The FACT (facilitates chromatin transcription) complex is required for transcript elongation through nucleosomes by RNA polymerase II (Pol II) in vitro. Here, we show that FACT facilitates Pol II-driven transcription by destabilizing nucleosomal structure so that one histone H2A-H2B dimer is removed during enzyme passage. We also demonstrate that FACT possesses intrinsic histone chaperone activity and can deposit core histones onto DNA. Importantly, FACT activity requires both of its constituent subunits and is dependent on the highly acidic C terminus of its larger subunit, Spt16. These findings define the mechanism by which Pol II can transcribe through chromatin without disrupting its epigenetic status.

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.

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.

RNA polymerase II (Pol II) must transcribe genes in a chromatin environment in vivo. We examined transcription by Pol II through nucleosome cores in vitro. At physiological and lower ionic strengths, a mononucleosome imposes a strong block to elongation, which is relieved at increased ionic strength. Passage of Pol II causes a quantitative loss of one H2A/H2B dimer but does not alter the location of the nucleosome. In contrast, bacteriophage SP6 RNA polymerase (RNAP) efficiently transcribes through the same nucleosome under physiological conditions, and the histone octamer is transferred behind SP6 RNAP. Thus, the mechanisms for transcription through the nucleosome by Pol II and SP6 RNAP are clearly different. Moreover, Pol II leaves behind an imprint of disrupted chromatin structure.

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Remodeling machines play an essential role in the control of gene expression, but how their activity is regulated is not known. Here we report that the nuclear protein nucleolin possesses a histone chaperone activity and that this factor greatly enhances the activity of the chromatin remodeling machineries SWI/SNF and ACF. Interestingly, nucleolin is able to induce the remodeling by SWI/SNF of macroH2A, but not of H2ABbd nucleosomes, which are otherwise resistant to remodeling. This new histone chaperone promotes the destabilization of the histone octamer, helping the dissociation of a H2A-H2B dimer, and stimulates the SWI/SNF-mediated transfer of H2A-H2B dimers. Furthermore, nucleolin facilitates transcription through the nucleosome, which is reminiscent of the activity of the FACT complex. This work defines new functions for histone chaperones in chromatin remodeling and regulation of transcription and explains how nucleolin could act on transcription.

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...