RNAi-Independent Heterochromatin Nucleation by the Stress-Activated ATF/CREB Family Proteins

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Science  25 Jun 2004:
Vol. 304, Issue 5679, pp. 1971-1976
DOI: 10.1126/science.1099035


At the silent mating-type interval of fission yeast, the RNA interference (RNAi) machinery cooperates with cenH, a DNA element homologous to centromeric repeats, to initiate heterochromatin formation. However, in RNAi mutants, heterochromatin assembly can still occur at low efficiency. Here, we report that Atf1 and Pcr1, two ATF/CREB family proteins, act in a parallel mechanism to the RNAi pathway for heterochromatin nucleation. Deletion of atf1 or pcr1 alone has little effect on silencing at the mating-type region, but when combined with RNAi mutants, double mutants fail to nucleate heterochromatin assembly. Moreover, deletion of atf1 or pcr1 in combination with cenH deletion causes loss of silencing and heterochromatin formation. Furthermore, Atf1 and Pcr1 bind to the mating-type region and target histone H3 lysine-9 methylation and the Swi6 protein essential for heterochromatin assembly. These analyses link ATF/CREB family proteins, involved in cellular response to environmental stresses, to nucleation of constitutive heterochromatin.

Heterochromatin governs diverse processes ranging from gene regulation and chromosome segregation to suppression of deleterious recombination in repetitive sequences. In the fission yeast Schizosaccharomyces pombe, heterochromatin is present mainly at the centromeres, telomeres, and a 20-kb silent domain at the mating-type region, the loci that are preferentially enriched in histone H3 lysine-9 (H3-K9) methylation and the Swi6 protein (a homolog of mammalian HP1 proteins) (1). The mechanisms that define these chromosomal regions as preferred sites of heterochromatin formation are not fully understood. Recent studies have implicated RNAi pathway in targeting of heterochromatin to repetitive DNA sequences in S. pombe and in other organisms including Tetrahymena, Arabidopsis, and Drosophila (27). It has been demonstrated that deletions of factors involved in the RNAi pathway such as Dicer (dcr1), RNA-dependent RNA polymerase (rdp1), and Argonaute (ago1) disrupt heterochromatin assembly at centromeres (4). Moreover, cenH sequence [96% similar to dg and dh centromeric repeats (8)] that is present at the silent mating-type (mat2/3) region (see Fig. 1A) serves as an RNAi-dependent heterochromatin nucleation center at the endogenous locus and at an ectopic location (2). Although RNAi machinery cooperates with cenH to initiate heterochromatin formation at the mat2/3 region, the RNAi pathway is dispensable for the subsequent maintenance of heterochromatin at this locus (2). Moreover, deletion of cenH causes specific defects in the establishment of the silenced state. In strains where cenH is replaced with the ura4+ reporter gene (KΔ::ura4+), cells carrying an ura4-on epigenetic state switch to the ura4-off epigenetic state inefficiently; however, once established, the ura4-off state is stably inherited and rarely switches back to the ura4-on state (8). This establishment of the silenced state is coupled to H3-K9 methylation and Swi6 recruitment at the mat2/3 region (9). Furthermore, Swi6 overexpression can efficiently convert the ura4-on state to the ura4-off state, which suggests that Swi6 can cooperate with elements outside of cenH to nucleate heterochromatin assembly independent of RNAi.

Fig. 1.

Atf1 is required for heterochromatin formation at the silent mating-type region. (A) A schematic diagram of the mating-type region. Black arrows represent IR-L and IR-R boundary elements. Shaded box denotes cenH sequence and striped box represents REIII sequence. Arrowheads indicate binding sites for Atf1/Pcr1. (B) Δatf1 in combination with RNAi mutants affect maintenance of heterochromatin at Kint2::ura4+. Serial dilution plating assays in the presence and absence of FOA were performed to measure Kint2::ura4+ expression. Levels of Swi6 and H3-K9 methylation at Kint2::ura4+ were determined by ChIP. DNA isolated from immunoprecipitated chromatin fractions or from whole-cell extract (WCE) was quantitatively analyzed by competitive polymerase chain reaction (PCR), in which one primer pair amplifies differentsized PCR products from the Kint2::ura4+ and the control ura4DS/E minigene at the endogenous location. The ratios of intensities of ura4+ to ura4DS/E signals in the ChIP and WCE lanes were used to calculate the relative fold enrichment, shown below each lane. (C) Δatf1 in combination with RNAi mutants completely abolished the establishment of heterochromatin. Cells were grown in medium containing 35 μg/ml TSA for 10 generations and were allowed to recover in the absence of TSA for an additional 10 generations. Expression of Kint2::ura4+ and levels of Swi6 and H3-K9 methylation at Kint2::ura4+ after recovery from TSA treatment are shown.

We hypothesized that the RNAi-independent pathway of heterochromatin nucleation might involve factors that localize to specific site(s) within the mat2/3 region and could act upstream of H3-K9 methylation and Swi6 localization. In a swi6 mutant, H3-K9 methylation is asymmetrically enriched around the cenH repeat with more methylation on the mat3 side of the cenH locus (2), which suggests that unknown factors binding between cenH and mat3 may function to recruit heterochromatin. Analysis of a 2.1-kb region between cenH and the mat3 loci revealed two heptamer sequences ATGACGT, a well-characterized binding site for the transcription factor Atf1/Pcr1 (10). One of these sites overlaps with a silencer element involved in Swi6-independent local repression of the mat3 locus (10).

Both Atf1 and Pcr1 contain a bZIP DNA binding domain that shares strong homology with the activating transcription factor/cAMP response element–binding protein (ATF/CREB) family of proteins and they regulate gene expression during sexual differentiation and environmental stress (1113). To determine whether Atf1 and Pcr1 are involved in heterochromatin formation at the silent mating-type region, we generated atf1 and pcr1 deletion strains carrying a Kint2::ura4+ reporter (see Fig. 1A). Deletion of either of these factors had little effect on the maintenance of heterochromatin at this region (Figs. 1B and 2A). However, when aft1 or pcr1 deletion was combined with RNAi mutants, we observed a striking decrease in the silencing of the ura4+ reporter, which correlated with significant reduction in histone H3-K9 methylation levels and Swi6 localization (Figs. 1B and 2A). We also investigated the effect of these mutations on the initial establishment of heterochromatin at the mat2/3 region. Treatment of cells with the histone deacetylase inhibitor trichostatin A (TSA) is known to derepress the mating-type region; however, silencing is efficiently reestablished once cells are allowed to recover in the absence of the drug (2). In contrast, in ago1, dcr1, or rdp1 mutants, reestablishment of the silent state after TSA treatment is impaired, although heterochromatin formation still occurred at a slower rate (Figs. 1C and 2B) (2). When deletion of RNAi components was combined with either Δatf1 or Δpcr1, double mutants strikingly failed to reestablish silencing, which was coupled with complete loss of H3-K9 methylation and the Swi6 protein at the mat locus (Figs. 1C and 2B). The residual silencing observed in double mutants before TSA treatment could reflect the ability of heterochromatin complexes to promote their own reassembly in the absence of de novo nucleation (9). Taken together, these results demonstrate that Atf1/Pcr1 and RNAi machinery act in parallel pathways to nucleate heterochromatin formation at the mat2/3 region.

Fig. 2.

Pcr1 is required for heterochromatin formation at the silent mating-type region. (A) Δpcr1 in combination with RNAi mutants affect heterochromatin at Kint2::ura4+. Expression of ura4+ and levels of Swi6 and H3-K9 methylation at Kint2::ura4+ are shown. (B) Δpcr1 in combination with RNAi mutants completely abolished the establishment of heterochromatin. Cells were treated with TSA as described in Fig. 1C. Expression of ura4+ and levels of Swi6 and H3-K9 methylation at Kint2::ura4+ after recovery from TSA treatment are shown.

In support of these findings, deletion of either atf1 or pcr1, but not RNAi mutants, in combination with cenH deletion (Fig. 3A) resulted in loss of silencing and heterochromatin assembly at the mat2/3 locus. As shown in Fig. 3B (top), deletion of aft1 or pcr1 in the KΔ::ura4+ strain resulted in the efficient conversion of the ura4-off state to the ura4-on state. This change was accompanied by loss of H3-K9 methylation and Swi6 localization at the mat locus (Fig. 3C). Moreover, Δatf1 or Δpcr1 inhibited the stochastic conversion of the ura4-on state to the ura4-off state (Fig. 3B, middle). In contrast, RNAi mutants combined with KΔ::ura4+ ura4-off epiallele had no effect on silencing, H3-K9 methylation, or Swi6 localization at the mat2/3 region (Fig. 3, D and E). Therefore, we conclude that cenH and RNAi machinery function in the same pathway to silence the mat locus. Cells that lack this heterochromatin assembly pathway rely completely on a second pathway mediated by Atf1/Pcr1.

Fig. 3.

atf1 and pcr1 act in a redundant manner as the RNAi pathway to silence the mat2/3 region. (A) Schematic diagram of the mat locus in KΔ::ura4+ strain carrying the replacement of the cenH with ura4+. (B) KΔ::ura4+ marker showing the ura4-off or the ura4-on state was combined with Δatf1 or Δpcr1 and serial dilution plating assay was used to measure ura4+ expression. (C) Levels of Swi6 and H3-K9 methylation at KΔ::ura4+ were determined by ChIP. (D) KΔ::ura4+ura4-off epiallele was combined with mutations in RNAi components and expression of ura4+ was measured using serial dilution plating assay. (E) Levels of Swi6 and H3-K9 methylation at KΔ::ura4+ determined by ChIP.

To investigate whether Swi6-induced establishment of silencing in the absence of cenH (9) requires Atf1/Pcr1, KΔ::ura4+ ura4-on cells were transformed with a plasmid carrying the swi6+ gene. Unlike wild-type background cells, Swi6 overexpression in Δatf1 or Δpcr1 strains failed to convert the ura4-on to the ura4-off state (Fig. 3B, bottom) and no detectable increases in H3-K9 methylation or the Swi6 protein were observed at the mat locus (Fig. 3C). These data demonstrate that in the absence of RNAi, Atf1 and Pcr1 are indispensable for heterochromatin nucleation at the mat2/3 region and that they may participate in the creation of a second nucleation site for heterochromatin formation. To test this idea, a strain carrying deletion of one of the Atf1/Pcr1-binding sites (ΔREIII) at the mat2/3 region was constructed together with a Kint2::ura4+ reporter (Fig. 1A) (10). As expected, deletions of RNAi components in combination with ΔREIII greatly reduced establishment of silencing of Kint2::ura4+ after TSA treatment (Fig. 4A), concurrent with dramatic reduction of H3-K9 methylation and the Swi6 protein at the mat2/3 region when compared with either single mutants or a wild-type strain (Fig. 4A).

Fig. 4.

REIII sequence containing Atf1/Pcr1-binding site is required for heterochromatin formation at the mat2/3 region. (A) Effect of ΔREIII on heterochromatin nucleation in combination with RNAi mutants. Cells were treated with TSA as in Fig. 1C. Expression of Kint2::ura4+ and levels of Swi6 and H3-K9 methylation at Kint2::ura4+ after recovery from TSA treatment are shown. (B) High-resolution mapping of Atf1 and Pcr1 at the mating-type region. Strains expressing Atf1 and Pcr1 tagged with 3× FLAG (the DYDDDK epitope) were used to perform ChIP with FLAG-specific antibody. DNA isolated from ChIP or WCE fractions was subjected to multiplex PCR to amplify DNA fragments from the mat locus (indicated below the scheme at the top), as well as an act1 control fragment. Asterisks indicate DNA fragments that contain Atf1/Pcr1-binding sites. (C) Atf1 and Pcr1 interact with Swi6 and Clr4. In vitro translated and 35S-labeled luciferase (Luc), Atf1, and Pcr1 were incubated with equal amounts of glutathione S-transferase (GST), GST-Swi6, or GST-Clr4 immobilized on glutathione beads and washed extensively. The eluted proteins were resolved by SDS–polyacrylamide gel electrophoresis and imaged by autoradiography.

We next tested whether Atf1 and Pcr1 bind directly to their putative binding sites in vivo. Indeed, mapping of Atf1 and Pcr1 by chromatin immunoprecipitation (ChIP) revealed that both proteins specifically localize to regions containing their binding sites and that no binding was detected at surrounding mat2/3 sequences (Fig. 4B). Note that the localization of Atf1/Pcr1 to the mat locus was not dependent on the Swi6 protein (Fig. 4B). These findings suggest that Atf1/Pcr1 function upstream of Swi6 and could directly recruit heterochromatin machinery to the mating-type region.

Collectively, these data indicate that binding of Atf1/Pcr1 to their recognition sequences in the mating-type region creates a nucleation site for constitutive heterochromatic structures. To explore the mechanism of Atf1/Pcr1-mediated heterochromatin formation, we examined whether Atf1/Pcr1 could directly recruit factors involved in heterochromatin formation. We found that Atf1 or Pcr1 bind to the Swi6 and Clr4 proteins (Fig. 4C), consistent with our observation that Swi6-induced establishment of silencing requires Atf1/Pcr1 (Fig. 3B). This mechanism may resemble the recruitment of facultative heterochromatin by KRAB/KAP-1 or retinoblastoma (Rb) protein in mammals, which can recruit histone modifying activities and heterochromatin proteins such as HP1 to silence specific genes located in otherwise euchromatic territories (1416). It should be noted however that, unlike Rb or KRAB/KAP-1, Atf1/Pcr1-mediated constitutive heterochromatin spreads throughout the silent mating-type interval and this causes regional silencing.

The involvement of factors required for the cellular response to environmental stress in heterochromatin assembly suggests the possibility that these factors might modify chromatin structure as a part of a programmed sequence of events that serves to cushion against the effects of environmental stresses (17). In this respect, it is notable that Atf1 activity is regulated by Sty1/Spc1 protein kinases in response to environmental stress (18), and defects in heterochromatin assembly under elevated temperature conditions have been observed in different systems (1922). In mammals, heat shock can also trigger the loss of heterochromatin structures at pericentric regions and elevates transcription of embedded satellite III repeat sequences (23). Considering the importance of heterochromatin in diverse cellular functions such as gene silencing, genome stability, and chromosome segregation, it is not surprising that cells have evolved multiple pathways of heterochromatin assembly.

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