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| Status |
Public on Jun 07, 2024 |
| Title |
Drosophila melanogaster Set8 and L(3)mbt function in gene expression independently of histone H4 lysine 20 methylation [GFP-L3mbt_CUT&RUN] |
| Organism |
Drosophila melanogaster |
| Experiment type |
Genome binding/occupancy profiling by high throughput sequencing
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| Summary |
Mono-methylation of Lysine 20 of histone H4 (H4K20me1) is catalyzed by Set8 and thought to play important roles in many aspects of genome function that are mediated by H4K20me-binding proteins. We interrogated this model in a developing animal by comparing in parallel the transcriptomes of Set8null, H4K20R/A, and l(3)mbt mutant Drosophila melanogaster. We found that the gene expression profiles of H4K20A and H4K20R larvae are markedly different than Set8null larvae despite similar reductions in H4K20me1. Set8null mutant cells have a severely disrupted transcriptome and fail to proliferate in vivo, but these phenotypes are not recapitulated by mutation of H4K20 indicating that the developmental defects of Set8null animals are largely due to H4K20me1-independent effects on gene expression. Further, the H4K20me1 binding protein L(3)mbt is recruited to the transcription start sites of most genes independently of H4K20me even though genes bound by L(3)mbt have high levels of H4K20me1. Moreover, both Set8 and L(3)mbt bind to purified H4K20R nucleosomes in vitro. We conclude that gene expression changes in Set8null and H4K20 mutants cannot be explained by loss of H4K20me1 or L(3)mbt binding to chromatin, and therefore that H4K20me1 does not play a large role in gene expression.
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| Overall design |
To assess L(3)mbt binding in Drosophila, we engineered an N-terminal GFP tagged l(3)mbt allele. To determine whether L(3)mbt recruitment to the genome required H4K20me we utilized this allele and performed CUT&RUN with a GFP-specific antibody in 3rd instar wandering imaginal wing discs in H4K20R, His4rnull, GFP-L(3)mbt, where all H4K20 is mutated, along with GFP-L(3)mbt and HWT, His4rnull, GFP-L(3)mbt control genotypes.
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| Contributor(s) |
Crain AT, Butler MB, Hill CA, Huynh M, McGinty RK, Duronio RJ |
| Citation missing |
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| Submission date |
May 31, 2024 |
| Last update date |
Jun 07, 2024 |
| Contact name |
Robert J. Duronio |
| E-mail(s) |
duronio@med.unc.edu
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| Organization name |
UNC Chapel Hill
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| Street address |
250 Bell Tower Dr., Gen. Sci. Bldg. 3356, CB7100
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| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599 |
| Country |
USA |
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| Platforms (1) |
| GPL30203 |
NextSeq 2000 (Drosophila melanogaster) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA1118785 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE268820_GFP-L3mbt.vs.OregonR.peaks.bed.gz |
31.2 Kb |
(ftp)(http) |
BED |
| GSE268820_GFP-L3mbt_spikeNorm_GFP_allReps_avg.bw |
5.9 Mb |
(ftp)(http) |
BW |
| GSE268820_HWT_His4rnull_GFP-L3mbt_spikeNorm_GFP_allReps_avg.bw |
2.9 Mb |
(ftp)(http) |
BW |
| GSE268820_K20R_His4rnull_GFP-L3mbt_spikeNorm_GFP_allReps_avg.bw |
31.9 Mb |
(ftp)(http) |
BW |
| GSE268820_OregonR_spikeNorm_GFP_allReps_avg.bw |
16.4 Mb |
(ftp)(http) |
BW |
| GSE268820_RAW.tar |
44.2 Mb |
(http)(custom) |
TAR (of BW) |
SRA Run Selector |
| Raw data are available in SRA |
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