lncRNA transcription induces meiotic recombination through chromatin remodelling in fission yeast

Abstract

Noncoding RNAs (ncRNAs) are involved in various biological processes, including gene expression, development, and disease. Here, we identify a novel consensus sequence of a cis-element involved in long ncRNA (lncRNA) transcription and demonstrate that lncRNA transcription from this cis-element activates meiotic recombination via chromatin remodeling. In the fission yeast fbp1 gene, glucose starvation induces a series of promoter-associated lncRNAs, referred to as metabolic-stress-induced lncRNAs (mlonRNAs), which contribute to chromatin remodeling and fbp1 activation. Translocation of the cis-element required for mlonRNA into a well-characterized meiotic recombination hotspot, ade6-M26, further stimulates transcription and meiotic recombination via local chromatin remodeling. The consensus sequence of this cis-element (mlon-box) overlaps with meiotic recombination sites in the fission yeast genome. At one such site, the SPBC24C6.09c upstream region, meiotic double-strand break (DSB) formation is induced in an mlon-box-dependent manner. Therefore, mlonRNA transcription plays a universal role in chromatin remodeling and the regulation of transcription and recombination.

Fig. 1. mlon-IE works at other loci and conditions in addition to its role in the fbp1 upstream region.

a Schematic representation of the fbp1 upstream region containing upstream activating sequence 1 and 2 (UAS1 and UAS2), the binding sites for Atf1 and Rst2, respectively. The numbers indicate the transcription start site of the fbp1 transcripts and the distances of UAS1, UAS2 and the TATA box from the first ATG of fbp1 open reading frame (ORF). b Representative images of northern blot showing stepwise expression of mlonRNAs and fbp1 mRNA and blot of the chromatin analysis showing stepwise chromatin remodeling during glucose starvation. Wild-type haploid cells were grown to 2.0 × 10⁷ cells/ml in YER medium, then transferred to YED medium. Cells were harvested at the indicated times. cam1 transcript was used as an internal control. c M26 and M375 mutations in ade6 (bold italic) and inserted mlon-IE sequences are shown. d ade6 transcription in indicated cells. Diploid cells were cultured to induce meiosis, as described in the Methods section. 18S rRNA stained by ethidium bromide is shown as the loading control.

Fig. 2. Transcription from mlon-IE activates meiotic DSB formation and recombination in ade6-M26, the meiotic recombination hotspot.

a Recombination rates at the M26 or M375 control allele (indicated as ade⁺/10⁴ spores) were examined, as described in the Methods section (left). Recombination between leu1-32 and his3-D1 (indicated as leu⁺/his⁺ percentage) was measured as the control (right). Error bars represent standard deviations. n = 3 or 5 biologically independent experiments. P values were calculated using the unpaired one-sided Student’s t-test: *P < 0.05, n.s. (not significant). b Indicated haploid cells possessing the pat1-114 rad50s genotype were cultured to induce meiosis, and DNA was prepared as described in the Methods section. Meiotic DSBs were detected by Southern blotting. The dotted line indicates DSBs around M26 and inserted mlon-IE. c Meiotic chromatin remodeling in indicated cells. Diploid cells were cultured as in Fig. 1c. The black arrowhead and the dotted line indicate MNase-sensitive sites at the M26 mutation and the inserted mlon-IE, respectively.

Fig. 3. mlon-IE works downstream of binding sequences for transcription factors, CCAAT-binding factor and Rst2.

a Schematic representation of ade6-4002, ade6-4099, ade6-4095 and ade6-4156. 4002 insertion, 4099 mutation, 4095 mutation and 4156 mutation are shown (bold italic). mlon-IE was translocated 200 bp downstream from the 4002/4099/4095/4156 mutation. b ade6 transcripts of indicated cells in meiosis. Cells were cultured, as described in Fig. 1c. c Recombination rates of indicated cells were assessed, as described in Fig. 2a. Error bars represent standard deviations. n = 3 or 6 biologically independent experiments. P values were calculated using the unpaired one-sided Student’s t-test: *P < 0.05. d, e Meiotic DSBs of indicated cells were detected by Southern blotting, as described in Fig. 2b.

Fig. 4. Determination of a consensus sequence of mlon-IE.

a Schematic representation of the fbp1 upstream region, including three distinct lncRNAs (mlonRNA-a, mlonRNA-b, and mlonRNA-c) and UAS1 and UAS2. The open circle indicates mlon-IE at 110 bp upstream from the mlonRNA-c TSS. The ten nucleotides of the mlon-IE (5′-ATCTTATGTA-3′) sequence were comprehensively replaced with each of the three other nucleotides. b Relative mlonRNA-c transcript levels of indicated cells at 30 min after glucose starvation compared to wild-type cells. The band intensity of mlonRNA-c transcripts in Supplementary Fig. 6 was quantified. n = 2 biologically independent experiments. c mlon-box, a consensus sequence of mlon-IE.

Fig. 5. DSB formation at the natural meiotic recombination hotspot in the SPBC24C6.09c upstream region is mediated by mlon-box-dependent intergenic transcription.

a Relationship between mlon-box sites and meiotic DSB distribution (Rec12-oligos). The X axis indicates the distance from the nearest mlon-box, and the Y axis indicates copy numbers of the Rec12-oligos-signal. The strongest plot (arrowhead) represents the meiotic recombination hotspot in the SPBC24C6.09c upstream region. b Schematic representation of the mlon-box (open circle) in the intergenic region between SPBC24C6.09c and SPNCRNA.1506. The entire mlon-box sequence comprising 10 nucleotides was replaced with a 10 nucleotide sequence from act1 ORF in mlon-box-replacement cells (bold italic). Location of probes for northern blot was indicated by bold bar. c Detection of meiotic DSBs in the intergenic region between SPBC24C6.09c and SPNCRNA.1506. Indicated cells were cultured and analyzed, as described in Fig. 2b. The dotted line indicates DSB sites around the mlon-box. d Northern analysis to detect the SPBC24C6.09c transcript in wild-type and mlon-box-replacement cells. The left probe indicated in Fig. 4b was used. Indicated cells were cultured to induce meiosis, as described in Fig. 2b. The cam1 transcript is shown as a loading control. e, ChIP analysis to examine histone H3 binding at the mlon-box in indicated cells. Cells were cultured to induce meiosis, as described in Fig. 2b. The relative increase in the ratio at the indicated time after the onset of meiosis is indicated. n = 2 biologically independent experiments. P-values were calculated using Student’s t-test: *P < 0.05.