PRMT6 activates cyclin D1 expression in conjunction with the transcription factor LEF1

The establishment of cell type specific gene expression by transcription factors and their epigenetic cofactors is central for cell fate decisions. Protein arginine methyltransferase 6 (PRMT6) is an epigenetic regulator of gene expression mainly through methylating arginines at histone H3. This way it influences cellular differentiation and proliferation. PRMT6 lacks DNA-binding capability but is recruited by transcription factors to regulate gene expression. However, currently only a limited number of transcription factors have been identified, which facilitate recruitment of PRMT6 to key cell cycle related target genes. Here, we show that LEF1 contributes to the recruitment of PRMT6 to the central cell cycle regulator CCND1 (Cyclin D1). We identified LEF1 as an interaction partner of PRMT6. Knockdown of LEF1 or PRMT6 reduces CCND1 expression. This is in line with our observation that knockdown of PRMT6 increases the number of cells in G1 phase of the cell cycle and decreases proliferation. These results improve the understanding of PRMT6 activity in cell cycle regulation. We expect that these insights will foster the rational development and usage of specific PRMT6 inhibitors for cancer therapy.


Supplementary
. LEF1 ChIP-Seq analysis in K562 cells. A Distribution of the aggregated peak numbers around the transcription start site. The distance from the TSS is shown in bp. The peak count shows that most LEF1 peaks are located near a transcription start site. B Peak distribution over different genomic features. 24.2% of the peaks are located in promoter regions or introns. 32.9% are located in intergenic regions and 42.9% in UTRs, Exons and Introns.
C GO-term analysis of genes with altered expression upon PRMT6 knock down in K562 cells. PRMT6 was knocked down by shRNA and gene expression changes were measured by Agilent whole human genome expression microarrays. Altered genes were grouped according to their GO-terms using DAVID. Enriched GO-terms and the number of genes in the groups are shown. Command line arguments of the R script. The MACS peak coordinates were reduced to a 3 columns BED file and transferred to Granges with the Bioconductor package ChIPpeakAnno, (V.3.10). Only peaks which overlap +/-500 bp around the transcription start site were extracted. The peaks were annotated with entrez IDs and the enriched GO-terms were calculated.

Glutathione S-transferase (GST) pulldown assay
For GST-pulldown, GST-fusion proteins were constructed using pGEX-4T1 (Amersham Biosciences). GST or GST-fusion proteins were co-expressed with chaperone plasmid pGro7 (TAKARA) in E. coli BL21 (DE3) (NEB). Induction was performed by using 0.05% L-Arabinose (pGro7) and 1 mM IPTG (pGEX-4T1) for 4 hours at 37°C. Cells were harvested in lysis buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 10 % Glycerol, 0.1 % Triton X-100, 1 mM DTT, 1 mM EDTA, protease inhibitor), disrupted by sonification and the supernatant was incubated with glutathione beads (Pierce TM , Thermo Scientific) for 4 hours with rotation at 4°C. Glutathione beads were washed for three times with lysis buffer and then incubated in dissociation buffer (50 mM Tris, pH 7.4, 100 mM KCl, 10 mM MgCl2, 5 mM ATP) for 2 hours at 4°C to remove the non-specific binding of co-expressed chaperone proteins. The Beads were washed two times with lysis buffer and equal amounts of protein bound to beads were employed in GST-pulldown assay. GST-pulldown with GST-PRMT6 as bait was either performed with LEF1 protein expressed in HEK293T cells or with in vitro translated LEF1.
For expression of LEF1 protein in HEK293T cells, 2 x 10 6 cells were seeded into a 6cm dish. Cells were transfected with 6 µg Plasmid and 18 µl of Metafectene transfection reagent. After 48 hours, cells were harvested and lysed. For each pulldown reaction 500 µg of cell lysate was used.
In vitro translation was performed using the TNT T7 Quick coupled transcription/translation system (Promega). For pulldown reaction, 10 µl of in vitro translate was incubated with protein beads in 250 µl lysis buffer for 3 hours at 4°C.
With the same buffer, protein beads were washed for four times and boiled in 20 µl of SDS loading dye. The eluted proteins were analysed with Western Blot.
For radioactive labelling of protein, in vitro transcription/translation was performed in the presence of 35 S-methionine (10 mCi/ml; 1000 Ci/mmol; Hartmann Analytic).
Proteins were pulled out with glutathione beads (see above), detected by SDS-PAGE and autoradiography as described 39 .

Mass spectrometry
Transduced cells expressing the avi-PRMT6 protein were grown in heavy (H) SILAC medium and the control cells with avi-tag only were grown in light (L) SILAC medium.
Nuclear extracts of 1 × 10 8 K562 avi-PRMT6 and Bio-tag only control cells were Carbamidomethylation of cysteine was defined as a fixed modification. Minimal peptide length was set to seven amino acids, allowing up to two missed cleavages. Both on the peptide and protein level the false discovery rate (FDR) was set to 1% on using a forward-and-reverse concatenated decoy database approach. SILAC multiplicity was set to double labeling (Lys+0/Arg+0, Lys+8/Arg+10) requiring at least two ratio counts for peptide quantitation and enabling the "re-quantify" option.