FACT and the Proteasome Promote Promoter Chromatin Disassembly and Transcriptional Initiation

Abstract

The packaging of the eukaryotic genome into chromatin represses gene expression by blocking access of the general transcription machinery to the underlying DNA sequences. Accordingly, eukaryotes have developed a variety of mechanisms to disrupt, alter, or disassemble nucleosomes from promoter regions and open reading frames to allow transcription to occur. Although we know that chromatin disassembly from the yeast PHO5 promoter is triggered by the Pho4 activator, the mechanism is far from clear. Here we show that the Pho4 activator can occupy its nucleosome-bound DNA binding site within the PHO5 promoter. In contrast to the role of Saccharomyces cerevisiae FACT (facilitates chromatin transcription) complex in assembling chromatin within open reading frames, we find that FACT is involved in the disassembly of histones H2A/H2B from the PHO5 promoter during transcriptional induction. We have also discovered that the proteasome is required for efficient chromatin disassembly and transcriptional induction from the PHO5 promoter. Mutants of the degradation function of the proteasome have a defect in recruitment of the Pho4 activator, whereas mutants of the ATPase cap of the proteasome do recruit Pho4 but are still delayed for chromatin assembly. Finally, we rule out the possibility that the proteasome or ATPase cap is driving chromatin disassembly via a potential ATP-dependent chromatin remodeling activity.Eukaryotic chromatin is made up of a fundamental repeating unit, termed the nucleosome, which consists of 147 bp of DNA wrapped around the outside of an octamer of histone proteins (1). The histone octamer in turn comprises a heterotetramer of histone proteins H3/H4 and two heterodimers of histones H2A/H2B. In order to allow the transcription machinery to gain access to the DNA, the chromatin structure is altered by the concerted action of three processes (2): (i) post-translational modifications on the histones, (ii) the breakage of histone-DNA contacts by ATP-dependent chromatin remodeling machines, and (iii) the ultimate removal of histones from the DNA by histone chaperones. The sequence-specific transcriptional activators trigger these chromatin alterations occurring at promoters during transcriptional induction, but it is unclear whether transcriptional activators can first bind to their nucleosome-buried DNA recognition sequences to start this cascade of chromatin dynamics. Similarly, once transcription is complete, transcriptional activators leave the DNA, and promoters are repackaged with histones via the process of chromatin assembly.The assembly and disassembly of nucleosomes appear to occur in a stepwise manner (3). This is due to the peripheral positions of the H2A/H2B dimers within the nucleosome (1), necessitating the removal of H2A/H2B prior to removal of the central H3/H4 tetramer. Conversely, H3/H4 must be deposited onto the DNA prior to H2A/H2B in order to assemble a nucleosome. Accordingly, histone chaperones exist that bind to either H2A/H2B or H3/H4 to mediate chromatin assembly and disassembly...

High mobility group protein-mediated transcription requires DNA damage marker γ-H2AX

“…In addition, displacement of H1 during the initial steps of gene activation has been reported [32]. Furthermore, the histone chaperone complex FACT (facilitates chromatin transcription) catalyzes integration of H2AX into nucleosomes [33], induces disassembly of promoter-associated nucleosomes, and enhances transcriptional activation [34]. Interestingly, yeast FACT is a heterodimeric protein complex of Spt16 and Pob3 that interacts and functionally cooperates with the HMG1-like protein Nhp6 [35].…”

The FACT complex interacts with the E3 ubiquitin ligase Psh1 to prevent ectopic localization of CENP-A

“…Moreover, CENP-A mislocalizes to promoter regions that have high histone turnover (Krassovsky et al 2012;Lacoste et al 2014), and FACT can evict histones at promoters (Ransom et al 2009;Takahata et al 2009). In the future, it will be important to determine whether polymerases or other factors exist in the Psh1-associated FACT complex to couple CENP-A Cse4 degradation to chromatin-based processes.…”

Disassembly of synthetic Agrobacterium T-DNA–protein complexes via the host SCF VBF ubiquitin–ligase complex pathway

“…Furthermore, it is tempting to speculate that, once at the chromatin, the SCF VBF pathway can expose not only the T-DNA by degrading VirE2 attached to VIP1, but also the target host DNA by degrading histone molecules to which VIP1 also attaches (21,22). Thus, disassembly of the T-complex via the host UPS may represent a more general mechanism for uncoating of DNA molecules within eukaryotic cells; for example, proteasomal degradation of H2A/ H2B histones from promoter regions has been shown to promote chromatin disassembly during transcriptional activation (39).…”