Promoter for the human ferritin heavy chain-encoding gene (FERH): structural and functional characterization

doi:10.1016/0378-1119(92)90696-M

Gene

Volume 111, Issue 2, 15 February 1992, Pages 255-260

https://doi.org/10.1016/0378-1119(92)90696-M Get rights and content

Abstract

We conducted a functional analysis of the promoter for the human ferritin heavy chain-encoding gene (pFERH) in HepG2 and HeLa cells. The activity of pFERH is equivalent in both cell types, despite their different ferritin (Fer) isotypes. Transfections of a series of 5′-deletion mutants indicate that pFERH activity is essentially dependent on two motifs. One of them, accounting for about 50% of the total transcriptional activity, is recognized by the RNA polymerase II transcription factor, Sp1, and the other by a low-affinity factor present in both the cell types analyzed.

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Cited by (35)

PCR-based profiling of transcription factor activity in vivo by a virus-based reporter battery
2022, iScience
Citation Excerpt :
This replacement result in the 6 constructs expressing different set of Rep and Ref genes (pLVTFrep#1 to pLVTFrep#6) for which TFBSs were inserted between the BamHI and HindIII site. A reporter-construct backbone without TFBS (mini, Promega pGL4.23), and a synthetic promotor element composed from human ferritin H promoter (Bevilacqua et al., 1992), or a synthetic Arc/Arg3.1 enhancer/promoter (Kawashima et al., 2009) were used as control promoters. To construct AAV-based TF activity reporter constructs, we synthesized a double floxed cassette consisting of loxP and lox2272, as described before (Saunders et al., 2012), and cloned the Rep-genes in the reverse orientation using NheI and AscI sites.
Understanding the molecular mechanisms of gene regulation is pivotal for understanding how cells establish and modify their identities and functions. Multiple transcription factors (TFs) coordinate to alter gene expression in cells; however, a method to quantitatively analyze the activity of each TF is lacking, particularly in vivo. Here, we introduce a viral-vector-based TF reporter battery that can be used to simultaneously analyze the activity of multiple TFs, visualized as the TF activity profile (TFAP) obtained by qPCR. We show that the cells possess distinct TFAPs that dynamically change according to experimental manipulation or physiological activity. We report a practical method to obtain the TFAP of a defined cell population and their experience-dependent changes in the mouse brain in vivo. The TFAP obtained by our method will help bridge the information gap between the genome and transcriptome and aid the multi-omics view of understanding the gene regulation system.
Identification of H ferritin-dependent and independent genes in K562 differentiating cells by targeted gene silencing and expression profiling
2014, Gene
Citation Excerpt :
The structural and functional differences between the two subunits and the diversity in tissue-specific distribution are also reflected at the transcriptional level. Besides the presence of distinct trans-acting factors acting on the two ferritin promoters, a robust body of evidence suggests that the FHC promoter activity is differentially regulated in different cell types as well as in different stages of cell life (Bevilacqua et al., 1992, 1995, 1997a, 1998; D'Agostino et al., 1995; Faniello et al., 1999). Moreover, the FHC promoter is positively regulated by cAMP (Bevilacqua et al., 1997b) and by cJun (Faniello et al., 2002) whereas E1A (Bevilacqua et al., 1997b) and p53 (Faniello et al., 2008) act as transcriptional repressors.
Ferritin is best known as the key molecule in intracellular iron storage, and is involved in several metabolic processes such as cell proliferation, differentiation and neoplastic transformation. We have recently demonstrated that the shRNA silencing of the ferritin heavy subunit (FHC) in a melanoma cell line is accompanied by a consistent modification of gene expression pattern leading to a reduced potential in terms of proliferation, invasiveness, and adhesion ability of the silenced cells.
In this study we sought to define the repertoire of genes whose expression might be affected by FHC during the hemin-induced differentiation of the erythromyeloid cell line K562. To this aim, gene expression profiling was performed in four different sets of cells: i) wild type K562; ii) sh-RNA FHC-silenced K562; iii) hemin-treated wild-type K562; and iv) hemin-treated FHC-silenced K562. Statistical analysis of the gene expression data, performed by two-factor ANOVA, identified three distinct classes of transcripts: a) Class 1, including 657 mRNAs whose expression is modified exclusively during hemin-induced differentiation of K562 cells, independently from the FHC relative amounts; b) Class 2, containing a set of 70 mRNAs which are consistently modified by hemin and FHC-silencing; and c) Class 3, including 128 transcripts modified by FHC-silencing but not by hemin.
Our data indicate that FHC may function as a modulator of gene expression during erythroid differentiation and add new findings to the knowledge of the complex gene network modulated during erythroid differentiation.
Ferritin H induction by histone deacetylase inhibitors
2010, Biochemical Pharmacology
Citation Excerpt :
HDAC inhibitors transcriptionally activate the ferritin H promoter by engaging elements of the human ferritin H promoter approximately 60 nucleotides 5′ of the transcriptional start site, a region known to be important in the transcriptional control of the human ferritin H gene. For example, two cis-acting elements in the proximal promoter of human ferritin H gene have previously been identified: a distal GC box which contains a potential Sp1/Sp3-binding site (−132 to −109 bp from the transcription start), and a proximal site containing an inverted CCAAT box (−65 to −45 bp) [31]. The latter is recognized by a protein complex composed of the trimeric transcription factor NF-Y, p300 (a member of HAT family) and p300/CBP-associated factor (pCAF) [32] which binds NF-Y [33].
Because both iron deficiency and iron excess are deleterious to normal cell function, the intracellular level of iron must be tightly controlled. Ferritin, an iron binding protein, regulates iron balance by storing iron in a bioavailable but nontoxic form. Ferritin protein comprises two subunits: ferritin H, which contains ferroxidase activity, and ferritin L. Here we demonstrate that ferritin H mRNA and protein are induced by histone deacetylase inhibitors (HDAC inhibitors), a promising class of anti-cancer drugs, in cultured human cancer cells. Deletion analysis and EMSA assays reveal that the induction of ferritin H occurs at a transcriptional level via Sp1 and NF-Y binding sites near the transcriptional start site of the human ferritin H promoter. Classically, HDAC inhibitors modulate gene expression by increasing histone acetylation. However, ChIP assays demonstrate that HDAC inhibitors induce ferritin H transcription by increasing NF-Y binding to the ferritin H promoter without changes in histone acetylation. These results identify ferritin H as a new target of HDAC inhibitors, and recruitment of NF-Y as a novel mechanism of action of HDAC inhibitors.
p53-Mediated downregulation of H ferritin promoter transcriptional efficiency via NF-Y
2008, International Journal of Biochemistry and Cell Biology
The tumor suppressor protein p53 triggers many of the cellular responses to DNA damage by regulating the transcription of a series of downstream target genes. p53 acts on the promoter of the target genes by interacting with the trimeric transcription factor NF-Y. H ferritin promoter activity is tightly dependent on a multiprotein complex called Bbf; on this complex NF-Y plays a major role.
The aim of this work was to study the modulation of H ferritin expression levels by p53. CAT reporter assays indicate that: (i) p53 overexpression strongly downregulates the transcriptional efficiency driven by an H ferritin promoter construct containing only the NF-Y recognition sequence and that the phenomenon is reverted by p53 siRNA; (ii) the p53 C-terminal region is sufficient to elicitate this regulation and that a correct C-terminal acetylation is also required. The H ferritin promoter displays no p53-binding sites; chromatin immunoprecipitation assays indicate that p53 is recruited on this promoter by NF-Y. The p53–NF-Y interaction does not alter the NF-Y DNA-binding ability as indicated by electrophoretic mobility shift assay (EMSA) analysis.
These results demonstrate that the gene coding for the H ferritin protein belongs to the family of p53-regulated genes, therefore adding a new level of complexity to the regulation of the H ferritin transcription and delineate a role for this protein in a series of cellular events triggered by p53 activation.
High level expression of proteins using sequences from the ferritin heavy chain gene locus
2007, Journal of Biotechnology
An expression vector has been generated using a gene highly expressed under conditions found in a typical fed-batch bioreactor process. The ferritin heavy chain (HC) gene exhibits higher levels of expression in the late stages of a fed-batch bioreactor than in the early stages. This property was considered advantageous for an expression vector, since the maximal cell density would coincide with maximal expression. The rat ferritin HC genomic region was isolated and converted into an expression vector where large segments of 5′ and 3′ flanking regions were included in an attempt to recreate the same high level of expression in stably transfected cells. Expression from the resulting ferritin HC vector was compared to vectors containing the commonly used strong promoters, CMV IE, and SV40 early promoter/enhancer, in the generation of stable transfectants. The ferritin HC vector was able to generate cell lines with significantly higher expression levels than those under the control of the viral promoters.
Induction of H-ferritin synthesis by oxalomalate is regulated at both the transcriptional and post-transcriptional levels
2006, Biochimica et Biophysica Acta - Molecular Cell Research
Ferritin gene expression is complex and is controlled at transcriptional level in response to a variety of stimuli such as hormones, cytokines and cAMP. Iron, hemin and several compounds, chemically different, also activate the transcription of the ferritin gene. Ferritin biosynthesis is mainly regulated at post-transcriptional level by iron regulatory proteins (IRP1 and IRP2). We previously reported that oxalomalate, a competitive inhibitor of aconitase, remarkably decreases the IRP1 RNA-binding activity and induces a significant increase of ferritin expression. Here, we examined in cells cultured in presence of OMA the IRP1 intracellular content, ferritin biosynthesis and the transcriptional efficiency of H-ferritin gene promoter. Our results demonstrate a peculiar role of OMA that rapidly inactivates IRP1 without affecting IRP1 protein content and subsequently activates H-ferritin gene transcription leading to an overall increase of ferritin biosynthesis. We conclude that OMA regulates H-ferritin biosynthesis acting early at the post-transcriptional level and later on at transcriptional level.

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^∗: Permanent address: Dipartimento di Genetica, Biologia e Chimica Medica, Università degli Studi di Torino, Via Santena 5, 10126 Turin (Italy) Tel. (39-11)6963106.

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Promoter for the human ferritin heavy chain-encoding gene (FERH): structural and functional characterization

Abstract

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Cell

J. Biol. Chem.

Cell

J. Biol. Chem.

J. Biol. Chem.

Carcinofetal isoferritins

Nature

Iron regulates ferritin mRNA translation through a segment of its 5′ untranslated region

Structure and expression of ferritin genes in a human promyelocytic cell line that differentiates in vitro

Mol. Cell. Biol.

Structure of gene and pseudogenes of human apoferritin H

Nucleic Acids Res.

The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter

Cell