Transcription of eukaryotic genes by RNA polymerase II (Pol II) is typically accompanied by nucleosome survival and minimal exchange of histones H3/H4. The mechanism of 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 was conducted. When Pol II approaches the 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/H4 histones from DNA during enzyme passage.
Nucleosomes uniquely positioned on high-affinity DNA sequences present a polar barrier to transcription by human and yeast RNA polymerase II (Pol II). In one transcriptional orientation, these nucleosomes provide a strong, factor- and salt-insensitive barrier at the entry into the H3/H4 tetramer that can be recapitulated without H2A/H2B dimers. The same nucleosomes transcribed in the opposite orientation form a weaker, more diffuse barrier that is largely relieved by higher salt, TFIIS, or FACT. Barrier properties are therefore dictated by both the local nucleosome structure (influenced by the strength of the histone-DNA interactions) and the location of the high-affinity DNA region within the nucleosome. Pol II transcribes DNA sequences at the entry into the tetramer much less efficiently than the same sequences located distal to the nucleosome dyad. Thus, entry into the tetramer by Pol II facilitates further transcription, perhaps due to partial unfolding of the tetramer from DNA.
FACT (facilitates chromatin transcription) is a histone chaperone that promotes chromatin recovery during transcription, with additional roles in cell differentiation. Although several models of the action of FACT during transcription have been proposed, they remain to be experimentally evaluated. Here we show that human FACT (hFACT) facilitates transcription through chromatin and promotes nucleosome recovery in vitro. FACT action depends on the presence of histone H2A/H2B dimers in the nucleosome. Kinetic analysis suggests that hFACT decreases the lifetime of nonproductive RNA polymerase II (Pol II)-nucleosome complexes and facilitates the formation of productive complexes containing nucleosomal DNA partially uncoiled from the octamer. Taken together, our data suggest that hFACT interacts with DNA-binding surfaces of H2A/H2B dimers, facilitating uncoiling of DNA from the histone octamer. Thus, hFACT-H2A/ H2B interactions play a key role in overcoming the nucleosomal barrier by Pol II and promoting nucleosome survival during transcription.elongation | dynamics | DNA uncoiling | mechanism F ACT (facilitates chromatin transcription) is the transcription and replication factor (1, 2) involved in cell differentiation (3), and is also an important target for anticancer drugs (4). Human FACT (hFACT) is a heterodimer protein complex composed of two subunits [suppressor of Ty 16 homolog (Spt16) and structure specific recognition protein 1 (SSRP1)] that has histone chaperone activity (5, 6). FACT stimulates transcript elongation through nucleosomes in vitro (2, 6). In vivo, FACT colocalizes with RNA polymerase II (Pol II) and displays similar kinetics of recruitment and chromosome tracking (7,8). FACT is also essential for maintenance of chromatin structure during transcript elongation by Pol II (8-11).Different models have been proposed to describe the mechanism of FACT's action. FACT can induce global accessibility of nucleosomal DNA without histone H2A/H2B displacement (12, 13) and thus can facilitate action of processive enzymes on DNA. On the other hand, it has been suggested that FACT destabilizes nucleosomes by facilitating dissociation of histone H2A/H2B dimer from nucleosomes, thereby facilitating transcription through chromatin (6). The mechanism of FACT action during transcription remains to be experimentally evaluated.In this study, we systematically examined the effect of FACT on transcription through a nucleosome by Pol II in vitro. Our results show that FACT alleviates nucleosomal pausing, and that the presence of H2A/H2B dimers is required for FACT action. Kinetic studies suggest that the alternating FACT-dimer interactions result in an increased rate of conversion from the nonproductive to productive Pol II-nucleosome complexes and thus facilitate transcription and nucleosome survival. ResultsH2A/H2B Dimers Mediate FACT-Dependent Transcription Through a Nucleosome. In our experiments, we used yeast Pol II and DNA fragments bearing single nucleosomes assembled on DNA sequences having high affinity for the...
DNA accessibility to regulatory proteins is significantly affected by nucleosome structure and dynamics. FACT (facilitates chromatin transcription) increases the accessibility of nucleosomal DNA but the mechanism and extent of this nucleosome reorganization are unknown. We report here the effects of FACT on single nucleosomes revealed with spFRET microscopy. FACT binding results in a dramatic, ATP-independent, and reversible uncoiling of DNA that affects at least 70% of the DNA in a nucleosome. A mutated version of FACT is defective in this uncoiling, and a histone mutation that suppresses phenotypes caused by this FACT mutation in vivo restores the uncoiling activity in vitro. Thus FACT-dependent nucleosome unfolding modulates the accessibility of nucleosomal DNA, and this is an important function of FACT in vivo.
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