Transcriptionally active heterochromatin in rye B chromosomes

Abstract

B chromosomes (Bs) are dispensable components of the genomes of numerous species. Thus far, there is a lack of evidence for any transcripts of Bs in plants, with the exception of some rDNA sequences. Here, we show that the Giemsa banding-positive heterochromatic subterminal domain of rye (Secale cereale) Bs undergoes decondensation during interphase. Contrary to the heterochromatic regions of A chromosomes, this domain is simultaneously marked by trimethylated H3K4 and by trimethylated H3K27, an unusual combination of apparently conflicting histone modifications. Notably, both types of B-specific high copy repeat families (E3900 and D1100) of the subterminal domain are transcriptionally active, although with different tissue type-dependent activity. No small RNAs were detected specifically for the presence of Bs. The lack of any significant open reading frame and the highly heterogeneous size of mainly polyadenylated transcripts indicate that the noncoding RNA may function as structural or catalytic RNA.

Figure 1.

Cytological Organization of the B-Specific Domain during Development. (A) Giemsa C-banded metaphase chromosomes of rye with Bs (arrows and inset). (B) Root tip meristematic c-metaphase cell of Lindström wheat with six Bs hybridized with D1100 (red) and E3900 (green) probes. At this stage, the D1100 terminal domain displays a high level of condensation, and the B-specific domain is defined as two contiguous blocks (one centromere-proximal and the other distal); E3900 labels only the distal block (yellow signal due to superimposition of red and green channels). (C) Organization of the root tip showing the location of meristematic and protoxylem nuclei. (D) and (E) Heterogeneous organization of the B-specific domain defined by D1100 (red), showing condensed and diffuse regions in 2B meristematic cells. The start of the arrow indicates the start of the B domain, and the end of the arrow indicates the telomeric end of the domain. (D) Two interphase nuclei hybridized with D1100 and E3900 (green; yellow signal is due to superimposition with D1100 red signal). An unlabeled gap frequently separates the condensed and decondensed regions. The bottom of the figure shows the single channel images of the same nuclei for E3900 (left) and D1100 (right). (E) to (H) B-specific domain during mitoses in meristematic cells with two Bs: (E) at prophase, (F) at metaphase showing the B-specific domain fully condensed (only one B is visible), (G) at anaphase, and (H) at telophase (only one of the Bs is visible over its full length due to the orientation of projection). (I) D1100 pattern in a nucleus from a developing xylem cell (see [C] for location of cell type). (J) to (N) organization of the B-specific domain in anther tissues: (J) location of meiocytes and tapetal cells, (K) binucleate tapetal nuclei, (L) and (M) pachytene nuclei with two Bs fully paired (L) and six Bs with irregular pairing (M). The curved arrow in (L) indicates the full length of B-specific domain, and the intercalary cut shows the extended gap in D1100 labeling. (N) shows the magnification of the B-specific domain from the six B pachytene cells in (M), showing two fully paired Bs forming a bivalent (right) and four Bs with irregular pairing forming a tetravalent. DAPI staining for DNA is in blue. Bars = 10 μm.

Figure 2.

Histone Marks of As and Bs. Metaphase cells of rye with Bs (arrows) after immunostaining with antibodies specific for H3K4me1,2,3; H3K9me1,2,3; and H3K27me1,2,3.

Figure 3.

Comparative Analysis of B Variants. (A) to (F) Root tip meristematic metaphase cells of JNK rye ([A] and [B]) with four Bs (arrowheads) and of Lindström wheat ([C] and [D]) with two Bs (arrowheads) and an interphase nucleus of JNK rye with three Bs ([E] and [F]) after immunodetection of H3K4me3 ([A], [C], [E] in red) following in situ hybridization with the B-specific D1100 repeat ([B], [D], and [F] in green). In (A), note the absence of H3K4me3 in the heterochromatic subtelomeric blocks of rye As. Bars = 10 μm. (G) Individual standard B and deleted B (delB) after immunodetection of H3K4me3 (red) and in situ hybridization of D1100 (green). DNA is stained with DAPI (blue).

Figure 4.

Transcriptional Analysis of E3900 and D1100. (A) and (B) RNA gel blot analysis of E3900 (A) and D1100 (B) transcripts isolated from roots (R), leaves (L), and anthers (A) of 0B/+B rye. Schemata indicates the different regions (0N to 5N) of E3900 used as hybridization probes. Arrows indicate B-specific signals of small size (<200 bases). (C) Ethidium bromide–stained RNA gel that was used as a loading control. Size marker is the RiboRuler RNA ladder (Fermentas).

Figure 5.

Transcriptional Analysis of E3900 and D1100 Reveals Tissue Type–Specific Activity. (A) and (B) E3900 and D1100 RNA abundance assessed by semiquantitative RT-PCR on root (R), leaf (L), and anther (A) tissue of 0B/+B rye and 0B/+B wheat. Schematas indicate the different regions of E3900 and D1100 amplified by RT-PCR. (C) Controls: positive control using B-independent primers (eEF1-α elongation factor). Negative control using RNA of all samples without an initial reverse transcription step to demonstrate the absence of genomic DNA contamination, (n) PCR without template DNA and (g) PCR on genomic +B DNA. Each sample contained approximately the same amount of RNA.

Figure 6.

D1100 and E3900 Undergo Polyadenylation. RNA gel blot hybridization of D1100 and E3900 with total RNA separated into poly(A)⁺ and poly(A)⁻ fractions. A probe specific for actin was used as a poly(A)⁺ positive probe.