Sequential deregulation of histone marks, chromatin accessibility and gene expression in response to PROTAC-induced degradation of ASH2L.

The trithorax protein ASH2L is essential for organismal and tissue development. As a subunit of COMPASS/KMT2 complexes, ASH2L is necessary for methylation of histone H3 lysine 4 (H3K4). Mono- and tri-methylation at this site mark active enhancers and promoters, respectively, although the functional relevance of H3K4 methylation is only partially understood. ASH2L has a long half-life, which results in a slow decrease upon knockout. This has made it difficult to define direct consequences. To overcome this limitation, we employed a PROTAC system to rapidly degrade ASH2L and address direct effects. ASH2L loss resulted in inhibition of proliferation of mouse embryo fibroblasts. Shortly after ASH2L degradation H3K4me3 decreased with its half-life varying between promoters. Subsequently, H3K4me1 increased at promoters and decreased at some enhancers. H3K27ac and H3K27me3, histone marks closely linked to H3K4 methylation, were affected with considerable delay. In parallel, chromatin compaction increased at promoters. Of note, nascent gene transcription was not affected early but overall RNA expression was deregulated late after ASH2L loss. Together, these findings suggest that downstream effects are ordered but relatively slow, despite the rapid loss of ASH2L and inactivation of KMT2 complexes. It appears that the systems that control gene transcription are well buffered and strong effects are only beginning to unfold after considerable delay.

Loss of the Ash2l subunit of histone H3K4 methyltransferase complexes reduces chromatin accessibility at promoters.

Changes in gene expression programs are intimately linked to cell fate decisions. Post-translational modifications of core histones contribute to control gene expression. Methylation of lysine 4 of histone H3 (H3K4) correlates with active promoters and gene transcription. This modification is catalyzed by KMT2 methyltransferases, which require interaction with 4 core subunits, WDR5, RBBP5, ASH2L and DPY30, for catalytic activity. Ash2l is necessary for organismal development and for tissue homeostasis. In mouse embryo fibroblasts (MEFs), Ash2l loss results in gene repression, provoking a senescence phenotype. We now find that upon knockout of Ash2l both H3K4 mono- and tri-methylation (H3K4me1 and me3, respectively) were deregulated. In particular, loss of H3K4me3 at promoters correlated with gene repression, especially at CpG island promoters. Ash2l loss resulted in increased loading of histone H3 and reduced chromatin accessibility at promoters, accompanied by an increase of repressing and a decrease of activating histone marks. Moreover, we observed altered binding of CTCF upon Ash2l loss. Lost and gained binding was noticed at promoter-associated and intergenic sites, respectively. Thus, Ash2l loss and reduction of H3K4me3 correlate with altered chromatin accessibility and transcription factor binding. These findings contribute to a more detailed understanding of mechanistic consequences of H3K4me3 loss and associated repression of gene transcription and thus of the observed cellular consequences.

Induction of senescence upon loss of the Ash2l core subunit of H3K4 methyltransferase complexes.

Gene expression is controlled in part by post-translational modifications of core histones. Methylation of lysine 4 of histone H3 (H3K4), associated with open chromatin and gene transcription, is catalyzed by type 2 lysine methyltransferase complexes that require WDR5, RBBP5, ASH2L and DPY30 as core subunits. Ash2l is essential during embryogenesis and for maintaining adult tissues. To expand on the mechanistic understanding of Ash2l, we generated mouse embryo fibroblasts (MEFs) with conditional Ash2l alleles. Upon loss of Ash2l, methylation of H3K4 and gene expression were downregulated, which correlated with inhibition of proliferation and cell cycle progression. Moreover, we observed induction of senescence concomitant with a set of downregulated signature genes but independent of SASP. Many of the signature genes are FoxM1 responsive. Indeed, exogenous FOXM1 was sufficient to delay senescence. Thus, although the loss of Ash2l in MEFs has broad and complex consequences, a distinct set of downregulated genes promotes senescence.

Ultraviolet Mutagenesis Coupled with Next-Generation Sequencing as a Method for Functional Interrogation of Powdery Mildew Genomes.

Powdery mildews are obligate biotrophic fungal pathogens causing important diseases of plants worldwide. Very little is known about the requirements for their pathogenicity at the molecular level. This is largely due to the inability to culture these organisms in vitro or to modify them genetically. Here, we describe a mutagenesis procedure based on ultraviolet (UV) irradiation to accumulate mutations in the haploid genome of the barley powdery mildew pathogen Blumeria graminis f. sp. hordei. Exposure of B. graminis f. sp. hordei conidia to different durations of UV-C radiation (10 s to 12 min) resulted in a reduced number of macroscopically visible fungal colonies. B. graminis f. sp. hordei colony number was negatively correlated with exposure time and the total number of consecutive cycles of UV irradiation. Dark incubation following UV exposure further reduced fungal viability, implying that photoreactivation is an important component of DNA repair in B. graminis f. sp. hordei. After several rounds of UV mutagenesis, we selected two mutant isolates in addition to the parental B. graminis f. sp. hordei K1 isolate for whole-genome resequencing. By combining automated prediction of sequence variants and their manual validation, we identified unique UV-induced mutations in the genomes of the two isolates. Most of these mutations were in the up- or downstream regions of genes or in the intergenic space. Some of the variants detected in genes led to predicted missense mutations. As an additional insight, our bioinformatic analyses revealed a complex population structure within supposedly clonal B. graminis f. sp. hordei isolates.

The Parauncinula polyspora Draft Genome Provides Insights into Patterns of Gene Erosion and Genome Expansion in Powdery Mildew Fungi.

Due to their comparatively small genome size and short generation time, fungi are exquisite model systems to study eukaryotic genome evolution. Powdery mildew fungi present an exceptional case because of their strict host dependency (termed obligate biotrophy) and the atypical size of their genomes (>100 Mb). This size expansion is largely due to the pervasiveness of transposable elements on 70% of the genome and is associated with the loss of multiple conserved ascomycete genes required for a free-living lifestyle. To date, little is known about the mechanisms that drove these changes, and information on ancestral powdery mildew genomes is lacking. We report genome analysis of the early-diverged and exclusively sexually reproducing powdery mildew fungus Parauncinula polyspora, which we performed on the basis of a natural leaf epiphytic metapopulation sample. In contrast to other sequenced species of this taxonomic group, the assembled P. polyspora draft genome is surprisingly small (<30 Mb), has a higher content of conserved ascomycete genes, and is sparsely equipped with transposons (<10%), despite the conserved absence of a common defense mechanism involved in constraining repetitive elements. We speculate that transposable element spread might have been limited by this pathogen's unique reproduction strategy and host features and further hypothesize that the loss of conserved ascomycete genes may promote the evolutionary isolation and host niche specialization of powdery mildew fungi. Limitations associated with this evolutionary trajectory might have been in part counteracted by the evolution of plastic, transposon-rich genomes and/or the expansion of gene families encoding secreted virulence proteins.IMPORTANCE Powdery mildew fungi are widespread and agronomically relevant phytopathogens causing major yield losses. Their genomes have disproportionately large numbers of mobile genetic elements, and they have experienced a significant loss of highly conserved fungal genes. In order to learn more about the evolutionary history of this fungal group, we explored the genome of an Asian oak tree pathogen, Parauncinula polyspora, a species that diverged early during evolution from the remaining powdery mildew fungi. We found that the P. polyspora draft genome is comparatively compact, has a low number of protein-coding genes, and, despite the absence of a dedicated genome defense system, lacks the massive proliferation of repetitive sequences. Based on these findings, we infer an evolutionary trajectory that shaped the genomes of powdery mildew fungi.

The Integration of Algal Carbon Concentration Mechanism Components into Tobacco Chloroplasts Increases Photosynthetic Efficiency and Biomass.

Increasing the productivity of crops is a major challenge in agricultural research. Given that photosynthetic carbon assimilation is necessary for plant growth, enhancing the efficiency of photosynthesis is one strategy to boost agricultural productivity. The authors attempted to increase the photosynthetic efficiency and biomass of tobacco plants by expressing individual components of the Chlamydomonas reinhardtii carbon concentration mechanism (CCM) and integrating them into the chloroplast. Independent transgenic varieties are generated accumulating the carbonic anhydrase CAH3 in the thylakoid lumen or the bicarbonate transporter LCIA in the inner chloroplast membrane. Independent homozygous transgenic lines showed enhanced CO2 uptake rates (up to 15%), increased photosystem II efficiency (by up to 18%), and chlorophyll content (up to 19%). Transgenic lines produced more shoot biomass than wild-type and azygous controls, and accumulated more carbohydrate and amino acids, reflecting the higher rate of photosynthetic CO2 fixation. These data demonstrate that individual algal CCM components can be integrated into C3 plants to increase biomass, suggesting that transgenic lines combining multiple CCM components could further increase the productivity and yield of C3 crops.