In eukaryotes the assembly of DNA into highly condensed heterochromatin is critical for a broad range of functions related to genome integrity. The methylation of histone H3 on lysine 9 (H3K9me) is central to the formation of heterochromatin by creating binding sites for a range of chromatin proteins important for silencing transposable elements, chromosome segregation, and epigenetic inheritance. S. pombe is an excellent model that has been used extensively to study the molecular mechanisms that generate and regulate heterochromatin. Centromeres, subtelomeres, and the mating-type region are packaged into constitutive heterochromatin while meiosis genes are silenced by facultative heterochromatin until cells are starved for nitrogen. Importantly, Clr4, the histone H3K9 methyltransferase is recruited to heterochromatin regions by multiple mechanisms. Constitutive heterochromatin results from RNAi factors that include Ago1 containing RNA-induced transcriptional silencing complex (RITS). Facultative heterochromatin at meiosis genes is independent of RNAi and relies on RNA elimination factors Red1, Mmi1, and the nuclear exosome. However, gaps exist in understanding how different processing activities function to target heterochromatin. A new approach for identifying gene function is the high throughput sequencing of integration profiles also known as Tn-Seq which identifies genes important for growth in selective conditions. Genes necessary to sustain growth under a specific condition do not tolerate insertions in that condition. Tn-seq has been applied to identify pathogenic genes in bacteria. However, we developed this method for identifying essential genes in yeast and subsequently others have applied the strategy. With the goal of identifying novel factors important for heterochromatin we produced dense profiles of integrations using the Hermes transposable element and a silencing reporter (ura4) positioned in the outer repeats of centromere 1. Inserts that disrupted genes important for heterochromatin activated ura4 and thus the cells were unable to grow when passaged in 5-fluoroorotic acid (FOA). Genes with established roles in heterochromatin assembly had significantly fewer insertions in the cells with the centromere reporter otr1R::ura4 relative to cells lacking the reporter. The list of candidates contained a total of 199 genes and importantly, 65 are known to be essential for viability. These essential genes were candidates because they tolerated many insertions in their 3sequences that reduced heterochromatin but not viability. The high number of essential genes is significant in that most proteins found to be important for heterochromatin are identified in screens of deletion strains that cannot include essential genes. The 199 candidates showed highly significant enrichments for functions in silencing at centromere outer repeats and included all four factors that produce siRNA. Other RNA processing factors were identified that were not previously linked to heterochromatin structure. Strikingly, four of the RNA processing candidates form an interaction module of canonical mRNA polyadenylation and cleavage factors (CPF) as predicted from highly homologous proteins in S. cerevisiae. To determine whether polyadenylation and cleavage contributes to heterochromatin structure at the centromere repeats we focused on the function of Iss1, a subunit of CPF. We generated a C-terminal truncation of Iss1 (Iss1-C) removing 38 amino acids that, based on the Hermes insertions, were not important for viability. Iss1-C showed no growth restriction on non-selective medium but exhibited a heterochromatin defect as demonstrated by growth in the absence of uracil and reduced levels of H3K9 dimethylation (H3K9me2) at otr1R::ura4. These results demonstrate the Hermes screen correctly identified Iss1 as important for heterochromatin structure at the otr1R::ura4 reporter. Interestingly, we found Iss1 contributes to heterochromatin of centromere repeats in cells that lack the otr1R::ura4 reporter but in this case the contribution to H3K9me2 was only observed when the RNAi pathway was disabled by deletion of ago1. This role at the outer centromere repeats is therefore independent or redundant with RNAi. We expanded our study of the Iss1-C mutation to evaluate changes in expression and transcription termination genome-wide. RNA-seq data revealed Iss1-C did not significantly impact canonical transcription termination but 73 genes were found to have higher expression. Importantly, these genes overlapped significantly with genes upregulated in cells lacking Rrp6, the 3-5 exonuclease subunit of the nuclear exosome. Rrp6, as a key subunit of the nuclear exosome plays an important role in RNA surveillance in the degradation of meiotic transcripts expressed during vegetative growth and the resulting formation of heterochromatin at these genes. The elimination of meiotic mRNAs depends on the RNA binding protein Mmi1 to bind the determinant of selective removal (DSR) sequence in order to recruit the exosome. Our co-IP experiments revealed Iss1 interacted with Rrp6, Mmi1, and the polyA polymerase Pla1, indicating Iss1 is associated with this network of elimination factors. Significantly, this interaction with Mmi1 was disrupted by the Iss1-C mutation. We tested whether Iss1 plays a direct role in the heterochromatin of meiotic genes by performing ChIP-seq of Iss1-FLAG. While a subset of Iss1-bound genes was highly-expressed and was associated with the canonical function of Iss1 in mRNA termination, most Iss1-bound peaks showed a strong correlation with genes regulated by RNA elimination and heterochromatin. Importantly, the iss1-C mutation caused significant increases in RNA levels of these genes. Taken together, these studies of RNA levels, Iss1 association with chromatin, and H3K9me2 indicate that Iss1 plays a direct role in the formation of heterochromatin at meiotic genes. Our application of Hermes profiles to identify genes important for heterochromatin formation demonstrates the significance of the approach especially given that we were able to identify large numbers of essential genes, a result that is not obtainable with other screens.
| Sangesland, Maya; Atwood-Moore, Angela; Rai, Sudhir K et al. (2016) Qualitative and Quantitative Assays of Transposition and Homologous Recombination of the Retrotransposon Tf1 in Schizosaccharomyces pombe. Methods Mol Biol 1400:117-30 |
