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| Status |
Public on May 19, 2016 |
| Title |
HiC of Wild Type Neurospora crassa and mutants deficient in heterochromatin formation |
| Organism |
Neurospora crassa |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
Other
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| Summary |
Eukaryotic genomes are organized into chromatin domains with distinct three-dimensional arrangements resulting from nucleic acid and protein factor interactions within the physical constraints of the nucleus. It is of obvious interest to determine interactions between various chromosomal regions defined by these nuclear constraints, and to identify important factors that limit the interactions. We used chromosome conformation capture (3C) followed by high-throughput sequencing (HiC) to improve our understanding of Neurospora crassa genome organization and to examine if known components of heterochromatin machinery influence nuclear organization. In heterochromatin establishment, DIM-5 tri-methylates histone H3 on lysine 9 (H3K9me3), and this mark is subsequently bound by Heterochromatin Protein-1 (HP1). NUP-6 is required to target DIM-5 to incipient A:T rich DNA and the dim-3 mutant of NUP-6 is deficient in DIM-5 localization. We performed HiC on chromatin from wildtype, Δdim-5, Δhpo, and dim-3 strains. The genome configuration of wild type nuclei revealed strong intra- and inter-chromosomal associations between both constitutive and facultative heterochromatic domains, with the strongest interactions among the centromeres, telomeres and interspersed heterochromatin. We found that loss of the H3K9me3 mark, as well as loss of HP1, minimally altered the chromatin organization found at these strongly interacting heterochromatic regions. Surprisingly, dim-3 chromatin was highly disorganized suggesting NUP-6 plays key role(s) in genome structure. Thus, our datasets suggest that while heterochromatic regions are critical to defining the genome conformation in Neurospora, non-canonical protein factors may play a key role in maintaining this organization.
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| Overall design |
We analyzed a total of four strains of Neurospora crassa by chromatin conformation capture followed by high throughput sequencing (HiC). A wild type strain, NMF39, serves as the reference for the mutants deficient in heterochromatin formation (delta dim-5; delta hpo; and dim-3). Each strain has at least two replicates, and the NMF39 wild type strain has three replicates. Strains were grown from conidia ~4 hours, crosslinked, and conidia were made into spheroplasts. Genomic DNA from spheroplasts was digested with HindIII, ends were filled in with dNTPs (including biotin-conjugated dCTP), blunt ends were ligated, crosslinks were reversed, DNA was soniciated, ligation products were purified with streptavidin beads, and libraries were prepared for sequencing.
Supplementary file 'GSE71024_H3K9me3_10kb-bins_wg_forCircos.txt' includes processed data obtained by using the raw data from Sample GSM1686146 of Series GSE68897, and supplementary file 'GSE71024_H3K27me3_10kb-bins_wg_forCircos.txt' includes processed data obtained by using the raw data from Sample GSM1686139 of Series GSE68897.
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| Contributor(s) |
Galazka JM, Klocko AD, Selker EU, Freitag M |
| Citation(s) |
27260477 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 GM035690 |
Genetic Aspects of DNA Methylation |
UNIVERSITY OF OREGON |
SELKER |
| R37 GM035690 |
Genetic Aspects of DNA Methylation |
UNIVERSITY OF OREGON |
SELKER |
| R01 GM097637 |
Assembly and Maintenance of Centromeres in Filamentous Fungi |
OREGON STATE UNIVERSITY |
FREITAG |
| F32 GM097821 |
Genetic analysis of DNA methylation in Neurospora crassa |
UNIVERSITY OF OREGON |
KLOCKO |
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| Submission date |
Jul 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Andrew David Klocko |
| E-mail(s) |
aklocko@uccs.edu
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| Phone |
719-255-3255
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| Organization name |
University of Colorado Colorado Springs
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| Department |
Chemistry and Biochemistry
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| Lab |
Klocko
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| Street address |
278 Centennial Hall, 1420 Austin Bluffs Pkwy
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| City |
Colorado Springs |
| State/province |
Colorado |
| ZIP/Postal code |
80918 |
| Country |
USA |
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| Platforms (1) |
| GPL20705 |
Illumina HiSeq 2500 (Neurospora crassa) |
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| Samples (5)
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| Relations |
| BioProject |
PRJNA290116 |
| SRA |
SRP061251 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE71024_H3K27me3_10kb-bins_wg_forCircos.txt.gz |
284.1 Kb |
(ftp)(http) |
TXT |
| GSE71024_H3K9me3_10kb-bins_wg_forCircos.txt.gz |
271.8 Kb |
(ftp)(http) |
TXT |
| GSE71024_RAW.tar |
797.1 Mb |
(http)(custom) |
TAR (of TDF, TXT) |
| GSE71024_neurospora_crassa_or74a_12_genome_FIXED.fasta.gz |
11.3 Mb |
(ftp)(http) |
FASTA |
| GSE71024_neurospora_crassa_or74a_12_transcripts_FIXED.gtf.gz |
755.6 Kb |
(ftp)(http) |
GTF |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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