DNA sequence-dependent epigenetic inheritance of gene silencing and histone H3K9 methylation

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Science  07 Apr 2017:
Vol. 356, Issue 6333, pp. 88-91
DOI: 10.1126/science.aaj2114

DNA sequence and inherited gene silencing

Cell fate decisions require a gene's transcriptional status, whether on or off, to be stably and heritably maintained over multiple cell generations. For silenced genes, heterochromatin domains are associated with specific histone posttranslational modifications, and these histone marks are maintained during DNA replication and chromosome duplication (see the Perspective by De and Kassis). Laprell et al. show that parental methylated histone H3 lysine 27 (H3K27) nucleosomes in Drosophila are inherited in daughter cells after replication and can repress transcription, but that they are not sufficient to propagate the mark. Trimethylation of newly incorporated nucleosomes requires recruitment of the methyltransferase Polycomb repressive complex 2 (PRC2) to neighboring cis-regulatory DNA elements. Coleman and Struhl demonstrate that H3K27 trimethylated nucleosomes play a causal role in transmitting epigenetic memory at a Drosophila HOX gene through anchoring of PRC2 at the Polycomb response element binding site. Wang and Moazed examine fission yeast and show that both sequence-dependent and chromodomain sequence-independent mechanisms are required for stable epigenetic inheritance of histone modifications and the epigenetic maintenance of silencing. These studies highlight the crucial role of DNA binding for heritable gene silencing during growth and development.

Science, this issue p. 85, p. eaai8236, p. 88; see also p. 28


Epigenetic inheritance mechanisms play fundamental roles in maintaining cellular memory of gene expression states. In fission yeast, histone H3 lysine 9 (H3K9) is methylated (H3K9me) at heterochromatic domains. These domains can be epigenetically inherited when epe1+, encoding an enzyme that promotes H3K9 demethylation, is deleted. How native epigenetic states are stably maintained in epe1+ cells remains unknown. Here, we developed a system to examine the role of DNA sequence and genomic context in propagation of a cis-heritable H3K9me-dependent silenced state. We show that in epe1+ cells, in addition to sequence-independent mechanisms that propagate H3K9me, epigenetic inheritance of silencing requires binding sites for sequence-dependent activating transcription factor (ATF)–adenosine 3′,5′-monophosphate (cAMP) response element–binding protein (CREB) family transcription factors within their native chromosomal context. Thus, specific DNA sequences contribute to cis inheritance of H3K9me and silent epigenetic states.

Large regions of eukaryotic DNA are packaged into heterochromatin, which is inaccessible to RNA polymerases and that results in repressed or “silenced” genes (1). Heterochromatin also plays a central role in the maintenance of genome stability by inhibiting transposons and recombination between repeated DNA elements (1). The formation of heterochromatic domains is associated with specific histone posttranslational modifications, and the transmission of these histone marks during cell division is thought to occur by sequence-independent cis copying mechanisms involving the transfer of parental histones to newly synthesized daughter DNA strands during DNA replication. Recognition of the parental histone marks by enzymes or complexes that catalyze the same modification on newly deposited histones then results in restoration of the epigenetic state (25). In support of this hypothesis, several groups have recently demonstrated that sequence-dependent establishment of heterochromatin can be uncoupled from its subsequent epigenetic maintenance (69). However, heterochromatin formation and maintenance also often require a continuous input from specificity factors, such as site-specific DNA binding proteins, which recruit histone-modifying complexes to target specific chromosome regions (1013).

The fission yeast, Schizosaccharomyces pombe, contains extensive domains of heterochromatin at its pericentromeric, telomeric, and mating-type chromosome regions (14). These domains share key features with heterochromatin in multicellular organisms, including (i) histone 3 lysine 9 (H3K9) methylation, which is catalyzed by Clr4, the homolog of human SUV39H1 and SUV39H2 enzymes; (ii) association with HP1 proteins; (iii) perinuclear clustering; and (iv) epigenetic heritability (5, 14). Two distinct mechanisms, involving the RNA interference (RNAi) machinery and site-specific DNA binding proteins, recruit Clr4 and mediate heterochromatin establishment in S. pombe (1517). At the mating-type (mat) locus, in addition to RNAi, activating transcription factor (ATF)–adenosine 3′,5′-monophosphate (cAMP) response element–binding protein (CREB) family transcription factors, the Atf1-Pcr1 heterodimer, and its cognate binding sites are required for heterochromatin establishment (17). However, whether or not these pathways also participate in epigenetic maintenance of heterochromatin, which is transmitted in cis at both the mating-type and pericentromeric DNA regions (18, 19), has not yet been addressed.

Ectopic recruitment of the Clr4 H3K9 methyltransferase to a normally euchromatic region results in the formation of a silent domain of methylated H3K9 (H3K9me), which is unstable and rapidly decays unless epe1+, which encodes a JmjC domain demethylase family member, is deleted (6, 7). This is in contrast to the native heterochromatic domain at the mat locus in which replacing the cenH or K region with a reporter gene results in stable ON and OFF expression states of the reporter gene that can be transmitted in cis through meiosis (18). In order to gain insight into how epigenetic states are stably maintained under conditions of normal H3K9me turnover, we generated cells that allow inducible heterochromatin establishment at the mat locus by replacing the cenH region of the mat locus with a ura4+ reporter gene containing cognate binding sites for the bacterial TetR protein inserted upstream of the ura4+ gene (cenH::9xtetO-ura4+), as well as a DNA fragment encoding TetR-Clr4∆CD (referred to as TetR-Clr4-initiator or TetR-Clr4-I) inserted at the trp1+ locus (6) (Fig. 1A and fig. S1A). The binding of TetR-Clr4-I to the above reporter can be reversed by the addition of tetracycline (Tetoff). First, to confirm that the cenH::9xtetO-ura4+ strains displays a cis-inheritance phenotype in the absence of TetR-Clr4-I, we inserted different 30–base pair (bp) barcodes downstream of the 3′ untranslated region (3′ UTR) of the ura4+ reporter (-B1 and -B2) in order to be able to track the genotype of ura4+ expressed (ON) and silenced (OFF) epialleles by using allele-specific polymerase chain reaction (PCR) (Fig. 1, A and B). Consistent with previous results (18), after crosses between ON and OFF cells, diploid formation, and sporulation, the ON and OFF states were maintained and segregated faithfully to the meiotic progeny with the expected 2:2 ratio (Fig. 1C), and histone H3K9me2 at both ura4+ and adjacent mat sequences was specifically associated with the OFF alleles (Fig. 1, D and E).

Fig. 1 Construction of a modified mating-type (mat) locus that allows examination of the role of specific DNA sequences in maintenance of cis epigenetic states.

(A) Schematic diagram of the mat locus in which the cenH RNAi-dependent heterochromatin nucleation region is replaced with nine tetracycline operators located upstream of the ura4+ promoter and coding region (cenH::9xtetO-ura4+). s1 and s2 (red rectangles) denote the two 7-bp Atf1-Pcr1–binding sites. Black arrows indicate the location of the boundary sequences, inverted repeat left (IR-L) and inverted repeat right (IR-R). Barcodes of 30 bp were inserted downstream of ura4+ 3′UTR, (ura4+-B1) or (ura4+-B2) to allow identification by PCR. (B) Silencing assays showing that the expression states of a cenH:9xtetO-ura4+-B1 ON allele and a cenH::9xtetO-ura4+-B2 OFF allele. Ethidium bromide–stained gel on the right shows PCR-based genotyping of the meiotic progeny. N/S, nonselective medium; 5-FOA, 5-fluoroorotic acid–containing medium; and -Ura, medium lacking uracil. (C) (Top) Schematic diagram of the experimental design for crossing cenH::9xtetO-ura4+-B1 ON with cenH::9xtetO-ura4+-B2 OFF allele to test cis epigenetic inheritance of silencing and H3K9me. (Bottom) Silencing assays showing that the epigenetic expression states of a cenH::9xtetO-ura4+-B1 ON allele and a cenHΔ::9xtetO-ura4+-B2 OFF alleles can be inherited in cis after mating, diploid formation, and meiosis. (D and E) ChIP-qPCR assays showing H3K9me2 at ura4+ (D) and adjacent mat region (E) associated with the epigenetic OFF state in the meiotic progeny of the cross shown in (C). Error bars in (D) and (E) indicate SD from three biological replicates.

We used the above inducible system to determine the role of specific DNA sequences in epigenetic maintenance. We constructed a series of strains in which the entire region between the inverted repeat left (IR-L) and inverted repeat right (IR-R) boundary elements was deleted, or in addition to cenH, either mat3 and flanking regions or mat2 and flanking regions were deleted and replaced with 9xtetO-ura4+ (Fig. 2A). We then induced heterochromatin formation and silencing by introduction of TetR-Clr4-I into cells containing each of the above deletions (Fig. 2, A and B). As a control, we also introduced TetR-Clr4-I into cells in which the euchromatic ura4+ locus was modified by the insertion of nine upstream tetracycline operators (ura4::9xtetO-ura4+ cells) (fig. S1A). As expected, TetR-Clr4-I induced silencing at both the endogenous ura4+ and the mat locus as evidenced by robust growth on medium containing 5-fluoroorotic acid (FOA) and lacking tetracycline (FOA – tet) (Fig. 2B, rows 1 and 2, and fig. S1). However, upon release of TetR-Clr4-I by growth on tetracycline-containing medium, silencing was only maintained at the mat locus, which indicated that sequences within the mat locus contributed to epigenetic maintenance (Fig. 2B, row 2, cenH::9xtetO-ura4+, and fig. S1). Consistent with this observation, deletion of the entire region between the boundary elements weakened establishment of, but completely abolished, epigenetic maintenance (Fig. 2B, row 3, mat::9xtetO-ura4+). However, deletion of either the mat2-cenH or the cenH-mat3 regions allowed both robust establishment and maintenance of silencing, indicating that redundant sequences within the mat locus contributed to epigenetic maintenance (Fig. 2B, rows 4 and 5, cenH-mat3::9xtetO-ura4+ and mat2-cenH::9xtetO-ura4+). The mat locus contains two 7-bp Atf1-Pcr1–binding sites, s1 and s2, which are located to the left of mat3 and have previously been shown to be required for establishment of silencing (Fig. 2A) (17). To determine whether these binding sites play a role in epigenetic maintenance, we made precise deletions of each seven nucleotide–binding site in mat2-cenHΔ::9xtetO-ura4+ cells. Deletion of these binding sites dramatically reduced epigenetic maintenance, as indicated by loss of growth on FOA medium containing tetracycline (Fig. 2B, row 6, mat2-cenH::9xtetO-ura4+ s1s2∆). Consistent with the silencing assays, after release of TetR-Clr4-I by growth on tetracycline-containing medium (+tet), chromatin immunoprecipitation (ChIP) combined with quantitative PCR (qPCR) showed that H3K9me2 and H3K9me3 at ura4+ and adjacent mat sequences were only efficiently maintained at the locus that contained the Atf1-Pcr1–binding sites (Fig. 2, C and D, and fig. S2, A and B; compare s1+s2+ with s1s2∆). As a control, H3K9me levels at the centromeric dg repeats did not change in any of the above strains (Fig. 2E). Furthermore, ChIP-qPCR experiments showed that similar levels of Atf1-3XFlag were associated with the s1 and s2 sites of ura4+ ON and OFF alleles, as well as with a euchromatic target of Atf1-Pcr1 (SPCC320.03) (Fig. 2F and fig. S2C). These results indicate that, once heterochromatin is established at the mat2-cenH mat locus, its epigenetic inheritance requires the Atf1-Pcr1–binding sites, even though similar levels of Atf1-Pcr1 are bound in the ON and OFF states.

Fig. 2 Sequences within the mating-type locus and binding sites for the ATF-CREB transcription factors mediate cis inheritance of silencing and H3K9me.

(A) Schematic diagram of the mating-type (mat) locus and its modifications used for analysis of the role of Atf1-Pcr1–binding sites in cis epigenetic inheritance. (B) Silencing assays using cells with the indicated genotypes showing that deletion of specific sequences within the mating-type locus abolishes epigenetic inheritance of silencing upon TetR-Clr4-I release with tetracycline. (C and D) ChIP-qPCR assays showing Atf1-Pcr1–binding site-dependent maintenance of H3K9me3 upon release of TetR-Clr4-I. (E) ChIP-qPCR assays showing that tetracycline-mediated release of TetR-Clr4-I does not affect H3K9me3 at the centromeric dg repeats, cen-dg. (F) ChIP-qPCR assays showing that s1/s2 site-dependent binding of Atf1-Flag in both the ON and OFF states in cenH::9xtetO-ura4+ cells. Error bars in (C) to (F) indicate SD from three biological replicates.

We next investigated whether the Atf1-Pcr1–binding sites and surrounding DNA region were sufficient to confer epigenetic maintenance at an ectopic locus (fig. S3A). As shown previously for inheritance of ade6+ reporter gene silencing (6, 7), the silent state of ura4::9xtetO-ura4+ was lost after release of TetR-Clr4-I in epe1+ cells, although it could be epigenetically maintained in epe1Δ cells (fig. S3B, FOA+tet, rows 1 and 2). The insertion of the 4.6-kb mat3 DNA fragment, spanning the region between cenH and the IR-R boundary element and containing both Atf1- and Pcr1-binding sites, into the native ura4+ locus in epe1+ cells expressing the TetR-Clr4-I initiator (ura4::9xtetO-ura4+-mat3, s1+s2+, epe1+) resulted in some growth on FOA+tet medium, which was above the background observed in the parental cells lacking the 4.6-kb insertion (fig. S3B, FOA+tet, compare rows 3 and 1). This demonstrates that the mat3 DNA fragment containing the Atf1- and Pcr1-binding sites has weak maintenance activity when located outside the mating-type locus and suggests that additional features at the mat locus are likely to also play important roles. Consistent with this hypothesis, deletion of the IR-L and IR-R boundary elements resulted in weaker epigenetic maintenance of silencing at the mat2-cenH::9xtetO-ura4+ locus (fig. S3, C and D; compare rows 1 and 2). The degree of maintenance at the mat locus lacking the boundary elements was similar to that conferred by the 4.6-kb mat2 insertion at the ura4+ locus (fig. S3D, compare rows 2 and 3). Together these results strongly suggest that the mat region containing the s1 and s2 Atf1-Pcr1–binding sites has similar epigenetic maintenance activity at the mat and ectopic ura4+ loci and that strong epigenetic maintenance at the mat locus requires the Atf1-Pcr1–binding sites, as well as the boundary elements.

The chromodomain of Clr4 is required for epigenetic maintenance of ectopically induced heterochromatin in epe1∆ cells by a mechanism that involves chromodomain-mediated recognition of H3K9me (6, 7). To determine whether this mechanism also contributes to sequence-dependent epigenetic maintenance, we compared the maintenance of silencing and H3K9me in cenH::9xtetO-ura4+, tetR-clr4-I cells expressing either wild-type or chromodomain mutant Clr4 proteins. As shown in Fig. 3A, cells that expressed Clr4 with either a point mutation in the chromodomain (clr4W31G), which disrupts H3K9me recognition (20), or a chromodomain deletion (clr4CD), failed to grow on FOA+tet medium; this failure indicated an inability to maintain silencing after release of TetR-Clr4-I, which correlated with loss of H3K9 methylation (Fig. 3, B and C). Thus, both sequence-dependent and Clr4 chromodomain sequence-independent mechanisms are required for epigenetic maintenance of silencing at the mat locus. We next investigated whether deletion of epe1+ could circumvent the requirement for s1 and s2 sites in inheritance of silencing and H3K9me at the mat locus. We observed efficient maintenance of silencing (Fig. 3D, compare rows 2 and 3, FOA+tet) and H3K9me in epe1∆ cells that lack the Atf1-Pcr1–binding sites (Fig. 3, E and F). Deletion of epe1+ can therefore suppress the continuous requirement for Atf1-Pcr1-binding sites in epigenetic maintenance of silencing and H3K9me at the mat locus. Finally, we showed that transient expression of TetR-Clr4-I mediated stable epigenetic inheritance of silencing and H3K9me at the mat locus only when Atf1- and Pcr1-binding sites were present (fig. S4). This observation suggests that Atf1-Pcr1–binding sites can act as components of an epigenetic switch that mediates the inheritance of heterochromatin after a transient establishment period.

Fig. 3 Contributions of chromatin versus sequence-dependent mechanisms to cis inheritance of silencing and H3K9me.

(A) Silencing assays for cells with the indicated genotypes showing that mutations of the Clr4 chromodomain abolish maintenance of cenH::9xtetO-ura4+ silencing after release of TetR-Clr4-I by the addition of tetracycline. (B and C) ChIP-qPCR experiments showing requirement for the Clr4 chromodomain in maintenance of H3K9me2 upon release of TetR-Clr4-I with tetracycline at ura4+ (B) and mat (C). (D) Silencing assays showing that deletion of epe1+ suppresses the requirement for Atf1-Pcr1–binding sites (s1+s2+ versus s1s2∆). (E and F) ChIP-qPCR assays showing that deletion of epe1+ allowed efficient epigenetic maintenance of H3K9me2 in the absence of Atf1-Pcr1–binding sites (s1s2∆). Error bars in (B), (C), (E), and (F) indicate SD from three biological replicates.

The role of specificity factors in maintenance of epigenetic states is likely to be broadly conserved. A requirement for DNA sequence in maintenance of silencing has been previously described in Saccaromyces cerevisiae and Drosophila, where binding sites for factors that initiate silencing, silencers and Polycomb response elements (PREs), respectively, are continuously required to maintain the silent state (1013). Our findings demonstrate a role for sequence-dependent mechanisms in cis inheritance of epigenetic states. Considering the requirement for analogous networks of histone modifications and histone-binding proteins in the yeast and Drosophila systems, both silencing mechanisms probably also involve cis inheritance. Although the mechanisms by which transcription factor–binding sites contribute to cis inheritance remain to be elucidated, our findings can be explained by models that posit a role for transcription factors in creating a permissive context in which parental histone modifications and epigenetic states can be restored after DNA replication (fig. S5). The coupling of epigenetic inheritance to the activity of specific DNA sequences is likely to serve a crucial function in maintaining the fidelity of epigenetic propagation and reducing spurious epigenetic inactivation events.

Supplementary Materials

Materials and Methods

Figs. S1 to S5

Tables S1 and S2

References (2124)

References and Notes

Acknowledgments: We thank G. Thon for a gift of strain SPY6707, members of the Moazed lab for helpful discussions, and R. Behrouzi, M. Currie, N. Iglesias, G. Jih, and K. Ragunathan for comments on the manuscript. This work was supported by a grant from the NIH (GM072805) to D.M. and a China Scholarship Council award to X.W. D.M. is an investigator of the Howard Hughes Medical Institute.
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