Related Experiment Video

Updated: Sep 23, 2026

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1
11:02

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Published on: May 27, 2016

Characterization of the murine Nramp1 promoter: requirements for transactivation by Miz-1

Holly Bowen1, Abigail Lapham, Emma Phillips

  • 1Division of Biochemistry and Molecular Biology, University of Southampton, Bassett Crescent East, Southampton SO16 7PX, United Kingdom.

The Journal of Biological Chemistry
|July 4, 2003
PubMed

Insights

Murine Nramp1, a divalent cation transporter in macrophages, controls pathogen growth. Its promoter is regulated by c-Myc, Miz-1, and Sp1, with E boxes inhibiting activity in proliferating cells.

Area of Science:

  • Molecular Biology
  • Immunology
  • Cell Biology

Background:

  • Murine Nramp1 (natural resistance-associated macrophage protein 1) is a divalent cation transporter crucial for macrophage-mediated intracellular pathogen control.
  • The Nramp1 gene promoter is TATA-deficient, featuring initiator elements and regulation by c-Myc, which represses transcription, and Miz-1 (Myc-interacting zinc-finger protein 1), which stimulates it.

Purpose of the Study:

  • To elucidate the regulatory mechanisms of the murine Nramp1 promoter, focusing on the roles of c-Myc, Miz-1, and Sp1 (Specificity protein 1).
  • To identify specific DNA elements and protein interactions that modulate Nramp1 gene expression in macrophages.

Main Methods:

  • Analysis of Nramp1 promoter activity using reporter assays in cell lines (COS-1, RAW264.7).
  • Site-directed mutagenesis of the Nramp1 promoter to assess the function of E box and Sp1-binding sites.
  • RNA interference (RNAi) targeting Miz-1 to confirm its role in Nramp1 regulation.
  • Chromatin immunoprecipitation (ChIP) assays to identify transcription factor binding to the Nramp1 promoter.

Main Results:

  • A non-canonical E box element was found to inhibit basal Nramp1 promoter activity and Miz-1-mediated activation.
  • A consensus Sp1-binding site (GC box) is essential for Miz-1-dependent transactivation but not repression.
  • Repression by c-Myc involves competition with p300/CBP (coactivators) for Miz-1 binding.
  • The Nramp1 promoter exhibits preferential expression in macrophages, with cell-type-specific effects of Sp1 site mutations.
  • c-Myc, Miz-1, and Sp1 were confirmed to bind the Nramp1 core promoter in macrophages, with binding dynamics influenced by interferon-gamma and lipopolysaccharide stimulation.

Conclusions:

  • The murine Nramp1 promoter activity is finely tuned by the interplay of E box elements, Sp1, Miz-1, and c-Myc.
  • GC box-binding factors stimulate Nramp1 expression in macrophages, while E box elements inhibit it in proliferating cells.
  • These findings provide insights into the transcriptional regulation of Nramp1, essential for innate immunity and macrophage function.

More Related Videos

Massively Parallel Reporter Assays in Cultured Mammalian Cells11:03

Massively Parallel Reporter Assays in Cultured Mammalian Cells

Published on: August 17, 2014

Related Concept Videos

Co-activators and Co-repressors02:04

Co-activators and Co-repressors

Gene transcription is regulated by the synergistic action of several proteins that form a complex at a gene regulatory site. This is observed in eukaryotes, where the regulation of gene expression is a complex process. Regulatory proteins in eukaryotes can broadly be classified into two types – regulators that bind directly to specific DNA sequences and co-regulators that associate with regulatory proteins but cannot directly bind to the DNA. These co-regulators are further divided into...
Master Transcription Regulators02:23

Master Transcription Regulators

Master transcription regulators are regulatory proteins that are predominantly responsible for regulating the expression of multiple genes. Often these genes work in concert to drive a  complex process. Activation of a master transcription regulator can lead to a cascade of transcriptional activation necessary for that outcome. These regulators can directly bind to the regulatory sequences of the various genes involved, or they can indirectly regulate transcription by binding to regulatory...
RNA Polymerase II Accessory Proteins02:36

RNA Polymerase II Accessory Proteins

Proteins that regulate transcription can do so either via direct contact with RNA Polymerase or through indirect interactions facilitated by adaptors, mediators, histone-modifying proteins, and nucleosome remodelers. Direct interactions to activate transcription is seen in bacteria as well as in some eukaryotic genes. In these cases, upstream activation sequences are adjacent to the promoters, and the activator proteins interact directly with the transcriptional machinery. For example, in...
Eukaryotic Transcription Activators02:42

Eukaryotic Transcription Activators

Transcription activators are proteins that promote the transcription of genes from DNA to RNA. In most cases, these proteins contain two separate domains ‒ a domain that binds to DNA and a domain for activating transcription; however, in some cases, a single domain is responsible for both binding and activation of transcription, as seen in the glucocorticoid receptor and MyoD.
The binding domains are capable of recognizing and interacting with regulatory sequences on the DNA. These domains are...