Single nucleotide polymorphism (SNP) discovery and genotyping are essential to genetic mapping. There remains a need for simple, inexpensive platform that allows high-density SNP in large populations. Here we describe the sequencing of restriction-site associated DNA (RAD) tags, which identified more than 13,000 SNPs, mapped three traits two model organisms, using less half capacity one Illumina run. We demonstrated different marker densities can be attained by choice restriction enzyme....

Trimethylated lysine 27 on histone H3 (H3K27me3) is present in Drosophila, Arabidopsis, worms, and mammals, but absent from yeasts that have been examined. We identified analyzed H3K27me3 the filamentous fungus Neurospora crassa other species. covers 6.8% of N. genome, encompassing 223 domains, including 774 genes, all which are transcriptionally silent. H3K27me3-marked genes less conserved than unmarked only ∼35% marked by also discreta tetrasperma. found three components Polycomb...

Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin domains, which may be stabilized by feedback loops involving chromatin proteins modifications histones DNA. Neurospora crassa sports features found higher eukaryotes, namely cytosine methylation (5mC), histone H3 lysine 9 (H3K9me), protein 1 (HP1), is a model to investigate maintenance. We mapped distribution HP1, 5mC, H3K9me3, H3K4me2 at 100 bp resolution explored their interplay....

Significance Regulators of chromatin structure play critical roles in DNA-based processes. Lysine (K) Methyltransferase 1 (KMT1) homologs perform methylation H3 lysine-9 and are best known for their essential role heterochromatin formation transcriptional silencing. Heterochromatin is also important maintenance genome stability, although the mechanisms not well understood. We report that altered activity Polycomb repressive complex-2 (PRC2), a histone lysine-27 methyltransferase complex,...

Abstract The segregation of eukaryotic genomes into euchromatin and heterochromatin represents a fundamental poorly understood process. Here, we demonstrate that genome-wide establishment is triggered by the maternal to zygotic transition (MZT) during zebrafish embryogenesis. We find prior MZT, lack hallmarks including histone H3 lysine 9 trimethylation (H3K9me3) condensed chromatin ultrastructure. Global heterochromatic features occurs following MZT requires both activation genome...

High-density microarrays were used to profile circadian gene expression in Neurospora crassa cultures grown constant darkness. We identified 145 clock-controlled genes (ccgs). The ccgs peaked mRNA accumulation at all phases of the day, with majority peaking late night early morning. predicted or known functions demonstrate that clock contributes a wide range cellular processes, including cell signaling, development, metabolism, and stress responses. Although period rhythm most was found...

Methylation of DNA and Lysine 9 on histone H3 (H3K9) is associated with gene silencing in many animals, plants, fungi. In Neurospora crassa, methylation H3K9 by DIM-5 directs cytosine recruiting a complex containing Heterochromatin Protein-1 (HP1) the DIM-2 methyltransferase. We report genetic, proteomic, biochemical investigations into how controlled. These studies revealed DCDC, previously unknown protein including DIM-5, DIM-7, DIM-9, CUL4, DDB1. Components DCDC are required for H3K9me3,...

Circadian-regulated gene expression is predominantly controlled by a transcriptional negative feedback loop, and it evident that chromatin modifications remodeling are integral to this process in eukaryotes. We previously determined multiple ATP–dependent chromatin-remodeling enzymes function at frequency (frq). In report, we demonstrate the Neurospora homologue of chd1 required for normal frq sustained rhythmicity. Surprisingly, our studies CHD1 also revealed DNA sequences within promoter...

Histone posttranslational modifications (HPTMs) are involved in chromatin-based regulation of fungal secondary metabolite biosynthesis (SMB) which the corresponding genes—usually physically linked co-regulated clusters—are silenced under optimal physiological conditions (nutrient-rich) but activated when nutrients limiting. The exact molecular mechanisms by HPTMs influence silencing and activation, however, still to be better understood. Here we show a combined approach quantitative mass...

The development of whole-genome bisulfite sequencing (WGBS) has led to a number exciting discoveries about the role DNA methylation leading plethora novel testable hypotheses. Methods for constructing sodium bisulfite-converted and amplified libraries have recently advanced point that bottleneck experiments use WGBS shifted data analysis interpretation. Here we present empirical evidence an over-representation reads from methylated in WGBS. This enrichment is exacerbated by higher cycles PCR...

Abstract DectiSomes are anti‐infective drug‐loaded liposomes targeted to pathogenic cells by pathogen receptors including the Dectins. We have previously used C‐type lectin (CTL) Dectin‐1, Dectin‐2, and DC‐SIGN target extracellular oligoglycans surrounding diverse fungi kill them. Dectin‐3 (also known as MCL, CLEC4D ) is a CTL receptor whose cognate ligands partly distinct from other CTLs. expressed purified truncated polypeptide (DEC3) comprised of its carbohydrate recognition domain stalk...

Aspergillus species cause pulmonary invasive aspergillosis resulting in nearly 100,000 deaths each year. Patients at the greatest risk of developing life-threatening have weakened immune systems and/or various lung disorders. are treated with antifungals such as amphotericin B (AmB), caspofungin acetate, or triazoles (itraconazole, voriconazole, etc.), but these antifungal agents serious limitations due to lack sufficient fungicidal effect and human toxicity. Liposomes AmB intercalated into...

Androgen deprivation therapy has led to elevated cases of androgen receptor (AR) pathway-independent prostate cancer with dysregulated fatty acid metabolism. However, it is unclear how cells sustain metabolism drive AR-independent cancer. Long-chain acyl-CoA synthetases (ACSL) catalyze the conversion acids into acyl-CoAs that are required for In this study, we demonstrate expression levels ACSL3 and 4 were oppositely regulated by androgen-AR signaling in cells. AR served as a transcription...

Abstract Polycomb group (PcG) proteins form chromatin modifying complexes that stably repress lineage- or context-specific genes in animals, plants, and some fungi. Repressive Complex 2 (PRC2) catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3) to assemble repressive chromatin. In the model fungus Neurospora crassa, H3K27me3 deposition is controlled by H3K36 methyltransferase ASH1 components constitutive heterochromatin including H3K9me3-binding protein HETEROCHROMATIN PROTEIN 1...

ABSTRACT The sialic acid Ig-like lectins Siglec-3 and Siglec-15 are pathogen receptors that bind acid-modified glycoproteins, best characterized in metastatic cancers. Because fungi produce sialoglycans sialo-glycoproteins, we wondered if Siglecs had the potential for targeted delivery of antifungal drugs. We purified extracellular V-region C2 ligand-binding domains stalk regions SIG3 SIG15. floated two polypeptides on surface liposomes loaded with amphotericin B (AmB) labeled rhodamine to...

Many processes in fungi are regulated by light, but the molecular mechanisms not well understood. The White Collar-1 (WC-1) protein is required for all known blue-light responses Neurospora crassa. In response to WC-1 levels increase, and transiently phosphorylated. To test hypothesis that increase after light treatment sufficient activate light-regulated gene expression, we used microarrays identify genes respond treatment. We then overexpressed dark-grown tissue an levels. found 3% of were...

Transposable elements are common in genomes and must be controlled. Many organisms use DNA methylation to silence such selfish DNA, but the mechanisms that restrict appropriate regions largely unknown. We identified a JmjC domain protein Neurospora , METHYLATION MODULATOR-1 (DMM-1), prevents aberrant spreading of histone H3K9 from inactivated transposons into nearby genes. Mutation conserved residue within Fe(II)-binding site abolished dmm-1 function, as did mutations cysteine-rich domains....

Invasive fungal diseases caused by Candida albicans , Cryptococcus neoformans and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals current use serious limitations due host toxicity and/or insufficient cell killing that results recurrent infections. Few new antifungal drugs been introduced last 2 decades. Hence, there is a critical need for improved therapeutics. By targeting antifungal-loaded...