Spo11 generates gaps through concerted cuts at sites of topological stress.

Meiotic recombination is essential for chromosome segregation at meiosis and fertility. It is initiated by programmed DNA double-strand breaks (DSBs) introduced by Spo11, a eukaryotic homologue of an archaeal topoisomerase (Topo VIA)1. Here we describe previously uncharacterized Spo11-induced lesions, 34 to several hundred base pair-long gaps, which are generated by coordinated pairs of DSBs termed double DSBs. Isolation and genome-wide mapping of the resulting fragments with single base-pair precision revealed enrichment at DSB hotspots but also a widely dispersed distribution across the genome. Spo11 prefers to cut sequences with similarity to a DNA-bending motif2, which indicates that bendability contributes to the choice of cleavage site. Moreover, fragment lengths have a periodicity of approximately (10.4n + 3) base pairs, which indicates that Spo11 favours cleavage on the same face of underwound DNA. Consistently, double DSB signals overlap and correlate with topoisomerase II-binding sites, which points to a role for topological stress and DNA crossings in break formation, and suggests a model for the formation of DSBs and double DSBs in which Spo11 traps two DNA strands. Double DSB gaps, which make up an estimated 20% of all initiation events, can account for full gene conversion events that are independent of both Msh2-dependent heteroduplex repair3,4 and the MutLγ endonuclease4. Because non-homologous gap repair results in deletions, and ectopically re-integrated double DSB fragments result in insertions, the formation of double DSBs is a potential source of evolutionary diversity and pathogenic germline aberrations.

Spt5 Phosphorylation and the Rtf1 Plus3 Domain Promote Rtf1 Function through Distinct Mechanisms.

Rtf1 is a conserved RNA polymerase II (RNAPII) elongation factor that promotes cotranscriptional histone modification, RNAPII transcript elongation, and mRNA processing. Rtf1 function requires the phosphorylation of Spt5, an essential RNAPII processivity factor. Spt5 is phosphorylated within its C-terminal domain (CTD) by cyclin-dependent kinase 9 (Cdk9), the catalytic component of positive transcription elongation factor b (P-TEFb). Rtf1 recognizes phosphorylated Spt5 (pSpt5) through its Plus3 domain. Since Spt5 is a unique target of Cdk9 and Rtf1 is the only known pSpt5-binding factor, the Plus3/pSpt5 interaction is thought to be a key Cdk9-dependent event regulating RNAPII elongation. Here, we dissect Rtf1 regulation by pSpt5 in the fission yeast Schizosaccharomyces pombe We demonstrate that the Plus3 domain of Rtf1 (Prf1 in S. pombe) and pSpt5 are functionally distinct and that they act in parallel to promote Prf1 function. This alternate Plus3 domain function involves an interface that overlaps the pSpt5-binding site and that can interact with single-stranded nucleic acid or with the polymerase-associated factor (PAF) complex in vitro We further show that the C-terminal region of Prf1, which also interacts with PAF, has a similar parallel function with pSpt5. Our results elucidate unexpected complexity underlying Cdk9-dependent pathways that regulate transcription elongation.

Chromatin Immunoprecipitation of Histone Modifications in Fission Yeast.

Chromatin immunoprecipitation (ChIP) is a sensitive, accurate, and reliable technique widely used to analyze protein-DNA interactions at specific binding sites in vivo. It has been a particularly powerful technique for mapping of histone modification patterns both at individual loci and genome-wide. Here we provide a detailed protocol for ChIP of histone modifications associated with active transcription in fission yeast (Schizosaccharomyces pombe).

Functional interaction of Rpb1 and Spt5 C-terminal domains in co-transcriptional histone modification.

Transcription by RNA polymerase II (RNAPII) is accompanied by a conserved pattern of histone modifications that plays important roles in regulating gene expression. The establishment of this pattern requires phosphorylation of both Rpb1 (the largest RNAPII subunit) and the elongation factor Spt5 on their respective C-terminal domains (CTDs). Here we interrogated the roles of individual Rpb1 and Spt5 CTD phospho-sites in directing co-transcriptional histone modifications in the fission yeast Schizosaccharomyces pombe. Steady-state levels of methylation at histone H3 lysines 4 (H3K4me) and 36 (H3K36me) were sensitive to multiple mutations of the Rpb1 CTD repeat motif (Y1S2P3T4S5P6S7). Ablation of the Spt5 CTD phospho-site Thr1 reduced H3K4me levels but had minimal effects on H3K36me. Nonetheless, Spt5 CTD mutations potentiated the effects of Rpb1 CTD mutations on H3K36me, suggesting overlapping functions. Phosphorylation of Rpb1 Ser2 by the Cdk12 orthologue Lsk1 positively regulated H3K36me but negatively regulated H3K4me. H3K36me and histone H2B monoubiquitylation required Rpb1 Ser5 but were maintained upon inactivation of Mcs6/Cdk7, the major kinase for Rpb1 Ser5 in vivo, implicating another Ser5 kinase in these regulatory pathways. Our results elaborate the CTD 'code' for co-transcriptional histone modifications.

The PAF complex and Prf1/Rtf1 delineate distinct Cdk9-dependent pathways regulating transcription elongation in fission yeast.

Cyclin-dependent kinase 9 (Cdk9) promotes elongation by RNA polymerase II (RNAPII), mRNA processing, and co-transcriptional histone modification. Cdk9 phosphorylates multiple targets, including the conserved RNAPII elongation factor Spt5 and RNAPII itself, but how these different modifications mediate Cdk9 functions is not known. Here we describe two Cdk9-dependent pathways in the fission yeast Schizosaccharomyces pombe that involve distinct targets and elicit distinct biological outcomes. Phosphorylation of Spt5 by Cdk9 creates a direct binding site for Prf1/Rtf1, a transcription regulator with functional and physical links to the Polymerase Associated Factor (PAF) complex. PAF association with chromatin is also dependent on Cdk9 but involves alternate phosphoacceptor targets. Prf1 and PAF are biochemically separate in cell extracts, and genetic analyses show that Prf1 and PAF are functionally distinct and exert opposing effects on the RNAPII elongation complex. We propose that this opposition constitutes a Cdk9 auto-regulatory mechanism, such that a positive effect on elongation, driven by the PAF pathway, is kept in check by a negative effect of Prf1/Rtf1 and downstream mono-ubiquitylation of histone H2B. Thus, optimal RNAPII elongation may require balanced action of functionally distinct Cdk9 pathways.