Transcriptional activation of HO involves sequential changes in chromatin structure along the promoter during the cell cycle. Nucleosome displacement at HO occurs stepwise, first at URS1, then in sequence at the left end of URS2, the right end of URS2, and finally at the TATA region when the gene is transcribed (). Concomitant with this chromatin disassembly, acetylation of the residual nucleosomes also occurs in waves along the promoter (). ChIP experiments show there is sequential recruitment of three coactivator complexes, Swi/Snf, SAGA, and Mediator, first to URS1 and subsequently to URS2 (). Importantly, these are dependent changes: the nucleosome eviction and coactivator binding must occur first at URS1 before chromatin changes and coactivator binding can occur at URS2 later in the cell cycle. Finally, two chromatin factors that have not been previously described as binding to upstream promoter elements, FACT and Asf1, are specifically required for the chromatin disassembly and coactivator binding events at URS2 (). FACT binding to URS2 occurs before Asf1 binding, and FACT and Asf1 are each required for nucleosome eviction in distinct regions of URS2 ().
Model of events at the HO promoter during the cell cycle
The Swi5 DNA-binding protein is required for all subsequent changes at the HO
promoter, and thus can be considered a “pioneer” factor (Cirillo et al., 2002
). Swi5 is cell cycle regulated, entering the nucleus after anaphase (Moll et al., 1991
). Swi5 interacts directly with the Swi/Snf, SAGA, and Mediator coactivators, and recruits them to the URS1 region of the HO
promoter. Swi5 is rapidly degraded by ubiquitin mediated proteolysis (Kishi et al., 2008
), and binding of the three coactivators quickly disappears. There are two Swi5 binding sites at URS1, separated by 500 bp. Both binding sites are required for HO
activation, and experiments suggest an interaction between the two sites (McBride et al., 1997
). Swi5 binds more strongly in vitro to Site B at −1300 (McBride et al., 1997
), and ChIP experiments also show stronger Swi5 binding to Site B in vivo (Suppl Fig S4
). Swi/Snf and SAGA bind to both Site A and Site B within URS1, with stronger binding to Site B; Mediator appears to bind only to Site B. We suggest that there is an interaction between the Swi5 molecules bound to these two sites, and that it is the interaction of Swi5 and coactivators bound to the two sites that leads to eviction of the nucleosomes in between.
The three coactivators are recruited to URS1 by Swi5, and 10–15 min later they are present at the URS2 region of the promoter, which we call coactivator re-recruitment (). FACT and Asf1 are required for this recruitment of coactivators to URS2, and FACT and Asf1 both bind to URS2 before the peak of coactivator binding here (). We think of URS1 as the “staging area,” where coactivators are first recruited. Subsequently, the coactivators move from the staging area to the URS2 region where the “traditional” activation occurs. This two part activation scheme allows greater stringency of control over HO expression by imposing additional requirements before activation of expression. The requirement for FACT and Asf1 for coactivator re-recruitment suggests that mobilization of coactivators from the staging area to the activation site is blocked by an inhibitory chromatin structure.
Previous work showed FACT binding to transcribed open reading frames, but not to upstream promoter elements (Mason and Struhl, 2003
; Saunders et al., 2003
), with FACT recruited by ubiquitylated H2B (Fleming et al., 2008
). FACT had not been previously shown to interact with sequence-specific DNA-binding proteins, but here we show that FACT interacts with SBF to facilitate binding to HO
(). At the HO
URS2 promoter region SBF and FACT are both required for nucleosome eviction and coactivator binding. Importantly, FACT binding precedes the nucleosome eviction which precedes coactivator binding.
Disassembly of chromatin can occur at promoters when a gene is transcriptionally activated (Boeger et al., 2003
; Reinke and Horz, 2003
; Workman, 2006
). Nucleosome displacement at certain promoters requires Asf1 (Adkins et al., 2004
), Gcn5 (Verdone et al., 2002
), or Swi/Snf (Biddick et al., 2008
; Schwabish and Struhl, 2007
). Nucleosome eviction is often accompanied by histone acetylation (Boeger et al., 2003
; Erkina and Erkine, 2006
; Zhao et al., 2005
), as we observe at HO
. Remarkably, nucleosome eviction at HO
occurs stepwise during the cell cycle, moving across the promoter (). Swi/Snf is required for efficient nucleosome eviction at URS1, as there is only weak and transient nucleosome loss in the swi2
mutant (). This residual nucleosome eviction may be facilitated by SAGA or Mediator; the swi2
mutation sharply reduces, but does not completely eliminate, binding of these coactivators. This histone eviction at URS1 at 20 min is followed by repopulation of nucleosomes over the next 20–40 min (). Interestingly, this histone loss at URS1 persists in a number of the mutants, including gcn5
(). The mechanism underlying this defect in nucleosome repopulation at URS1 is not clear; the gcn5
, and asf1
mutants are defective in nucleosome loss at URS2 while the gal11
mutant is not.
In contrast to URS1, chromatin disassembly at URS2 occurs before the coactivators bind to this region. Nucleosome loss here requires FACT and Asf1, but these factors are differentially required at two regions of URS2 (). Chromatin disassembly at the left end of URS2 requires FACT but not Asf1, and FACT binds primarily to this part of URS2 (Suppl Fig S5A
) shortly before the chromatin disassembly. Asf1 binds later to URS2 (), primarily at the right end of URS2 (Suppl Fig S5B
) when the Asf1-dependent nucleosome eviction occurs here. FACT and Swi/Snf may contribute to this chromatin disassembly, but both factors are required for Asf1 binding to HO
. Importantly, FACT and Asf1 are not required for nucleosome displacement at URS1, and we therefore conclude that chromatin disassembly at URS1 and the two regions of URS2 are all mechanistically different. The Asf1 histone chaperone may receive the H3-H4 tetramers evicted from URS1 or the left end of URS2, but stable Asf1 binding is only detected by ChIP slightly later in the cell cycle. Finally, the absence of RNA polymerase II at URS1 or URS2 at any time during the cell cycle () eliminates the possibility that nucleosome eviction or coactivator recruitment are due to either poised polymerase or expression of a noncoding transcript.
The yeast HO gene encodes an endonuclease that initiates mating type switching. Inappropriate expression of an endonuclease could be disastrous in natural populations, and HO expression is therefore tightly regulated. Multiple activators and coactivators are required to overcome the repression at HO. Here we show that different factors are required to evict nucleosomes from different regions of the HO promoter. Rather than being uniform, the chromatin of the HO promoter appears to contain a series of nucleosomes with individual properties, much like a series of padlocks that each requires a different key and that must be unlocked in a defined order.