RACK7 recognizes H3.3G34R mutation to suppress expression of MHC class II complex components and their delivery pathway in pediatric glioblastoma


HA-RACK7 Full length HA-RACK7 Full length HA-RACK7 Full length D -2 10 -1 10 0 10 1 10 2 10 3 10 4 PHD RACK7(D104A) -H3.3G34R(22-44) Fnorm [1/1000] Concentration (µM) 

RACK7 recognizes H3.3G34R mutation. (A)

In vitro peptide pull-down assays using various H3.3 peptides and GST-tagged BS69 50-401 purified from E. coli. Left panel: the concentration of the peptides used in the peptide pull-down assays. Right panel: the biotinylated peptides were immobilized on streptavidin beads and the bound proteins were subject to SDS-PAGE and visualized by Coomassie blue staining. (B) Peptide pull-down assays using indicated biotinylated peptides and the HA-tagged full length RACK7. Glycine at position 34 was replaced with various amino acids as indicated. (C) Peptide pull-down assays using the indicated biotinylated peptides and the HA-tagged full length RACK7. (D) MicroScale Thermophoresis analysis of the interaction between GST-tagged PHD RACK7(D104A) protein purified from E.coli with H3. 3G34R(22-44) Western blot of RACK7 level in R6, R6 Table S3. 
List of DNA oligonucleotides used in ChIP-qPCR assay. 

DNA oligonucleotides for ChIP-qPCR

Intergenic region1 F TGTGTCTGTCCATCCCCTCTG 
Intergenic region1 R AGAGAAACGGGCCTCCCTTAG Intergenic region2 F GGAAGTCATGGGCATGAGGAT Intergenic region2 R GTAGGTCTCTTCTCACGGCAG ZNF75A F GCTTCAGCGGGTCTGGATAC ZNF75A R AGTGGAAGTGACGCTTTCCG SCAPER F GGGAGGTGTGAGGGTTGATT SCAPER R ACGAGAAAGACCTGTGTGGC MRTO4 F CACGGGAAGTTGCCCTACAG MRTO4 R GTGCACCCCGTTAGGACC PRKCA F GCACAGTCCTCACCTGGTTTC PRKCA R GCATTTCTGAGCATCCCTTCTG CIITA F TTGGCATTAGGTCGCACTGT CIITA R CAGTACCCAAGCACTCAGCA HLA-DRA F GCCTTTCATCCTTCTCCAGTGT HLA-DRA R TTGGCAGTACTGAGGACATAGC HLA-DQA1 F GCTACCACCAGCAGAGATCC HLA-DQA1 R GCCCTTTCTCTGGCTTCTGT CD74 F CAGGCAAATGTCCACTCCCT CD74 R TCCTCCTGCGTGTTTGAGAC Negative control F CCATTCAACCCAAGGATGAGC Negative control R TCTGCAATCATGGTCCTCACT GFAP F TCGAGTTCCCACACATCAGC GFAP R CCAGGTCTCAGGCTCCTAGT QKI F CATTTCGAGCACGACTGACG QKI R GATCTTGTTGCACCTTCCCG VIM F AAGTCGATGGACAGAGGCG VIM R AGATCTGAAGTCGCGGAGAA OCIAD2 F CTGCCAGGACTGACTGAGTG OCIAD2 R GGTACAGGGACCAATTCGCA COL6A1 F CACGCTGGTTTTCAGACGTT COL6A1 R TGCAGTTCAGTTCCCGTGTC Table S4.
List of DNA oligonucleotides used in RT-qPCR assay. 

DNA oligonucleotides for RT-qPCR


CIITA F TGAGGCTGTGTGCTTCTGAG


F

  Fig. S1. 




  Fig. 1H and Fig. 1I. (G) Peptide pull-down assays using the indicated biotinylated peptides 




  Fig. S3. 




  Fig. S4. 




  Fig. S6. 


Table S1 .

 S1 Effects of treatments on MHC class II level in tumor cells 


		[37-40] .
	Treatment	Type of tumor cells	Effects
	DNA-alkylating agents	lymphoma cell line	Activates CIITA and induces MHC-II
	JAK1/2 inhibitor ruxolitinib (RUX)	myeloid leukemia (CML) cells	Activates CIITA and induces MHC-II
	BRAF inhibitors	melanoma cells	Enhances IFN-inductibility of MHC-II
	HDAC inhibitors	various cancer cells	Upregulates MHC-II
	Demethylating agents-5-azacytidine	various cancer cells	Upregulates MHC-II
	IFN-γ	various cancer cells	Upregulates MHC-II
	Bacillus Calmette-Gue´rin	bladder cancer	Induces expression of MHC-II
	TLR7 agonists-Imiquimod	cutaneous cancer cells	Enhances expression of MHC-II
	TLR7/8 agonists-Resiquimod (R-848)	AML blasts	Upregulates MHC-II
	CpG oligodeoxynucleotide	tumor B cells	Upregulates MHC-II
	CD40 agonists	malignant B cells	Enhances expression of MHC-II

Table S2 .

 S2 List of antibodies used in the study. 


	Antibodies for immunoblotting		
	Primary Abs	Clone	Source	Catalog number Dilution
	Rabbit anti-RACK7 Antibody	Polyclonal	Bethyl	A302-089A	1:6000
	Mouse anti-LAMIN B1 Antibody	3C10G12 Proteintech	66095-1-Ig	1:3000
	Mouse anti-FLAG-tag Antibody	M2	Sigma	F1804	1:3000
	Rabbit anti-GFAP Antibody	Polyclonal Proteintech	16825-1-AP	1:1000
	Rabbit anti-HLA-DRA Antibody	Polyclonal Proteintech	17221-1-AP	1:2000
	Rabbit anti-HLA-DRB5 Antibody Polyclonal Proteintech	21702-1-AP	1:500
	Antibodies for immunofluorescent (IF)	
	Primary Abs	Clone	Source	Catalog number Dilution
	Rabbit anti-RACK7 Antibody	Polyclonal	Bethyl	A302-089A	1:500
	Rabbit anti-GFAP Antibody	Polyclonal Proteintech	16825-1-AP	1:250
	Rabbit anti-CD74 Antibody	D5N3I	Cell	77274	1:200
			signaling		
			technology		

Antibody for Chromatin immunoprecipitation (ChIP) and ChIP-Seq

  
	Antibody	Clone	Source	Catalog number Amount
	Rabbit anti-RACK7 Antibody	Polyclonal	Bethyl	A302-089A	2μg
	Rabbit anti-histone H3K4me1	Polyclonal	Active	39297	5μl
	Antibody (pAb)		motif		
	Antibody for Flow Cytometry		
	Antibody	Clone	Source	Catalog number Amount
	APC Mouse Anti-Human HLA-DR	G46-6	BD	560896	20μl
			bioscience
Fangfang Jiao, Ze Li, Chen He, Wenqi Xu, Gensheng Yang, Tingting Liu, Hongjie Shen, Jiajun Cai, Jamie N Anastas, Ying Mao, Yongchun Yu, Yujiang Fei Lan, Yujiang Geno Shi, Chris Jones, Yanhui Xu, Suzanne J Baker, Yang Shi, Rui Guo
RACK7 recognizes H3.3G34R mutation to suppress expression of MHC class II complex components and their delivery pathway in pediatric glioblastoma