An Iron Enhancer Element in the FTN-1 Gene Directs Iron-dependent Expression in Caenorhabditis elegans Intestine

Abstract

Ferritin is a ubiquitous protein that sequesters iron and protects cells from iron toxicity. Caenorhabditis elegans express two ferritins, FTN-1 and FTN-2, which are transcriptionally regulated by iron. To identify the cis-acting sequences and proteins required for iron-dependent regulation of ftn-1 and ftn-2 expression, we generated transcriptional GFP reporters corresponding to 5-upstream sequences of the ftn-1 and ftn-2 genes. We identified a conserved 63-bp sequence, the iron-dependent element (IDE), that is required for iron-dependent regulation of a ftn-1 GFP reporter in intestine. The IDE contains two GATAbinding motifs and three octameric direct repeats. Site-directed mutagenesis of the GATA sequences, singly or in combination, reduces ftn-1 GFP reporter expression in the intestine. In vitro DNA mobility shift assays show that the intestine-specific GATA protein ELT-2 binds to both GATA sequences. Inhibition of ELT-2 function by RNA interference blocks ftn-1 GFP reporter expression in vivo. Insertion of the IDE into the promoter region of a heterologous reporter activates iron-dependent transcription in intestine. These data demonstrate that the activation of ftn-1 and ftn-2 transcription by iron requires ELT-2 and that the IDE functions as an iron-dependent enhancer in intestine.Iron is essential in many biological processes, including DNA synthesis, respiration, nitrogen fixation, oxygen transport, heme synthesis, and photosynthesis. At high levels, however, iron can be toxic because of its ability to react with molecular oxygen to generate free radicals that oxidize DNA and proteins and initiate lipid peroxidation, all of which can lead to cell injury and death (1). Consequently, organisms have developed mechanisms to sense, acquire and store this metal within a narrow physiologic range.Ferritin sequesters iron in a form that is biologically available but unable to catalyze free radical formation (2, 3). Mammalian ferritin has a molecular mass of ϳ480,000 and consists of 24 related subunits of two types, a light subunit (L) 2 and a heavy subunit (H). These subunits assemble to form a shell surrounding a cavity that can accommodate up to 4500 iron atoms. The H-subunits oxidize ferrous iron to ferric iron within the cavity; the L-subunits lack ferroxidase activity and function with the H-subunits in iron nucleation. Mutations in either the ferritin H or the ferritin L gene can be deleterious. Ferritin H knock-out mice die early in embryogenesis (4), while mutations in ferritin H or ferritin L genes are associated with hereditary conditions, such as hyperferritinemia cataract syndrome (5, 6), adult onset basal ganglia disease (7), and autosomal dominant iron-overload disease (8).Ferritin induction by iron is regulated by transcriptional and post-transcriptional mechanisms that are organism-specific (9 -11). In vertebrates, ferritin expression is primarily regulated at the translational level by cytosolic proteins that bind to ironresponsive elements (IREs) in the 5Ј-or 3Ј-untranslated regions of mRNAs of...

Iron Overload Coordinately Promotes Ferritin Expression and Fat Accumulation in Caenorhabditis elegans

“…Consistent with previous reports (Gourley et al 2003;Romney et al 2008), FAC induced the expression of FTN-1::GFP ( Figure 1A) and ftn-1 messenger RNA (mRNA) ( Figure 1B) in wildtype N2 . Concurrently, FAC promoted fat accumulation, as indicated by an increased size of LDs and increased abundance of large-sized LDs ( Figure 1C and Supplemental Material, Figure S1, A and B, and Figure S2).…”

“…Nevertheless, FAC led to increased expression of sgk-1 mRNA as well as increased fluorescence intensity in both strains (Figure 1, F and G), suggesting that FAC induces the transcriptional expression of sgk-1. Conversely, restriction of iron by BP (2,29-bipyridyl, 0.1 mM), an iron chelator (Breuer et al 1995;Romney et al 2008Romney et al , 2011, also represses the expression of SGK-1::GFP (MQD862, N2;Psgk1::sgk-1::gfp) ( Figure S5). Altogether, these studies show that iron overload induces the expression of sgk-1, which promotes the expression of ftn-1 and fat accumulation.…”

“…The wild-type strain was N2. The strains used in this study were as follows: acs-20(tm3232)IV, age-1(hx546)II, akt-1(ok525)V, cyp-23A1(gk253)II, daf-2(e1370)III, ftn-1:: GFP{XA6900, pha-1 (e2123ts)III;qaEx01[ftn-1::4pes-10::GFP-his, pha-1(+)]} (Romney et al 2008), cyp-23A1(gk253)II;ftn-1:: GFP{XA6900, pha-1(e2123ts)III;qaEx01[ftn-1::4pes-10 ::GFP-his, pha-1(+)]}, sgk-1(ft15)X, sgk-1(ok538)X, sgk-1(ok538)X;cyp-23A1(gk253), sgk-1::gfp(MQD862, N2;Psgk-1::sgk-1:: gfp [hqIs150]) (Hertweck et al 2004;Zhu et al 2015), rsks-1(ok1255)III, vit-2(ok3211)X, vit-3(ok2348)X, vit-2::gfp {RT130,unc-199(ed3);pwIs23 The Psgk-1::gfp transgenes were created using methods as previously described by Frokjaer-Jensen et al (2008), and the transgenic strain was created by microinjection. Briefly, an extrachromosomal transgenic strain was made by injection into the EG4322 (ttTi5605;unc-119(ed3).…”

“…FAC supplementation was performed as described previously (Romney et al 2008). In brief, synchronized L1 worms were placed and cultivated on NGM plates supplemented with…”

Tissue- and Expression Level–Specific Chromatin Looping at Maizeb1Epialleles

“…The presence of repeated sequences in enhancers and other regulatory elements appears to influence transcription in other systems as well Greene et al, 2007;Shadley et al, 2007;Romney et al, 2008;Espley et al, 2009). Intriguingly, two of the newly identified b1 regions that seem to have regulatory activity contain repeated sequences as well (Figure 4), in a direct (;43 kb upstream) and an inverted orientation (;15 kb upstream).…”