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EBNA2 binding selects RNAPII at the DEXI promoter within costs from CIITA

By 2022년 08월 06일No Comments

EBNA2 binding selects RNAPII at the DEXI promoter within costs from CIITA

I confirmed new deletion of EBF1 and PU

one hundred kb of the CIITA TSS. I recognized one significant joining webpages located at the fresh 3′ end of your own CIITA gene transcript (Fig 6A). I made use of CRISPR/Cas9 gene modifying so you can mutate new EBNA2 binding internet sites into the LCLs. The fresh new EBNA2 binding web site overlaps predicted joining websites to possess EBF1 and you can PU.1. Several independent pairs regarding book RNAs (gRNAs) had been built to carry out

200bp removal at EBNA2 joining webpages. 1 binding site of the PCR investigation of genomic DNA (S2 Fig). Processor chip assay exhibited that binding away from EBF1, PU.1, and you can EBNA2 have been somewhat low in CRISPR edited LCLs (Fig 6B). We 2nd assayed transcription inside the CRISPR EBNA2_BS ko vs manage cells. I unearthed that CRISPR EBNA2_BS ko structure got a rise in CIITA and HLA-DRA, DQA1, DPA1, DPB1 (Fig 6C). EBNA2 responsive gene HES1 was not affected by CRISPR ko from the EBNA2 binding site in the CIITA locus. On the other hand, new DEXI gene oriented downstream along with the contrary positioning to help you CIITA are downregulated into the tissues devoid of the new EBNA2 joining web site (Fig 6D). Because a processing, an identical CRISPR ko is actually performed into the BJAB structure, an EBV- and you will EBNA2-negative lymphoma telephone together with no influence on CIITA, HLA-II, otherwise DEXI gene transcription (Fig 6E). These types of findings strongly recommend brand new EBNA2 binding site within 3′ area off CIITA gene is important towards repression regarding CIITA and you may the brand new activation from DEXI in the EBV positive 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 read the the fresh new regulation regarding DEXI from the EBNA2, we re also-checked out the newest gene business for the CIITA and you will DEXI genes and you will brand new relative ranks of its understood supporter-booster factors (Fig 7A). DEXI is positioned from the contrary orientation and you can direct-to-lead having CIITA. I indexed one multiple CTCF binding sites had been discovered amongst the promoters of any gene. I second queried the RNA-seq analysis and found that DEXI is actually highly induced during EBV immortalization out-of B-tissue (Fig 7B). I and additionally found that DEXI transcription are upregulated because of the EBNA2 phrase during the Akata T1 and you may T2 cells (Fig 7C and you may 7D). I second requested if EBNA2 induction changed brand new relative joining of RNA polymerase II (RNAPII) at DEXI promoter prior to CIITA supporter III (CIITA-pIII) that drives the newest constitutive term of CIITA inside B structure . We learned that EBNA2 term resulted in a rise in RNAPII in the DEXI supporter, which have a matching reduced total of joining from the CIITA-pIII, in Akata T1 and you will T2 muscle (Fig 7E). Also, the new histone amendment H3K4me3 that is directly coordinated that have promoter activation try graced during the DEXI and you can depleted on CIITA-pIII (Fig 7F).

These types of results suggest that EBNA2 binding upstream of the DEXI supporter serves as a traditional transcriptional activator and you can reorganizes RNAPII localization and you can direction taste having DEXI at the expense 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.


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