M. E. Valieva

Stabilization of Nucleosomes by Histone Tails and by FACT Revealed by spFRET Microscopy

A correct chromatin structure is important for cell viability and is tightly regulated by numerous factors. Human protein complex FACT (facilitates chromatin transcription) is an essential factor involved in chromatin transcription and cancer development. Here FACT-dependent changes in the structure of single nucleosomes were studied with single-particle Förster resonance energy transfer (spFRET) microscopy using nucleosomes labeled with a donor-acceptor pair of fluorophores, which were attached to the adjacent gyres of DNA near the contact between H2A-H2B dimers. Human FACT and its version without the C-terminal domain (CTD) and the high mobility group (HMG) domain of the structure-specific recognition protein 1 (SSRP1) subunit did not change the structure of the nucleosomes, while FACT without the acidic C-terminal domains of the suppressor of Ty 16 (Spt16) and the SSRP1 subunits caused nucleosome aggregation. Proteolytic removal of histone tails significantly disturbed the nucleosome structure, inducing partial unwrapping of nucleosomal DNA. Human FACT reduced DNA unwrapping and stabilized the structure of tailless nucleosomes. CTD and/or HMG domains of SSRP1 are required for this FACT activity. In contrast, previously it has been shown that yeast FACT unfolds (reorganizes) nucleosomes using the CTD domain of SSRP1-like Pol I-binding protein 3 subunit (Pob3). Thus, yeast and human FACT complexes likely utilize the same domains for nucleosome reorganization and stabilization, respectively, and these processes are mechanistically similar.

Development of fluorescently labeled mononucleosomes for the investigation of transcription mechanisms by single complex microscopy

Fluorescence microscopy of single molecules and complexes is an increasingly popular method for research on nucleosomes and functionally important processes involving these biological objects. Precisely positioned mononucleosomes have been developed in the present work using a fluorescently labeled DNA template; such nucleosomes are novel tools for the investigation of structural rearrangements of chromatin during transcription by RNA polymerase (RNAP). Two fluorophores, the donor Cy3 and the acceptor Cy5, were introduced into the nontranscribed DNA strand. DNA coiling around the histone octamer resulted in the positioning of both fluorophores on adjacent DNA coils in the middle part of the nucleosome. The distance between the fluorophores was less than 60 Å, and, therefore, Förster resonance energy transfer (FRET) could occur. Structural rearrangements in the nucleosomes were detected using the changes in FRET efficiency measured in fluorescence microscopic studies of individual complexes of nucleosomes with RNAP. Labeling had no effect on the ability of RNAP to transcribe DNA in nucleosomes. An open complex with RNAP and elongation complexes arrested in positions–39 and–5 relatively to the nucleosome border were obtained and characterized. More than 80% of the nucleosomes have been shown to retain their structure (that is, recover the initial positioning of DNA on the histone octamer) after the completion of transcription. The experimental system developed opens up new possibilities for research on nucleosome structure and its modulation by various protein chaperones and chromatin remodeling complexes.

Functional roles of the DNA-binding HMGB domain in the histone chaperone FACT in nucleosome reorganization

The essential histone chaperone FACT (facilitates chromatin transcription) promotes both nucleosome assembly and disassembly. FACT is a heterodimer of Spt16 with either SSRP1 or Pob3, differing primarily by the presence of a high-mobility group B (HMGB) DNA-binding domain furnished only by SSRP1. Yeast FACT lacks the intrinsic HMGB domain found in SSRP1-based homologs such as human FACT, but yeast FACT activity is supported by Nhp6, which is a freestanding, single HMGB-domain protein. The importance of histone binding by FACT domains has been established, but the roles of DNA-binding activity remain poorly understood. Here, we examined these roles by fusing single or multiple HMGB modules to Pob3 to mimic SSRP1 or to test the effects of extended DNA-binding capacity. Human FACT and a yeast mimic both required Nhp6 to support nucleosome reorganization in vitro, indicating that a single intrinsic DNA-binding HMGB module is insufficient for full FACT activity. Three fused HMGB modules supported activity without Nhp6 assistance, but this FACT variant did not efficiently release from nucleosomes and was toxic in vivo. Notably, intrinsic DNA-binding HMGB modules reduced the DNA accessibility and histone H2A–H2B dimer loss normally associated with nucleosome reorganization. We propose that DNA bending by HMGB domains promotes nucleosome destabilization and reorganization by exposing FACTs histone-binding sites, but DNA bending also produces DNA curvature needed to accommodate nucleosome assembly. Intrinsic DNA-bending activity therefore favors nucleosome assembly by FACT over nucleosome reorganization, but excessive activity impairs FACT release, suggesting a quality control checkpoint during nucleosome assembly.

Structure and function of histone chaperone FACT

FACT is heterodimer protein complex and histone chaperone that plays an important role in maintaining and modifying the chromatin structure during various DNA-dependent processes. FACT is involved in nucleosome assembly de novo and in the preservation and recovery of the nucleosome structure during and after transcription, replication and DNA repair. During transcript elongation, FACT reduces the height of the nucleosome barrier and supports the maintenance of nucleosomes during and after the passage of RNA polymerase II. In this process, FACT interacts with histone H2A–H2B dimer within nucleosomes, thus, facilitating uncoiling of nucleosomal DNA from the octamer of histones. It also facilitates the subsequent recovery of the canonical nucleosome structure after transcription. FACT also plays an important role in the transformation of human cells and in maintaining the viability of tumor cells.

Inhibiting the pro-tumor and transcription factor FACT: Mechanisms

Conventional antitumor therapy is often complicated by the emergence of the so-called cancer stem cells (CSCs), which are characterized by low metabolic rates and high resistance to almost all existing therapies. Many problems of clinical oncology and a poor efficacy of current treatments in particular are ascribed to CSCs. Therefore, it is important to develop new compounds capable of eliminating both rapidly proliferating tumor cells and standard treatment-resistant CSCs. Curaxins have been demonstrated to manifest various types of antitumor activity. Curaxins simultaneously affect at least three key molecular cascades involved in tumor development, including the p53, NF-κB, and HSF1 metabolic pathways. In addition, studies of some curaxins indicate that they can inhibit the transcriptional induction of the genes for matrix metalloproteinases 1 and 8 (MMP1 and MMP8); the PI3K/AKT/mTOR signaling cascades; cIAP-1 (apoptosis protein 1) inhibitor activity; topoisomerase II; and a number of oncogenes, such as c-MYC and others. In vivo experiments have shown that the CSC population increases on gemcitabine monotherapy and is reduced on treatment with curaxin CBL0137. The data support the prospective use of FACT inhibitors as new anticancer drugs with multiple effects on cell metabolism.

Structure and function of the histone chaperone FACT – Resolving FACTual issues

FAcilitates Chromatin Transcription (FACT) has been considered essential for transcription through chromatin mostly based on cell-free experiments. However, FACT inactivation in cells does not cause a significant reduction in transcription. Moreover, not all mammalian cells require FACT for viability. Here we synthesize information from different organisms to reveal the core function(s) of FACT and propose a model that reconciles the cell-free and cell-based observations. We describe FACT structure and nucleosomal interactions, and their roles in FACT-dependent transcription, replication and repair. The variable requirements for FACT among different tumor and non-tumor cells suggest that various FACT-dependent processes have significantly different levels of relative importance in different eukaryotic cells. We propose that the stability of chromatin, which might vary among different cell types, dictates these diverse requirements for FACT to support cell viability. Since tumor cells are among the most sensitive to FACT inhibition, this vulnerability could be exploited for cancer treatment.

Purification of protein–DNA complexes by native gel electrophoresis for electron microscopy study

Electrophoretic separation under native conditions may be used for purification of protein molecules and their complexes with DNA and other ligands. Here, we employed this approach to separate protein-DNA complexes with a molecular weight of approximately 200 kDa: mono- and dinucleosomes. The purified mononucleosomes were subjected to single particle electron microscopy study using negative stain contrasting, and the two-dimensional projections of the nucleosomes at 25 Å resolution were obtained. A comparison of the nucleosome projections before and after separation in the native PAGE revealed different orientation of particles on the carbon film.

Histone Chaperones: Variety and Functions

Histone chaperones are required for the formation of the nucleosome—the basic unit of chromatin that consists of the DNA and histones. In this review, participation of histone chaperones CAF-1, ASF1, NAP1, and FACT in key cellular processes is discussed. Being multifunctional factors, histone chaperones take part in DNA replication, transcription, and repair. During replication, histone chaperones are required to form chromatin structure on both the mother and daughter DNA. They are involved at different stages of genome packaging: from histone transport into the nucleus to nucleosome formation. During transcription, histone chaperones reduce a nucleosome barrier for RNA polymerases accelerating the rate of RNA synthesis and promote nucleosome reassembly. During DNA repair, histone chaperones provide access to the damaged genome region for the repair enzymes, and participate in the chromatin assembly after DNA repair. Mutations in histone chaperones typically result in multiple defects in the cell, underlying the functional importance of these proteins.