Biology subjects

Selker, E. U.

Publications and source records attributed to Selker, E. U..

7 recordsLinked to original sources

bioRxiv2022

Tup1 is Required for Transcriptional Repression Necessary in Quiescence in S. cerevisiae

Upon glucose starvation, S. cerevisiae shows a dramatic alteration in transcription, resulting in wide-scale repression of most genes and activation of some others. This coincides with an arrest of cellular proliferation. A subset of such cells enters quiescence, a reversible non-dividing state. Here, we demonstrate that the conserved transcriptional corepressor Tup1 is critical for transcriptional repression after glucose depletion. We show that Tup1-Ssn6 binds new targets upon glucose depletion, where it remains as the cells enter the G0 phase of the cell cycle. In addition, we show that Tup1 represses a variety of glucose metabolism and transport genes. We explored how Tup1 mediated repression is accomplished and demonstrated that Tup1 coordinates with the Rpd3L complex to deacetylate H3K23. We found that Tup1 coordinates with Isw2 to affect nucleosome positions at glucose transporter HXT family genes during G0. Finally, microscopy revealed that a quarter of cells with a Tup1 deletion contain multiple DAPI puncta. Taken together, these findings demonstrate the role of Tup1 in transcriptional reprogramming in response to environmental cues leading to the quiescent state.

molecular biology↗

bioRxiv2021

The ACF Chromatin Remodeling Complex is Essential for Polycomb Repression

Establishing and maintaining appropriate gene repression is critical for the health and development of multicellular organisms. Histone H3 lysine 27 (H3K27) methylation is a chromatin modification associated with repressed facultative heterochromatin, but the mechanism of this repression remains unclear. We used a forward genetic approach to identify genes involved in transcriptional silencing of H3K27-methylated chromatin in the filamentous fungus Neurospora crassa. We found that the N. crassa homologs of ISWI (NCU03875) and ACF (NCU00164) are required for repression of a subset of H3K27-methylated genes and that they form an ACF chromatin remodeling complex. This N. crassa ACF complex interacts with chromatin throughout the genome, yet association with facultative heterochromatin is specifically promoted by the H3K27 methyltransferase, SET-7. H3K27-methylated genes that are upregulated when iswi or acf1 are deleted show a downstream shift of the +1 nucleosome, suggesting that proper nucleosome positioning is critical for repression of facultative heterochromatin. Our findings support a direct role for the ACF complex in Polycomb repression.

genetics↗

bioRxiv2020

Identification of a PRC2 accessory subunit required for subtelomeric H3K27 methylation in Neurospora

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 lysine 27 (H3K27) in genomic regions of most eukaryotes and is critical for maintenance of the associated transcriptional repression. However, the mechanisms that shape the distribution of H3K27 methylation, such as recruitment of PRC2 to chromatin and/or stimulation of PRC2 activity, are unclear. Here, using a forward genetic approach in the model organism Neurospora crassa, we identified two alleles of a gene, NCU04278, encoding an unknown PRC2 accessory subunit (PAS). Loss of PAS resulted in losses of H3K27 methylation concentrated near the chromosome ends and derepression of a subset of associated subtelomeric genes. Immunoprecipitation followed by mass spectrometry confirmed reciprocal interactions between PAS and known PRC2 subunits, and sequence similarity searches demonstrated that PAS is not unique to N. crassa. PAS homologs likely influence the distribution of H3K27 methylation, and underlying gene repression, in a variety of fungal lineages.

molecular biology↗

bioRxiv2019

Rapid and Inexpensive Preparation of Genome-Wide Nucleosome Footprints from Model and Non-Model Organisms

Eukaryotic DNA is packaged into nucleosomes, the smallest repeating unit of chromatin. The positions of nucleosomes determine the relative accessibility of genomic DNA. Several protocols exist for mapping nucleosome positions in eukaryotic genomes in order to study the relationship between chromatin structure and DNA-dependent processes. These nucleosome mapping protocols can be laborious and, at minimum, require two to three days to isolate nucleosome-protected DNA fragments. We have developed a streamlined protocol for mapping nucleosomes from S. cerevisiae liquid culture or from patches on solid agar. This method isolates nucleosome-sized footprints in three hours using 1.5 ml tubes with minimal chemical waste. We validate that these footprints match those produced by previously published methods and we demonstrate that our protocol works for N. crassa and S. pombe. A slightly modified protocol can be used for isolation of nucleosome-protected DNA fragments from a variety of wild fungal specimens thereby providing a simple, easily multiplexed and unified strategy to map nucleosome positions in model and non-model fungi. Finally, we demonstrate recovery of nucleosome footprints from the diploid myeloid leukemia cell line PLB-985 in less than three hours using an abbreviated version of the same protocol. With reduced volume and incubation times and a streamlined workflow, the described method should be compatible with high-throughput, automated creation of MNase-seq libraries. We believe this simple validated method for rapidly producing sequencing-ready nucleosome footprints from a variety of organisms will make nucleosome mapping studies widely accessible to researchers globally.

genetics↗

bioRxiv2019

Evolutionarily ancient BAH-PHD protein mediates Polycomb silencing

Methylation of histone H3 lysine 27 (H3K27) is widely recognized as a transcriptionally repressive chromatin modification but the mechanism of repression remains unclear. We devised and implemented a forward genetic scheme to identify factors required for H3K27 methylation-mediated silencing in the filamentous fungus Neurospora crassa and identified a bromo-adjacent homology (BAH)-plant homeodomain (PHD)-containing protein, EPR-1 (Effector of Polycomb Repression 1; NCU07505). EPR-1 associates with H3K27 methylation in vivo and in vitro, and loss of EPR-1 de-represses H3K27-methylated genes without loss of H3K27 methylation. EPR-1 is not fungal-specific; orthologs of EPR-1 are present in a diverse array of eukaryotic lineages, suggesting an ancestral EPR-1 was a component of a primitive Polycomb repression pathway. SignificancePolycomb group (PcG) proteins are employed by a wide variety of eukaryotes for the maintenance of gene repression. Polycomb repressive complex 2 (PRC2), a multimeric complex of PcG proteins, catalyzes the methylation of histone H3 lysine 27 (H3K27). In the filamentous fungus, Neurospora crassa, H3K27 methylation represses scores of genes, despite the absence of canonical H3K27 methylation effectors that are present in plants and animals. We report the identification and characterization of an H3K27 methylation effector, EPR-1, in N. crassa and demonstrate its widespread presence and early eukaryotic origins with phylogenetic analyses. These findings indicate that an ancient EPR-1 may have been part of a nascent Polycomb repression system in eukaryotes.

molecular biology↗

bioRxiv2019

Marked Neurospora crassa strains for competition experiments and Bayesian methods for fitness estimates

The filamentous fungus Neurospora crassa, a model microbial eukaryote, has a life cycle with many features that make it suitable for studying experimental evolution. However, it has lacked a general tool for estimating relative fitness of different strains in competition experiments. To remedy this need, we constructed N. crassa strains that contain a modified csr-1 locus and developed an assay for detecting the proportion of the marked strain using a post PCR high resolution melting assay. DNA extraction from spore samples can be performed on 96-well plates, followed by a PCR step, which allows many samples to be processed with ease. Furthermore, we suggest a Bayesian approach for estimating relative fitness from competition experiments that takes into account the uncertainty in measured strain proportions. We show that there is a fitness effect of the mating type locus, as mating type mat a has a higher competitive fitness than mat A. The csr-1* marker also has a small fitness effect, but is still a suitable marker for competition experiments. As a proof of concept, we estimate the fitness effect of the qde-2 mutation, a gene in the RNA interference pathway, and show that its competitive fitness is lower than what would be expected from its mycelial growth rate alone.

genetics↗

bioRxiv2017

Telomere repeats induce domains of H3K27 methylation in Neurospora

Development in higher organisms requires selective gene silencing, directed in part by di-/tri-methylation of lysine 27 on histone H3 (H3K27me2/3). Knowledge of the cues that control formation of such repressive Polycomb domains is extremely limited. We exploited natural and engineered chromosomal rearrangements in the fungus Neurospora crassa to elucidate the control of H3K27me2/3. Analyses of H3K27me2/3 in strains bearing chromosomal rearrangements revealed both position-dependent and position-independent facultative heterochromatin. We found that proximity to chromosome ends is necessary to maintain, and sufficient to induce, transcriptionally repressive, subtelomeric H3K27me2/3. We ascertained that such telomere-proximal facultative heterochromatin requires native telomere repeats and found that a short array of ectopic telomere repeats, (TTAGGG)17, can induce a large domain ([~]225 kb) of H3K27me2/3. This provides an example of a cis-acting sequence that directs H3K27 methylation. Our findings provide new insight into the relationship between genome organization and control of heterochromatin formation.

genetics↗