Abstract P-23: Histone N-terminal Tails Reduce Early Nucleosomal Pausing during Transcription

Background: Nucleosomes are the barriers to transcript elongation by RNA polymerase 2 (Pol 2) in vitro and in vivo. Formation and overcoming the barrier are important for transcription regulation. N-terminal tails of core histones do not affect the inner structure of nucleosomal core. However, strongly positively charged tails can interact with the DNA, thereby impeding polymerase progression through the template. Removal of histone tails was shown to facilitate transcription through a nucleosome by both yeast and human Pol 2, and the effect was most noticeable at lower ionic strength (40 mM KCl). In vivo experiments established a new mechanism of overcoming of +1 nucleosomal barrier by removal of histone tails by specific regulative proteinase. As +1 nucleosomal barrier is formed mostly by the promoter-proximal part of the nucleosomal DNA, here we address the effects of histone tails on elongation through this part of the nucleosome. Methods: We have studied the effect of histone tails on transcription by yeast Pol 2 and model enzyme E. coli RNA polymerase utilizing very similar mechanisms of elongation through chromatin. 603 nucleosomes were transcribed in vitro using purified proteins and components. To focus on the proximal part of the nucleosome, transcript elongation was conducted for a limited time and at low ionic strength. Results: For the phosphorylated form of yeast Pol 2 and E. coli RNAP, histone tail removal significantly reduces the strong nucleosome-specific pausing that the yeast polymerase encounters ∼15 bp within the 603 nucleosome and further downstream, leading to both increased traversal of the pause and the accumulation of complexes paused at more distal locations. However, tail removal did not lead to a significant increase in full traversal of either nucleosomal template. The effect of histone tails removal was cognate for both enzymes but differs in detailed effect on the barrier. Conclusion: Histone tails provide a significant part of the nucleosomal barrier to transcript elongation by Pol 2-type mechanism. The effect is very pronounced in the promoter-proximal part of the nucleosomal DNA, suggesting that histone tails could play a role during the regulation of the +1 nucleosomal barrier. The role of Pol 2 CTD phosphorylation and formation of the intranucleosomal loops in the regulation of +1 nucleosomal barrier will also be addressed

Abstract P-24: Microscopic Analyses of Liquid-Liquid Phase Separation Induced by Linker Histone H1.0

Background: Liquid-liquid phase separation (LLPS) that leads to the formation of temporary functional domains in cells plays an important role in the processes of chromatin condensation and gene regulation. Earlier, it was demonstrated that histone H1.4 can form LLPS droplets with DNA. In the present work, LLPS was studied for histone H1.0, which is mainly expressed in differentiated and non-dividing cells. H1.0 is involved in cancer development: its amount decreases with the progression of tumor cells to malignancy. Methods: LSM710 confocal microscope (Zeiss) equipped with the 40x/1.2W objective was used to image mixtures of H1.0 with Cy3/Cy5 labeled DNA or nucleosomes in fluorescent and transmitted-light channels at the excitation of 514 nm. The formation of condensates as a result of LLPS was confirmed by salt-jump and FRAP/FLIP experiments. Results: Condensates were not observed when the ratio of negative to positive charges (N/P) in the samples was >1. At N/P~0.7, optically homogeneous droplet-like condensates were found. The appearance of condensates, their size and shape depended on concentrations of H1.0 and DNA. LLPS condensates but not aggregates disappeared by salt-jump to 650 mM NaCl. FRAP/FLIP experiments revealed a moderate rate of fluorescence recovery (τ½22s) indicating moderate DNA mobility of the H1.0-mediated condensates. The appearance of condensates was also observed in the mixtures of H1.0, DNA and Cy3/Cy5-labeled nucleosomes. Nucleosomes were involved in the condensate formation and found to be 2-fold more mobile (τ½10 s) than DNA. Conclusion: LLPS-related properties of H1.0 were studied for DNA and nucleosomes in vitro. Comparison with H1.4 shows that H1.0 forms liquid condensates of approximately the same size. Our result also may indicate that chromatin retains pronounced dynamic properties in H1.0-induced droplets despite the fact that H1.0 induces the formation of more compact chromatin

Abstract P-28: Structural Dynamics of DNA-Associated Chaperon Facilitates Chromatine Transcription

Background: Histone chaperon FACT (“FAcilitates Chromatin Transcription”) is a multifunctional and conserved eukaryotic protein involved in DNA transcription, replication and repair; which can reversibly unfold nucleosomes in presence of ATP. FACT is necessary for the viability and growth of breast tumor cells meanwhile in normal cells it can be knocked out without loss of vitality. Human FACT (hFACT) is a target for promising anticancer drug curaxins, which causes FACT trapping in chromatin of cancer cells and destabilizes the nucleosome. The nucleosome-unfolding activity is an important function of hFACT in vivo; however, the mechanism of FACT-dependent nucleosome unfolding remains unknown. Methods: Here, we studied negative stained hFACT structure using single particle electron microscopy using JEOL 2100 TEM. Micrographs were captured with 25k magnification, and 4.1 Å pixel size. EM images pre-processing and single particles collection were performed in EMAN2.3, followed by 2D-particles analysis in RELION2.0. Final 2D-classes included ~70 000 single particles images. Results: Based on 2D-classess data analysis we evaluated several states of hFACT reflecting its conformational flexibility: the “closed” complex is characterized by four domains localized close to each other and forming a compact structure; “intermediate” state represented by classes with identified three domains having compact structure and more disordered fourth domain, and the “open” complex, represented by three domains forming almost linear structure. The “closed” and “open” states are present in comparable amounts and significantly outnumber the “intermediate” state. It has been shown that hFACT domains are connected through flexible linkers and SPT16 and SSRP1 dimerization domains (DDs) form the “joint”-like connection between the two subunits. In the “сlosed” conformation, the DNA-binding surface of FACT is covered by its two C-terminal and middle domains (MDs). The N-terminal domain (NTD) of SPT16 was not resolved previously, but it is the best candidate for the forth domain that is clearly visible only in the “closed” conformation of hFACT, based on its dimensions and the longest linker length. Conclusion: We propose that during conversion to the “open” complexes SPT16 NTD is moving away from the other subunits leading to formation of the first intermediate state with the NTD domain poorly resolved or not resolved, while less mobile DDs and MDs maintain more compact structure and the DNA-binding site is still protected by the CTDs. In the “open” state SPT16/SSRP1 visible MDs and DDs form almost linear structure, unmasking the DNA-binding sites and making them accessible for the interaction with a nucleosome

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

Mechanism of histone survival during transcription by RNA polymerase II

Transcription of eukaryotic genes by RNA polymerase II is typically accompanied by minimal exchange of histones H3/H4 carrying various covalent modifications. In vitro studies suggest that histone survival is accompanied by the formation of a small transient DNA loop on the surface of the histone octamer including a molecule of transcribing enzyme

PARP-1-Associated Pathological Processes: Inhibition by Natural Polyphenols

Poly (ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme involved in processes of cell cycle regulation, DNA repair, transcription, and replication. Hyperactivity of PARP-1 induced by changes in cell homeostasis promotes development of chronic pathological processes leading to cell death during various metabolic disorders, cardiovascular and neurodegenerative diseases. In contrast, tumor growth is accompanied by a moderate activation of PARP-1 that supports survival of tumor cells due to enhancement of DNA lesion repair and resistance to therapy by DNA damaging agents. That is why PARP inhibitors (PARPi) are promising agents for the therapy of tumor and metabolic diseases. A PARPi family is rapidly growing partly due to natural polyphenols discovered among plant secondary metabolites. This review describes mechanisms of PARP-1 participation in the development of various pathologies, analyzes multiple PARP-dependent pathways of cell degeneration and death, and discusses representative plant polyphenols, which can inhibit PARP-1 directly or suppress unwanted PARP-dependent cellular processes

Role of Histone Tails and Single Strand DNA Breaks in Nucleosomal Arrest of RNA Polymerase

Transcription through nucleosomes by RNA polymerases (RNAP) is accompanied by formation of small intranucleosomal DNA loops (i-loops). The i-loops form more efficiently in the presence of single-strand breaks or gaps in a non-template DNA strand (NT-SSBs) and induce arrest of transcribing RNAP, thus allowing detection of NT-SSBs by the enzyme. Here we examined the role of histone tails and extranucleosomal NT-SSBs in i-loop formation and arrest of RNAP during transcription of promoter-proximal region of nucleosomal DNA. NT-SSBs present in linker DNA induce arrest of RNAP +1 to +15 bp in the nucleosome, suggesting formation of the i-loops; the arrest is more efficient in the presence of the histone tails. Consistently, DNA footprinting reveals formation of an i-loop after stalling RNAP at the position +2 and backtracking to position +1. The data suggest that histone tails and NT-SSBs present in linker DNA strongly facilitate formation of the i-loops during transcription through the promoter-proximal region of nucleosomal DNA