The methyltransferase enzymes KMT2D, SETD1B, and ASH1L are key mediators of both metabolic and epigenetic changes during cellular senescence

Abstract

Cellular senescence is a terminal cell fate characterized by growth arrest and a metabolically active state characterized by high glycolytic activity. Human fibroblasts were placed in a unique metabolic state using a combination of methionine restriction (MetR) and rapamycin (Rapa). This combination induced a metabolic reprogramming that prevented the glycolytic shift associated with senescence. Surprisingly, cells treated in this manner did not undergo senescence but continued to divide at a slow rate even at high passage, in contrast with either Rapa treatment or MetR, both of which extended life span but eventually resulted in growth arrest. Transcriptome-wide analysis revealed a coordinated regulation of metabolic enzymes related to one-carbon metabolism including three methyltransferase enzymes (KMT2D, SETD1B, and ASH1L), key enzymes for both carnitine synthesis and histone modification. These enzymes appear to be involved in both the metabolic phenotype of senescent cells and the chromatin changes required for establishing the senescence arrest. Targeting one of these enzymes, ASH1L, produced both a glycolytic shift and senescence, providing proof of concept. These findings reveal a mechanistic link between a major metabolic hallmark of senescence and nuclear events required for senescence.

FIGURE 1:

Combined MetR and Rapa treatment increases replicative life span in HCFs beyond MetR or Rapa alone. Panel (A) contains a representative life span analysis of HCFs maintained under the following conditions with or without 1 nM Rapa: standard culture conditions at 30 mg/l methionine (Ctrl), methionine-restricted at 3 mg/l methionine (MetR 3), methionine-restricted at 1 mg/l methionine (MetR 1), and methionine-deficient (MetDEF; no methionine added to the culture medium). Treatments were initiated at cumulative population doubling (cPDL) 27, as indicated by the arrow, using cultures maintained under control conditions from a cPDL of 3. Protein and/or RNA samples were collected at three points. Experimental Point 1 is the time when 50–60% of the life span is completed for the Ctrl cultures and all cultures have high proliferative potential. Experimental Point 2 is the time when 50–60% of the life span is completed for Rapa, MetR, and the combination Rapa+MetR. Ctrl cultures are entering senescence at this point. At Experimental Point 3, samples were taken from the combination Rapa+MetR for RNA sequence analysis. Life span analysis was performed three times with similar results and was performed at 6% oxygen with similar results. Panel (B) contains maximum life span as assessed by logistic regression analysis with 95% confidence intervals. In this graph. Conditions that show a significant difference (P < 0.05) from Ctrl for the average maximum cPDL were assessed from three independent experiments as assessed by T test are labeled with an asterisk. Growth rates at early passage of the MetR (0 mg/l and 1 mg/l) cultures were significantly reduced relative to Ctrl cultures (difference in growth rate over 5 wk, P = 0.004 for MetR 1 mg/l and P = 0.002 for MetR 0 mg/l, as assessed by two-tailed, unpaired T test), which revealed an acute negative impact on proliferation of these cultures. The addition of Rapa to these early passage cultures increased the growth rate such that there was no significant difference from controls. Life span curves were completed three times under ambient oxygen conditions and once in 5% oxygen. In all cases, the results were comparable. The combination of Rapa treatment and MetR increased life span beyond all other conditions and prevented senescence as judged by continued slow proliferation until termination of the experiment. In Panel (B), average data ± SD from three independent life span curves is presented. Maximum cPDLs were calculated using the logistic growth function in GraphPad to provide predicted maximal population doublings. Asterisks denote values that are statistically significant relative to control life span at P < 0.05.

FIGURE 2:

Markers of senescence are reduced with Rapa treatment and MetR. Steady-state protein (A, B) and mRNA (C–E) levels of senescence markers were examined by immunoblot and NanoString analysis, respectively. HCFs were grown under the indicated conditions and protein levels of p16, p53, p21, lamin B1, phosphorylated S6rp [p(S235/236)S6rp], and S6rp were determined by immunoblot analysis. Actin and EEF2 were used as loading controls. Panel (A) includes samples taken from early passage cultures compared with samples taken from senescent cells. The samples in (B) were taken at Experimental Point 2 in Figure 1. At this time, the control cultures (30 mg/l methionine without Rapa) were entering senescence and displaying characteristic changes in gene expression associated with senescence, such as increased p16^INK4A and p21^Cip1/Waf1 levels and reduced lamin B1. A senescent cell sample is used as control for the data set in (B). The bar labels are as follows: standard conditions 30 mg/l methionine (Ctrl); 1 mg/l methionine (MetR); 1 nM Rapa treatment (Rapa); and a combination of MetR plus Rapa treatment (MetR + Rapa). Assessment of mRNA levels for p21^Cip1/Waf1 (C), p16^INK4A (D), and lamin B1 (E) was performed at Week 4 of life span while protein levels were assessed at Week 23. Bars marked with an asterisk represent values that are significantly different from relative control values (without Rapa) at P < 0.05 and bars marked with a pound sign represent values that are significantly different between conditions with or without Rapa within the same methionine group (i.e., Ctrl, MetR) as evaluated using a two-tailed unpaired T test. Immunoblot analyses were repeated a minimum of three times and the Nanostring results are representative of two independent experiments. Nanostring results were verified by quantitative real-time RT-PCR.

FIGURE 3:

HCFs exposed to MetR plus Rapa treatment do not undergo replicative senescence. Panel (A) contains a volcano plot showing differential gene expression analysis of late passage control cells compared with early-passage control cultures. The graph compares the log [2-fold change] in sequencing counts with the (–)log 10 of the P value for each comparison. A total of 789 mRNA sequences met the criteria of P < 0.05 and a log 2-fold change of 2. A subset of genes whose expression levels are known to be altered during the senescence program were identified in the analysis and these genes are presented in the bar graph to the right of the plot. Three senescence-related genes from this list are labeled in the volcano plot. Panel (B) contains an identical comparison as in (A) comparing early passage cells with the very late-passage MethR + Rapa-treated cells at PD 91. Note that the senescence-associated genes show a completely different pattern of expression from the results in (A). Panel (C) contains the results of distance matrix analysis for all cultures in the study. Early passage cells were used at Week 4, while late passage, Rapa-treated, MetR, and Rapa+MetR were used at Week 23 (Experimental Points 1 and 2 in Figure 1). Samples labeled EarlyPass represent values from early passage (cPDL 25) cells. Samples labeled RapaCtrl contain values from cultures treated with only Rapa. Samples labeled MetR contain values from methionine-restricted (1 mg/l) cultures. Samples labeled RapaMetR contain values from cultures exposed to MetR plus Rapa. Samples labeled LatePass contain values from senescent control cultures. The Rapa+MetR samples were used at Week 47 (Experimental Point 3 in Figure 1). These late passage (cPDL 91) cultures maintained under the combination of MetR plus Rapa treatment are labeled OldRapaMR. The gene count profile from each RNA-Seq sample was compared with all other RNA-Seq samples to evaluate general similarity. Darker blue shading indicates a shorter Euclidean distance (greater similarity) compared with lighter blue shading which indicates a longer distance. Each block contains the numerical Euclidean distance for the given comparison with a higher number indicating a greater distance. The diagonal shows zeros indicating that a sample compared with itself gives an identical value, as similarity between samples is inversely related to distance. Samples with names ending in a letter were included in RNA-Seq Run 1. Samples with names ending in a number were included in Run 2. Panel (C) contains the results of a PCA of the RNA-Seq data. The plot represents the general similarity of each sample based on the top 500 differentially expressed genes. Circles represent samples run in the first round of sequencing and triangles represent samples run in the second round of sequencing. Each treatment is color coded. Note that the samples from the very late passage MethR plus Rapa treatment cultures cluster separately from all other samples in accordance with the SDM results.

FIGURE 4:

MetR plus Rapa treatment preserves chromatin accessibility. Panel (A) contains chromatin accessibility as assessed by ATAC seq analysis for a representative chromosome (chromosome 9). Two independent replicates from cultures at early passage, late passage (senescent), and late passage treated with MetR plus Rapa are presented. In (A), peaks are presented as black lines, resulting in dark regions denoting high accessibility, while regions of reduced accessibility lack coverage and appear as white areas (indicated by red circles on senescent tracks). For comparison, UCSC tracks denoting ENCODE generated open chromatin marks (DNAse hypersensitivity and Histone H2K27ac) are presented below the ATAC seq analysis. Panel (B) contains an analysis of the relative peak density in intergenic, intron, exon, 5′ UTR, and promoter regions in each sample set.

FIGURE 5:

The combination of Rapa treatment plus MetR increases fatty acid utilization. Metabolic flexibility is defined as the ability to maintain oxygen consumption following treatment with metabolic pathway pharmacological inhibitor(s). Panel (A) compares the OCR in response to palmitate (palm) and an inhibitor of CPT1 (etomoxir [eto]) in early-passage versus late passage (senescent) cultures. Panel (B) shows results comparing late passage cultures versus late passage cultures treated with Rapa. Panel (C) shows the results of a comparison between methionine-restricted cultures and cultures subjected to the combination ofRapa plus MetR (1 mg/l) treatment. Panels (D) and (E) contain results of glucose utilization rates and ECARs, respectively. Results are representative of at least two independent experiments and significance (P < 0.05) in unpaired T tests between samples and is indicated by asterisks.

FIGURE 6:

The combination of MetR and Rapa increases utilization of alternative carbon sources following inhibition of specific metabolic pathways. Metabolic flexibility, defined as the ability to maintain oxygen consumption following treatment with metabolic pathway pharmacological inhibitor(s), was tested under conditions of Rapa and Rapa plus MetR at 1 mg/l. Panels (A) and (B) contain the results of a comparison of Rapa-treated cultures and cultures treated with the combination of MetR and Rapa. Panels (C) and (D) contain the results of a comparison of methionine-restricted cultures and cultures treated with the combination of MetR and Rapa. Pyruvate oxidation was determined following the addition of UK5099 (inhibitor of mitochondrial pyruvate transporter), while the capacity for pyruvate oxidation was determined following sequential treatment of the glutaminase inhibitor BPTES (10 μM) and Etomoxir (15 μM) followed by UK5099. Glutamine/palmitate oxidation was determined following treatment with BPTES/Etomoxir (left panels in [A] and [C]), while the capacity for glutamine/palmitate oxidation was determined following sequential treatment of UK5099, and the combination of BPTES and Etomoxir (right panels in [A] and [C]). The measurement of spare capacity was based on the difference between capacity and the rate of oxidation for pyruvate oxidation (left panels in [B] and [D]) and glutamine/fatty acid oxidation (right panels in [B] and [D]). OCR and ECAR were normalized to cell number by counting cells in each well at the completion of the assay using flow cytometry. Asterisks represent values that are significantly (P < 0.05) different versus control. Significance is based on unpaired, two-tailed, Student’s T test. Results are representative of two independent experiments.

FIGURE 7:

Reduced expression of histone methyltransferase ASH1L induces premature senescence while histone H3 levels are maintained by MetR plus Rapa. In Panel (A), quantitative RT-PCR analysis of mRNA levels for the methyltransferases ASH1L, SETD1B, and KMT2D is shown. Values were normalized relative to GAPDH. Bars labeled Ctrl show values from early passage cultures in comparison to early passage cultures under MetR (1 mg/l), Rapa treatment, or the combination of MetR + Rapa or senescent from cultures grown to late passage (>50 population doublings) that have entered senescence. Panel (B) contains quantitative real-time RT-PCR analysis of ASH1L mRNA levels in HCF cells following infection with two independent lentiviral vectors expressing shRNA constructs targeting the ASH1L mRNA. Panel (C) shows the percentage of cells stained positive for SA-β-galactosidase activity in the 2 shRNA-expressing cultures examined in (B). Panel (D) contains representative photographs of HCF cells stained for SA-β-galactosidase harboring either the pLKO vector or the pLKO expressing one of the two shRNA molecules directed against ASH1L. Induction of senescence was repeated in two independent experiments. Differences marked with a single asterisk represent significance at P < 0.05. Differences marked with a double asterisk represent significance at P < 0.01. Panel (E) shows the histone H3 content of late passage cells and late passage cells cultured under MetR + Rapa conditions. Panel (F) shows relative levels of H3K36me1, 2, and 3. Relative quantities for the modifications are presented as absorbance units per ng H3 protein. Panel (G) shows the graphic output of the ECAR generated from a Seahorse extracellular flux analysis system. Panel (H) contains an integrated quantification of the ECAR from the Seahorse extracellular flux analysis. The flux analysis results are representative of three independent experiments and two independent lentiviral vectors expressing shRNA constructs targeting the ASH1L mRNA shown in (B). Differences marked with a single asterisk are significant at P < 0.05 and differences marked with a double asterisk are significant at P < 0.01.

FIGURE 8:

Schematic representation of dual roles for the KMT2D, SETD1B, and ASH1L methyltransferases in beta-oxidation and histone modification. The schematic highlights a potential role for the regulation of both metabolic and epigenetic changes during senescence by the three enzymes KMT2D, SETD1B, and ASH1L. Rate limiting steps in both carnitine synthesis and histone 3 methylation at lysine 4, a critical site for transcriptional activation of gene promoters. Targeting the most differentially expressed of these enzymes, ASH1L, results in a dramatic decrease in transcription, a metabolic shift toward glycolysis, and senescence. We propose that these three enzymes help coordinate metabolic status with epigenetic modifications during senescence.