Aaron R. Hieb

B2 RNA binds directly to RNA polymerase II to repress transcript synthesis

B2 RNA is a small noncoding RNA polymerase III transcript that represses mRNA transcription in response to heat shock in mouse cells. Here we define the mechanism by which B2 RNA inhibits RNA polymerase II (Pol II) transcription. Using a purified Pol II transcription system, we found that B2 RNA potently inhibits transcription by binding to core Pol II with high affinity and specificity. Through this interaction, B2 RNA assembles into preinitiation complexes at the promoter and blocks RNA synthesis. Once B2 RNA is removed from preinitiation complexes, transcriptional activity is restored. Our studies describe a previously unobserved mechanism of transcriptional repression by a small RNA and suggest that B2 RNA associates with Pol II at promoters in heat shocked cells to actively inhibit transcription.

Nucleosome accessibility governed by the dimer/tetramer interface

Nucleosomes are multi-component macromolecular assemblies which present a formidable obstacle to enzymatic activities that require access to the DNA, e.g. DNA and RNA polymerases. The mechanism and pathway(s) by which nucleosomes disassemble to allow DNA access are not well understood. Here we present evidence from single molecule FRET experiments for a previously uncharacterized intermediate structural state before H2A–H2B dimer release, which is characterized by an increased distance between H2B and the nucleosomal dyad. This suggests that the first step in nucleosome disassembly is the opening of the (H3–H4)2 tetramer/(H2A–H2B) dimer interface, followed by H2A–H2B dimer release from the DNA and, lastly, (H3–H4)2 tetramer removal. We estimate that the open intermediate state is populated at 0.2–3% under physiological conditions. This finding could have significant in vivo implications for factor-mediated histone removal and exchange, as well as for regulating DNA accessibility to the transcription and replication machinery.

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.

Stability of Nucleosomes Containing Homogenously Ubiquitylated H2A and H2B Prepared Using Semisynthesis

Post-translational modifications (PTMs) of histones are an essential feature in the dynamic regulation of chromatin. One of these modifications, ubiquitylation, has been speculated to directly influence the stability of the nucleosome, which represents the basic building block of chromatin. Here we report a strategy for the semisynthesis of site-specifically ubiquitylated histone H2A (uH2A). This branched protein was generated through a three-piece expressed protein ligation approach including a traceless ligation at valine. uH2A could be efficiently incorporated into nucleosomes, thereby opening the way to detailed biochemical and biophysical studies on the function of this PTM. Accordingly, we used uH2A, as well as a previously generated ubiquitylated H2B, in chaperone-coupled nucleosome stability assays to demonstrate that the direct effect of ubiquitylated histones on nucleosomal stability is in fact modest.

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.

Fluorescence strategies for high-throughput quantification of protein interactions

Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein–DNA and protein–protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A–H2B heterodimer.

Deconstructing the late phase of vimentin assembly by total internal reflection fluorescence microscopy (TIRFM).

Quantitative imaging of intermediate filaments (IF) during the advanced phase of the assembly process is technically difficult, since the structures are several µm long and therefore they exceed the field of view of many electron (EM) or atomic force microscopy (AFM) techniques. Thereby quantitative studies become extremely laborious and time-consuming. To overcome these difficulties, we prepared fluorescently labeled vimentin for visualization by total internal reflection fluorescence microscopy (TIRFM). In order to investigate if the labeling influences the assembly properties of the protein, we first determined the association state of unlabeled vimentin mixed with increasing amounts of labeled vimentin under low ionic conditions by analytical ultracentrifugation. We found that bona fide tetrameric complexes were formed even when half of the vimentin was labeled. Moreover, we demonstrate by quantitative atomic force microscopy and electron microscopy that the morphology and the assembly properties of filaments were not affected when the fraction of labeled vimentin was below 10%. Using fast frame rates we observed the rapid deposition of fluorescently labeled IFs on glass supports by TIRFM in real time. By tracing their contours, we have calculated the persistence length of long immobilized vimentin IFs to 1 µm, a value that is identical to those determined for shorter unlabeled vimentin. These results indicate that the structural properties of the filaments were not affected significantly by the dye. Furthermore, in order to analyze the late elongation phase, we mixed long filaments containing either Alexa 488- or Alexa 647-labeled vimentin. The ‘patchy’ structure of the filaments obtained unambiguously showed the elongation of long IFs through direct end-to-end annealing of individual filaments.

Quantifying Chromatin-Associated Interactions: The HI-FI System

Chromatin plays a vital role in regulating cellular processes that occur on the DNA. Modulation of chromatin structure is conducted through interactions with binding factors that direct critical actions such as posttranslational modifications, nucleosome remodeling, and incorporation of histone variants. Specific factors recognize and act upon the various physical states of chromatin to modulate DNA accessibility. The ability to quantitatively characterize these interactions in vitro can provide valuable insight into the mechanisms that dictate chromatin architecture. Here, we describe in detail fluorescence methodologies for quantifying the thermodynamic principles that guide interactions between nucleosomal arrays, mononucleosomes, or nucleosome components and chromatin-associated factors through application of the HI-FI (High-throughput Interactions by Fluorescence Intensity) system. These measurements utilize fluorescence (de)quenching and FRET assays performed in 384-well microplates, making the assays suitable for high-throughput characterization of interactions at low concentrations. Further, this system can be used to determine the stoichiometric composition of complexes and specific sites of interaction. After quantification on a plate reader or similar instrument, the solution-based assays can be directly transferred to native gels for visualization of interaction(s). We also highlight procedural details on the efficient attachment of fluorescent dyes to histones and DNA. In all, the HI-FI system of assays can be used to elucidate mechanistic details of how specific chromatin-associated factors function at the molecular level.

Quantifying Chromatin-Associated Interactions

Chromatin plays a vital role in regulating cellular processes that occur on the DNA. Modulation of chromatin structure is conducted through interactions with binding factors that direct critical actions such as posttranslational modifications, nucleosome remodeling, and incorporation of histone variants. Specific factors recognize and act upon the various physical states of chromatin to modulate DNA accessibility. The ability to quantitatively characterize these interactions in vitro can provide valuable insight into the mechanisms that dictate chromatin architecture. Here, we describe in detail fluorescence methodologies for quantifying the thermodynamic principles that guide interactions between nucleosomal arrays, mononucleosomes, or nucleosome components and chromatin-associated factors through application of the HI-FI (High-throughput Interactions by Fluorescence Intensity) system. These measurements utilize fluorescence (de)quenching and FRET assays performed in 384-well microplates, making the assays suitable for high-throughput characterization of interactions at low concentrations. Further, this system can be used to determine the stoichiometric composition of complexes and specific sites of interaction. After quantification on a plate reader or similar instrument, the solution-based assays can be directly transferred to native gels for visualization of interaction(s). We also highlight procedural details on the efficient attachment of fluorescent dyes to histones and DNA. In all, the HI-FI system of assays can be used to elucidate mechanistic details of how specific chromatin-associated factors function at the molecular level.

A quantitative investigation of linker histone interactions with nucleosomes and chromatin

Linker histones such as H1 are abundant basic proteins that bind tightly to nucleosomes, thereby acting as key organizers of chromatin structure. The molecular details of linker histone interactions with the nucleosome, and in particular the contributions of linker DNA and of the basic C-terminal tail of H1, are controversial. Here we combine rigorous solution-state binding assays with native gel electrophoresis and Atomic Force Microscopy, to quantify the interaction of H1 with chromatin. We find that H1 binds nucleosomes and nucleosomal arrays with very tight affinity by recognizing a specific DNA geometry minimally consisting of a solitary nucleosome with a single ~18 base pair DNA linker arm. The association of H1 alters the conformation of trinucleosomes so that only one H1 can bind to the two available linker DNA regions. Neither incorporation of the histone variant H2A.Z, nor the presence of neighboring nucleosomes affects H1 affinity. Our data provide a comprehensive thermodynamic framework for this ubiquitous chromatin architectural protein.