A Novel Transcription-Interference Mechanism To Suppress Checkpoint

Meiosis is a linear differentiation pathway remarkable for deliberate DNA damage. During meiotic prophase, DNA damage checkpoint is repressed, which may promote homologous recombination and repair. Meiotic breaks are known to influence meiotic chromosome segregation, preventing aneuploidy in gametes. Convergent genes cause transcriptional interference through overlapping transcripts, resulting in gene suppression. This general mechanism of gene silencing has been noted in both yeast and metazoans. In Schizosaccharomyces pombe, chk1⁺ is convergent with the meiotically upregulated meu27⁺ gene. Through gain- and loss-of-function assays we are developing a model of checkpoint regulation during nitrogen stress. We find that altered meu27⁺ expression impacts chk1⁺ mRNA levels, DNA segregation, and cell cycle progression. S. pombe encodes several DNA damage checkpoint genes that are convergent with stress-inducible transcripts. Therefore, we investigate the possibility that convergent transcription is a mechanism altering DNA damage repair during other stresses and differentiation programmes which may trigger unregulated cell division.

A Novel Transcription-Interference Mechanism To Suppress Checkpoint

Meiosis is a linear differentiation pathway remarkable for deliberate DNA damage. During meiotic prophase, DNA damage checkpoint is repressed, which may promote homologous recombination and repair. Meiotic breaks are known to influence meiotic chromosome segregation, preventing aneuploidy in gametes. Convergent genes cause transcriptional interference through overlapping transcripts, resulting in gene suppression. This general mechanism of gene silencing has been noted in both yeast and metazoans. In Schizosaccharomyces pombe, chk1⁺ is convergent with the meiotically upregulated meu27⁺ gene. Through gain- and loss-of-function assays we are developing a model of checkpoint regulation during nitrogen stress. We find that altered meu27⁺ expression impacts chk1⁺ mRNA levels, DNA segregation, and cell cycle progression. S. pombe encodes several DNA damage checkpoint genes that are convergent with stress-inducible transcripts. Therefore, we investigate the possibility that convergent transcription is a mechanism altering DNA damage repair during other stresses and differentiation programmes which may trigger unregulated cell division.

Epigenetic Requirements for Triggering Heterochromatinization and Piwi-Interacting RNA Production from Transgenes in the Drosophila Germline

Transgenes containing a fragment of the I retrotransposon represent a powerful model of piRNA cluster de novo formation in the Drosophila germline. We revealed that the same transgenes located at different genomic loci form piRNA clusters with various capacity of small RNA production. Transgenic piRNA clusters are not established in piRNA pathway mutants. However, in the wild-type context, the endogenous ancestral I-related piRNAs heterochromatinize and convert the I-containing transgenes into piRNA-producing loci. Here, we address how the quantitative level of piRNAs influences the heterochromatinization and piRNA production. We show that a minimal amount of maternal piRNAs from ancestral I-elements is sufficient to form the transgenic piRNA clusters. Supplemental piRNAs stemming from active I-element copies do not stimulate additional chromatin changes or piRNA production from transgenes. Therefore, chromatin changes and piRNA production are initiated by a minimum threshold level of complementary piRNAs, suggesting a selective advantage of prompt cell response to the lowest level of piRNAs. It is noteworthy that the weak piRNA clusters do not transform into strong ones after being targeted by abundant I-specific piRNAs, indicating the importance of the genomic context for piRNA cluster establishment. Analysis of ovarian transcription profiles suggests that regions facilitating convergent transcription favor the formation of transgenic piRNA clusters.

Trafficking of siRNA precursors by the dsRBD protein Blanks in Drosophila

RNA interference targets aberrant transcripts with cognate small interfering RNAs, which derive from double-stranded RNA precursors. Several functional screens have identified Drosophila blanks/lump (CG10630) as a facilitator of RNAi, yet its molecular function has remained unknown. The protein carries two dsRNA binding domains (dsRBD) and blanks mutant males have a spermatogenesis defect. We demonstrate that blanks selectively boosts RNAi triggered by dsRNA of nuclear origin. Blanks binds dsRNA via its second dsRBD in vitro, shuttles between nucleus and cytoplasm and the abundance of siRNAs arising at many sites of convergent transcription is reduced in blanks mutants. Since features of nascent RNAs - such as introns and transcription beyond the polyA site – contribute to the small RNA pool, we propose that Blanks binds dsRNA formed by cognate nascent RNAs in the nucleus and fosters its export to the cytoplasm for dicing. We refer to the resulting small RNAs as blanks exported siRNAs (bepsiRNAs). While bepsiRNAs were fully dependent on RNA binding to the second dsRBD of blanks in transgenic flies, male fertility was not. This is consistent with a previous report that linked fertility to the first dsRBD of Blanks. The role of blanks in spermatogenesis appears thus unrelated to its role in dsRNA export.

Principles of Meiotic Chromosome Assembly

During meiotic prophase, chromosomes organise into a series of chromatin loops emanating from a proteinaceous axis, but the mechanisms of assembly remain unclear. Here we elucidate how this elaborate three-dimensional chromosome organisation is underpinned by genomic sequence in Saccharomyces cerevisiae. Entering meiosis, strong cohesin-dependent grid-like Hi-C interaction patterns emerge, reminiscent of mammalian interphase organisation, but with distinct regulation. Meiotic patterns agree with simulations of loop extrusion limited by barriers, yet are patterned by convergent transcription rather than binding of the mammalian interphase factor, CTCF, which is absent in S. cerevisiae—thereby both challenging and extending current paradigms of local chromosome organisation. While grid-like interactions emerge independently of meiotic chromosome synapsis, synapsis itself generates additional compaction that matures differentially according to telomere proximity and chromosome size. Collectively, our results elucidate fundamental principles of chromosome assembly and demonstrate the essential role of cohesin within this evolutionarily conserved process.

Transcription-coupled genetic instability marks acute lymphoblastic leukemia structural variation hotspots

Progression of malignancy to overt disease requires multiple genetic hits. Activation-induced deaminase (AID) can drive lymphomagenesis by generating off-target DNA breaks at loci that harbor highly active enhancers and display convergent transcription. The first active transcriptional profiles from acute lymphoblastic leukemia (ALL) patients acquired here reveal striking similarity at structural variation (SV) sites. Specific transcriptional features, namely convergent transcription and Pol2 stalling, were detected at breakpoints. The overlap was most prominent at SV with recognition motifs for the recombination activating genes (RAG). We present signal feature analysis to detect vulnerable regions and quantified from human cells how convergent transcription contributes to R-loop generation and RNA polymerase stalling. Wide stalling regions were characterized by high DNAse hypersensitivity and unusually broad H3K4me3 signal. Based on 1382 pre-B-ALL patients, the ETV6-RUNX1 fusion positive patients had over ten-fold elevation in RAG1 while high expression of AID marked pre-B-ALL lacking common cytogenetic changes.