Journal: Theranostics

Article Title: Proteasome activator PA200 maintains stability of histone marks during transcription and aging

doi: 10.7150/thno.48744

Figure Lengend Snippet: PA200 promotes degradation of core histones primarily in actively-transcribed regions. (A) Gene plot of core histone enrichment signals in PA200 +/+ (WT) and PA200 -/- (KO) MEFs. (B) The IGV genome browser view of H3K4me3 and H3K56ac enrichment in WT and PA200 KO MEFs for the selected chromosomes, while other regions between them are hidden as indicated by grey dash lines. The levels of DNA methylation are shown in parallel. Yellow dash lines indicate equal heights at y axes in each pair of WT and PA200 KO samples. Squares with green dash lines highlighted the differences in the levels of histones H3 and H4 in the selected clusters of gene loci. The signal track of each sample was calculated by MACS2 and normalized by sequencing depth. (C) The distribution of PA200-dependent histone degradation regions genome-wide by collectively analyzing DNA sequencing and WGBS data. The PA200-dependent histone degradation regions include 5972 hypo-methylation regions and 550 hyper-methylation regions, which are defined in the Methods. The average CG levels within the “hyper-methylation” regions are >0.5. To analyze the distribution of hypo-/hyper-methylation areas genome-wide, the areas whose average CG levels are>0.5 are defined as “hyper-methylation areas”, whereas whose average CG levels are<0.5 are defined as “hypo-methylation areas” as described in the Methods. The numbers of hyper-methylation and hypo-methylation areas are 684 and 123, respectively.

Article Snippet: After pulse-chase analysis as described as above, the DNA fragments associated with the newly-synthesized H3/H4 proteins were then purified with DNA purification columns (CST, #14209) and processed for ChIP-seq as described later.

Techniques: DNA Methylation Assay, Sequencing, Genome Wide, DNA Sequencing, Methylation

Journal: Theranostics

Article Title: Proteasome activator PA200 maintains stability of histone marks during transcription and aging

doi: 10.7150/thno.48744

Figure Lengend Snippet: Deletion of PA200 disrupts genome-wide transcription. (A) Hierarchical clustering of the differentially-expressed genes (DEGs) in the PA200 +/+ (WT) and PA200 -/- (KO) mouse liver. DEGs were defined according to the combination of the absolute value of log2-Ratio ≥1 and diverge probability ≥0.8. Coloring indicates the log2-transformed fold change. (B-C) Enrichment of H3K4me3 (B) and H3K56ac (C) on the gene bodies and 21 kb upstream of TSS and 21kb downstream of TTS regions in the MEF genome. (D-I) The genome average plot for the change in H3K4me3 (D-F) or H3K56ac (G-I) (normalized to H3) in the PA200 -/- (KO) over the wild-type MEF cells (WT). (J-K) The IGV genome browser view of H3K4me3 (J) or H3K56ac (K) enrichment in PA200 +/+ (WT) and PA200 -/- (KO) MEF chromosomes. The levels of DNA methylation and polymerase II were shown in parallel. (L-M) The profile analysis of RNA polymerase II on the gene bodies and 1.5 kb upstream of TSS and 1.5 kb downstream of TTS regions of the genes enriched with the H3K4me3 (L) or H3K56ac (M) in the PA200 -/- (KO) MEF cells normalized to those in the wild-type group (WT).

Article Snippet: After pulse-chase analysis as described as above, the DNA fragments associated with the newly-synthesized H3/H4 proteins were then purified with DNA purification columns (CST, #14209) and processed for ChIP-seq as described later.

Techniques: Genome Wide, Transformation Assay, DNA Methylation Assay

Journal: Theranostics

Article Title: Proteasome activator PA200 maintains stability of histone marks during transcription and aging

doi: 10.7150/thno.48744

Figure Lengend Snippet: PA200/Blm10 promotes degradation of the core histones during aging and extends cellular lifespan. (A) SA-β-gal staining of primary WT and PA200 -/- (KO) MEF cells at the indicated days. The percentage of β-galactosidase-positive cells in each group was displayed in bar graph. Scale bar: 50 µm. ** p< 0.01, *** p< 0.001; one-way ANOVA. (B) Heat map showing the expression (normalized reads per kilo base per million mapped-reads (RPKM)) of all DEGs in PA200 -/- (KO) MEF cells cultured for 0 or 30 days (normalized to WT MEFs). Each treatment has two independent biological replicates which were clustered into one group. Coloring indicates the log2 transformed fold change. (C) Immunoblotting of the whole-cell extracts from primary MEF cells at indicated days. The levels of HMGB1, a biomarker for young cells, decreased during aging. Protein levels were quantified by densitometry (normalized to β-actin). * p< 0.05; one-way ANOVA. (D) Bud scars of the wild-type, Blm10-deficient (Blm10Δ) or Blm10-overexpressing (Blm10 O/E) yeast at the indicated times. The bar graph showed the bud scar numbers, and images from the calcofluor 28 staining showed the sizes of yeast cells and bud scars. A scar is pointed by an arrow. More than 30 cells were counted for each group. ** p< 0.01; two-way ANOVA. (E) Immunoblotting of the whole-cell extracts of the wild-type or Blm10 O/E at the indicated times. Total sonicated DNA on 1% agarose gel was used as control. The total proteins were visualized by Coomassie staining following SDS-PAGE. Quantitation of histone levels was carried out by densitometry (normalized to total DNA). *** p< 0.001; two-way ANOVA. (F) Immunoblotting of the whole-cell extracts of the wild-type or Blm10Δ yeast as analyzed similarly to (E). Quantitation of histone levels was carried out by densitometry (normalized to total DNA). *** p< 0.001; two-way ANOVA. The relative histone levels in different groups at time point 0 were set to 1. Data represent three independent biological replicates (mean ± SEM).

Article Snippet: After pulse-chase analysis as described as above, the DNA fragments associated with the newly-synthesized H3/H4 proteins were then purified with DNA purification columns (CST, #14209) and processed for ChIP-seq as described later.

Techniques: Staining, Expressing, Cell Culture, Transformation Assay, Western Blot, Biomarker Assay, Sonication, Agarose Gel Electrophoresis, SDS Page, Quantitation Assay