Histone chaperone FACT action during transcription through chromatin by RNA polymerase II

Abstract

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...

Integrated molecular mechanism directing nucleosome reorganization by human FACT

“…In addition, the previous structural reports indicate competitive relationships between the nucleosomal DNA and several histone chaperones, including FACT, during the association with internal histones (Hu et al 2011;Winkler et al 2011;Elsässer et al 2012;Liu et al 2012;Chen et al 2015;Huang et al 2015;Kemble et al 2015;Richet et al 2015). Thus, we assumed that the interaction of hFACT with nucleosomes would involve local detachments of the nucleosomal DNA from histones (Winkler et al 2011;Hsieh et al 2013;Kemble et al 2015). Our preliminary analysis revealed that hFACT forms stable complexes with nucleosomes treated with DNase I (data not shown).…”

“…4). However, in the previously reported mechanisms for nucleosome reorganization by FACT, H2A-H2B displacement is not obligatory (Xin et al 2009;Winkler et al 2011;Hsieh et al 2013;Zheng et al 2014). This scientific disagreement could be explained by the alternative interpretation of the previous data.…”

“…In all of these mechanisms, FACT and DNA exhibit mutually competitive binding to H2A-H2B within nucleosomes (Winkler et al 2011;Hsieh et al 2013;Zheng et al 2014;Kemble et al 2015). Thus, FACT shields DNA-binding surfaces of H2A-H2B within nucleosomes and partially peels DNA from histones.…”

The FACT Histone Chaperone Guides Histone H4 Into Its Nucleosomal Conformation in Saccharomyces cerevisiae

“…FACT therefore appears to induce or stabilize an alternative, reorganized nucleosome structure in which the normal components of a nucleosome remain associated with one another, but in a way that is less compact or stable than in the canonical form (Formosa 2012). Destabilization of nucleosomes is presumably the mechanism through which FACT decreases the barrier to RNA polymerase progression posed by chromatin (Orphanides et al 1998;Hsieh et al 2013), enhances binding of TATA Binding Protein to sites within nucleosomal DNA (Biswas et al 2005), and promotes displacement of nucleosomes from the promoters of inducible genes prior to the initiation of transcription (Schwabish and Struhl 2004;Ransom et al 2009;Takahata et al 2009b;Xin et al 2009). However, FACT is also important for preventing dispersal of existing nucleosomes during transcription and for promoting rapid reestablishment of chromatin-based repression (Jamai et al 2009;Hainer et al 2012).…”

FACT and the H2B N Tail

“…Importantly, it also found that FACT competes with DNA for binding to H2A-H2B with no effect on DNA-tetramer interactions, indicating that FACT mediates H2A-H2B exchange with DNA. In support of this model, Reinberg, Studitsky, and their colleagues recently provided data in support of a role for FACT in displacing DNA from the H2A-H2B dimer during RNA Pol IImediated transcription on chromatin in vitro (10). As Pol II enters a nucleosome, it partially uncoils DNA from the surface of the octamer.…”