EBNA2 joining picks RNAPII from the DEXI promoter at expense out of CIITA
I confirmed the fresh removal of EBF1 and you can PU
a hundred kb of your own CIITA TSS. We recognized one to biggest joining website located at the newest 3′ end of the CIITA gene transcript (Fig 6A). We utilized CRISPR/Cas9 gene editing to mutate brand new EBNA2 binding sites for the LCLs. The latest EBNA2 binding web site overlaps predicted binding sites getting EBF1 and you will PU.step 1. Several independent pairs off book RNAs (gRNAs) was basically made to carry out
200bp deletion in the EBNA2 joining webpages. step one joining webpages of the PCR data regarding genomic DNA (S2 Fig). Processor assay presented that binding out-of EBF1, PU.1, and you will EBNA2 have been notably lower in CRISPR modified LCLs (Fig 6B). I 2nd assayed transcription when you look at the CRISPR EBNA2_BS ko compared to control cells. We found that CRISPR EBNA2_BS ko structure got a boost in CIITA and HLA-DRA, DQA1, DPA1, DPB1 (Fig 6C). EBNA2 responsive gene HES1 was not impacted by CRISPR ko out of new EBNA2 binding website during the CIITA locus. On the other hand, the fresh new DEXI gene centered downstream along with the contrary orientation to help you CIITA try downregulated in cells without having the new EBNA2 binding webpages (Fig 6D). Since the a handling, an equivalent CRISPR ko try chathour performed inside BJAB tissue, an EBV- and EBNA2-bad lymphoma cellphone along with no affect CIITA, HLA-II, otherwise DEXI gene transcription (Fig 6E). These types of results suggest brand new EBNA2 joining webpages during the 3′ region away from CIITA gene is very important into the repression regarding CIITA and you can the fresh new activation regarding DEXI when you look at the EBV self-confident LCLs.
(A) Screenshot of UCSC genome browser with ChIP-seq tracks of EBNA2, EBF1, PU.1, ETS1, RBPJ and GeneHancer interactions at CIITA region. gRNA-targeted region is indicated by a red box. (B) ChIP-qPCR in Ctrl or EBNA2_BS KO EREB2.5 cells with antibodies to either EBNA2, EBF1, PU.1 or IgG. (C) Expression of CIITA, HLA-DRA, DQA1, DPA1, DPB1, and HES1 in Ctrl and EBNA2_BS KO EREB2.5 cells was measured by ??CT method (2-tailed student t test; *** p<0.001 or ns (not significant)). (D) Same as in panel C showing DEXI gene only. (E) Expression of CIITA, HLA-DRA, DQA1, DPA1, DPB1, and DEXI in Ctrl and EBNA2_BS KO BJAB cells was measured by ??CT method (2-tailed student t test; ns (not significant)).
To help expand have a look at the newest regulation out of DEXI because of the EBNA2, i re also-examined the newest gene organization to the CIITA and you can DEXI family genes and new cousin ranks of the known supporter-enhancer factors (Fig 7A). DEXI is regarding reverse positioning and you may head-to-direct which have CIITA. I listed one several CTCF joining internet sites was indeed receive between the promoters of any gene. We 2nd queried our RNA-seq research and found you to DEXI try strongly triggered through the EBV immortalization out of B-structure (Fig 7B). We along with discovered that DEXI transcription try upregulated by the EBNA2 expression in Akata T1 and you may T2 tissues (Fig 7C and you will 7D). We second expected whether EBNA2 induction changed brand new cousin binding of RNA polymerase II (RNAPII) during the DEXI promoter relative to CIITA promoter III (CIITA-pIII) which drives new constitutive expression from CIITA into the B structure . We learned that EBNA2 term resulted in a rise in RNAPII on DEXI supporter, which have a matching reduced total of joining during the CIITA-pIII, in Akata T1 and T2 tissue (Fig 7E). Also, new histone amendment H3K4me3 that’s directly synchronised having supporter activation was enriched in the DEXI and you will exhausted within CIITA-pIII (Fig 7F).
These types of conclusions suggest that EBNA2 joining upstream of the DEXI supporter functions as an ancient transcriptional activator and you can reorganizes RNAPII localization and you will direction liking to own DEXI at the cost of CIITA
(A) ChIP-Seq for CTCF, EBNA2, EBF1 and RBPJ shown on UCSC browser. ChIP-primers position for CIITA-PIII and DEXI-promoter are indicated. (B) RNA-seq read-count quantification of DEXI transcripts during EBV infection of primary B-cells for 2 donors. (C) Akata T1 and T2 cells induced with estradiol for 48 hrs and assayed by RT-qPCR using the ??CT method. (D) Western blot of EBNA2 expression in Akata T1 and T2 cells without (-) or with (+) E2 addition for 48 hrs. (E) RNAPII ChIP assay in Akata T1 or T2 cells with (+) or without (-) E2 induction at primer positions for CIITA-pIII or DEXI promoter. (F) Same as in panel E, except for H3K4me3 ChIP. Error bars are SDM, and * p<0.05, ** p<0.01, *** p < .001 or ns (not significant) by 2-tailed student t-test.