Genetics最新文献

Precise regulation of chromatin structure is essential for ensuring genome stability and cellular function. In Saccharomyces cerevisiae, the YEATS (Yaf9-ENL-AF9-Taf14-Sas5) domain protein Yaf9 is a shared component of the NuA4 acetyltransferase and the SWR1 chromatin remodelling complexes. We investigated the function of Yaf9 and discovered that it becomes essential for survival when histone H4 acetylation is impaired. The loss of Yaf9 in a strain with impaired H4 acetylation led to cell cycle arrest in the G2/M phase and activation of the homologous recombination pathway. This synthetic lethality was not recapitulated by inactivating the Yaf9 YEATS domain, suggesting that it is independent of Yaf9's ability to recognise acyl-modified lysine residues. We also found that Yaf9 was required in both NuA4 and SWR1 complexes to ensure cell viability in the absence of H4 acetylation. Together, these findings reveal a compensatory relationship between Yaf9 and histone H4 acetylation, suggesting that Yaf9 acts as a functional link between chromatin remodelling and histone modification pathways to maintain genome integrity under conditions of chromatin stress.

Histone H1 and related linker histones play critical roles in chromosome organization in eukaryotic cells. Although histone H1 is essential for compacting nucleosomes into chromatin fibers and is a major structural component of chromosomes, its association with chromatin is highly dynamic. Histone H1 exchange modulates the accessibility of regulatory proteins to DNA and has been implicated in the regulation of gene expression and cellular pluripotency. Relatively little is known, however, about how histone H1 binding, exchange and function is regulated in vivo. In this study, we investigated the regulation of histone H1 function in Drosophila using live analysis and confocal microscopy. A gain-of-function genetic screen identified several factors that affect chromosome structure, histone H1 binding or histone H1 exchange, including the ATP-dependent chromatin-remodeling factor XNP, the hypoxia-induced factor Scylla, the winged helix transcription factor Jumeau and the microRNA bantam. Our findings show that altered expression of single factors can have surprisingly global effects on higher-order chromatin structure and histone H1 binding in vivo, with the potential to trigger large scale changes in genome organization and accessibility.

The optimal choice of parents and crosses and, therefore, the prediction of the segregation variance are of high relevance to maximize genetic gain in breeding programs. Several methods have been developed for the prediction of segregation variance, includingcorrelation with genotypic diversity, progeny simulations, or algebraic derivations in case of a diploid inheritance. To the best of our knowledge, no algebraic derivation using parental genotypic information is available to predict segregation variance for autotetraploid species. The objectives of our study were to (1) derive algebraic derivation based on linkage disequilibrium (LD) between linked loci to predict the segregation variance in autotetraploid species; (2) compare the performance of segregation variance estimated based on simulated progenies and the algebraic derivations; (3) investigate by simulations how experimental parameters affect the accuracy of segregation variance prediction; and (4) compare the segregation variance estimated in empirical data of potato and the one based on the algebraic derivations. The segregation variance estimated by the developed derivations showed very high correlations with the one observed in large simulated progenies, but those were lower when phased parental haplotypes were not available or family size decreased. The correlation between segregation variance estimated by the developed derivation and the empirical data was low. This could be attributed to the small family size used in the study, which we could show to increase LD between unlinked loci. The proposed algebraic derivations promise to be a precise alternative to simulations to help breeders in optimizing their family choices and sizes considering the segregation variance.

Many mutations have detrimental effects. The mutation load in a population depends on the efficacy of purifying selection in removing deleterious genetic variation. Here, we estimated the proportion of deleterious mutations segregating in 24 population samples of 19 bird species. Exploiting the conserved avian karyotype with high variation in recombination rate and GC content, we quantified the joint effects of effective population size (Ne), recombination (r) and GC-biased gene-conversion (gBGC). In agreement with the nearly-neutral theory of molecular evolution, mutation load was substantially higher in populations with small Ne. Purging efficacy increased with recombination rate resulting in more than a two-fold difference of genetic load between large and small chromosomes. GC-biased mutations contributed about one third to the pool of deleterious mutations. Their expected accumulation in regions of high recombination was offset by purging efficacy in large, but not small populations. This study provides insight into how the interaction of evolutionary processes shapes mutation load. It suggests that genetic risk factors in small populations are fueled by gBGC and cluster in regions of low recombination.

Transcription initiation by RNA Pol II is driven by a preinitiation complex (PIC) comprising Pol II and general transcription factors (GTFs): TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH. In Saccharomyces cerevisiae, transcription start site (TSS) selection proceeds by a promoter scanning mechanism where the PIC scans downstream for TSSs. To determine which factors participate in TSS selection by promoter scanning, we designed and implemented forward genetic selections using initiation-sensitive genetic reporters. These reporters were of two types, either being sensitive to upstream or downstream TSS shifts, allowing detection of alleles that affect promoter scanning in different directions. From >1000 candidates, we identified three primary classes of mutants: existing and novel mutants within known PIC components, including novel alleles of multiple TFIIH subunits; mutants that alter promoter scanning indirectly through cellular conditions (GTP levels and Mn2+ levels); and mutants in chromatin and transcription elongation-related factors. Genome-wide analysis using TSS mapping shows that tested PIC mutants alter TSS usage globally across promoters while tested chromatin and transcription elongation mutants had much more limited effects. These studies expand the spectrum of mutants able to perturb initiation by promoter scanning and suggest potential plasticity in the TFIIH structure that may be part of the evolution of this initiation mechanism.

The abnormal oocyte (ao) gene of Drosophila melanogaster is a maternal-effect lethal gene previously identified as encoding a transcriptional regulator of core histones. However, background genetic mutations in existing ao mutant strains could compromise their utility in manipulating histone levels. To distinguish the true ao phenotype from background effects, we created two new ao reagents: a CRISPR/Cas9-mediated knockout of the ao allele for genetic and molecular analyses and an epitope-tagged ao allele for cytological experiments. Using these reagents, we confirm previous findings that loss of ao causes maternal-effect lethality, which can be rescued by either a decrease in the histone gene copy number or by Y chromosome heterochromatin. Our data indicate that ao genetically interacts with the heterochromatin, as previously suggested. However, contrary to a prior study, we detected neither Ao localization to histone genes nor ao repression of core histone transcript levels. Thus, the molecular basis for ao-associated maternal-effect lethality remains unknown.

The chromatin remodelling factor and histone chaperone Yta7 is a member of the ATAD2 family of AAA+ ATPases from Saccharomyces cerevisiae that has in vivo functions consistent with both nucleosome assembly and disassembly activity. At the centromere, Yta7 is required for proper deposition of the centromeric histone H3 variant CENP-A Cse4. Here, we performed a genetic screen to identify suppressors of the defect of a mutation in CENP-A Cse4 that impairs the interaction with the DNA of the centromeric nucleosome (cse4-S135A). This identified two suppressor alleles of YTA7, yta7-R483S and -D518E, which are in the AAA1 domain of Yta7. Interestingly, Yta7-R483S enhanced the deposition of CENP-A Cse4 at the centromere and showed a ∼40% increased ATPase activity, suggesting that the hyperactivity of the motor domain is responsible for suppression of the cse4-S135A growth defect. In contrast, Yta7-D518E showed reduced ATPase activity, but both Yta7-R483S and -D518E retained the interaction with CENP-A Cse4 and centromeric sequences as well as hexamer formation in vitro. Our analysis of in vivo interactions between Yta7 and CENP-A Cse4 further showed that the two AAA+ domains and the non-canonical bromodomain of Yta7 are necessary and sufficient for interaction with CENP-A Cse4. The genetic screen furthermore revealed a mutation in the chromatin remodeler Fun30 as a suppressor of the centromeric defect of cse4-S135A. Altogether, this work reveals unusual, hypermorphic properties of Yta7 variants and highlights the importance of nucleosome remodelers in establishing centromeric chromatin.

Maltose is one of the most abundant sugars in brewer's wort, and its efficient utilization is critical for successful fermentation. However, maltose consumption varies naturally among Saccharomyces eubayanus strains isolated from different host trees, such as Quercus and Nothofagus. To identify the genetic determinants underlying these phenotypic differences, we performed bulk segregant analysis (BSA) and quantitative trait loci (QTL) mapping using an F2 offspring derived from QC18 (Quercus-associated) and CL467.1 (Nothofagus-associated) strains. QTL mapping identified two significant genomic regions on subtelomeric loci of chromosomes V-R and XVI-L, each containing complete MAL loci composed of MAL32 (encoding maltase), MAL31 (transporter), and MAL33 (transcriptional activator) genes. Comparative polymorphism analyses identified mutations in MAL32 and MAL33 of QC18, including frameshift mutations resulting in premature stop codons. Functional validation demonstrated that the heterologous expression of MAL33ChrV from CL467.1 fully restored maltose utilization in QC18, indicating the functional presence of MAL33 cis-regulatory sequences and MAL32 and MAL31 genes in QC18. While structural protein predictions identified truncation and impaired functionality in the maltose-responsive activation domain of Mal33p from QC18, overexpression of QC18's own MAL33ChrV allele also improved maltose metabolism, suggesting dosage-dependent transcriptional limitations rather than complete functional loss. These results indicate that allelic variations in the maltose-responsive activation domain of Mal33p result in differences in maltose consumption between strains. We hypothesized that reduced maltose metabolism in QC18 is an adaptive response to the distinct sugar composition in Quercus robur bark, contrasting with the starch-rich environment of Nothofagus pumilio. These findings highlight subtelomeric MAL gene diversity as a reservoir of genetic variation, representing a key evolutionary mechanism that influences maltose adaptation among natural Saccharomyces isolates.

The genome is compacted in the nucleus through a hierarchical chromatin organization, ranging from chromosome territories to compartments, topologically associating domains (TADs), and individual nucleosomes. Nucleosome remodeling complexes hydrolyze ATP to translocate DNA and thereby mobilize histone proteins. While nucleosome remodeling complexes have been extensively studied for their roles in regulating nucleosome positioning and accessibility, their contributions to higher-order chromatin architecture remain less well understood. Here, we investigate the roles of two key nucleosome remodelers, esBAF and INO8°C, in shaping 3D genome organization in mouse embryonic stem cells. Using Hi-C, we find that loss of either remodeler has minimal effects on global compartment or TAD structures. In contrast, subcompartment organization is notably altered, suggesting that esBAF and INO8°C contribute to finer-scale chromatin topology. To overcome the limited resolution of Hi-C for detecting regulatory loops, we employed promoter capture Micro-C (PCMC), which revealed that the loss of esBAF or INO8°C alters a subset of promoter anchored looping interactions. Although these changes occur at distinct genomic loci for each remodeler, the affected sites are commonly enriched for bivalent chromatin regions bound by OCT4, SOX2, and NANOG (OSN), as well as BRG1 and INO80 themselves. Together, our findings reveal that esBAF and INO8°C selectively influence subcompartment identity and enhancer-promoter communication at key regulatory loci, highlighting a previously underappreciated role for nucleosome remodelers in higher-order chromatin organization.

Ligand-gated acetylcholine receptors (AChRs) are pentameric transmembrane proteins that display extensive molecular diversity through combinatorial assembly of subunits. In the nematode Caenorhabditis elegans, this diversity is exceptionally expanded, with nearly 60 AChR subunit genes, yet the composition and function of many receptor subtypes remain poorly defined. At neuromuscular junctions (NMJs), L-AChRs are heteromeric AChRs sensitive to levamisole, a nematode-specific L-AChR agonist, that play a crucial role in locomotion. Forward genetic screens for mutants resistant to levamisole have identified the L-AChR canonical subunits (LEV-1, UNC-29, LEV-8, UNC-63 and UNC-38) along with key biosynthetic regulators. ACR-8 is an alternative L-AChR subunit that has been poorly characterized, largely because acr-8 mutants remain sensitive to levamisole. Using genetic approaches and in vivo imaging, we show that ACR-8-containing L-AChRs (ACR-8*) are present at the NMJ and, surprisingly, they are more abundant than the well-characterized LEV-8-containing L-AChRs (LEV-8*). Although both LEV-8* and ACR-8* are enriched at synapses and rely on the same clustering machinery, they segregate into distinct postsynaptic nanodomains. The two populations play a redundant function in worm locomotion, and in mutant conditions, one receptor population can compensate for the loss of the other. Interestingly, ACR-8* are already present at high levels at the NMJ from early developmental stages, whereas LEV-8* levels gradually increase throughout development. Our work sheds new light on the molecular landscape of AChRs of the C. elegans NMJ.