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. 2022 Apr 25;11(9):1450.
doi: 10.3390/cells11091450.

The Ash2l SDI Domain Is Required to Maintain the Stability and Binding of DPY30

Mengjie Ma  1 , Jiafeng Zhou  1 , Zhihua Ma  1 , Hanxue Chen  1 , Liang Li  1 , Lin Hou  1 , Bin Yin  1 , Boqin Qiang  1 , Pengcheng Shu  1   2 , Xiaozhong Peng  1   3
Affiliations

The Ash2l SDI Domain Is Required to Maintain the Stability and Binding of DPY30

Mengjie Ma et al. Cells. .

Abstract

ASH2L and DPY30 are important for the assembly and catalytic activity of the complex associated with SET1 (COMPASS), which catalyzes histone methylation and regulates gene expression. However, the regulations among COMPASS components are not fully understood. Here, we leveraged a mouse model and cell lines to observe the outcome of Ash2l depletion and found a significant decrease in DPY30. Analyzing ASH2L ChIP-seq and RNA-seq data excluded transcriptional and translational regulation of ASH2L to DPY30. The decrease in DPY30 was further attributed to the degradation via the ubiquitin-mediated proteasomal pathway. We also verified that three amino acids in the ASH2L Sdc1 DPY30 interaction (SDI) domain are essential for the recognition and binding of DPY30. Lastly, we unexpectedly observed that overexpression of DPY30 in Ash2l-depleted cells rescued the decrease in Ccnd1 and the abnormal cell cycle, which indicates that DPY30 can participate in other complexes to regulate gene expression. Overall, our results, for the first time, reveal that the existence of DPY30 relies on the binding with ASH2L, with degradation of DPY30 via the ubiquitin-proteasome system, and they further indicate that the function of DPY30 can be independent of ASH2L.

Keywords: Ash2l; COMPASS; Dpy30; protein degradation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Ash2l is crucial for the expression of DPY30 both in vivo and in vitro. (A) Immunofluorescence of ASH2L and DPY30 in the E14.5 dorsal cortex. Scale bars = 100 μm. (B) Immunofluorescence of RbBP5 in the E14.5 dorsal neocortex. (C) Western blot to detect the expression of ASH2L and WDR5 in the E14.5 neocortex. (D) Detection of DPY30 and ASH2L in HEK293T cells transfected with Ash2l-shRNAs, harvested 72 h after transfection. (C,D) were biologically repeated three times.
Figure 2
Figure 2
ASH2L does not regulate the transcription of Dpy30. (A) H3K4me3 and ASHS2L ChIP-seq visualized by gene track snapshot at Dpy30 gene loci. (B) RNA-seq result visualized by gene track snapshot at Ash2l, Dpy30, and Actb gene loci. (C) RT-qPCR to detect the expression of DPY30 after Ash2l knocked down in HEK293T cell line. Statistical analysis: ** p < 0.01.
Figure 3
Figure 3
ASH2L is necessary for the stability of DPY30. (A,B) mRNA stability assays to detect the relative quantity of Dpy30 mRNA by RT-qPCR after knockdown and overexpression of Ash2l followed with actinomycin D (10 μg/mL) treatment. Relative quantity is normalized by GAPDH at each timepoint. The experiment was repeated three times, and the standard deviation is shown. The t-test at each timepoint indicates no significant difference. (C) HEK293T cells were transfected with Ash2l-shRNAs for 36 h and treated with cycloheximide (50 μg/mL) for the duration shown in hours. The expression levels of ASH2L and DPY30 were tested by Western blotting. The experiment was biologically repeated three times.
Figure 4
Figure 4
The stability and degradation of DPY30 (A) Construction of the ASH2L-WT, ASH2L-ΔSDI, and ASH2L-3R mutations. (B) Coimmunoprecipitation assay using FALG-tagged ASH2L-WT, ASH2L-SDI, and ASH2L-3R to detect their binding with DPY30. (C) Gradient doses of MG132 (upper panel) and chloroquine (lower panel) were used to inhibit the cell proteasome and lysosome, respectively, and the relative expression level of DPY30 in different cell lines was observed by Western blot. Experiments in different cell lines were repeated three times. (D) Coimmunoprecipitation assay using FLAG-tagged DPY30 to detect the binding of ubiquitin after knockdown of ASH2L.
Figure 5
Figure 5
DPY30 ameliorates the outcome of Ash2l depletion (A) Immunofluorescence of Cyclin D1 in the dorsal neocortex of WT and Ash2l-cKO mice at E12.5, E14.5, and E16.5. (B) Ash2l-shRNA was used to interfere with the expression of Ash2l in the N1E-115 cell line, and the expression of Cyclin D1 was decreased compared to that of the control group. (C) RT-qPCR to detect the expression of Ccnd1 after overexpression of DPY30. The siAsh2l group and siAsh2l + Dpy30-pCIG group were compared with yjr siN.C. group. Statistical analysis: * p < 0.05; ** p < 0.01; *** p < 0.001. (C’) Western blotting was biologically repeated three times and applied to detect Cyclin D1 protein after the overexpression of DPY30.
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