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...

Efficient maintenance of chromatin structure during passage of RNA polymerase II (Pol II) is critical for cell survival and functioning. Moderate-level transcription of eukaryotic genes by Pol II is accompanied by nucleosome survival, extensive exchange of histones H2A/H2B and minimal exchange of histones H3/H4. Complementary in vitro studies have shown that transcription through chromatin by single Pol II complexes is uniquely coupled with nucleosome survival via formation of a small intranucleosomal DNA loop (Ø-loop) containing the transcribing enzyme. In contrast, transient displacement and exchange of all core histones are observed during intense transcription. Indeed, multiple transcribing Pol II complexes can efficiently overcome the high nucleosomal barrier and displace the entire histone octamer in vitro. Thus, various Pol II complexes can remodel chromatin to different extents. The mechanisms of nucleosome survival and displacement during transcription and the role of DNA-histone interactions and various factors during this process are discussed.

Maintenance of the chromatin states and histone modification patterns during transcription is essential for proper gene regulation and cell survival. Histone octamer survives moderate transcription, but is evicted during intense transcription in vivo by RNA polymerase II (Pol II). Previously we have shown that nucleosomes can survive transcription by single Pol II complexes in vitro. To study the mechanism of histone displacement from DNA, the encounter between multiple complexes of RNA polymerase and a nucleosome was analyzed in vitro. Multiple transcribing Pol II complexes can efficiently overcome the high nucleosomal barrier and displace the entire histone octamer, matching the observations in vivo. DNA-bound histone hexamer left behind the first complex of transcribing enzyme is evicted by the next Pol II complex. Thus transcription by single Pol II complexes allows survival of the original H3/H4 histones, while multiple, closely spaced complexes of transcribing Pol II can induce displacement of all core histones along the gene.elongation | rate | cooperation | subnucleosome | eviction

Gene programs that control differentiation are regulated through the interplay between DNA, RNA, and protein. Cooperation among these fundamental cellular components can occur through highly structured interactions connecting domains formed by specific sequences of nucleotides, ribonucleotides, and/or amino acids and also through the assembly of biomolecular condensates. Here, we show that endoderm differentiation is regulated through the interaction of the long noncoding (lnc) RNA DIGIT and the bromodomain and extra-terminal (BET) domain family protein BRD3. BRD3 forms phase-separated condensates that contain DIGIT, occupies enhancers of endoderm transcription factors, and is required for endoderm differentiation. Purified BRD3 binds to acetylated histone H3 lysine 18 (H3K18ac) in vitro and occupies regions of the genome enriched in H3K18ac during endoderm differentiation, including the key transcription factors that regulate endoderm differentiation. DIGIT is also enriched in regions of H3K18ac, and depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings support a model where cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest a broader role for protein-lncRNA phase-separated condensates as regulators of transcription in development.