is an important player in the response to DNA damage. Recently, Histone PARylation Factor (HPF1) was shown to be a critical modulator of the activity of PARP1 by facilitating PARylation of histones and redirecting the target amino acid specificity from acidic to serine residues. Here, we investigate the mechanism and specific consequences of HPF1-mediated PARylation using nucleosomes as both activators and substrates for PARP1. HPF1 provides that catalytic base Glu284 to substantially redirect PARylation by PARP1 such that the histones in nucleosomes become the primary recipients of PAR chains. Surprisingly, HPF1 partitions most of the reaction product to free ADP-ribose (ADPR), resulting in much shorter PAR chains compared to reactions in the absence of HPF1. This HPF1-mediated switch from polymerase to hydrolase has important implications for the PARP1-mediated response to DNA damage and raises interesting new questions about the role of intracellular ADPR and depletion of NAD+.

Analytical Ultracentrifugation (AUC): An Overview of the Application of Fluorescence and Absorbance AUC to the Study of Biological Macromolecules.

The biochemical and biophysical investigation of proteins, nucleic acids, and the assemblies that they form yields essential information to understand complex systems. Analytical ultracentrifugation (AUC) represents a broadly applicable and information-rich method for investigating macromolecular characteristics such as size, shape, stoichiometry, and binding properties, all in the true solution-state environment that is lacking in most orthogonal methods. Despite this, AUC remains underutilized relative to its capabilities and potential in the fields of biochemistry and molecular biology. Although there has been a rapid development of computing power and AUC analysis tools in this millennium, fewer advancements have occurred in development of new applications of the technique, leaving these powerful instruments underappreciated and underused in many research institutes. With AUC previously limited to absorbance and Rayleigh interference optics, the addition of fluorescence detection systems has greatly enhanced the applicability of AUC to macromolecular systems that are traditionally difficult to characterize. This overview provides a resource for novices, highlighting the potential of AUC and encouraging its use in their research, as well as for current users, who may benefit from our experience. We discuss the strengths of fluorescence-detected AUC and demonstrate the power of even simple AUC experiments to answer practical and fundamental questions about biophysical properties of macromolecular assemblies. We address the development and utility of AUC, explore experimental design considerations, present case studies investigating properties of biological macromolecules that are of common interest to researchers, and review popular analysis approaches. © 2020 The Authors.

Bridging of nucleosome-proximal DNA double-strand breaks by PARP2 enhances its interaction with HPF1.

Poly(ADP-ribose) Polymerase 2 (PARP2) is one of three DNA-dependent PARPs involved in the detection of DNA damage. Upon binding to DNA double-strand breaks, PARP2 uses nicotinamide adenine dinucleotide to synthesize poly(ADP-ribose) (PAR) onto itself and other proteins, including histones. PAR chains in turn promote the DNA damage response by recruiting downstream repair factors. These early steps of DNA damage signaling are relevant for understanding how genome integrity is maintained and how their failure leads to genome instability or cancer. There is no structural information on DNA double-strand break detection in the context of chromatin. Here we present a cryo-EM structure of two nucleosomes bridged by human PARP2 and confirm that PARP2 bridges DNA ends in the context of nucleosomes bearing short linker DNA. We demonstrate that the conformation of PARP2 bound to damaged chromatin provides a binding platform for the regulatory protein Histone PARylation Factor 1 (HPF1), and that the resulting HPF1•PARP2•nucleosome complex is enzymatically active. Our results contribute to a structural view of the early steps of the DNA damage response in chromatin.

Single and double box HMGB proteins differentially destabilize nucleosomes.

Nucleosome disruption plays a key role in many nuclear processes including transcription, DNA repair and recombination. Here we combine atomic force microscopy (AFM) and optical tweezers (OT) experiments to show that high mobility group B (HMGB) proteins strongly disrupt nucleosomes, revealing a new mechanism for regulation of chromatin accessibility. We find that both the double box yeast Hmo1 and the single box yeast Nhp6A display strong binding preferences for nucleosomes over linker DNA, and both HMGB proteins destabilize and unwind DNA from the H2A-H2B dimers. However, unlike Nhp6A, Hmo1 also releases half of the DNA held by the (H3-H4)2 tetramer. This difference in nucleosome destabilization may explain why Nhp6A and Hmo1 function at different genomic sites. Hmo1 is enriched at highly transcribed ribosomal genes, known to be depleted of histones. In contrast, Nhp6A is found across euchromatin, pointing to a significant difference in cellular function.

Biochemical and Biophysical Methods for Analysis of Poly(ADP-Ribose) Polymerase 1 and Its Interactions with Chromatin.

Poly (ADP-Ribose) Polymerase I (PARP-1) is a first responder to DNA damage and participates in the regulation of gene expression. The interaction of PARP-1 with chromatin and DNA is complex and involves at least two different modes of interaction. In its enzymatically inactive state, PARP-1 binds native chromatin with similar affinity as it binds free DNA ends. Automodification of PARP-1 affects interaction with chromatin and DNA to different extents. Here we describe a series of biochemical and biophysical techniques to quantify and dissect the different binding modes of PARP-1 with its various substrates. The techniques listed here allow for high throughput and quantitative measurements of the interaction of different PARP-1 constructs (inactive and automodified) with chromatin and DNA damage models.

Bivalent interaction of the PZP domain of BRPF1 with the nucleosome impacts chromatin dynamics and acetylation.

BRPF1 (bromodomain PHD finger 1) is a core subunit of the MOZ histone acetyltransferase (HAT) complex, critical for normal developmental programs and implicated in acute leukemias. BRPF1 contains a unique assembly of zinc fingers, termed a PZP domain, the physiological role of which remains unclear. Here, we elucidate the structure-function relationship of this novel epigenetic reader and detail the biological and mechanistic consequences of its interaction with nucleosomes. PZP has a globular architecture and forms a 2:1 stoichiometry complex with the nucleosome, bivalently interacting with histone H3 and DNA. This binding impacts the nucleosome dynamics, shifting the DNA unwrapping/rewrapping equilibrium toward the unwrapped state and increasing DNA accessibility. We demonstrate that the DNA-binding function of the BRPF1 PZP domain is required for the MOZ-BRPF1-ING5-hEaf6 HAT complex to be recruited to chromatin and to acetylate nucleosomal histones. Our findings reveal a novel link between chromatin dynamics and MOZ-mediated acetylation.

Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone.

Poly [ADP-ribose] polymerase 1 (PARP-1) is a highly abundant chromatin-associated enzyme. It catalyzes the NAD(+)-dependent polymerization of long chains of poly-ADP ribose (PAR) onto itself in response to DNA damage and other cues. More recently, the enzymatic activity of PARP-1 has also been implicated in the regulation of gene expression. The molecular basis for the functional switch from chromatin architectural protein to transcription factor and DNA damage responder, triggered by PARP-1 automodification, is unknown. Here, we show that unmodified PARP-1 engages in at least two high-affinity binding modes with chromatin, one of which does not involve free DNA ends, consistent with its role as a chromatin architectural protein. Automodification reduces PARP-1 affinity for intact chromatin but not for nucleosomes with exposed DNA ends. Automodified (AM) PARP-1 has the ability to sequester histones (both in vitro and in cells) and to assemble nucleosomes efficiently in vitro. This unanticipated nucleosome assembly activity of AM-PARP-1, coupled with the fast turnover of the modification, suggests a model in which DNA damage or transcription events trigger transient histone chaperone activity.

The histone variant H2A.W defines heterochromatin and promotes chromatin condensation in Arabidopsis.

Histone variants play crucial roles in gene expression, genome integrity, and chromosome segregation. We report that the four H2A variants in Arabidopsis define different genomic features, contributing to overall genomic organization. The histone variant H2A.W marks heterochromatin specifically and acts in synergy with heterochromatic marks H3K9me2 and DNA methylation to maintain transposon silencing. In vitro, H2A.W enhances chromatin condensation by promoting fiber-to-fiber interactions via its conserved C-terminal motif. In vivo, H2A.W is required for heterochromatin condensation, demonstrating that H2A.W plays critical roles in heterochromatin organization. Similarities in conserved motifs between H2A.W and another H2A variant in metazoans suggest that plants and animals share common mechanisms for heterochromatin condensation.

Molecular functions of the TLE tetramerization domain in Wnt target gene repression.

Wnt signaling activates target genes by promoting association of the co-activator ß-catenin with TCF/LEF transcription factors. In the absence of ß-catenin, target genes are silenced by TCF-mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF-dependent repression is unclear. We describe the unusual three-dimensional structure of the N-terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. We find that differences in repression potential of TCF/LEFs correlates with their affinities for TLE-Q, rather than direct competition between ß-catenin and TLE for TCFs as part of an activation-repression switch. Structure-based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression.

Assembly of nucleosomal arrays from recombinant core histones and nucleosome positioning DNA.

Core histone octamers that are repetitively spaced along a DNA molecule are called nucleosomal arrays. Nucleosomal arrays are obtained in one of two ways: purification from in vivo sources, or reconstitution in vitro from recombinant core histones and tandemly repeated nucleosome positioning DNA. The latter method has the benefit of allowing for the assembly of a more compositionally uniform and precisely positioned nucleosomal array. Sedimentation velocity experiments in the analytical ultracentrifuge yield information about the size and shape of macromolecules by analyzing the rate at which they migrate through solution under centrifugal force. This technique, along with atomic force microscopy, can be used for quality control, ensuring that the majority of DNA templates are saturated with nucleosomes after reconstitution. Here we describe the protocols necessary to reconstitute milligram quantities of length and compositionally defined nucleosomal arrays suitable for biochemical and biophysical studies of chromatin structure and function.

The role of the nucleosome acidic patch in modulating higher order chromatin structure.

Higher order folding of chromatin fibre is mediated by interactions of the histone H4 N-terminal tail domains with neighbouring nucleosomes. Mechanistically, the H4 tails of one nucleosome bind to the acidic patch region on the surface of adjacent nucleosomes, causing fibre compaction. The functionality of the chromatin fibre can be modified by proteins that interact with the nucleosome. The co-structures of five different proteins with the nucleosome (LANA, IL-33, RCC1, Sir3 and HMGN2) recently have been examined by experimental and computational studies. Interestingly, each of these proteins displays steric, ionic and hydrogen bond complementarity with the acidic patch, and therefore will compete with each other for binding to the nucleosome. We first review the molecular details of each interface, focusing on the key non-covalent interactions that stabilize the protein-acidic patch interactions. We then propose a model in which binding of proteins to the nucleosome disrupts interaction of the H4 tail domains with the acidic patch, preventing the intrinsic chromatin folding pathway and leading to assembly of alternative higher order chromatin structures with unique biological functions.

Alternative modes of binding of poly(ADP-ribose) polymerase 1 to free DNA and nucleosomes.

Poly(ADP-ribose) polymerase 1 (PARP-1) is an abundant nuclear protein that binds chromatin and catalyzes the transfer of ADP-ribose groups to itself and to numerous target proteins upon interacting with damaged DNA. The molecular basis for the dual role of PARP-1 as a chromatin architectural protein and a first responder in DNA repair pathways remains unclear. Here, we quantified the interactions of full-length PARP-1 and its N-terminal half with different types of DNA damage and with defined nucleosome substrates. We found that full-length PARP-1 prefers nucleosomes with two linker DNA extensions over any other substrate (including several free DNA models) and that the C-terminal half of PARP-1 is necessary for this selectivity. We also measured the ability of various substrates to activate PARP-1 activity and found that the most important feature for activation is one free DNA end rather than tight interaction with the activating nucleic acid. Our data provide insight into the different modes of interaction of this multidomain protein with nucleosomes and free DNA.

Histone chaperone FACT coordinates nucleosome interaction through multiple synergistic binding events.

In eukaryotic cells, DNA maintenance requires ordered disassembly and re-assembly of chromatin templates. These processes are highly regulated and require extrinsic factors such as chromatin remodelers and histone chaperones. The histone chaperone FACT (facilitates chromatin transcription) is a large heterodimeric complex with roles in transcription, replication, and repair. FACT promotes and subsequently restricts access to DNA as a result of dynamic nucleosome reorganization. However, until now, there lacked a truly quantitative assessment of the critical contacts mediating FACT function. Here, we demonstrate that FACT binds histones, DNA, and intact nucleosomes at nanomolar concentrations. We also determine roles for the histone tails in free histone and nucleosome binding by FACT. Furthermore, we propose that the conserved acidic C-terminal domain of the FACT subunit Spt16 actively displaces nucleosomal DNA to provide access to the histone octamer. Experiments with tri-nucleosome arrays indicate a possible mode for FACT binding within chromatin. Together, the data reveal that specific FACT subunits synchronize interactions with various target sites on individual nucleosomes to generate a high affinity binding event and promote reorganization.

The linker region of macroH2A promotes self-association of nucleosomal arrays.

MacroH2A is a histone variant found in higher eukaryotes localized at the inactive X chromosome and is known to maintain heterochromatic regions in the genome. MacroH2A consists of a conserved histone domain and a macro domain connected by a linker region. To understand the contributions of the three domains to chromatin condensation, we incorporated various constructs of macroH2A into defined nucleosomal arrays and analyzed their impact on in vitro chromatin compaction. The folding and oligomerization properties of arrays containing full-length macroH2A (macroH2A(FL)), macroH2A(1-161) (encompassing the histone domain and linker region), and macroH2A(1-122) (histone domain only) were compared with major-type H2A arrays. Analytical ultracentrifugation and atomic force microscope imaging indicate that macroH2A(1-161)-containing arrays favor condensation under conditions where major-type arrays are nearly fully extended. In contrast, arrays with macroH2A(FL) exhibit behavior similar to that of major-type arrays. This suggests that the linker region of macroH2A facilitates array condensation and that this behavior is inhibited by the macro domain. Furthermore, chimeric major-type H2A arrays containing the macroH2A linker domain (H2A(ML)) exhibited the same condensation properties as macroH2A(1-161) arrays, thus emphasizing the intriguing behavior of the macroH2A linker region.

Biophysical analysis and small-angle X-ray scattering-derived structures of MeCP2-nucleosome complexes.

MeCP2 is a highly abundant chromatin architectural protein with key roles in post-natal brain development in humans. Mutations in MeCP2 are associated with Rett syndrome, the main cause of mental retardation in girls. Structural information on the intrinsically disordered MeCP2 protein is restricted to the methyl-CpG binding domain; however, at least four regions capable of DNA and chromatin binding are distributed over its entire length. Here we use small angle X-ray scattering (SAXS) and other solution-state approaches to investigate the interaction of MeCP2 and a truncated, disease-causing version of MeCP2 with nucleosomes. We demonstrate that MeCP2 forms defined complexes with nucleosomes, in which all four histones are present. MeCP2 retains an extended conformation when binding nucleosomes without extra-nucleosomal DNA. In contrast, nucleosomes with extra-nucleosomal DNA engage additional DNA binding sites in MeCP2, resulting in a rather compact higher-order complex. We present ab initio envelope reconstructions of nucleosomes and their complexes with MeCP2 from SAXS data. SAXS studies also revealed unexpected sequence-dependent conformational variability in the nucleosomes themselves.

The transcription factor Spn1 regulates gene expression via a highly conserved novel structural motif.

Spn1/Iws1 plays essential roles in the regulation of gene expression by RNA polymerase II (RNAPII), and it is highly conserved in organisms ranging from yeast to humans. Spn1 physically and/or genetically interacts with RNAPII, TBP (TATA-binding protein), TFIIS (transcription factor IIS), and a number of chromatin remodeling factors (Swi/Snf and Spt6). The central domain of Spn1 (residues 141-305 out of 410) is necessary and sufficient for performing the essential functions of SPN1 in yeast cells. Here, we report the high-resolution (1.85 Å) crystal structure of the conserved central domain of Saccharomyces cerevisiae Spn1. The central domain is composed of eight α-helices in a right-handed superhelical arrangement and exhibits structural similarity to domain I of TFIIS. A unique structural feature of Spn1 is a highly conserved loop, which defines one side of a pronounced cavity. The loop and the other residues forming the cavity are highly conserved at the amino acid level among all Spn1 family members, suggesting that this is a signature motif for Spn1 orthologs. The locations and the molecular characterization of temperature-sensitive mutations in Spn1 indicate that the cavity is a key attribute of Spn1 that is critical for its regulatory functions during RNAPII-mediated transcriptional activity.

Nucleosome-binding affinity as a primary determinant of the nuclear mobility of the pioneer transcription factor FoxA.

FoxA proteins are pioneer transcription factors, among the first to bind chromatin domains in development and enable gene activity. The Fox DNA-binding domain structurally resembles linker histone and binds nucleosomes stably. Using fluorescence recovery after photobleaching, we found that FoxA1 and FoxA2 move much more slowly in nuclei than other transcription factor types, including c-Myc, GATA-4, NF-1, and HMGB1. We find that slower nuclear mobility correlates with high nonspecific nucleosome binding, and point mutations that disrupt nonspecific binding markedly increase nuclear mobility. FoxA's distinct nuclear mobility is consistent with its pioneer activity in chromatin.