scholarly journals A Light-Inducible Strain for Genome-Wide Histone Turnover Profiling in Neurospora crassa

Genetics ◽  
2020 ◽  
Vol 215 (3) ◽  
pp. 569-578
Author(s):  
William K. Storck ◽  
Sabrina Z. Abdulla ◽  
Michael R. Rountree ◽  
Vincent T. Bicocca ◽  
Eric U. Selker
Keyword(s):  
Neurospora Crassa ◽  
Histone H3 ◽  
Chromatin Accessibility ◽  
Histone Octamer ◽  
Genome Wide ◽  
Incubation Periods ◽  
Mutant Strains ◽  
Functional Consequences ◽  
Dynamic Structures ◽  
Histone Exchange

In chromatin, nucleosomes are composed of ∼146 bp of DNA wrapped around a histone octamer, and are highly dynamic structures subject to remodeling and exchange. Histone turnover has previously been implicated in various processes including the regulation of chromatin accessibility, segregation of chromatin domains, and dilution of histone marks. Histones in different chromatin environments may turnover at different rates, possibly with functional consequences. Neurospora crassa sports a chromatin environment that is more similar to that of higher eukaryotes than yeasts, which have been utilized in the past to explore histone exchange. We constructed a simple light-inducible system to profile histone exchange in N. crassa on a 3xFLAG-tagged histone H3 under the control of the rapidly inducible vvd promoter. After induction with blue light, incorporation of tagged H3 into chromatin occurred within 20 min. Previous studies of histone turnover involved considerably longer incubation periods and relied on a potentially disruptive change of medium for induction. We used this reporter to explore replication-independent histone turnover at genes and examine changes in histone turnover at heterochromatin domains in different heterochromatin mutant strains. In euchromatin, H3-3xFLAG patterns were almost indistinguishable from that observed in wild-type in all mutant backgrounds tested, suggesting that loss of heterochromatin machinery has little effect on histone turnover in euchromatin. However, turnover at heterochromatin domains increased with loss of trimethylation of lysine 9 of histone H3 or HP1, but did not depend on DNA methylation. Our reporter strain provides a simple yet powerful tool to assess histone exchange across multiple chromatin contexts.

scholarly journals Reduced Gene Dosage of Histone H4 Prevents CENP-A Mislocalization in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Jessica R. Eisenstatt ◽  
Kentaro Ohkuni ◽  
Wei-Chun Au ◽  
Olivia Preston ◽  
Evelyn Suva ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Chromosomal Instability ◽  
Budding Yeast ◽  
Gene Dosage ◽  
Histone H3 ◽  
Histone H4 ◽  
Genome Wide ◽  
A Genome ◽  
Mutant Strains ◽  
Histone H3 Variant

ABSTRACTMislocalization of the centromeric histone H3 variant (Cse4 in budding yeast, CID in flies, CENP-A in humans) to non-centromeric regions contributes to chromosomal instability (CIN) in yeast, fly, and human cells. Overexpression and mislocalization of CENP-A has been observed in cancers, however, the mechanisms that facilitate the mislocalization of overexpressed CENP-A have not been fully explored. Defects in ubiquitin-mediated proteolysis of overexpressed Cse4 (GALCSE4) leads to its mislocalization and synthetic dosage lethality (SDL) in mutants for E3 ubiquitin ligases (Psh1, Slx5, SCFMet30, SCFCdc4), Doa1, Hir2, and Cdc7. In contrast, defects in sumoylation of GALcse4K215/216/A/R prevent its mislocalization and do not cause SDL in a psh1Δ strain. Here, we used a genome-wide screen to identify factors that facilitate the mislocalization of overexpressed Cse4 by characterizing suppressors of the psh1Δ GALCSE4 SDL. Deletions of histone H4 alleles (HHF1 or HHF2), which were among the most prominent suppressors, also suppress slx5Δ, cdc4-1, doa1Δ, hir2Δ, and cdc7-4 GALCSE4 SDL. Reduced dosage of H4 contributes to defects in sumoylation and reduced mislocalization of overexpressed Cse4. We determined that the hhf1-20, cse4-102, and cse4-111 mutants, which are defective in the Cse4-H4 interaction, also exhibit reduced sumoylation of Cse4 and do not display psh1Δ GALCSE4 SDL. In summary, we have identified genes that contribute to the mislocalization of overexpressed Cse4 and defined a role for the gene dosage of H4 in facilitating Cse4 sumoylation and mislocalization to non-centromeric regions, contributing to SDL when Cse4 is overexpressed in mutant strains.

scholarly journals Regulatory DNA in A. thaliana can tolerate high levels of sequence divergence

2017 ◽  
Author(s):  
C.M. Alexandre ◽  
J.R. Urton ◽  
K. Jean-Baptiste ◽  
M.W. Dorrity ◽  
J.C. Cuperus ◽  
...  
Keyword(s):  
Sequence Variation ◽  
Sequence Divergence ◽  
Purifying Selection ◽  
Chromatin Accessibility ◽  
Dna Variation ◽  
Genome Wide ◽  
Functional Consequences ◽  
Species Comparisons ◽  
Regulatory Dna ◽  
Regulatory Sites

ABSTRACTVariation in regulatory DNA is thought to drive evolution. Cross-species comparisons of regulatory DNA have provided evidence for both weak purifying selection and substantial turnover in regulatory regions. However, disruption of transcription factor binding sites can affect the expression of neighboring genes. Thus, the base-pair level functional annotation of regulatory DNA has proven challenging. Here, we explore regulatory DNA variation and its functional consequences in genetically diverse strains of the plant Arabidopsis thaliana, which largely maintain the positional homology of regulatory DNA. Using chromatin accessibility to delineate regulatory DNA genome-wide, we find that 15% of approximately 50,000 regulatory sites varied in accessibility among strains. Some of these accessibility differences are associated with extensive underlying sequence variation, encompassing many deletions and dramatically hypervariable sequence. For the majority of such regulatory sites, nearby gene expression was similar, despite this large genetic variation. However, among all regulatory sites, those with both high levels of sequence variation and differential chromatin accessibility are the most likely to reside near genes with differential expression among strains. Unexpectedly, the vast majority of regulatory sites that differed in chromatin accessibility among strains show little variation in the underlying DNA sequence, implicating variation in upstream regulators.

scholarly journals Changes in chromatin accessibility landscape and histone H3 core acetylation during valproic acid-induced differentiation of embryonic stem cells

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Claudia Baumann ◽  
Xiangyu Zhang ◽  
Ling Zhu ◽  
Yuhong Fan ◽  
Rabindranath De La Fuente
Keyword(s):  
Stem Cells ◽  
Valproic Acid ◽  
Embryonic Stem Cells ◽  
Histone H3 ◽  
Embryonic Stem ◽  
Histone H1 ◽  
Chromatin Accessibility ◽  
Directed Differentiation ◽  
Induced Differentiation ◽  
Genome Wide

AbstractDirected differentiation of mouse embryonic stem cells (mESCs) or induced pluripotent stem cells (iPSCs) provides powerful models to dissect the molecular mechanisms leading to the formation of specific cell lineages. Treatment with histone deacetylase inhibitors can significantly enhance the efficiency of directed differentiation. However, the mechanisms are not well understood. Here, we use CUT&RUN in combination with ATAC-seq to determine changes in both histone modifications and genome-wide chromatin accessibility following valproic acid (VPA) exposure. VPA induced a significant increase in global histone H3 acetylation (H3K56ac), a core histone modification affecting nucleosome stability, as well as enrichment at loci associated with cytoskeletal organization and cellular morphogenesis. In addition, VPA altered the levels of linker histone H1 subtypes and the total histone H1/nucleosome ratio indicative of initial differentiation events. Notably, ATAC-seq analysis revealed changes in chromatin accessibility of genes involved in regulation of CDK serine/threonine kinase activity and DNA duplex unwinding. Importantly, changes in chromatin accessibility were evident at several key genomic loci, such as the pluripotency factor Lefty, cardiac muscle troponin Tnnt2, and the homeodomain factor Hopx, which play critical roles in cardiomyocyte differentiation. Massive parallel transcription factor (TF) footprinting also indicates an increased occupancy of TFs involved in differentiation toward mesoderm and endoderm lineages and a loss of footprints of POU5F1/SOX2 pluripotency factors following VPA treatment. Our results provide the first genome-wide analysis of the chromatin landscape following VPA-induced differentiation in mESCs and provide new mechanistic insight into the intricate molecular processes that govern departure from pluripotency and early lineage commitment.

scholarly journals High-throughput single-cell chromatin accessibility CRISPR screens enable unbiased identification of regulatory networks in cancer

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Sarah E. Pierce ◽  
Jeffrey M. Granja ◽  
William J. Greenleaf
Keyword(s):  
Transcription Factor ◽  
Single Cell ◽  
Cancer Cells ◽  
High Throughput ◽  
Regulatory Networks ◽  
Temporal Dynamics ◽  
Chromatin Accessibility ◽  
Regulatory Regions ◽  
Factor Binding ◽  
Genome Wide

AbstractChromatin accessibility profiling can identify putative regulatory regions genome wide; however, pooled single-cell methods for assessing the effects of regulatory perturbations on accessibility are limited. Here, we report a modified droplet-based single-cell ATAC-seq protocol for perturbing and evaluating dynamic single-cell epigenetic states. This method (Spear-ATAC) enables simultaneous read-out of chromatin accessibility profiles and integrated sgRNA spacer sequences from thousands of individual cells at once. Spear-ATAC profiling of 104,592 cells representing 414 sgRNA knock-down populations reveals the temporal dynamics of epigenetic responses to regulatory perturbations in cancer cells and the associations between transcription factor binding profiles.

scholarly journals An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition

PLoS ONE ◽  
2016 ◽  
Vol 11 (11) ◽  
pp. e0166438 ◽  
Author(s):  
Brian Egan ◽  
Chih-Chi Yuan ◽  
Madeleine Lisa Craske ◽  
Paul Labhart ◽  
Gulfem D. Guler ◽  
...  
Keyword(s):  
Histone H3 ◽  
Genome Wide ◽  
Alternative Approach

scholarly journals Sexuality in Neurospora crassa I. Mutations to male sterility

1972 ◽  
Vol 19 (3) ◽  
pp. 191-204 ◽  
Author(s):  
J. Weijer ◽  
N. V. Vigfusson
Keyword(s):  
Neurospora Crassa ◽  
Male Sterility ◽  
Wide Spectrum ◽  
Female Parent ◽  
Behaviour Pattern ◽  
Type Locus ◽  
Development Cycle ◽  
Ultraviolet Light Irradiation ◽  
Mutant Strains ◽  
Aberrant Segregation

SUMMARYMutations giving rise to sexual sterility were induced in Neurospora crassa macroconidia by ultraviolet-light irradiation. Thirty mutants were isolated on the basis of their male sterility in crosses with a wild-type strain. When used as the male parent these mutants exhibited a wide spectrum of sexual behaviour patterns ranging from the production of only small brown protoperithecia (complete male sterility) to the production of large and normally pigmented perithecia but with an undeveloped ostiole and very few if any spores. For many of the mutants the behaviour pattern is different when the strain is used as the female parent. Segregation data reveal that none of these mutants represent mutations of the mating-type locus. These findings suggest that the sexual development cycle is blocked at various stages in the different mutant strains. All attempts to restore fertility by supplying various additives to the medium or by varying the incubation time and temperature were unsuccessful. Conidial viability tests carried out on many of the strains revealed no abnormality in this respect. The aberrant segregation patterns exhibited by many of the mutants are discussed.

A histone H3 methyltransferase controls DNA methylation in Neurospora crassa

Nature ◽  
2001 ◽  
Vol 414 (6861) ◽  
pp. 277-283 ◽  
Author(s):  
Hisashi Tamaru ◽  
Eric U. Selker
Keyword(s):  
Dna Methylation ◽  
Neurospora Crassa ◽  
Histone H3

scholarly journals Adenosine kinase-deficient mutant of Neurospora crassa

1982 ◽  
Vol 152 (3) ◽  
pp. 1292-1294
Author(s):  
J M Magill ◽  
P Dalke ◽  
T S Lyda ◽  
C W Magill
Keyword(s):  
Neurospora Crassa ◽  
Resistant Mutant ◽  
Adenosine Kinase ◽  
Deficient Mutant ◽  
Wild Type ◽  
Purine Nucleotides ◽  
Mutant Strains

Tubercidin-resistant mutant strains of Neurospora crassa were isolated, and at least one appeared to be deficient in adenosine kinase. No significant differences in [8-14C]adenosine labeling of purine nucleotides or nucleosides were found between the wild type and the adenosine kinase-deficient strains.

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