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

Abstract

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.

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 (cenHΔ::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 cenHΔ:9xtetO-ura4⁺-B1 ON allele and a cenHΔ::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 cenHΔ::9xtetO-ura4⁺-B1 ON with cenHΔ::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 cenHΔ::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.

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 cenHΔ::9xtetO-ura4⁺ cells. Error bars in (C) to (F) indicate SD from three biological replicates.

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 cenHΔ::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 s1Δs2Δ). (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 (s1Δs2Δ). Error bars in (B), (C), (E), and (F) indicate SD from three biological replicates.