Journal: bioRxiv

Article Title: A novel antiviral lncRNA EDAL shields a T309 O -GlcNAcylation site to promote EZH2 degradation

doi: 10.1101/824813

Figure Lengend Snippet: A. N2a cells were transfected with pcDNA3.1 or pcDNA-EDAL for 12 h and then infected with RABV at MOI 1 for 48 h. Total RNA was isolated and subjected to RNA-seq analysis ( n =2; 2 fold change (FC) and 0.01 p-value ). B. N2a cells were transfected with pcDNA3.1 or pcDNA-EDAL for 48 h and then ChIP-seq analysis was performed. Volcano plot showed the peaks enriched in negative control (NC) cells and EDAL overexpression cells. X axis was the log2 ratio of EDAL versus NC signals for each peak, and Y axis was the significance of the differences (−log10 ( P-values )). C. Six up-regulated and loss of H3K27me3 mark genes were cloned into the mammalian expression vector pCAGGS and overexpressed in N2a cells. At 12 h post transfection, the cells were infected with RABV for 48 h at MOI 0.01, and virus titers in the supernatant were measured. D. N2a cells were transfected with pCAGGS- Pcp4l1 (pC- Pcp4l1 ) at indicated dose for 12 h, and then infected with RABV at MOI 0.01. At 48 hpi, the virus load in the cell supernatant was measured. PCP4L1 expression level was analyzed by Western blotting. E. pcDNA-RABV-N, pcDNA-RABV-P together with pCAGGS or pC-Pcp4l1-flag was transfected into N2a cells for 48 h. The level of RABV-N protein and RABV-P protein was analyzed by Western blotting and normalized to GAPDH. F. N2a cells were transfected with pCAGGS- Pcp4l1 (pC- Pcp4l1 ) for 12 h, and then infected with VSV at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. G. N2a cells were transfected with pC- Pcp4l1 for 24 h, and then infected with SFV at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. H. N2a cells were transfected with pC- Pcp4l1 for 24 h, and then infected with HSV-1 at MOI 0.01. At indicated hpi, the virus load in the cell supernatant was measured. I. Sequencing profile of Pcp4l1 for ChIP-seq. The two tracks show H3K27me3 signals for pcDNA3.1 and pcDNA-EDAL samples after removing input background. The brown rectangle indicates the predicted promoter region of Pcp4l1 . J. N2a cells were transfected with pcDNA-EDAL or pcDNA3.1 for 48 h, and then ChIP-qPCR were performed with H3K27me3 antibody in the promoter region of Pcp4l1 . K. N2a cells were treated with 4 µM gsk126 or DMSO (mock) for 48 h and Pcp4l1 mRNA level was analyzed by qPCR. L. Proposed model for EDAL-induced EZH2 lysosomal degradation, and the potential subsequent impact on EZH2-mediated epigenetic silencing of Pcp4l1 . Statistical analysis of grouped comparisons was carried out by student’s t test(**P<0.01; ***P<0.001). Bar graph represents means ± SD, n = 3.

Article Snippet: The primary antibodies were against RABV N protein (prepared by our lab, 1:5000), H3K27me3 (Abclonal Technology, Wuhan, China, A2363, 1:2000), H3 (Abclonal Technology, A2348, 1:2000), EZH2 (CST, #5246, 1:2000), Flag tag (MBL, M185-3L, 1:10000), HA tag (MBL, M180-3, 1:10000), PCP4L1 (ProteinTech, 25933-1-AP, 1:2000) or GAPDH (ProteinTech, 60004-1-Ig, 1:5000).

Techniques: Transfection, Infection, Isolation, RNA Sequencing Assay, ChIP-sequencing, Negative Control, Over Expression, Clone Assay, Expressing, Plasmid Preparation, Western Blot, Sequencing

Journal: Nature Protocols

Article Title: Engineering proteins for allosteric control by light or ligands

doi: 10.1038/s41596-019-0165-3

Figure Lengend Snippet: Fig. 2 | Strategies for engineered control of protein activity. a, In rapamycin-regulated control (RapR), an insertable FKBP domain (iFKBP) is inserted at a surface loop, where it allosterically inhibits the activity of the protein. Heterodimerization of iFKBP with co-expressed FRB is induced by addition of rapamycin (pink circle) to the medium, leading to activation of the target protein and productive interaction with downstream targets (P). Typically, constitutively active proteins are used so that activity is solely controlled by the user. b, The single-chain unimolecular version of this approach, called uniRapR, does not require co-expression of FRB, as the inserted domain (U) is a fusion of iFKBP and FRB. c, RapR-TAP enables allosteric activation of the target protein in a specified subcellular region, or when it is interacting specifically with one downstream target. Here, iFKBP is inserted into the target, and the FRB domain required for activation is fused to a subcellular targeting sequence (left) or to the specific target protein (right). d, In protein photoactivation, the LOV2 domain (L) is fused to the target protein, where it sterically blocks the active site. Blue light induces a LOV2 conformational change that reversibly uncovers the active site. e, In allosteric photoinhibition, LOV2 is inserted at a site where it allosterically perturbs the active site only in the light. f, A structural model of uniRapR13 bound to rapamycin (top) and the LOV2 domain from the crystal structure of PA- Rac1 (PDB ID 2WKP). The close proximity of the N and C termini of LOV2 and of the iFKBP portion of the uniRapR domain enables successful insertion into surface loops and subsequent allosteric regulation. hv, light.

Article Snippet: This plasmid is used to design single-chain rapamycin-induced allosteric proteins ● DNA encoding residues 1–87 of the iFKBP domain (Addgene, plasmid no. 25933) and residues 1–96 of FRB (Addgene, plasmid no. 25920) These plasmids are used to design dual-chain rapamycin-induced allosteric proteins ● DNA encoding residues 1–143 of the LOV2 domain (Addgene, plasmid no. 86974).

Techniques: Control, Activity Assay, Activation Assay, Expressing, Sequencing