Daria A. Gaykalova

Using DNA mechanics to predict in vitro nucleosome positions and formation energies

In eukaryotic genomes, nucleosomes function to compact DNA and to regulate access to it both by simple physical occlusion and by providing the substrate for numerous covalent epigenetic tags. While competition with other DNA-binding factors and action of chromatin remodeling enzymes significantly affect nucleosome formation in vivo, nucleosome positions in vitro are determined by steric exclusion and sequence alone. We have developed a biophysical model, DNABEND, for the sequence dependence of DNA bending energies, and validated it against a collection of in vitro free energies of nucleosome formation and a set of in vitro nucleosome positions mapped at high resolution. We have also made a first ab initio prediction of nucleosomal DNA geometries, and checked its accuracy against the nucleosome crystal structure. We have used DNABEND to design both strong and weak histone- binding sequences, and measured the corresponding free energies of nucleosome formation. We find that DNABEND can successfully predict in vitro nucleosome positions and free energies, providing a physical explanation for the intrinsic sequence dependence of histone–DNA interactions.

Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II

Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3 and H4. The mechanism of nucleosome survival and recovery of chromatin structure remains obscure. Here we show how transcription through chromatin by Pol II is uniquely coupled with nucleosome survival. Structural modeling and functional analysis of the intermediates of transcription through a nucleosome indicated that when Pol II approaches an area of strong DNA-histone interactions, a small intranucleosomal DNA loop (zero-size or Ø-loop) containing transcribing enzyme is formed. During formation of the Ø-loop, the recovery of DNA-histone interactions behind Pol II is tightly coupled with their disruption ahead of the enzyme. This coupling is a distinct feature of the Pol II–type mechanism that allows further transcription through the nucleosome, prevents nucleosome translocation and minimizes displacement of H3 and H4 histones from DNA during enzyme passage.

Structural analysis of nucleosomal barrier to transcription

Significance On the majority of eukaryotic genes RNA polymerase II meets nucleosomes during transcription of every ∼200 bp of DNA. The key features of Pol II–nucleosome encounter are conserved from yeast to human, but the molecular mechanism of this process remains unknown. Our data suggest a mechanism of formation of the high nucleosomal barrier to Pol II that participates in regulation of transcript elongation in eukaryotes. The proposed mechanism explains the remarkable efficiency of nucleosome survival during transcription, important for maintenance of epigenetic and regulatory histone modifications. Similar mechanisms are likely used during various other DNA transactions, including DNA replication and ATP-dependent chromatin remodeling. Some factors involved in chromatin transcription (e.g., FACT and PARP) participate in cancer development/aging. Thousands of human and Drosophila genes are regulated at the level of transcript elongation and nucleosomes are likely targets for this regulation. However, the molecular mechanisms of formation of the nucleosomal barrier to transcribing RNA polymerase II (Pol II) and nucleosome survival during/after transcription remain unknown. Here we show that both DNA–histone interactions and Pol II backtracking contribute to formation of the barrier and that nucleosome survival during transcription likely occurs through allosterically stabilized histone–histone interactions. Structural analysis indicates that after Pol II encounters the barrier, the enzyme backtracks and nucleosomal DNA recoils on the octamer, locking Pol II in the arrested state. DNA is displaced from one of the H2A/H2B dimers that remains associated with the octamer. The data reveal the importance of intranucleosomal DNA–protein and protein–protein interactions during conformational changes in the nucleosome structure on transcription. Mechanisms of nucleosomal barrier formation and nucleosome survival during transcription are proposed.

Preparation and Analysis of Uniquely Positioned Mononucleosomes

Short DNA fragments containing single, uniquely positioned nucleosome cores have been extensively employed as simple model experimental systems for analysis of many intranuclear processes, including binding of proteins to nucleosomes, transcription, DNA repair and ATP-dependent chromatin remodeling. In many cases such simple model templates faithfully recapitulate numerous important aspects of these processes. Here we describe several recently developed procedures for obtaining and analysis of mononucleosomes that are uniquely positioned on 150-600 bp DNA fragments.

A polar barrier to transcription can be circumvented by remodeler-induced nucleosome translocation

Many eukaryotic genes are regulated at the level of transcript elongation. Nucleosomes are likely targets for this regulation. Previously, we have shown that nucleosomes formed on very strong positioning sequences (601 and 603), present a high, orientation-dependent barrier to transcription by RNA polymerase II in vitro. The existence of this polar barrier correlates with the interaction of a 16-bp polar barrier signal (PBS) with the promoter-distal histone H3–H4 dimer. Here, we show that the polar barrier is relieved by ISW2, an ATP-dependent chromatin remodeler, which translocates the nucleosome over a short distance, such that the PBS no longer interacts with the distal H3–H4 dimer, although it remains within the nucleosome. In vivo, insertion of the 603 positioning sequence into the yeast CUP1 gene results in a modest reduction in transcription, but this reduction is orientation-independent, indicating that the polar barrier can be circumvented. However, the 603-nucleosome is present at the expected position in only a small fraction of cells. Thus, the polar barrier is probably non-functional in vivo because the nucleosome is not positioned appropriately, presumably due to nucleosome sliding activities. We suggest that interactions between PBSs and chromatin remodelers might have significant regulatory potential.

Experimental Analysis of the Mechanism of Chromatin Remodeling by RNA Polymerase II

The vital process of transcription by RNA polymerase II (Pol II) occurs in chromatin environment in eukaryotic cells; in fact, moderately transcribed genes retain nucleosomal structure. Recent studies suggest that chromatin structure presents a strong barrier for transcribing Pol II in vitro, and that DNA-histone interactions are only partially and transiently disrupted during transcript elongation on moderately active genes. Furthermore, elongating Pol II complex is one of the major targets during gene regulation. Below, we describe a highly purified, defined experimental system that recapitulates many important properties of transcribed chromatin in vitro and allows detailed analysis of the underlying mechanisms.