Interaction of HapX with the CCAAT-binding complex--a novel mechanism of gene regulation by iron

Abstract

Iron homeostasis requires subtle control systems, as iron is both essential and toxic. In Aspergillus nidulans, iron represses iron acquisition via the GATA factor SreA, and induces iron-dependent pathways at the transcriptional level, by a so far unknown mechanism. Here, we demonstrate that iron-dependent pathways (e.g., heme biosynthesis) are repressed during iron-depleted conditions by physical interaction of HapX with the CCAAT-binding core complex (CBC). Proteome analysis identified putative HapX targets. Mutual transcriptional control between hapX and sreA and synthetic lethality resulting from deletion of both regulatory genes indicate a tight interplay of these control systems. Expression of genes encoding CBC subunits was not influenced by iron availability, and their deletion was deleterious during iron-depleted and iron-replete conditions. Expression of hapX was repressed by iron and its deletion was deleterious during iron-depleted conditions only. These data indicate that the CBC has a general role and that HapX function is confined to iron-depleted conditions. Remarkably, CBC-mediated regulation has an inverse impact on the expression of the same gene set in A. nidulans, compared with Saccharomyces cerevisae.

Figure 1

Iron-regulated gene expression in A. nidulans wild-type, ΔsreA, ΔhapX and ΔhapC strains. For Northern analysis, the total RNA was isolated from A. nidulans strains grown for 24 h under +Fe and −Fe conditions. As a control for loading and RNA quality, blots were hybridized with the γ-actin encoding acnA gene. (A) Expression of hapX but not hapC is partially controlled by SreA-mediated iron regulation. (B) Deletion of hapX or hapC causes derepression of sreA, during −Fe conditions but not the SreA regulon, during +Fe conditions. (C) Deletion of hapX or of hapC causes derepression of iron-dependent pathways during iron starvation. (D) A. nidulans siderophores. (E) A. nidulans siderophore metabolism. Known genes involved in siderophore biosynthesis and uptake are shaded in gray.

Figure 2

Deletion of hapX or genes encoding CBC subunits leads to cellular accumulation of PpIX, decreased TAFC synthesis and increased FC production, during −Fe conditions. (A) Mycelia of A. nidulans strains after growth for 24 h during +Fe and –Fe conditions. (B) Characteristic red auto-fluorescence caused by PpIX accumulation during −Fe conditions. During +Fe conditions, no auto-fluorescence was detectable in any strain (data not shown). (C) Quantification of PpIX accumulation. (D) Representative chromatograms of porphyrin analysis of wild-type, ΔhapX and ΔhapC strains after growth under −Fe conditions. C8, C7, C6, C5, C4 and C2 denote uroporphyrin, heptacarboxylporphyrin, hexacarboxylporphyrin, pentacarboxylporphyrin, coproporphyrin and protoporphyrin, respectively. (E) Quantification of siderophore production during −Fe conditions normalized to that of the wild type. The data represent the mean±s.d. of three simultaneously harvested flasks.

Figure 3

HapX and HapB interact in vivo. The interaction was observed after 24 h of growth, using BiFC in A. nidulans strains producing HapX and HapB fused with the C-terminal and N-terminal split fragments of eYFP, respectively. Panels 1, light microscopy; panels 2 and 3, fluorescence microscopy of DAPI-stained nuclei and BiFC, respectively. HapX and HapB interact during −Fe (B) but not +Fe (A) conditions in strain yXB. (C) HapX/HapB interaction is abolished by deletion of hapC in strain yXBΔC and is (D) reconstituted after complementation of yXBΔC with hapC in yXBC^c.

Figure 4

Deletion of the non-conserved N-terminal region or the conserved A-domain of HapE phenocopies hapX deletion. (A) Schematic representation of the HapE versions investigated. (B) Growth rates and production of TAFC, FC and PpIX after growth for 48 h during+Fe and −Fe conditions. For induction of amylase promoter-driven genes (Tanoue et al, 2006), strains were grown in medium containing starch as the sole carbon source. During +Fe conditions, production of TAFC, FC and PpIX was wild type-like in all strains (data not shown).

Figure 5

SPR analysis of iron-regulated HapX binding to DNA-bound CBC. (A) SDS–PAGE analysis of 1.5 μg of purified HapB, HapC, HapE, HapE-ΔNΔA and HapX proteins. (B) Concentration-dependent, steady-state binding of the CBC to biosensor-bound CCAAT boxes derived from the 5′-upstream regions of sreA and lysF, respectively. (C) Schematic representation of the SPR analysis of HapX/CBC interaction. HapX was injected onto preformed CBC/DNA complexes after reaching the steady-state level (D) Concentration-dependent association of HapX (12.5, 25, 50 and 100 nM, respectively) to the CBC (6.25 nM) bound to the biosensor-linked sreA CCAAT box. ‘CBC(−HapX)' shows the steady-state association of the CBC to the CCAAT box without application of HapX. ‘(−CBC)' shows the unspecific interaction of HapX (50 nM) with sensor-bound DNA. (E) Comparison of the interaction of HapX (100 nM) with the CBC and with the CBC containing HapE-ΔNΔA (CBC^*). Note that 12.5 nM CBC^* was necessary to reach an equilibrium response equivalent to 6.25 nM CBC. (F) Interaction of HapX (100 nM) after preincubation with iron (1, 2.5, 5 and 10 μM FeCl₃, respectively) or without iron (−Fe), with the DNA-bound CBC (6.25 nM). (G) Interaction of HapX (100 nM) after preincubation with 10 μM FeCl₃, 10 μM (NH₄)₂Fe(SO₄)₂, 10 μM CuCl₂, or without any metal (−metal), with DNA-bound CBC (6.25 nM). (H) Proposed model for HapX/CBC-mediated regulation of iron-dependent pathways and sreA.