Transcription Promotes the Interaction of the FAcilitates Chromatin Transactions (FACT) Complex with Nucleosomes inSaccharomyces cerevisiae

Abstract

The FACT (FAcilitates Chromatin Transactions) complex is a conserved complex that maintains chromatin structure on transcriptionally active genes. Consistent with this, FACT is enriched on highly expressed genes, but how it is targeted to these regions is unknown. In vitro, FACT binds destabilized nucleosomes, supporting the hypothesis that FACT is targeted to transcribed chromatin through recognition of RNA polymerase (RNAP)-disrupted nucleosomes. In this study, we used high-resolution analysis of FACT occupancy in Saccharomyces cerevisiae to test this hypothesis. We demonstrate that FACT interacts with nucleosomes in vivo and that its interaction with chromatin is dependent on transcription by any of the three RNAPs. Deep sequencing of micrococcal nucleaseresistant fragments shows that FACT-bound nucleosomes exhibit differing nuclease sensitivity compared to bulk chromatin, consistent with a modified nucleosome structure being the preferred ligand for this complex. Interestingly, a subset of FACT-bound nucleosomes may be "overlapping dinucleosomes," in which one histone octamer invades the 147-bp territory normally occupied by the adjacent nucleosome. While the differing nuclease sensitivity of FACT-bound nucleosomes could also be explained by the demonstrated ability of FACT to alter nucleosome structure, transcription inhibition restores nuclease resistance, suggesting that it is not due to FACT interaction alone. Collectively, these results are consistent with a model in which FACT is targeted to transcribed genes through preferential interaction with RNAP-disrupted nucleosomes.

Prevention of chromatin destabilization by FACT is crucial for malignant transformation

“…Our current model of FACT function is consistent with published structural studies that showed that mammalian FACT could only bind nucleosomal components or partially disassembled nucleosomes (e.g., lacking the H2A/H2B dimer) because the FACT binding epitopes are hidden within the folded nucleosome (Tsunaka et al 2016;Safina et al 2017;Wang et al 2018). It is also consistent with studies in yeast that showed that FACT is targeted to transcribed chromatin through its recognition of RNA polymerase-disrupted nucleosomes (Martin et al 2018) If the above model is correct, why is transcription increased following the loss of FACT? There are two potential explanations for this phenomenon, which will require further investigation.…”

Establishment and Maintenance of Chromatin Architecture Are Promoted Independently of Transcription by the Histone Chaperone FACT and H3-K56 Acetylation in Saccharomyces cerevisiae

“…Initial models suggested that FACT's ability to destabilize nucleosomes is required to support transcription elongation. However, FACT mutations do not appear to affect the rate of elongation in vivo (Mason and Struhl 2005;Biswas et al 2006;Kuryan et al 2012), and recent results show that FACT localizes to transcription units primarily as a response to exposure of its binding sites by ongoing transcription (Martin et al 2018). In this emerging view, FACT has a more prominent role in recovering from chromatin disruptions caused by transcription than in supporting the progression of transcription.…”

“…However, attempts to detect the predicted decrease in the rate of RNA Pol II elongation in FACT mutants have had mixed results, with some reporting indirect effects (Fleming et al 2008) but others finding no changes (Mason and Struhl 2005;Biswas et al 2006;Kuryan et al 2012). Recent work indicates that FACT occupancy is driven by exposure of its binding sites as nucleosomes are disrupted by transcription, suggesting that FACT responds to transcription rather than facilitating it (Martin et al 2018). If FACT or H3-K56ac promote polymerase progression, mutations could affect initiation (the ability to convert an initiation complex to an elongation complex by overcoming the initial barrier posed by the +1 nucleosome) or elongation (the ability to progress efficiently through each successive nucleosome encountered throughout transcription units).…”

“…Consistent with a role in transcription, FACT occupancy is generally proportional to the level of transcription (Mason and Struhl 2003;Pelechano et al 2009;Mayer et al 2010;Feng et al 2016). However, this occupancy profile can also be explained by a more passive model in which FACT localization is driven by exposure of its binding sites during any process that disrupts nucleosomes, including transcription (Kemble et al 2015;Tsunaka et al 2016;Martin et al 2018;Wang et al 2018). In this model, FACT's role is to rescue disrupted nucleosomes by reversing the reorganization pathway.…”

The nucleosome acidic patch directly interacts with subunits of the Paf1 and FACT complexes and controls chromatin architecture in vivo

“…Although no current crystal structures of Spt16 and histones show an interaction between Spt16 and the nucleosome acidic patch, it is likely that FACT recognition of the nucleosome is highly dynamic in vivo and interactions between Spt16 and the acidic patch may primarily occur in the context of active transcription. Indeed, a recent study found that transcription-disrupted nucleosomes enhance FACT binding to chromatin (89).…”