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Identification of an NF-?B p50/p65-responsive site in the human MIR155HG promoter.

ABSTRACT: MicroRNA-155 (miR-155) is the diced product of the MIR155HG gene. miR-155 regulates the expression of many immune-specific transcripts, is overexpressed in many human lymphomas, and has oncogenic activity in mouse transgenic models. MIR155HG has been proposed to be a target gene for transcription factor NF-?B largely due to the positive correlation between high nuclear NF-?B activity and increased miR-155 expression following treatment with NF-?B inducers or in subsets of hematopoietic cancers. Nevertheless, direct regulation of the human MIR155HG promoter by NF-?B has not been convincingly demonstrated previously.This report shows that induction of NF-?B activity rapidly leads to increased levels of both primary MIR155HG mRNA and mature miR-155 transcripts. We have mapped an NF-?B-responsive element to a position approximately 178 nt upstream of the MIR155HG transcription start site. The -178 site is specifically bound by the NF-?B p50/p65 heterodimer and is required for p65-induced reporter gene activation. Moreover, the levels of miR-155 in nine human B-lymphoma cell lines generally correlate with increased nuclear NF-?B proteins.Overall, the identification of an NF-?B-responsive site in the MIR155HG proximal promoter suggests that MIR155HG is a direct NF-?B target gene in vivo. Understanding NF-?B-mediated regulation of miR-155 could lead to improved immune cell-related diagnostic tools and targeted therapies.

SUBMITTER: Thompson RC

PROVIDER: S-EPMC3849010 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Identification of an NF-κB p50/p65-responsive site in the human MIR155HG promoter.

Thompson Ryan C RC Vardinogiannis Iosif I Gilmore Thomas D TD

BMC molecular biology 20130923

<h4>Background</h4>MicroRNA-155 (miR-155) is the diced product of the MIR155HG gene. miR-155 regulates the expression of many immune-specific transcripts, is overexpressed in many human lymphomas, and has oncogenic activity in mouse transgenic models. MIR155HG has been proposed to be a target gene for transcription factor NF-κB largely due to the positive correlation between high nuclear NF-κB activity and increased miR-155 expression following treatment with NF-κB inducers or in subsets of hema ...[more]

PMID: 24059932

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Project description:The family of NF-κB transcriptional activators controls the expression of many genes, including those involved in cell survival and development. The family consists of homo- and heterodimers constituted by combinations of five subunits. Subunit p50 includes 13 tyrosine residues, but the relationship between specific tyrosine phosphorylations and p50 function is not well understood. Subunits of p50 and p65 prepared in vitro formed a heterodimer, but this NF-κB would not bind to the interleukin-2 (IL-2) promoter DNA. Treatment of p50 with guanosine triphosphate (GTP) and a lysate from activated Jurkat cells, effected rapid p50 phosphorylation, and, in the presence of wild-type subunit p65, was accompanied on the same time scale by IL-2 promoter DNA binding. Modified p50s containing one of seven stoichiometrically phosphorylated tyrosines in NF-κB p50/p65 heterodimers, included three that facilitated binding to the IL-2 DNA promoter region to a greater extent than the wild type. One of these three stoichiometrically phosphorylated p50/p65 heterodimers of NF-κB, containing pTyr60 in the p50 subunit, was treated with a lysate from activated Jurkat cells + GTP and shown to be phosphorylated on the same time scale as wild-type p50. This modified NF-κB also developed IL-2 promoter DNA binding activity on the same time scale as the wild type but exhibited greater binding to the IL-2 DNA promoters than the wild type. The nature of this enhanced binding was studied in greater detail using a metabolically stable pTyr derivative at position 60 of p50 and cellular phosphatases. We suggest that enhanced DNA binding of modified NF-κB containing pTyr60 in the p50 subunit may reflect stoichiometric NF-κB phosphorylation at a site that is not normally fully phosphorylated, or not phosphorylated at all, and is relatively resistant to the effects of Jurkat cell tyrosine phosphatase activity. This conclusion was reinforced by demonstrating that modification of Tyr60 of p50 with a metabolically stable methylenephosphonate moiety further increased the stability of the formed NF-κB p50/p65 heterodimer against the action of activated Jurkat cell phosphatases.

Dataset Information

Identification of an NF-?B p50/p65-responsive site in the human MIR155HG promoter.

Publications

Identification of an NF-κB p50/p65-responsive site in the human MIR155HG promoter.

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