Biology subjects

Stutz, F.

Publications and source records attributed to Stutz, F..

10 recordsLinked to original sources

bioRxiv2024

Refined mechanism of promoter Nucleosome-Depleted Regions resetting after replication

Replication disrupts chromatin organization. Thus, the rapid resetting of nucleosome positioning is essential to maintain faithful gene expression. The initial step of this reconfiguration occurs at Nucleosome-Depleted Regions (NDRs). While studies have elucidated the role of Transcription Factors (TFs) and Chromatin Remodelers (CRs) in vitro or in maintaining NDRs in vivo, none has addressed their in vivo function shortly after replication. Through purification of nascent chromatin in yeast, we dissected the choreography of events governing the proper positioning of the -1/+1 nucleosomes flanking promoter NDRs. Our findings reveal that CRs are the primary contributors of -1/+1 repositioning post-replication, with RSC acting upstream of INO80. Surprisingly, while Reb1 and Abf1 TFs are not essential for NDR resetting, they are required for NDR maintenance via the promotion of H3 acetylations. Altogether, we propose a two-step model for NDR resetting in S. cerevisiae: first, CRs alone reset promoter NDRs after replication, while a combination of TFs and CRs is required for subsequent maintenance. TeaserRSC acts upstream of INO80 for NDR re-establishment after replication followed by a combined action of CRs and TFs for NDR maintenance.

molecular biology↗

bioRxiv2022

Self-Regulation of Visual Word Form Area activation with real-time fMRI neurofeedback

The Visual Word Form Area (VWFA) is a key region of the brains reading network and its activation has been shown to be strongly associated with reading skills. Here, for the first time, we investigated whether voluntary regulation of VWFA activity is feasible using real-time fMRI neurofeedback. 40 adults with typical reading skills were instructed to either upregulate (UP group) or downregulate (DOWN group) their own VWFA activity during six neurofeedback training runs. The VWFA target region was individually defined based on a functional localizer task. Before and after training, also regulation runs without feedback ("no-feedback runs") were performed. When comparing the two groups, we found stronger activity across the whole reading network, including the VWFA, for the UP than the DOWN group. Crucially, we observed a significant interaction of group and time (pre, post) for the no-feedback runs: The two groups did not differ in their VWFA activity before neurofeedback training, but the UP group showed significantly stronger activity than the DOWN group after neurofeedback training. Our results indicate that self-regulation of the VWFA activity is feasible and that, once learned, successful self-regulation can even be performed in the absence of feedback. These results are a crucial step toward to development of a potential clinical intervention to improve reading skills in individuals with reading impairments.

neuroscience↗

bioRxiv2022

Ubx5-Cdc48 assists the protease Wss1 at DNA-protein crosslink sites in yeast

DNA-protein crosslinks (DPCs) pose a serious threat to genome stability. The yeast proteases Wss1, 26S proteasome and Ddi1 are safeguards of genome integrity by acting on a plethora of DNA-bound proteins in different cellular contexts. The AAA ATPase Cdc48/p97 is known to assist Wss1/SPRTN in clearing DNA-bound complexes, however its contribution to DPC proteolysis remains unclear. Here, we show that the Cdc48 adaptor Ubx5 is detrimental in yeast mutants defective in DPC processing. Using an inducible site-specific crosslink, we show that Ubx5 accumulates at persistent DPC lesions in the absence of Wss1, which prevents their efficient removal from the DNA. Abolishing Cdc48 binding or complete loss of Ubx5 suppresses the sensitivity of wss1{Delta} cells to DPC inducing agents by favoring alternate repair pathways. We provide evidence for cooperation of Ubx5-Cdc48 and Wss1 in the genotoxin-induced degradation of RNAPII, a described candidate substrate of Wss1. We propose that Ubx5-Cdc48 assists Wss1 for proteolysis of a subset of DNA-bound proteins. Together, our findings reveal a central role for Ubx5 in DPC clearance and repair.

molecular biology↗

bioRxiv2022

Antisense non-coding transcription represses the PHO5 model gene via remodelling of promoter chromatin structure

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.

molecular biology↗

bioRxiv2021

Antisense-mediated repression of SAGA-dependent genes involves the HIR histone chaperone

Eukaryotic genomes are pervasively transcribed by RNA polymerase II (RNAPII), and transcription of long non-coding RNAs often overlaps with coding gene promoters. This might lead to coding gene repression in a process named Transcription Interference (TI). In Saccharomyces cerevisiae (S. cerevisiae), TI is mainly driven by antisense non-coding transcription and occurs through re-shaping of promoter Nucleosome-Depleted Regions (NDRs). In this study, we developed a genetic screen to identify new players involved in Antisense-Mediated Transcription Interference (AMTI). Among the candidates, we found the HIR histone chaperone complex known to be involved in de novo histone deposition. Using genome-wide approaches, we reveal that HIR-dependent histone deposition represses the promoters of SAGA-dependent genes via antisense non-coding transcription. However, while antisense transcription is enriched at promoters of SAGA-dependent genes, this feature is not sufficient to define the mode of gene regulation. We further show that the balance between HIR-dependent nucleosome incorporation and transcription factor binding at promoters directs transcription into a SAGA- or TFIID-dependent regulation. This study sheds light on a new connection between antisense non-coding transcription and the nature of coding transcription initiation.

molecular biology↗

bioRxiv2020

SUMO orchestrates multiple alternative DNA-protein crosslink repair pathways

Several endogenous metabolites, environmental agents, and therapeutic drugs promote formation of covalent DNA-protein crosslinks (DPCs). Persistent DPCs pose a serious threat to genome integrity and are eliminated by multiple repair pathways. Aberrant Top1 crosslinks to DNA, or Top1ccs, are processed by Tdp1 and Wss1 functioning in parallel pathways in Saccharomyces cerevisiae. It remains obscure how cells choose between these diverse mechanisms of DPC repair. Here we show that several SUMO biogenesis factors - Ulp1, Siz2, Slx5, Slx8 - control repair of Top1cc or an analogous DPC lesion. Genetic analysis reveals that SUMO promotes Top1cc processing in the absence of Tdp1 but has an inhibitory role if cells additionally lack Wss1. In the tdp1{Delta} wss1{Delta} mutant, the E3 SUMO ligase Siz2 stimulates sumoylation in the vicinity of the DPC, but not SUMO conjugation to Top1. This Siz2-dependent sumoylation delays DPC repair when cells progress through S and G2 phases. Our findings suggest that SUMO tunes available repair pathways to facilitate faithful DPC repair.

molecular biology↗

bioRxiv2019

Fine chromatin-driven mechanism of transcription interference by antisense noncoding transcription

Eukaryotic genomes are almost entirely transcribed by RNA polymerase II (RNAPII). Consequently, the transcription of long noncoding RNAs (lncRNAs) often overlaps with coding gene promoters triggering potential gene repression through a poorly characterized mechanism of transcription interference. In this study, we propose a global model of chromatin-based transcription interference in Saccharomyces cerevisiae (S. cerevisiae). By using a noncoding transcription inducible strain, we analyzed the relationship between antisense elongation and coding sense repression, nucleosome occupancy and transcription-associated histone modifications using near-base pair resolution techniques. We show that antisense noncoding transcription leads to the deaceylation of a subpopulation of -1/+1 nucleosomes associated with increased H3K36 trimethylation (H3K36me3). Reduced acetylation results in decreased binding of the RSC chromatin remodeler at -1/+1 nucleosomes and subsequent sliding into the Nucleosome-Depleted Region (NDR) hindering Pre-Initiation Complex (PIC) association. Finally, we extend our model by showing that natural antisense noncoding transcription significantly represses around 20% of S. cerevisiae genes through this chromatin-based transcription interference mechanism.\n\nHighlightsO_LIInduction of antisense noncoding transcription leads to -1/+1 nucleosome sliding that competes with sense transcription PIC deposition.\nC_LIO_LIAntisense induction leads to a subpopulation of H3K36me3 nucleosomes differently positioned compared to H3K18ac nucleosomes.\nC_LIO_LIRSC chromatin remodeler recruitment to -1/+1 nucleosomes is modulated by histone acetylation levels.\nC_LIO_LI20% of S. cerevisiae genes are significantly repressed by this antisense-dependent chromatin-based transcription interference mechanism.\nC_LI

molecular biology↗

bioRxiv2019

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not timely repaired. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including topoisomerase-1 trapped in covalent crosslinks (Top1ccs). Here we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S-phase dependent manner to assist eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitize cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among potential Ddi1 targets we found the core component of RNAP II and show that its genotoxin-induced degradation is impaired in ddi1. Together, we propose that the Ddi1 protease contributes to DPC proteolysis.

molecular biology↗

bioRxiv2018

The mRNA export adaptor Yra1 contributes to DNA double-strand break repair through its C-box domain

Yra1 is an mRNA export adaptor involved in mRNA biogenesis and export in S. cerevisiae. Yra1 overexpression was recently shown to promote accumulation of DNA:RNA hybrids favoring DNA double strand breaks (DSB), cell senescence and telomere shortening, via an unknown mechanism. Yra1 was also identified at an HO-induced DSB and Yra1 depletion causes defects in DSB repair. Previous work from our laboratory showed that Yra1 ubiquitination by Tom1 is important for mRNA export. Interestingly, we found that Yra1 is also ubiquitinated by the SUMO-targeted ubiquitin ligases Slx5-Slx8 implicated in the interaction of irreparable DSB with nuclear pores. Here we show that Yra1 binds an HO-induced irreparable DSB. Importantly, a Yra1 mutant lacking the evolutionarily conserved C-box is not recruited to an HO-induced irreparable DSB and becomes lethal under DSB induction in a HO-cut reparable system. Together, the data provide evidence that Yra1 plays a crucial role in DSB repair via homologous recombination. Unexpectedly, while the Yra1 C-box is essential, Yra1 sumoylation and/or ubiquitination are dispensable in this process.

molecular biology↗

bioRxiv2018

Pervasive transcription-dependent chromatin remodeling influences the replication initiation program

In Eukaryotic organisms, replication initiation follows a temporal program. Among the parameters that regulate this program in Saccharomyces cerevisiae, chromatin structure has been at the center of attention without considering the contribution of transcription. Here, we revisit the replication initiation program in the light of pervasive transcription. We find that noncoding RNA transcription termination in the vicinity of replication origins or ARS (Autonomously Replicating Sequences) maximizes replication initiation by restricting transcriptional readthrough into ARS. Consistently, high natural nascent transcription correlates with low ARS efficiency and late replication timing. High readthrough transcription is also linked to chromatin features such as high levels of H3K36me3 and deacetylated nucleosomes. Moreover, forcing ARS readthrough transcription promotes these histone modifications. Finally, replication initiation defects induced by increased transcriptional readthrough are partially rescued in the absence of H3K36 methylation. Altogether, these observations indicate that natural pervasive transcription into ARS influences replication initiation through chromatin remodeling.

molecular biology↗