Chromatin Transcription: Epigenetics, Cancer & Aging
Development and functioning of living organisms critically depends on proper gene expression. Regulation of genes involves a very complicated and fine-tuned interplay between specific nucleotide sequences, protein factors and dynamic changes in the spatial organization of the genome. In higher organisms regulation of gene expression occurs primarily at the step of transcription.
The majority of eukaryotic genes are transcribed by RNA polymerase II (Pol II). In the nucleus of eukaryotic cells DNA is organized in chromatin – tightly compacted DNA-protein complexes (nucleosomes) forming repeating units of genome organization on every ~200-bp region of the genome. Pol II meets these DNA-protein complexes during transcription, and they are likely targets for gene regulation at the level of transcript elongation. Nucleosomes present a high polar barrier for the enzyme in vitro1, but various protein factors allow efficient transcription through chromatin and facilitate histone survival2,3. Overall, the Pol II-specific mechanism of transcription through chromatin is likely used for maintenance of chromatin structure and the “histone code” that is particularly important during genomic transcription. The key features of Pol II–nucleosome encounter are conserved from yeast to human, highlighting the importance of this mechanism for the cell.
In our studies we have established a “minimal”, highly purified and efficient experimental system maintaining single- and multiple-round transcription through various defined mono- and polynucleosomes by yeast and human RNA polymerase II faithfully recapitulating numerous features of transcribed chromatin described in vivo and allowing their molecular analysis in vitro4.
Recent structural analysis of intermediates formed during transcription through chromatin allowed us to propose the mechanisms of nucleosomal barrier formation and nucleosome survival during transcription5,6. It indicated that both DNA–histone interactions and Pol II backtracking contribute to formation of the high nucleosomal barrier to transcription. Nucleosome survival during transcription likely occurs through allosterically stabilized histone–histone interactions. The data reveal the importance of intranucleosomal DNA–protein and protein–protein interactions during conformational changes in the nucleosome structure on transcription. The proposed mechanism suggests that the high nucleosomal barrier to Pol II participates in regulation of gene expression in eukaryotes and explains the remarkable efficiency of nucleosome survival during transcription, important for maintenance of epigenetic and regulatory histone modifications. Our recent structural analysis of other intermediates formed during transcription through chromatin will be presented.
Nucleosome structure and dynamics significantly affect DNA accessibility to other regulatory and effector proteins, for example, to DNA repair systems. In our studies we have shown that transcription through chromatin is accompanied by formation of small intranucleosomal DNA loops. Pol II captured within a small loop drives accumulation of DNA supercoiling, facilitating further transcription. DNA breaks relieve supercoiling and induce Pol II arrest, allowing detection of DNA damages hidden in chromatin structure7. Our recent studies of this novel chromatin-targeted mechanism of DNA repair will be presented.
FACT (facilitates chromatin transcription) is an essential and highly conserved histone chaperone that assists nucleosome assembly and disassembly, and plays an important role in cell differentiation and cancer development. Our previous studies have suggested that human FACT (hFACT) facilitates transcription through chromatin and promotes nucleosome recovery in vitro3. FACT also increases the accessibility of nucleosomal DNA to regulatory proteins, but the mechanism and extent of this nucleosome reorganization are unknown. Our recent studies using single particle FRET microscopy revealed a dramatic and reversible uncoiling of nucleosomal DNA by FACT. This FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and plays an important function of in vivo. Similar mechanisms are likely used during various other processes involving DNA, including DNA replication and ATP-dependent chromatin remodeling.
Our most recent studies are conducted using a combination of molecular genetics, genomics, biochemical, single-particle, structural and computational modeling approaches and are focused on the mechanisms of transcription through chromatin and action of various elongation factors and histone chaperones (hFACT and hPARP1, both are involved in cancer development and aging and are important targets for anti-cancer drugs) facilitating histone survival during this process. The developed experimental models allow construction of test systems for new anti-cancer drug screening and further analysis of the complicated mechanisms of regulation of gene expression.
1. Bondarenko VA, Steele LM, Ujvári A, Gaykalova DA, Kulaeva OI, Polikanov YS, Luse DS, Studitsky VM. Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II. Mol Cell 2006; 24:469–79.
2. Kulaeva OI, Gaykalova DA, Pestov NA, Golovastov VV, Vassylyev DG, Artsimovitch I, Studitsky VM. Mechanism of chromatin remodeling and recovery during passage of RNA polymerase II. Nat Struct Mol Biol 2009; 16:1272–8.
3. Hsieh F-K, Kulaeva OI, Patel SS, Dyer PN, Luger K, Reinberg D, Studitsky VM. Histone chaperone FACT action during transcription through chromatin by RNA polymerase II. Proc Natl Acad Sci U S A 2013; 110:7654–9.
4. Gaykalova DA, Kulaeva OI, Pestov NA, Hsieh F-K, Studitsky VM. Experimental analysis of the mechanism of chromatin remodeling by RNA polymerase II. Methods Enzymol 2012; 512:293–314.
5. Chang H-W, Kulaeva OI, Shaytan AK, Kibanov M, Kuznedelov K, Severinov KV, Kirpichnikov MP, Clark DJ, Studitsky VM. Analysis of the mechanism of nucleosome survival during transcription. Nucleic Acids Res 2014; 42:1619–27.
6. Gaykalova DA, Kulaeva OI, Volokh O, Shaytan AK, Hsieh F-K, Kirpichnikov MP, Sokolova OS, Studitsky VM. Structural analysis of nucleosomal barrier to transcription. Proc Natl Acad Sci U S A 2015; 112:E5787–95.
7. Pestov NA, Gerasimova NS, Kulaeva OI, Studitsky VM. Structure of transcribed chromatin is a sensor of DNA damage. Sci Adv 2015; 1:e1500021.