Identification of noncoding transcripts from within CENP-A chromatin at fission yeast centromeres

Abstract

The histone H3 variant CENP-A is the most favored candidate for an epigenetic mark that specifies the centromere. In fission yeast, adjacent heterochromatin can direct CENP-A(Cnp1) chromatin establishment, but the underlying features governing where CENP-A(Cnp1) chromatin assembles are unknown. We show that, in addition to centromeric regions, a low level of CENP-A(Cnp1) associates with gene promoters where histone H3 is depleted by the activity of the Hrp1(Chd1) chromatin-remodeling factor. Moreover, we demonstrate that noncoding RNAs are transcribed by RNA polymerase II (RNAPII) from CENP-A(Cnp1) chromatin at centromeres. These analyses reveal a similarity between centromeres and a subset of RNAPII genes and suggest a role for remodeling at RNAPII promoters within centromeres that influences the replacement of histone H3 with CENP-A(Cnp1).

FIGURE 1.

Chromatin-remodeling factor Hrp1^Chd1 contributes to CENP-A^Cnp1 chromatin formation. A, silencing of cen1:arg3⁺ in WT, cnp1-169, and hrp1Δ cells. The growth assay was performed on non-selective (N/S) or arginine-depleted (−Arg) plates at 25, 32, and 36 °C. B, viability of cells bearing hrp1Δ combined with mis6-302 (upper panels) or with cnp1-87 (lower panels) compared with wild-type and single mutants grown at 25, 32, or 36 °C. C, CENP-A^Cnp1 ChIP analyses in WT, hrp1Δ, cnp1-87, and hrp1Δ cnp1-87 cells grown at 36 °C. The enrichment of the cnt1 product was compared with input DNA relative to the act1⁺ control by quantitative PCR. D, genome browser view of cen1 showing ChIP-chip binding profiles for CENP-A^Cnp1 (purple) and H3 (green) in WT and hrp1Δ cells (as indicated) at 30 °C. The relative ratios of CENP-A^Cnp1 and H3 (hrp1Δ/WT) are indicated (black). Data on the y axis are presented in log 2 scale, and the x axis shows genome positions in base pairs.

FIGURE 2.

Genome-wide CENP-A^Cnp1 localization correlates with Hrp1^Chd1 occupancy and H3 density changes in hrp1Δ cells. A, Venn diagram illustrating the overlap of CENP-A^Cnp1 enrichment with Hrp1^Chd1 occupancy and H3 density increase in hrp1Δ cells (16). A cutoff of 1.5 was used for CENP-A^Cnp1 enrichment. The p values indicate the probability of overlap generated by a hypergeometric probability test using R. B, genome browser view of gene SPBC839.06 with reduced CENP-A^Cnp1 (purple) and increased H3 (green) levels at its promoter in hrp1Δ cells. The arrow indicates the direction of transcription. The relative ratios of CENP-A^Cnp1 and H3 are indicated (black). Data on the y axis are presented in log 2 scale, and the x axis shows genome positions in base pairs. C, Venn diagram showing the overlap between promoters where CENP-A^Cnp1 is reduced, promoters where H3 is increased, and promoters where H3 is reduced in hrp1Δ cells. A cutoff of 1.2 was used for CENP-A^Cnp1 reduction. D, Venn diagram showing the overlap between promoters where CENP-A^Cnp1 is increased, promoters where H3 is increased, and promoters where H3 is reduced in hrp1Δ cells. A cutoff of 1.2 was used for the CENP-A^Cnp1 increase.

FIGURE 3.

Accumulation of central domain transcripts in RNA-processing mutants. A, schematic of fission yeast cen1, indicating the central core (cnt), innermost repeat (imr), and outer repeats (otr/dg-dh). Regions amplified by primer pairs (PP1–PP10) used in RT-PCR are indicated below (black bars). B, RT-PCR analysis of transcripts from cen1 and the act1⁺ control. Wild-type, dhp1-1, and pfs2-11 cells were grown at 25 °C and then shifted to 36 °C for 6 h before RNA extraction. Wild-type and dis3-54 cells were grown at 36 °C and shifted to 18 °C for 9 h before RNA extraction. −RT, negative control performed without reverse transcriptase; *, unspecific products. PCR with a chromosomal DNA (chr. DNA) template was included as a positive control. C, RT-PCR analysis of transcripts from cnt2, cnt3, and the act1⁺ control. PP-cnt2 and PP-cnt3 are specific for the central domain of cen2 and cen3, respectively.

FIGURE 4.

Northern and 5′-RACE/PCR analyses of central domain transcripts. A, Northern analysis of total RNAs in RNA-processing and kinetochore mutants. An RNA probe complementary to cnt1 was used. Cells grown at the permissive temperature (25 °C for WT, pfs2-11, dhp1-1, mis6-302, and cnp1-1 cells and 36 °C for WT and dis3-54 cells) were shifted to the restrictive temperature (6 h at 36 °C for WT, pfs2-11, dhp1-1, mis6-302, and cnp1-1 cells and 9 h at 18 °C for WT and dis3-54 cells) before RNA extraction. act1⁺ was used as a loading control. *, rRNA interference with hybridization. For additional EtBr images, see supplemental Fig. S5A. B, the same cnt1 probe used on poly(A) RNA. For additional EtBr images, see supplemental Fig. S5B. C and D, Northern analysis of total or poly(A) RNAs from WT and cnp1-1 cells with an RNA probe complementary to the reverse strand (forward probe; C) or the forward strand (reverse probe; D) of the cc1/tm1 sequence, which is shared by cnt1 and cnt3. *, nonspecific band. For additional EtBr images, see supplemental Fig. S5 (C and D). E, schematic representation of transcription start sites determined by 5′-RACE/PCR in WT and cnp1-1 cells. Black arrows, transcription start sites identified in WT and cnp1-1 cells; gray arrows, transcription start sites identified only in cnp1-1 cells. F and G, Northern blots showing transcripts complementary to the TM-forward (F) or TM-reverse (G) probes in WT, mis6-302, cnp1-1, mis12-537, mis16-53, and mis18-262 cells grown at permissive (25 °C) and restrictive (36 °C) temperatures. *, nonspecific band. EtBr staining confirmed equal loading.

FIGURE 5.

Proposed model for the role of Hrp1 in the exchange of histone H3 with CENP-A^Cnp1 during RNAPII transcription. Transcription from the promoters within centromeric chromatin acts to promote the replacement of histone H3 with CENP-A^Cnp1 through the associated remodeling factor Hrp1^Chd1. Similarly, Hrp1^Chd1 acts at some euchromatic promoters to replace H3 with CENP-A^Cnp1. The resulting centromeric transcripts are degraded by the exosome.