Project description:Gcn5/PCAF dobule knockout (dKO) in MEFs leads to loss of the global H3K9ac, but only affect expression of a small nubmer of genes according to RNA-Seq data. To examine the function of H3K9ac on gene activation, H3K9ac ChIP-Seq was performed. We found that H3K9ac at Transcriptional Start sites (TSSs) of no-changed, up-regulated and down-regulated genes are all dramatially decreased in Gcn5/PCAF dKO cells, suggesting that H3K9ac is largely not required for gene activation. PCAF-/-;Gcn5f/D MEFs were infected with retroviral Cre to delete Gcn5 to generate Gcn5/PCAF dKO cells, followed by H3K9ac ChIP-Seq analysis
Project description:Histone acetyltransferases (HATs) GCN5/PCAF and CBP/p300 are transcription coactivators. However, how these HATs regulate ligand-induced nuclear receptor target gene expression remains unclear. Here we show in mouse embryonic fibroblasts (MEFs), deletion of GCN5/PCAF specifically eliminates acetylation on H3K9 (H3K9Ac) while deletion of CBP/p300 selectively reduces acetylation on H3K18 and H3K27 (H3K18/27Ac). Treating MEFs with a specific ligand for nuclear receptor PPARdelta induces sequential increases of H3K18/27Ac and H3K9Ac on the promoter of PPARdelta target gene Angptl4, which correlates with a robust ligand-induced Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9Ac but not H3K18/27Ac on Angptl4 promoter. Finally, we show CBP/p300 and their HAT activities are required, while GCN5/PCAF and H3K9Ac are dispensable, for ligand-induced PPARdelta target gene expression in MEFs. These results highlight the substrate and site specificities of HATs in cells, and suggest that GCN5/PCAF- and CBP/p300-mediated histone acetylations play distinct roles in regulating ligand-induced nuclear receptor target gene expression. PCAF and GCN5 have some redundant function. To identify PCAF/GCN5-regulated genes, immortalized MEFs with PCAF knockout and GCN5 conditional knockout were infected with retroviruses expressing either Cre recombinase or vector alone. We prepared duplicated RNAs from either vector or Cre infected cells (PCAF-/-;GCN5+/- or PCAF-/-;GCN5+/-) and RNAs from either Vector or Cre infected the other independently immortalized cells for 6 affymetrix microarray.
Project description:Gcn5/PCAF dobule knockout (dKO) in MEFs leads to loss of the global H3K9ac, but only affect expression of a small nubmer of genes according to RNA-Seq data. To examine the function of H3K9ac on gene activation, H3K9ac ChIP-Seq was performed. We found that H3K9ac at Transcriptional Start sites (TSSs) of no-changed, up-regulated and down-regulated genes are all dramatially decreased in Gcn5/PCAF dKO cells, suggesting that H3K9ac is largely not required for gene activation.
Project description:Gcn5/PCAF double knockout (dKO) leads to loss of the global H3K9ac. RNA-Seq was performed to define the changes of gene expression in response to Gcn5/PCAF deletion and H3K9ac loss PCAF-/-;Gcn5f/D MEFs were infected with retroviral Cre to delete Gcn5 to generate Gcn5/PCAF dKO cells, followed by RNA-Seq analysis using spike-in RNA as controls
Project description:Histone acetyltransferases (HATs) GCN5/PCAF and CBP/p300 are transcription coactivators. However, how these HATs regulate ligand-induced nuclear receptor target gene expression remains unclear. Here we show in mouse embryonic fibroblasts (MEFs), deletion of GCN5/PCAF specifically eliminates acetylation on H3K9 (H3K9Ac) while deletion of CBP/p300 selectively reduces acetylation on H3K18 and H3K27 (H3K18/27Ac). Treating MEFs with a specific ligand for nuclear receptor PPARdelta induces sequential increases of H3K18/27Ac and H3K9Ac on the promoter of PPARdelta target gene Angptl4, which correlates with a robust ligand-induced Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9Ac but not H3K18/27Ac on Angptl4 promoter. Finally, we show CBP/p300 and their HAT activities are required, while GCN5/PCAF and H3K9Ac are dispensable, for ligand-induced PPARdelta target gene expression in MEFs. These results highlight the substrate and site specificities of HATs in cells, and suggest that GCN5/PCAF- and CBP/p300-mediated histone acetylations play distinct roles in regulating ligand-induced nuclear receptor target gene expression.
Project description:Gcn5/PCAF double knockout (dKO) leads to loss of the global H3K9ac. RNA-Seq was performed to define the changes of gene expression in response to Gcn5/PCAF deletion and H3K9ac loss
Project description:Introgressed variants from other species can be an important source of genetic variation because they may arise rapidly, can include multiple mutations on a single haplotype, and have often been pretested by selection in the species of origin. Although introgressed alleles are generally deleterious, several studies have reported introgression as the source of adaptive alleles-including the rodenticide-resistant variant of Vkorc1 that introgressed from Mus spretus into European populations of Mus musculus domesticus. Here, we conducted bidirectional genome scans to characterize introgressed regions into one wild population of M. spretus from Spain and three wild populations of M. m. domesticus from France, Germany, and Iran. Despite the fact that these species show considerable intrinsic postzygotic reproductive isolation, introgression was observed in all individuals, including in the M. musculus reference genome (GRCm38). Mus spretus individuals had a greater proportion of introgression compared with M. m. domesticus, and within M. m. domesticus, the proportion of introgression decreased with geographic distance from the area of sympatry. Introgression was observed on all autosomes for both species, but not on the X-chromosome in M. m. domesticus, consistent with known X-linked hybrid sterility and inviability genes that have been mapped to the M. spretus X-chromosome. Tract lengths were generally short with a few outliers of up to 2.7 Mb. Interestingly, the longest introgressed tracts were in olfactory receptor regions, and introgressed tracts were significantly enriched for olfactory receptor genes in both species, suggesting that introgression may be a source of functional novelty even between species with high barriers to gene flow.
Project description:PArtner and Localizer of BRCA2 (PALB2) is essential to maintain genome stability in human cells. Upon DNA damage by double DNA strand breaks, PALB2 is required to repair DNA by homologous recombination. In undamaged conditions, PALB2 protects coding regions, by preventing DNA stress due to collisions between transcription and replication machineries. PALB2 associates with chromatin, which is essential to fulfill its function in genome stability maintenance, however molecular mechanisms regulating PALB2 chromatin association remain unknown. In our previous published study describing the KAT2A/B(GCN5/PCAF)-acetylome (Fournier et al., Nat.Comm. 2016, doi: 10.1038/ncomms13227) we have identified PALB2 as an acetylated protein target of the acetyltransferases KAT2A (GCN5) and KAT2B (PCAF) in vivo. KAT2A/B-acetylated sites of PALB2 were mapped within its DNA/Chromatin association domain. In this current study, we have conducted in vitro acetyltransferase (AT) assays, by mixing purified recombinant GST-tagged PALB2 full-length (FL) and PALB2 fragment P2.2 which associates with DNA/chromatin (from residues 295-610), with Flag-PCAF, Flag-GCN5 and Flag-GCN5 catalytic mutant (mut), followed by proteomics analysis to map PALB2 acetylated lysines by KAT2A(GCN5) and KAT2B(PCAF) in vitro. PALB2 FL and P2.2 alone, or PALB2 FL and P2.2 mixed with GCN5 catalytic mutant, were used as negative controls.
Project description:Translational research is commonly performed in the C57B6/J mouse strain, chosen for its genetic homogeneity and phenotypic uniformity. Here, we evaluate the suitability of the white-footed deer mouse (Peromyscus leucopus) as a model organism for aging research, offering a comparative analysis against C57B6/J and diversity outbred (DO) Mus musculus strains. Our study includes comparisons of body composition, skeletal muscle function, and cardiovascular parameters, shedding light on potential applications and limitations of P. leucopus in aging studies. Notably, P. leucopus exhibits distinct body composition characteristics, emphasizing reduced muscle force exertion and a unique metabolism, particularly in fat mass. Cardiovascular assessments showed changes in arterial stiffness, challenging conventional assumptions and highlighting the need for a nuanced interpretation of aging-related phenotypes. Our study also highlights inherent challenges associated with maintaining and phenotyping P. leucopus cohorts. Behavioral considerations, including anxiety-induced responses during handling and phenotyping assessment, pose obstacles in acquiring meaningful data. Moreover, the unique anatomy of P. leucopus necessitates careful adaptation of protocols designed for Mus musculus. While showcasing potential benefits, further extensive analyses across broader age ranges and larger cohorts are necessary to establish the reliability of P. leucopus as a robust and translatable model for aging studies.