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.

Keywords: DNA uncoiling; dynamics; elongation; mechanism.

Fig. 1.

FACT facilitates transcription through nucleosomes: The role of the promoter-distal H2A/H2B dimer. (A) (Upper) Nucleosomal template for Pol II transcription. (Lower) Experimental approach. (B) 603 and 601R nucleosomes were transcribed by Pol II at 150 mM KCl in the absence or presence of FACT, followed by analysis of pulse-labeled RNA by denaturing PAGE. Pol II pausing regions that are positively or negatively affected by FACT are indicated by green and red dotted lines, respectively. Blue and black asterisks indicate DNA-specific pause and labeled DNA fragments, respectively. nc, no chase. (C) Quantitation of the run-off transcripts (Fig. 1B). Averages from three experiments and SDs are shown. (D) The promoter-distal (D) dimer is required for FACT-dependent transcription through the +(35–48) region. (E) The proposed mechanism of FACT-dependent relief of nucleosomal +(35–48) pausing.

Fig. 2.

Disruption of dimer–tetramer interface is not required for FACT action. (A) Contacting residues in the dimer–tetramer interfaces of the core histone octamer. H2A, H2B, H3, and H4 are shown in yellow, red, blue and green, respectively. (B) 147-bp 601 DNA fragments were assembled into nucleosomes using WT and mutant (H3-I51N, H3-I51A, or H4-Y98H) histones. A point mutant of H2B (H2BT112C) was labeled with Alexa Fluor 488. Nucleosomes were run on 5% native PAGE, scanned on a Typhoon Imager at a wavelength of 520 nm (H2A/H2B fluorescence), and then stained with ethidium bromide (for DNA) or Imperial stain (for protein). (C) Effect of mutations disrupting the dimer–tetramer interface on transcription through chromatin in the absence or presence of FACT, as shown by analysis of pulse-labeled RNA by denaturing PAGE. (D) Effect of cross-linking of histone octamer by DMS on transcription through the nucleosome in the absence or presence of FACT, as shown by analysis of pulse-labeled RNA by denaturing PAGE. (E) The effect of FACT on transcription of intact and cross-linked nucleosomes. Averages from three experiments and SDs are shown.

Fig. 3.

FACT facilitates formation of productive intranucleosomal Pol II complexes. (A) 603 nucleosomes were transcribed by Pol II at 150 mM KCl in the absence or presence of FACT for indicated time intervals, followed by analysis of pulse-labeled RNA by denaturing PAGE. The intranucleosomal pauses (from A to K) were quantified using a Cyclone Phosphor Imager (PerkinElmer). (B) The quantified data were analyzed using an elongation model that produces a good fit of the experimental data to the calculated curves (Fig. S10). (C) KinTek Kinetic Explorer software (25) was used to determine the rate constants of each step of transcription through the nucleosome. Averages from three experiments are shown. The rate constants positively affected by FACT by more than sevenfold are shown in green. Expected complexes formed at each region (15) are shown.

Fig. 4.

FACT prevents histone octamer displacement during Pol II transcription. (A) During single-round transcription without FACT, ∼50% of templates lose histone octamer (pathway 1 → 2), likely owing to displacement of promoter-proximal H2A/H2B dimer (P-dimer) by Pol II (15). FACT–P-dimer interaction likely stabilizes the dimer–tetramer interaction and prevents loss of the octamer (pathway 1 → 2′). (B) The presence of FACT reduces the amount of histone-free DNA produced during transcription by Pol II. DNA-labeled 603 nucleosomes were transcribed at different KCl concentrations in the absence or presence of FACT, followed by sample analysis by native PAGE. (C) The amounts of histone-free DNA (presented as a fraction of transcribed templates) produced after transcription.

Fig. 5.

Proposed mechanism of FACT action during transcription by Pol II. (A) Pol II transcription through the nucleosome (complexes 1–4) (15) is accompanied by transient partial uncoiling of nucleosomal DNA from the octamer. (B) Proposed mechanism of FACT action during moderate-level transcription of genes; see the text for details.