Biology subjects

Carlier, F.

Publications and source records attributed to Carlier, F..

5 recordsLinked to original sources

bioRxiv2026

SAD-6/ATRX enables broad genome surveillance and defense in fungi

The chromatin remodeler ATRX and its orthologs maintain genome function by regulating repetitive DNA and dynamic chromatin, and their activities have been canonically associated with replication-independent deposition of the histone H3.3 variant. This model is difficult to reconcile with fungi, which encode ATRX orthologs but lack H3 variants that may separately support replication-coupled and replication-independent deposition. Here we show that the fungal ATRX ortholog SAD-6 instead relies on a highly divergent histone H4 variant (H4v) to mediate broad genome surveillance and defense. Deposition of H4v is strictly SAD-6-dependent and thus provides a sensitive genome-wide readout of SAD-6 activity, revealing its functions at telomeres, tRNA and rDNA loci, AT-rich DNA, artificial transgenes, decaying mobile elements, and many genic regions. We further show that SAD-6 is required for a pathway of repeat-induced point mutation (RIP) that also requires DIM-5, a conserved SUV39 methyltransferase that mediates trimethylation of histone H3 lysine-9 in heterochromatin. Together, these findings establish ATRX-like remodelers as broad regulators of genome surveillance and defense in fungi that act through a highly divergent histone H4 variant rather than H3.3. Given that RIP is proposed to recognize repetitive DNA via recombination-independent homologous pairing, the requirement for SAD-6 in RIP suggests that ATRX-like remodelers may couple DNA pairing to heterochromatin nucleation on repeats.

molecular biology↗

bioRxiv2024

Chromatin remodeling initiates transcriptional and post-transcriptional silencing in Neurospora

In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6 (a fungal ortholog of the SWI/SNF chromatin remodeler ATRX), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin. SIGNIFICANCE STATEMENTThis study addresses an enigmatic question of how transcriptional and post-transcriptional gene silencing can be initiated de novo in the absence of strong sequence-specific cues. Using the fungus Neurospora crassa as a model organism, we found that both types of silencing can be triggered in mitotic cells by the remodeling of a transiently perturbed (nucleosome-depleted) chromatin state. In this system, the initiation of silencing requires SAD-6, a conserved SWI/SNF chromatin remodeler orthologous to ATRX that has been already implicated in repetitive DNA silencing in fungi, plants, and animals. Thus, the model proposed in this study may underpin a range of gene-silencing phenomena observed in other eukaryotes.

molecular biology↗

bioRxiv2021

Regulation of DIM-2-dependent repeat-induced point mutation (RIP) by the recombination-independent homologous DNA pairing in Neurospora crassa

Repeat-induced point mutation (RIP) is a genetic process that creates cytosine-to-thymine (C-to-T) transitions in duplicated genomic sequences in fungi. RIP detects duplications irrespective of their origin, particular sequence, coding capacity, or genomic positions. Previous studies suggested that RIP involves a cardinally new mechanism of sequence recognition that operates on intact double-stranded DNAs. In the fungus Neurospora crassa, RIP can be mediated by a putative C5-cytosine methyltransferase (CMT) RID or/and a canonical CMT DIM-2. These distinct RIP pathways feature opposite substrate preferences: RID-dependent RIP is largely limited to the duplicated sequences, whereas DIM-2-dependent RIP preferentially mutates adjacent non-repetitive regions. Using DIM-2-dependent RIP as a principal readout of repeat recognition, here we show that GC-rich repeats promote stronger RIP compared to AT-rich repeats (independently of their intrinsic propensities to become mutated), with the relative contribution of AT base-pairs being close to zero. We also show that direct repeats promote much more efficient DIM-2-dependent RIP than inverted repeats; both the spacer DNA between the repeat units (the linker) and the flanking regions are similarly affected by this process. These and other results support the idea that repeat recognition for RIP involves formation of many short interspersed quadruplexes between homologous double-stranded DNAs, which need to undergo concomitant changes in their linking number to accommodate pairing. SUMMARYDuring repeat-induced point mutation (RIP) gene-sized duplications of genomic DNA are detected by a mechanism that likely involves direct pairing of homologous double-stranded DNAs. We show that DIM-2-dependent RIP, triggered by closely-positioned duplications, is strongly affected by their relative orientations (direct versus inverted). We also show that GC-rich repeats promote RIP more effectively than AT-rich repeats. These results support a model in which homologous dsDNAs can pair by establishing interspersed quadruplex-based contacts with concomitant changes in their supercoiling status.

genetics↗

bioRxiv2020

Loss of Polycomb Protein EZH2 causes major depletion of H3K27 and H3K9 tri-methylation and developmental defects in the fungus Podospora anserina

Selective gene silencing is key to development. The H3K27me3 enriched heterochromatin maintains transcription repression established during early development and regulates cell fate. Conversely, H3K9me3 enriched heterochromatin prevents differentiation but constitutes a permanent protection against transposable element. We exploited the fungus Podospora anserina, a valuable alternative to higher eukaryote models to question the biological relevance and interplay of these two distinct heterochromatin conformations. We found that H3K27me3 and H3K9me3 modifications are mutually exclusive within gene-rich regions but not within repeats. Lack of PaKmt6 EZH2-like enzyme resulted in loss of H3K27me3 and in significant H3K9me3 reduction, whereas lack of PaKmt1 SU(VAR)3-9-like enzyme caused loss of H3K9me3 only. We established that P. anserina developmental programs require H3K27me3 mediated silencing unlike most fungi studied to date. Our findings provide new insight into roles of these histone marks and into the relationship between chromatin modifications and development.

genetics↗

bioRxiv2019

A RID-like cytosine methyltransferase homologue controls sexual development in the fungus Podospora anserina

DNA methyltransferases are ubiquitous enzymes conserved in bacteria, plants and opisthokonta. These enzymes, which methylate cytosines, are involved in numerous biological processes, notably development. In mammals and higher plants, methylation patterns established and maintained by the cytosine DNA methyltransferases (DMTs) are essential to zygotic development. In fungi, some members of an extensively conserved fungal-specific DNA methyltransferase class are both mediators of the Repeat Induced Point mutation (RIP) genome defense system and key players of sexual reproduction. Yet, no DNA methyltransferase activity of these purified RID (RIP deficient) proteins could be detected in vitro. These observations led us to explore how RID-like DNA methyltransferase encoding genes would play a role during sexual development of fungi showing very little genomic DNA methylation, if any.\n\nTo do so, we used the model ascomycete fungus P. anserina. We identified the PaRid gene, encoding a RID-like DNA methyltransferase and constructed knocked-out {Delta}PaRid defective mutants. Crosses involving P. anserina {Delta}PaRid mutants are sterile. Our results show that, although gametes are readily formed and fertilization occurs in a {Delta}PaRid background, sexual development is blocked just before the individualization of the dikaryotic cells leading to meiocytes. Complementation of {Delta}PaRid mutants with ectopic alleles of PaRid, including GFP-tagged, point-mutated, inter-specific and chimeric alleles, demonstrated that the catalytic motif of the putative PaRid methyltransferase is essential to ensure proper sexual development and that the expression of PaRid is spatially and temporally restricted. A transcriptomic analysis performed on mutant crosses revealed an overlap of the PaRid-controlled genetic network with the well-known mating-types gene developmental pathway common to an important group of fungi, the Pezizomycotina.\n\nAuthor SummarySexual reproduction is considered to be essential for long-term persistence of eukaryotic species. Sexual reproduction is controlled by strict mechanisms governing which haploids can fuse (mating) and which developmental paths the resulting zygote will follow. In mammals, differential genomic DNA methylation patterns of parental gametes, known as DNA methylation imprints are essential to zygotic development, while in plants, global genomic demethylation often results in female-sterility. Although animal and fungi are evolutionary related, little is known about epigenetic regulation of gene expression and development in multicellular fungi. Here, we report on a gene of the model fungus Podospora anserina, encoding a protein called PaRid that looks like a DNA methyltrasferase. We showed that expression of the catalytically functional version of the PaRid protein is required in the maternal parental strain to form zygotes. By establishing the transcriptional profile of PaRid mutant strain, we identified a set of PaRid direct and/or indirect target genes. Half of them are also targets of a mating-type transcription factor known to be a major regulator of sexual development. So far, there was no other example of identified RID targets shared with a well-known developmental pathway that is common to an important group of fungi, the Pezizomycotina

genetics↗