MEP50/PRMT5 Reduces Gene Expression by Histone Arginine Methylation and this Is Reversed by PKCδ/p38δ Signaling

Abstract

Protein kinase C δ (PKCδ) and p38δ are key proteins in a cascade that stimulates keratinocyte differentiation. This cascade activates transcription of involucrin (hINV) and other genes associated with differentiation. Protein arginine methyltransferase 5 (PRMT5) is an arginine methyltransferase that symmetrically dimethylates arginine residues. This protein interacts with a cofactor, methylosome protein 50 (MEP50), and symmetrically dimethylates arginine eight of histone 3 (H3R8me2s) and arginine three of histone 4 (H4R3me2s) to silence gene expression. We use the hINV gene as a tool to understand the relationship between PKCδ/p38δ and PRMT5/MEP50 signaling. MEP50 suppresses hINV mRNA level and promoter activity. This is associated with increased arginine dimethylation of hINV gene-associated H3/H4. We further show that the PKCδ/p38δ keratinocyte differentiation cascade reduces PRMT5 and MEP50 expression, association with the hINV gene promoter, and H3R8me2s and H4R2me2s formation. We propose that PRMT5/MEP50-dependent methylation is an epigenetic mechanism that assists in silencing of hINV expression, and that PKCδ signaling activates gene expression by directly activating transcription and by suppressing PRMT5/MEP50-dependent arginine dimethylation of promoter-associated histones. This is an example of crosstalk between PKCδ/p38δ signaling and PRMT5/MEP50 epigenetic silencing.

Fig. 1

MEP50 and PRMT5 form a complex in keratinocytes. A Freshly isolated foreskin keratinocyte lysates (300 μg) were used for immunoprecipitation with Rabbit IgG or rabbit anti-MEP50, and 10 μg of total extract was electrophoresed. The antibodies for immunoblot are mouse anti-MEP50 and goat anti-PRMT5. Similar results were obtained in three separate experiments. The upper band (*) in the blot probed with MEP50 is non-specific. B/C Keratinocytes were electroporated with 3 μg of control-, MEP50- or PRMT5-siRNA. After 48h, RNA was isolated and MEP50 and PRMT5 mRNA levels were assessed by qRT-PCR. The values are mean ± SEM, n = 3. The asterisks indicate significant differences as determined by the students t-test (*, p<0.005). Extracts were also prepared to assess PRMT5 and MEP50 protein level. D MEP50 or PRMT5 overexpression. KERn were electroporated with 3 μg of control plasmid or plasmids encoding MEP50 or PRMT5, or MEP50-encoding adenovirus (10 MOI). After 48 h protein lysates were prepared for immunoblot with anti-MEP50 and anti-PRMT5. β-actin was used as the loading control. Similar results were observed in each of three experiments.

Fig. 2

MEP50 suppresses involucrin expression. A/B KERn were electroporated with the indicated plasmids. After 24 h RNA was isolated and MEP50, involucrin and filaggrin mRNA levels were assessed by qRT-PCR. The values are mean ± SEM (n = 3). The asterisks indicate a significant difference (p < 0.005). C/D KERn were transfected with 0.5 μg of the indicated involucrin promoter plasmids in the presence of 1 μg of pcDNA3 or pcDNA3-FLAG-MEP50. At 24 h post-transfection, extracts were prepared and assayed for promoter activity. E KERn were electroporated with 3 μg of control-siRNA or MEP50-siRNA. After 48 h, the cells were re-electroporated with 3 μg of endo-free involucrin promoter. After an additional 24 h, extracts were prepared and promoter activity (luciferase) assay.

Fig. 3

MEP50 suppression of hINV promoter activity. A/B Schematic showing key regulatory elements in the hINV promoter. hINV(-2473/-2088) is a construct in which the DRR (nucleotides -2473/-2088) is linked to the hINV minimal promoter. The dashed line indicates the fusion. hINV(-2473/2088)AP1-5M is identical, except that the AP1-5 site is mutated. hINV-2473 (full-length promoter), hINV-241 and hINV-41 (minimal promoter) comprise a truncation series. The functionally important AP1 (AP1-1 and AP1-5) sites and GC-rich (Sp1) element are indicated. The distances are in nucleotides relative to the transcription start site. C/D KERn were transfected with 0.5 μg of hINV-2473, which encodes the full-length wild-type human involucrin promoter, or the promoter harboring a mutant AP1-5 site (AP1-5m), or truncated promoters (hINV-241, hINV-41) and 1 μg of pcDNA3 or pcDNA3-FLAG-MEP50. At 24 h post-transfection cell extracts were prepared and assayed for promoter activity. The values are mean ± SEM, n = 3. The asterisks indicate a significant change, p < 0.005.

Fig. 4

MEP50 controls histone arginine methylation at the hINV promoter. A/B KERn were electroporated with 3 μg of control-siRNA or MEP50-siRNA. After 48 h, extracts were prepared for ChIP analysis. DNA from 1 million cells was sheared, collected and 100,000 cell equivalents of DNA was immunoprecipitated. The primers include nucleotides -2218/-2055 of the DRR region of the hINV promoter region that includes the AP1-5 site. The values are mean ± SEM, n = 3 and the asterisks indicate significant difference, p < 0.005.

Fig. 5

PKCδ/p38δ signaling reduces MEP50 and PRMT5 level and hINV promoter activity. A KERn were infected with 10 MOI of tAd5-EV or Ad5-PKCδ and at 48 h extracts were prepared for detection of PKCδ, MEP50, H3R8me2S and H4R3me2s. β-actin was used as a loading control. Similar results were obtained in three different experiments. B KERn were infected as above and after 48 h ChIP was performed using the Diagenode Low Cell ChIP Kit and primers spanning nucleotides -2218/-2055 if the hINV promoter region which includes the AP1-5 site. The values are mean ± SEM, n = 3. The asterisks indicate significant difference (p < 0.005).

Fig. 6

TPA suppresses MEP50 and PRMT5 level and activity. A/B KERn were treated with 50 ng/ml TPA for 48 h, RNA was isolated and MEP50 and PRMT5 mRNA levels were assessed by qRT-PCR. The values are mean ± SEM, n = 3, asterisk indicate a significant difference, p < 0.005. Simultaneously, protein extracts were prepared from identically treated cultures to detect MEP50, PRMT5, H3R8me2s and H4R3me2s. Similar results were obtained in three different experiments. C/D/E KERn were treated with 50 ng TPA/ml and after 48 h mRNA extracts were isolated for ChIP analysis and detection of MEP50 and PRMT5 interaction and H3R8-me2s and H4R3me2s formation at the hINV promoter. Similar results were obtained in three different experiments. The values are mean ± SEM, n = 3. The asterisks indicate significant difference (p < 0.005). F Proposed regulatory model – transcriptional activation and epigenetic de-repression. PKCδ activates the indicated p38δ MAPK cascade that triggers AP1, KLF4 and Sp1 transcription factor movement to the nucleus which activates hINV gene expression and other differentiation-related events. The PKCδ/p38δ cascade also suppresses PRMT5 and MEP50 level, leading to reduced histone arginine dimethylation of histone associated with the hINV promoter leading to de-repression of expression. This cascade can be triggered by expression of PKCδ, p38δ or by treatment with pro-differentiation agents (e.g., TPA).