The assembly of mammalian SWI/SNF chromatin remodeling complexes is regulated by lysine-methylation dependent proteolysis

Abstract

The assembly of mammalian SWI/SNF chromatin remodeling complexes is developmentally programed, and loss/mutations of SWI/SNF subunits alter the levels of other components through proteolysis, causing cancers. Here, we show that mouse Lsd1/Kdm1a deletion causes dramatic dissolution of SWI/SNF complexes and that LSD1 demethylates the methylated lysine residues in SMARCC1 and SMARCC2 to preserve the structural integrity of SWI/SNF complexes. The methylated SMARCC1/SMARCC2 are targeted for proteolysis by L3MBTL3 and the CRL4DCAF5 ubiquitin ligase complex. We identify SMARCC1 as the critical target of LSD1 and L3MBTL3 to maintain the pluripotency and self-renewal of embryonic stem cells. L3MBTL3 also regulates SMARCC1/SMARCC2 proteolysis induced by the loss of SWI/SNF subunits. Consistently, mouse L3mbtl3 deletion causes striking accumulation of SWI/SNF components, associated with embryonic lethality. Our studies reveal that the assembly/disassembly of SWI/SNF complexes is dynamically controlled by a lysine-methylation dependent proteolytic mechanism to maintain the integrity of the SWI/SNF complexes.

A Methylation-Phosphorylation Switch Controls EZH2 Stability and Hematopoiesis

“…silencing-induced EZH2 reduction is reversed by the treatment of 26S proteasome inhibitor, MG132, added in the last 6 hours of the experiment (Figure 1F and 1G) (Guo et al, 2022). Our studies indicate that LSD1 is required to maintain the protein stability of EZH2 to prevent the disassembly of PRC2.…”

“…Viral infected H1299 and G401 cells were selected by puromycin resistance as described before (Guo et al, 2022;Leng, Yu, et al, 2018).…”

“…Our previous studies have shown that L3MBTL3 recruits CRL4 DCAF5 ubiquitin E3 ligase complex to target substrates, such as DNMT1, SOX2, and SMARCC1, for ubiquitin dependent proteolysis (Guo et al, 2022;Leng, Yu, et al, 2018;Zhang et al, 2019). To determine whether DCAF5, a substrate specific subunit of the CRL4 ubiquitin E3 ligase complex (Leng, Yu, et al, 2018), is involved in regulating the levels of EZH2 and H3K27me3, we employed the CRISPR-Cas9 gene editing system to delete the exon 4 of the mouse Dcaf5 allele to establish the Dcaf5 deletion mutant mouse line with a stop codon to the remaining downstream read frame of the Dcaf5 allele (Figure 3A and 3B)(S. Kleinstiver et al, 2016;Slaymaker et al, 2016).…”

“…The unbound flow-through antibody fractions were then loaded onto the monomethylated K20 peptide column (0.5 ml), washed, and the bound antibody fractions were eluted by 5 ml of 100 mM glycine, pH2.5. The eluted antibodies (0.5 ml/fraction) were immediately neutralized by adding 100 l of 2M Tris, pH8.5, and tested for specificity towards the monomethylated K20 peptide but not to the unmethylated K420 peptide (Guo et al, 2022;Leng, Yu, et al, 2018;Zhang et al, 2019). Human LSD1 were cloned into pGEXKG vectors and purified by GSH-Sepharose (GE Healthcare).…”

“…For the analysis of proteins and DNA from embryos, the experimental procedures for embryo isolation were approved by the UNLV Institutional Animal Use and Care Committee (IACUC). The embryos from the euthanized pregnant female mice or dissected brains, spleens, or livers from the conditional knockout mice, washed with PBS, and lysed in the NP40 lysis buffer (Guo et al, 2022). For the nuclear and cytosolic fractions were separated by centrifugation.…”