Uma M. Muthurajan

Reconstitution of Nucleosome Core Particles from Recombinant Histones and DNA

Publisher Summary The ability to prepare nucleosome core particles (NCPs), or nucleosomal arrays, from recombinant histone proteins and defined-sequence DNA has become a requirement in many projects that address the role of histone modifications, histone variants, or histone mutations in nucleosome and chromatin structure. The cloning strategies for the construction of plasmids containing multiple repeats of defined DNA sequences, and the subsequent large-scale isolation of defined sequence DNA for nucleosome reconstitution are described. This chapter also describes adapted procedures to prepare nucleosomes with histones from other species, and for the refolding and reconstitution of (H2A– H2B) dimers and (H3–H4) 2 tetramers. Methods to reconstitute nucleosomes from different histone subcomplexes are described. A flow chart for all procedures involved in the preparation of synthetic nucleosomes is also presented.

Crystal structures of histone Sin mutant nucleosomes reveal altered protein–DNA interactions

Here we describe 11 crystal structures of nucleosome core particles containing individual point mutations in the structured regions of histones H3 and H4. The mutated residues are located at the two protein–DNA interfaces flanking the nucleosomal dyad. Five of the mutations partially restore the in vivo effects of SWI/SNF inactivation in yeast. We find that even nonconservative mutations of these residues (which exhibit a distinct phenotype in vivo) have only moderate effects on global nucleosome structure. Rather, local protein–DNA interactions are disrupted and weakened in a subtle and complex manner. The number of lost protein–DNA interactions correlates directly with an increased propensity of the histone octamer to reposition with respect to the DNA, and with an overall destabilization of the nucleosome. Thus, the disruption of only two to six of the ∼120 direct histone–DNA interactions within the nucleosome has a pronounced effect on nucleosome mobility and stability. This has implications for our understanding of how these structures are made accessible to the transcription and replication machinery in vivo.

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.

Histone Chaperone FACT Coordinates Nucleosome Interaction through Multiple Synergistic Binding Events

Background: The histone chaperone FACT binds and reorganizes nucleosomes during critical cellular processes. Results: FACT binds histones, DNA, and mono- and tri-nucleosomes with high affinity. FACT reduces non-nucleosomal histone/DNA interactions. Conclusion: Multiple regions of FACT strategically bind target sites on nucleosomes to coordinate (dis)assembly. Significance: The thermodynamic parameters guiding multiple FACT/nucleosome interaction(s) coincide with reorganization 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 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.

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

Significance Poly-ADP-ribosylation (PARylation) is an abundant posttranslational modification in eukaryotes. The responsible enzyme, poly [ADP-ribose] polymerase 1 (PARP-1), binds to chromatin and shapes its architecture. It is activated by DNA damage and other triggers, and catalyzes the addition of long chains of poly-ADP ribose (PAR) mainly to itself. The biological effects of PARylation are unknown. Here, we show that PARylation confers the ability to bind histones and to assemble nucleosomes upon PARP-1, and also affects its interaction with chromatin. The rapid turnover of PAR groups provides a mechanism for switching PARP-1 function from nucleosome binder to nucleosome assembler. This explains the involvement of active PARP-1 in both transcription and DNA damage repair, because histone chaperone activity is required during both processes. 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.

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

Wnt signaling activates target genes by promoting association of the co‐activator β‐catenin with TCF/LEF transcription factors. In the absence of β‐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 β‐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.

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.

Alternative Modes of Binding of Poly(ADP-ribose) Polymerase 1 to Free DNA and Nucleosomes

Background: Poly(ADP-ribose) polymerase 1 (PARP-1) modulates chromatin structure and is activated upon DNA damage. Results: PARP-1 differentiates between nucleosomes and DNA in its binding affinity and is activated to different degrees. Conclusion: PARP-1 engages different DNA-binding modules with nucleosomes and DNA. Significance: The role of PARP-1 as a chromatin architectural protein and responder in DNA repair is reflected in different binding modes. 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.

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 (macroH2AFL), 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 macroH2AFL 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 (H2AML) exhibited the same condensation properties as macroH2A(1–161) arrays, thus emphasizing the intriguing behavior of the macroH2A linker region.