Human Centromeres Produce Chromosome-Specific and Array-Specific Alpha Satellite Transcripts that Are Complexed with CENP-A and CENP-C

Abstract

Human centromeres are defined by alpha satellite DNA arrays that are distinct and chromosome specific. Most human chromosomes contain multiple alpha satellite arrays that are competent for centromere assembly. Here, we show that human centromeres are defined by chromosome-specific RNAs linked to underlying organization of distinct alpha satellite arrays. Active and inactive arrays on the same chromosome produce discrete sets of transcripts in cis. Non-coding RNAs produced from active arrays are complexed with CENP-A and CENP-C, while inactive-array transcripts associate with CENP-B and are generally less stable. Loss of CENP-A does not affect transcript abundance or stability. However, depletion of array-specific RNAs reduces CENP-A and CENP-C at the targeted centromere via faulty CENP-A loading, arresting cells before mitosis. This work shows that each human alpha satellite array produces a unique set of non-coding transcripts, and RNAs present at active centromeres are necessary for kinetochore assembly and cell-cycle progression.

Keywords: RNA; cell cycle; centromere; kinetochore; polymerase; repetitive DNA.

Figure 1. Human Centromeres Produce Array-Specific Non-Coding RNAs in cis

(A) Schematic of the human centromere, including core centromeric chromatin containing CENP-A and euchromatic H3 nucleosomes and flanking heterochromatin enriched for H3K9 and K27 methylation. These domains assemble on alpha satellite DNA, a 171bp monomer (open red arrows) tandemly arranged into higher order repeats (HORs, large red-filled arrows) that are extensively reiterated. A set number of monomers confers chromosome specificity so that alpha satellite DNA at each centromere is distinct. (B) The centromere of Homo sapiens chromosome X (HSAX) is defined by DXZ1, a single array based on a 12-mer HOR. Fluorescence images were obtained from diploid RPE1 interphase cells (bar, 5μm) and metaphase chromosomes (bar, 15μm) after RNA FISH with a probe spanning the entire DXZ1 HOR (green). Sequential DNA FISH with the same probe labeled in a different fluorophore (red) verified chromosomal location of RNA signal and permitted quantification of alpha satellite RNA:DNA ratio. In each experiment, control slides were treated with RNase to verify that FISH signals detected RNA. (C) Sequential RNA (green) – DNA (red) FISH on interphase and metaphase cells from male line HTD with a probe spanning DYZ3, the 34-mer alpha satellite HOR on HSAY and quantification of RNA:DNA ratio. In (B) and (C), each data point represents a single interphase centromere and mean ± SEM are shown. See also Figure S1.

Figure 2. Active and Inactive Alpha Satellite Arrays Are Transcribed

(A) HSA17 has three distinct alpha satellite arrays; either D17Z1 (red arrows) or D17Z1-B (green arrows) can be the active centromere. (B, B′) In RPE1, D17Z1 is active on both HSA17s. RNA FISH with D17Z1 (red) and D17Z1-B (green) HOR probes on interphase cells and metaphase chromosomes (B) and RNA-DNA FISH in interphase cells (B′). RNase treatment verified detection of RNA. Bars, 5μm. (C) Dot plots of RNA:DNA ratios of D17Z1 and D17Z1-B from RNA-DNA FISH in (B′) (mean ± SEM). (D) RT-qPCR of D17Z1, D17Z1-B, and DXZ1 RNA in RPE1 cells relative to qPCR of gDNA of same array. 45S rRNA and XIST lncRNA relative to gDNA are shown for comparison (mean ± SEM). Data represent two biological replicates that each contained three technical replicates. (E) RT-qPCR of D17Z1 and DXZ1 transcripts from synchronized RPE1 cells (mean ± SEM). No significant differences in RNA:DNA ratios were observed at D17Z1 or DXZ1 across the cell cycle. Data in this figure were statistically analyzed using a t-test. See also Figures S1–S3.

Figure 3. Chromosome-Specific Alpha Satellite RNAs Have Array-Specific Size Ranges

(A, A′) RNA FISH in RPE1 cells with PNA probe (green) recognizing the CENP-B box found in a subset of monomers within all alpha satellite arrays except for the Y. Multiple transcripts were detected in interphase (bar, 5μm), and at every centromere at metaphase (bar, 15μm). Asterisks denote HSAX. (B) Northern blot detection of D17Z1 RNA in total RNA isolated from multiple cells lines, with each cell line in duplicate. Ethidium bromide stained gel is shown under each blot. Bracket indicates size range for chromosome-specific, array-specific transcripts. A schematic of the entire D17Z1 higher order repeat (HOR) that was used as a probe is shown. (C) Northern blot of D17Z1-B RNA in multiple cell lines. A schematic of the entire D17Z1-B HOR that was used as a probe is shown. (D) Northern blot of DXZ1 RNA. A schematic of the entire DXZ1 HOR that was used as a probe is shown. (E) Northern blot of DXZ1 and D17Z1 RNA after rRNA depletion eliminated contaminating rRNA bands (*) observed in panels (B)–(D).

Figure 4. Active and Inactive Alpha Satellite RNAs Exhibit Different Stabilities

(A) Quantification of alpha satellite RNA:DNA ratios in RPE1 cells after RNA Polymerase I (RNAP I) inhibition with low concentrations of Actinomycin D (ActD) (mean ± SEM). (B, C) RNA (green) – DNA (red) FISH for DXZ1, D17Z1, and D17Z1-B after RNAP I/II inhibition with high concentrations of ActD. DXZ1 and D17Z1 are active centromere arrays; D17Z1-B is inactive. Bars, 5μm (C′) XIST RNA FISH (green) served as a control for efficacy of RNAP II inhibition. Bar, 15μm. (D, D′) Quantification of DXZ1 RNA:DNA ratios over 6 hours of ActD treatment, and D17Z1 and D17Z1-B over 16 hours of ActD treatment (mean ± SEM). Data were statistically analyzed using a t-test. In (A) and (D), each data point represents a single interphase centromere. See also Figure S4.

Figure 5. Alpha Satellite Transcripts Are Complexed with Centromere Proteins and are Unaffected by CENP-A Depletion

(A) Left, CENP-A immunostaining (red) and RNA FISH on RPE1 interphase cell using a CENP-B box PNA probe (green). CENP-A and ~50% of CENP-B box RNA FISH signals overlap. Inset emphasizes that chromosomes with multiple arrays produce transcripts from all arrays (arrows) yet only one array is active/associated with CENPs (arrowhead). Bar, 5μm. Right, CENP-A immunostaining (red) and RNA FISH with DXZ1 probe (green) on cell line LCL^XiX showing that CENP-A is associated with a portion of DXZ1 transcripts. Bar, 15μm. (B) Western blots of input, mock (no antibody), and native chromatin fractions from HAP1 cells immunoprecipitated (IP) with antibodies specific for CENP-A, CENP-B, CENP-C. (C) Native RNA-ChIP and RT-qPCR showing association of DXZ1 and D17Z1 transcripts with CENP-A and CENP-C (mean ± SEM; n=4). (D) Native RNA-ChIP and RT-qPCR showing that transcripts from the centromere inactive array D17Z1-B are associated with CENP-B that does not discriminate between active and inactive centromeres (mean ± SEM, n = 4). (E, F) Crosslinked RNA-ChIP and RT-qPCR showing that transcripts from active arrays DXZ1 and D17Z1, but not inactive D17Z1-B, are associated with CENP-A and CENP-C (mean ± SEM, n = 4). Active and inactive array transcripts are associated with CENP-B. (G, G′) CENP-A immunostaining (red) and RNA FISH for D17Z1 transcripts (green) after CENP-A depletion by siRNA in HT1080 cells. Nuclei with >50% CENP-A knockdown are denoted by dashed outlines; those with little CENP-A depletion have solid outlines. Quantitation of D17Z1 RNA:DNA ratio after CENP-A depletion (mean ± SEM). Each data point represents a single interphase HSA17 centromere. Data were statistically analyzed using a t-test. See also Figures S3 and S4.

Figure 6. Targeted Depletion of Alpha Satellite Transcripts Disrupts Centromere Assembly

(A) HT1080 cells were transfected with array-specific dsRNAs and assayed by immunostaining and RNA-DNA FISH after 6 days. Depletion of DXZ1 transcripts using array-specific dsRNAs decreases DXZ1 RNA and reduces CENP-A only at DXZ1 (red arrowhead). Transcripts and CENP-A (green arrowheads) at control array D7Z1 (HSA7) are not decreased. Bars, 5μm. (B, C) Quantitation of CENP-A and CENP-C at targeted DXZ1 array and control array D7Z1 (mean ± SEM). (D) RNA:DNA ratios of targeted DXZ1 and control array D7Z1 measured from RNA-DNA FISH experiments (mean ± SEM). (E) Depletion of active D17Z1-B transcripts in a somatic cell hybrid line Z12.3B containing a single epiallele HSA17 chromosome led to reduction of CENP-A (red arrowhead) and RNA at D17Z1-B (red) but not at inactive D17Z1 (green). (F) Quantitation of CENP-A at D17Z1-B after dsRNA depletion (mean ± SEM). Each data point represents a single interphase HSA17. (G) Quantitation of RNA:DNA ratios at targeted (D17Z1-B) and control (D17Z1) arrays after D17Z1-B dsRNA depletion (mean ± SEM). For (B), (D), (F), and (G) each data point represents a single centromere at interphase. Data were statistically analyzed using a t-test. See also Figure S5 for replicate data.

Figure 7. Cell Cycle Arrest and Impaired CENP-A Loading in the Absence of Alpha Satellite Transcripts

(A) Ki-67 immunostaining (red) determined cell cycle phase of individual nuclei from HT1080 cells mock-treated or depleted for DXZ1 transcripts. DXZ1 RNA (red) and DXZ1 DNA (green) were detected by sequential RNA-DNA FISH. (B) Quantitation of DXZ1 RNA:DNA ratios at targeted DXZ1 arrays (mean ± SEM). Each data point is a single centromere at interphase. (C) Quantitation of Ki-67-staged (G1/S/G2) interphase cells from mock-treated and DXZ1 RNA depleted cells. Bars, 10μm. N ≥ 60 cells analyzed for each treatment. (D) Schematic of experimental approach to measure nascent SNAP-tagged CENP-A incorporation on HSAX after DXZ1 dsRNA depletion in HT1080 cells. (E) Depletion of DXZ1 transcripts using array-specific dsRNAs decreases DXZ1 RNA (red) and reduces SNAP-CENP-A only at DXZ1 (green arrowhead). Total CENP-A is shown in far-right panel. Because DXZ1 dsRNA knockdown arrests cells [see (A)], dsRNA treatment was done for 52 hours to deplete transcripts by 50% while allowing cells to continue cycling to measure new CENP-A loading. Bars, 5μm. (F, G) Quantitation of new (SNAP) CENP-A and total CENP-A at mock-treated and targeted DXZ1 arrays (mean ± SEM). Each data point in (C) through (E) represents a single centromere at interphase. Replicate data is presented in Figure S6. (H) RNA:DNA ratios of mock treated and dsRNA-targeted DXZ1 (mean ± SEM). Each data point in (F) through (H) represents a single centromere at interphase. Data were statistically analyzed using a t-test, except for (C), in which the Chi-square test was used. See also Figure S6 for replicate data.