Genome-Wide Studies of Histone Demethylation Catalysed by the Fission Yeast Homologues of Mammalian LSD1

In order to gain a more global view of the activity of histone demethylases, we report here genome-wide studies of the fission yeast SWIRM and polyamine oxidase (PAO) domain homologues of mammalian LSD1. Consistent with previous work we find that the two S. pombe proteins, which we name Swm1 and Swm2 (after SWIRM1 and SWIRM2), associate together in a complex. However, we find that this complex specifically demethylates lysine 9 in histone H3 (H3K9) and both up- and down-regulates expression of different groups of genes. Using chromatin-immunoprecipitation, to isolate fragments of chromatin containing either H3K4me2 or H3K9me2, and DNA microarray analysis (ChIP-chip), we have studied genome-wide changes in patterns of histone methylation, and their correlation with gene expression, upon deletion of the swm1+ gene. Using hyper-geometric probability comparisons we uncover genetic links between lysine-specific demethylases, the histone deacetylase Clr6, and the chromatin remodeller Hrp1. The data presented here demonstrate that in fission yeast the SWIRM/PAO domain proteins Swm1 and Swm2 are associated in complexes that can remove methyl groups from lysine 9 methylated histone H3. In vitro, we show that bacterially expressed Swm1 also possesses lysine 9 demethylase activity. In vivo, loss of Swm1 increases the global levels of both H3K9me2 and H3K4me2. A significant accumulation of H3K4me2 is observed at genes that are up-regulated in a swm1 deletion strain. In addition, H3K9me2 accumulates at some genes known to be direct Swm1/2 targets that are down-regulated in the swm1¿ strain. The in vivo data indicate that Swm1 acts in concert with the HDAC Clr6 and the chromatin remodeller Hrp1 to repress gene expression. In addition, our in vitro analyses suggest that the H3K9 demethylase activity requires an unidentified post-translational modification to allow it to act. Thus, our results highlight complex interactions between histone demethylase, deacetylase and chromatin remodelling activities in the regulation of gene expression

Recombinogenic Conditions Influence Partner Choice in Spontaneous Mitotic Recombination

<div><p>Mammalian common fragile sites are loci of frequent chromosome breakage and putative recombination hotspots. Here, we utilized Replication Slow Zones (RSZs), a budding yeast homolog of the mammalian common fragile sites, to examine recombination activities at these loci. We found that rates of <i>URA3</i> inactivation of a <i>hisG-URA3-hisG</i> reporter at RSZ and non-RSZ loci were comparable under all conditions tested, including those that specifically promote chromosome breakage at RSZs (hydroxyurea [HU], <i>mec1Δ sml1Δ</i>, and high temperature), and those that suppress it (<i>sml1Δ</i> and <i>rrm3Δ</i>). These observations indicate that RSZs are not recombination hotspots and that chromosome fragility and recombination activity can be uncoupled. Results confirmed recombinogenic effects of HU, <i>mec1Δ sml1Δ</i>, and <i>rrm3Δ</i> and identified temperature as a regulator of mitotic recombination. We also found that these conditions altered the nature of recombination outcomes, leading to a significant increase in the frequency of <i>URA3</i> inactivation via loss of heterozygosity (LOH), the type of genetic alteration involved in cancer development. Further analyses revealed that the increase was likely due to down regulation of intrachromatid and intersister (IC/IS) bias in mitotic recombination, and that RSZs exhibited greater sensitivity to HU dependent loss of IC/IS bias than non RSZ loci. These observations suggest that recombinogenic conditions contribute to genome rearrangements not only by increasing the overall recombination activity, but also by altering the nature of recombination outcomes by their effects on recombination partner choice. Similarly, fragile sites may contribute to cancer more frequently than non-fragile loci due their enhanced sensitivity to certain conditions that down-regulate the IC/IS bias rather than intrinsically higher rates of recombination.</p></div

<i>sml1Δ</i> regulation of mitotic recombination.

<p><b>A, B.</b> Rates of <i>URA3</i> inactivation at the indicated locus in <i>sml1Δ</i> haploid and diploid strains grown in YPD. 95% Confidence Limits (CLs) for each value were calculated as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Wierdl1" target="_blank">[47]</a>. For each locus except for ORI, rate measurements from two independently derived strains were obtained by methods of median (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931.s002" target="_blank">Figure S2</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Lea1" target="_blank">[24]</a>. <b>C, D.</b> Effects of <i>sml1Δ</i> on rate of <i>URA3</i> inactivation. Black and grey circles correspond to rate measurements in <i>sml1Δ</i> (panels <b>A</b> and <b>B</b>) and WT (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen-1003931-g002" target="_blank">Figure 2A</a>), respectively. Capped lines indicate 95% CLs. The number at the top of each box is the ratio between average rate values in <i>sml1Δ</i> and WT at each locus. * denotes a statistically significant change (Chi square test, p<0.05). Numbers in red and blue denote statistically significant increase or decrease, respectively. <b>E.</b> Locus specific effects of <i>sml1Δ</i> on rate of <i>URA3</i> inactivation. ^a The average rate at the indicated locus in <i>sml1Δ</i> was normalized to the corresponding value in WT (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen-1003931-g002" target="_blank">Figure 2</a>). Numbers in red and blue denote statistically significant increase or decrease, respectively (Chi square test, p<0.05). NSS: Not Statistically Significant. <b>F.</b> Effect of <i>sml1Δ</i> on IC/IS bias. Fraction of 5FOA^R colonies that had undergone <i>URA3</i> inactivation via an IC/IS mediated event ^a The fraction in <i>sml1Δ</i> at each locus was normalized to the corresponding value in WT (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen-1003931-g003" target="_blank">Figure 3E</a>). ^b Statistical analysis (Fischer's exact test) was performed on the effects of <i>sml1Δ</i>. The number in red denotes statistically significant increase.</p

Rate of <i>URA3</i> inactivation in YPD and in HU.

<p><b>A, B.</b> Rate of <i>URA3</i> inactivation at the indicated locus in WT haploid- or diploid- strains grown in YPD (A) or in 10 mM HU (B). 95% Confidence Limits (CLs) for each value were calculated as previously described <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Wierdl1" target="_blank">[47]</a>. For each locus, rate measurements from two independently derived strains were obtained by the methods of median (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931.s002" target="_blank">Figure S2</a>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Lea1" target="_blank">[24]</a>. <b>C.</b> The average recombination rate (and 95% CLs) of the haploid- and diploid- strains analyzed in <b>A</b> and <b>B.</b> “HU/YPD”: The effects of HU over YPD on recombination rate was expressed as the ratio between the two average rates. <b>D, E.</b> Effects of HU on rate of <i>URA3</i> inactivation at each locus. Graphic representation of the data presented in <b>A</b> and <b>B.</b> Black and grey circles correspond to rate measurements in HU and YPD, respectively. Capped lines indicate 95% CLs. The red number at the top of each box is the ratio between the average rate values in HU and YPD at the indicated locus; the extent increase conferred by HU was statistically significant at every locus (Chi square test, p<0.05).</p

Experimental system.

<p><b>A.</b> Recombination activity at five different loci in chromosome III (ChrIII) was assessed by monitoring <i>URA3</i> inactivation in <i>hisG-URA3-hisG</i> reporter introduced at each of the five indicated site. The “kb from TEL” and “kb from CEN” are distances from the left telomere and the centromere (CEN) in kb, respectively. Grey boxes represent the six RSZs referred to as I through IV in ChrIII <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931-Cha1" target="_blank">[3]</a>. T: replication fork termination site; Open circle: active replication origin; Grey circle: tRNA gene; Triangle: Ty element. <b>B.</b> Summary of notable features at each locus examined. <b>C.</b> Mechanisms of <i>URA3</i> inactivation. Heterozygous diploid strains carrying a single copy of <i>hisG-URA3-hisG</i> (blue circle; <i>URA+</i>) were grown under specified conditions and selected for 5FOA resistance. “Molecular events” summarize possible mechanisms of <i>URA3</i> inactivation. Co-segregation of <i>ura3</i> chromatids at mitosis would lead to a 5FOA^R colony (pink circle) that no longer carries the <i>hisG-URA3-hisG</i> allele. The resulting diploid would carry either the “pop-out” and a single copy of WT (i, ii), two copies of WT (iii, iv), or just one copy of WT (v, vi). L and R; the immediate upstream and downstream sequences of each insertion locus utilized for targeted introduction of the tester construct via homologous recombination (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003931#pgen.1003931.s001" target="_blank">Figure S1</a>). INT: the allele containing the tester construct.</p