The leukaemia-associated transcription factors EVI-1 and MDS1/EVI1 repress transcription and interact with histone deacetylase.

EVI-1 and its variant form, MDS1/EVI1, have been reported to act in an antagonistic manner and be differentially regulated in samples from patients with acute myeloid leukaemia and rearrangements of the long arm of chromosome 3. Here, we show that both EVI-1 and MDS1/EVI1 can repress transcription from a reporter construct containing EVI-1 binding sites and interact with histone deacetylase in mammalian cells. This interaction can be recapitulated in vitro and is mediated by a previously characterized transcription repression domain, whose activity is alleviated by the histone deacetylase inhibitor trichostatin A.

Homo-oligomerisation and nuclear localisation of mouse histone deacetylase 1.

Reversible histone acetylation changes the chromatin structure and can modulate gene transcription. Mammalian histone deacetylase 1 (HDAC1) is a nuclear protein that belongs to a growing family of evolutionarily conserved enzymes catalysing the removal of acetyl residues from core histones and other proteins. Previously, we have identified murine HDAC1 as a growth factor-inducible protein in murine T-cells. Here, we characterise the molecular function of mouse HDAC1 in more detail. Co-immunoprecipitation experiments with epitope-tagged HDAC1 protein reveal the association with endogenous HDAC1 enzyme. We show that HDAC1 can homo-oligomerise and that this interaction is dependent on the N-terminal HDAC association domain of the protein. Furthermore, the same HDAC1 domain is also necessary for in vitro binding of HDAC2 and HDAC3, association with RbAp48 and for catalytic activity of the enzyme. A lysine-rich sequence within the carboxy terminus of HDAC1 is crucial for nuclear localisation of the enzyme. We identify a C-terminal nuclear localisation domain, which is sufficient for the transport of HDAC1 and of reporter fusion proteins into the nucleus. Alternatively, HDAC1 can be shuttled into the nucleus by association with another HDAC1 molecule via its N-terminal HDAC association domain. Our results define two domains, which are essential for the oligomerisation and nuclear localisation of mouse HDAC1.

Substrate binding is a prerequisite for stabilisation of mouse thymidine kinase in proliferating fibroblasts.

Thymidine kinase (TK) expression in mammalian cells is strictly growth regulated, with high levels of the enzyme present in proliferating cells and low levels in resting cells. We have shown that mouse TK expressed from a constitutive promoter is still subject to this regulation. The drastic decline in TK enzyme levels in resting cells is largely due to a pronounced reduction in the half-life of the protein. Deletion of the 30 C-terminal amino acid residues from TK abrogates growth regulation, rendering the enzyme very stable. Moreover, the substrate thymidine was sufficient to stabilise the labile TK protein in quiescent cells. Here, we report that the ability of TK to bind substrates is essential for both growth-dependent regulation and stabilisation by the substrate. By mutation or elimination of the binding sites for either of the two substrates, ATP and thymidine, we expressed TK proteins lacking enzymatic activity which abolished growth-regulated expression in both cases. Mutant TK proteins impaired in substrate binding were subject to rapid degradation in exponentially growing cells and thymidine was no longer sufficient to inhibit this rapid decay. A C-terminal truncation known to stabilise the TK wild-type protein in resting cells did not affect the rapid turnover of enzymatically inactive TK proteins. Proteasome inhibitors also failed to stabilise these substrate-binding mutants. By cross-linking experiments, we show that TK proteins with mutated substrate-binding sites exist only as monomers, whereas active TK enzyme forms dimers and tetramers. Our data indicate that, In addition to the C terminus intact substrate-binding sites are required for growth-dependent regulation of TK protein stability.

Histone deacetylase 1 can repress transcription by binding to Sp1.

The members of the Sp1 transcription factor family can act as both negative and positive regulators of gene expression. Here we show that Sp1 can be a target for histone deacetylase 1 (HDAC1)-mediated transcriptional repression. The histone deacetylase inhibitor trichostatin A activates the chromosomally integrated murine thymidine kinase promoter in an Sp1-dependent manner. Coimmunoprecipitation experiments with Swiss 3T3 fibroblasts and 293 cells demonstrate that Sp1 and HDAC1 can be part of the same complex. The interaction between Sp1 and HDAC1 is direct and requires the carboxy-terminal domain of Sp1. Previously we have shown that the C terminus of Sp1 is necessary for the interaction with the transcription factor E2F1 (J. Karlseder, H. Rotheneder, and E. Wintersberger, Mol. Cell. Biol. 16:1659-1667, 1996). Coexpression of E2F1 interferes with HDAC1 binding to Sp1 and abolishes Sp1-mediated transcriptional repression. Our results indicate that one component of Sp1-dependent gene regulation involves competition between the transcriptional repressor HDAC1 and the transactivating factor E2F1.

Characterization of the translation-dependent step during iron-regulated decay of transferrin receptor mRNA.

Iron regulates the stability of the mRNA encoding the transferrin receptor (TfR). When iron is scarce, iron regulatory proteins (IRPs) stabilize TfR mRNA by binding to the 3'-untranslated region. High levels of iron induce degradation of TfR mRNA; the translation inhibitor cycloheximide prevents this. To distinguish between cotranslational mRNA decay and a trans effect of translation inhibitors, we designed a reporter system exploiting the properties of the selectable marker gene thymidine kinase (TK). The 3'-untranslated region of human transferrin receptor, which contains all elements necessary for iron-dependent regulation of mRNA stability, was fused to the TK cDNA. In stably transfected mouse fibroblasts, the expression of the reporter gene was perfectly regulated by iron. Introduction of stop codons in the TK coding sequence or insertion of stable stem-loop structures in the leader sequence did not affect on the iron-dependent regulation of the reporter mRNA. This implies that global translation inhibitors stabilize TfR mRNA in trans. Cycloheximide prevented the destabilization of TfR mRNA only in the presence of active IRPs. Inhibition of IRP inactivation by cycloheximide or by the specific proteasome inhibitor MG132 correlated with the stabilization of TfR mRNA. These observations suggest that inhibition of translation by cycloheximide interferes with the rate-limiting step of iron-induced TfR mRNA decay in a trans-acting mechanism by blocking IRP inactivation.

Molecular cloning and characterization of the mouse histone deacetylase 1 gene: integration of a retrovirus in 129SV mice.

Reversible histone acetylation plays an important role for chromatin structure and gene expression. The acetylation state of core histones is controlled by histone acetyltransferases and histone deacetylases. Here we report the cloning and characterization of the mouse histone deacetylase 1 (HDAC1) gene. The mouse genome contains several HDAC1-related structures representing the HDAC1 gene and at least three pseudogenes. The HDAC1 gene comprises 14 exons ranging from 49 to 539 bp. Interestingly the murine HDAC1 gene strongly resembles the previously published mouse HDAC2 gene (Zeng et al., J. Biol. Chem. 273 (1998) 28921-28930). The sizes of ten of the 14 exons are identical for both genes and the splicing sites for 11 introns align in identical positions suggesting a gene duplication event. The HDAC1 gene is located only 128 bp downstream from the MARCKS-related protein (MRP) gene in a tail-to-tail orientation. The murine MRP gene was previously mapped to a conserved gene cluster on chromosome 4 sharing linkage homology to human chromosome 1p32-36. The genes for HDAC1 and MRP are co-expressed in a variety of cell types. In the genome of 129SV mice the largest intervening sequence of the HDAC1 gene, intron 3, harbors a complete copy of the endogenous retrovirus MuERV-L. In contrast the HDAC1 gene in other mouse strains such as C57B16, C3H/An and C-RY lacks the retrovirus. Our study provides useful tools for future targeted gene disruption studies.

Histone H4 acetylation during interleukin-2 stimulation of mouse T cells.

Proliferation and cell cycle progression of eukaryotic cells are closely linked to changes in chromatin structure and gene expression. By reversible histone acetylation the cell is able to modulate chromatin condensation and accessibility of specific regions within the chromatin. Here, we examined histone H4 acetylation patterns during growth induction of the murine interleukin-2 dependent T cell line B6.1. In order to detect acetylation on each of the four potential target residues we produced a set of antibodies recognizing specifically acetylated lysine 5, 8, 12 and 16 in the N-terminal tail of histone H4. Acetylation was generally low in resting T cells, but increased after stimulation with a specific kinetics for each lysine. Lysine 16 was acetylated during the G1 phase and deacetylated during S phase. H4 acetylation on lysine 5, 8 and 12, in contrast, was induced before cells started to replicate, and persisted until cells entered mitosis. Treatment of resting B6.1 cells with the specific deacetylase inhibitor trichostatin A (TSA) led to H4 hyperacetylation at all four lysine residues indicating that the histone modification can occur in the absence of replication. After release from TSA treatment normal H4 acetylation levels were reestablished by extremely rapid deacetylation of lysines 5, 8, 12 and 16. The deacetylation step was 60-100 times faster than TSA induced acetylation and equally efficient in resting and exponentially growing T cells. Our results indicate the presence of cell cycle regulated lysine specific acetylating and deacetylating activities in mouse T cells.

Growth-regulated antisense transcription of the mouse thymidine kinase gene.

The expression of the salvage pathway enzyme thymidine kinase (TK) is very low in resting mammalian cells, but increases dramatically when growth-stimulated cells enter S phase. The 30-fold rise in TK mRNA levels in response to growth factors is due to a well-characterized transcriptional activation and less defined post-transcriptional mechanisms. A minigene containing the murine TK promoter and the TK cDNA showed a 3-fold increase in TK mRNA levels after growth induction in stably transfected mouse TK-deficient L fibroblasts. Introduction of the first three TK introns resulted in a 10-fold regulation of TK expression which was predominantly due to repressed TK mRNA levels in serum-deprived cells. Removal of intron 3 from this construct or replacement of the TK promoter by a constitutive SV40 promoter led to a reduced, but still significant increase in TK mRNA levels during the onset of proliferation. These results indicate that both the TK promoter and specific TK introns contribute independently to the growth-dependent regulation of TK mRNA expression. To examine the regulatory mechanisms in more detail we analyzed TK transcription rates and steady-state levels of nuclear transcripts from an SV40 promoter-driven minigene that contains introns 2 and 3 of the TK gene. Using a set of single-stranded probes we detected TK-specific antisense transcription that was up-regulated in resting cells. Similarly, antisense transcription of the endogenous TK gene in Swiss 3T3 cells rose during serum deprivation while sense transcription was regulated in the opposite way. Luciferase reporter assays revealed the presence of a putative antisense promoter in intron 3 of the murine TK gene. These results suggest a negative role for intron-dependent antisense transcription in the regulation of TK mRNA expression in mouse fibroblasts.

Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia.

The transforming proteins of acute promyelocytic leukaemias (APL) are fusions of the promyelocytic leukaemia (PML) and the promyelocytic leukaemia zinc-finger (PLZF) proteins with retinoic acid receptor-alpha (RARalpha). These proteins retain the RARalpha DNA- and retinoic acid (RA)-binding domains, and their ability to block haematopoietic differentiation depends on the RARalpha DNA-binding domain. Thus RA-target genes are downstream effectors. However, treatment with RA induces differentiation of leukaemic blast cells and disease remission in PML-RARalpha APLs, whereas PLZF-RARa APLs are resistant to RA. Transcriptional regulation by RARs involves modifications of chromatin by histone deacetylases, which are recruited to RA-target genes by nuclear co-repressors. Here we show that both PML-RARalpha and PLZF-RARalpha fusion proteins recruit the nuclear co-repressor (N-CoR)-histone deacetylase complex through the RARalpha CoR box. PLZF-RARalpha contains a second, RA-resistant binding site in the PLZF amino-terminal region. High doses of RA release histone deacetylase activity from PML-RARalpha, but not from PLZF-RARalpha. Mutation of the N-CoR binding site abolishes the ability of PML-RARalpha to block differentiation, whereas inhibition of histone deacetylase activity switches the transcriptional and biological effects of PLZF-RARalpha from being an inhibitor to an activator of the RA signalling pathway. Therefore, recruitment of histone deacetylase is crucial to the transforming potential of APL fusion proteins, and the different effects of RA on the stability of the PML-RARalpha and PLZF-RARalpha co-repressor complexes determines the differential response of APLs to RA.

The inability of cells to grow in low iron correlates with increasing activity of their iron regulatory protein (IRP).

We studied the factors that determine the differing growth requirements of low-iron-tolerant (LIT) versus high-iron-dependent (HID) cells for extracellular nontransferrin iron. The growth of LIT cells HeLa and THP-1, when transferred from transferrin (5 micrograms/ml) medium into low-iron (5 microM ferric citrate) medium, was not significantly affected while HID cells Jiyoye and K562 showed nearly no growth. HeLa and THP-1 cells, as well as Jiyoye and K562 cells, do not produce transferrin in sufficient amounts to support their growth in low-iron medium. Surprisingly, similar rates of iron uptake in low-iron medium (0.033 and 0.032 nmol Fe/min and 10(6) cells) were found for LIT cells HeLa and HID cells K562. Furthermore, the intracellular iron level (4.64 nmol/10(6) cells) of HeLa cells grown in low-iron medium was much higher than iron levels (0.15 or 0.20 nmol/10(6) cells) of HeLa or K562 cells grown in transferrin medium. We demonstrated that the activity (ratio activated/total) of the iron regulatory protein (IRP) in HID cells Jiyoye and K562 increased more than twofold (from 0.32 to 0.79 and from 0.47 to 1.12, respectively) within 48 h after their transfer into low-iron medium. In the case of LIT cells HeLa and THP-1, IRP activity stayed at similar or slightly decreased levels (0.86-0.73 and 0.58-0.55, respectively). Addition of iron chelator deferoxamine (50 microM, i.e., about half-maximal growth-inhibitory dose) resulted in significantly increased activity of IRP also in HeLa and THP-1 cells. We hypothesize that the relatively higher bioavailability of nontransferrin iron in LIT cells, over that in HID cells, determines the differing responses observed under low-iron conditions.

Identification of mouse histone deacetylase 1 as a growth factor-inducible gene.

Reversible acetylation of core histones plays an important role in transcriptional regulation, cell cycle progression, and developmental events. The acetylation state of histones is controlled by the activities of acetylating and deacetylating enzymes. By using differential mRNA display, we have identified a mouse histone deacetylase gene, HD1, as an interleukin-2-inducible gene in murine T cells. Sequence alignments revealed that murine HD1 is highly homologous to the yeast RPD3 pleiotropic transcriptional regulator. Indirect immunofluorescence microscopy proved that mouse HD1 is a nuclear protein. When expressed in yeast, murine HD1 was also detected in the nucleus, although it failed to complement the rpd3delta deletion phenotype. HD1 mRNA expression was low in G0 mouse cells but increased when the cells crossed the G1/S boundary after growth stimulation. Immunoprecipitation experiments and functional in vitro assays showed that HD1 protein is associated with histone deacetylase activity. Both HD1 protein levels and total histone deacetylase activity increased upon interleukin-2 stimulation of resting B6.1 cells. When coexpressed with a luciferase reporter construct, HD1 acted as a negative regulator of the Rous sarcoma virus enhancer/promoter. HD1 overexpression in stably transfected Swiss 3T3 cells caused a severe delay during the G2/M phases of the cell cycle. Our results indicate that balanced histone acetylation/deacetylation is crucial for normal cell cycle progression of mammalian cells.

Cell growth inhibition by the Mad/Max complex through recruitment of histone deacetylase activity.

BACKGROUND: The organization of chromatin is crucial for the regulation of gene expression. In particular, both the positioning and properties of nucleosomes influence promoter-specific transcription. The acetylation of core histones has been suggested to alter the properties of nucleosomes and affect the access of DNA-binding transcriptional regulators to promoters. A recently identified mammalian histone deacetylase (HD1) shows homology to the yeast Rpd3 protein, which together with Sin3 affects the transcription of several genes. Mammalian Sin3 proteins interact with the Mad components of the Myc/Max/Mad network of cell growth regulators. Mad/Max complexes may recruit mammalian Rpd3-like enzymes, therefore, directing histone deacetylase activity to promoters and negatively regulating cell growth. RESULTS: We report the identification of a tetrameric complex composed of Max, Mad1, Sin3B and HD1. This complex has histone deacetylase activity which can be blocked by the histone deacetylase inhibitors trichostatin A and sodium butyrate. The inhibition of cell growth by Mad1 is enhanced by Sin3B and HD1, as measured by colony formation assays. Furthermore, a Mad1-induced block of S-phase progression can be overcome by trichostatin A, as shown in microinjection experiments. CONCLUSIONS: The recruitment of a histone deacetylase by sequence-specific DNA-binding proteins provides a mechanism by which the state of acetylation of histones in nucleosomes and hence the activity of specific promoters can be influenced. The finding that Mad/Max complexes interact with Sin3 and HD1 in vivo suggests a model for the role of Mad proteins in antagonizing the function of Myc proteins.

Thymidine inhibits the growth-arrest-specific degradation of thymidine kinase protein in transfected L fibroblasts.

The expression of murine thymidine kinase (TK) is strictly dependent on the growth state of the cell. Expressing epitope-tagged TK in LTK cells, we have previously shown that low TK enzyme levels in G0 cells are in part due to a dramatic decrease in TK protein stability. Here we report that thymidine, one of the substrates of TK, is able to counteract the growth-arrest-specific decrease of TK expression. While TK mRNA levels and TK translation rate are almost unaffected by thymidine, the TK protein half-life rose more than sixfold after addition of the nucleoside to resting cells. The effect of thymidine is reversible and is independent of its presence during the protein synthesis of TK. Dideoxythymidine, a specific inhibitor of the TK enzyme activity, also has the capacity to increase TK protein levels in G0 cells, indicating that the substrate itself exerts the stabilising effect on the TK protein.

Carboxy-terminal residues of mouse thymidine kinase are essential for rapid degradation in quiescent cells.

The expression of murine thymidine kinase (TK) is highly dependent on the growth state of the cell. The enzyme is nearly undetectable in resting (G0) cells, but TK protein levels rise dramatically when serum-stimulated cells reach the G1/S boundary. To study post-transcriptional regulation of TK expression, Ltk- cells were stably transfected with the coding region of the TK cDNA under the control of a constitutive SV40 promoter. While TK mRNA levels were growth independent in this cell line, TK protein expression and enzyme activity were low in resting cells but increased strongly after growth stimulation by serum. Measurements of translation efficiency and protein stability by immunoprecipitation and pulse-chase experiments indicated that a fourfold change in protein synthesis rate and a sevenfold rise in protein stability are responsible for the increase of TK expression. Progressive deletion of three, six, ten and 20 carboxy-terminal residues of the enzyme resulted in a stepwise loss of its growth-dependent regulation. In addition, a truncated protein lacking the last 30 amino acid residues was expressed at a level tenfold higher than the full-length polypeptide. Further analysis showed that removal of the C-terminal 30 residues did not affect the translation rate, but resulted in the drastic increase in protein half-life. These results demonstrate that residues at the carboxy terminus of the murine enzyme are essential for the growth-dependent regulation of TK protein stability.

Effect of transcription inhibitors on the iron-dependent degradation of transferrin receptor mRNA.

Transferrin receptor (TfR) mRNA expression is tightly linked to intracellular iron levels. Upon iron deprivation, the iron regulatory protein (IRP) stabilizes TfR mRNA by binding to stem-loop structures in its 3'-untranslated region, whereas increased iron levels result in inactivation of the mRNA-binding protein and rapid degradation of TfR mRNA. Although IRP and the regulation of its RNA binding activity have been studied intensively, little is known about the mechanism of TfR mRNA degradation. In order to get more information about factors involved in this process we investigated the in vivo IRP-RNA interaction and the effect of transcription inhibitors on the iron-dependent decay of TfR mRNA. Here we demonstrate that part of the active IRP co-localizes with TfR mRNA to the rough endoplasmic reticulum. High intracellular iron levels led to a drastic reduction of this active RNA-bound IRP in vivo, indicating that IRP dissociates prior to TfR mRNA decay. Furthermore, the transcription inhibitor actinomycin D and translation inhibitor cycloheximide suppressed TfR mRNA degradation but did not interfere with the IRP dissociation step. Other inhibitors of RNA polymerase II had no effect on iron-dependent degradation of TfR mRNA. However, high concentrations of alpha-amanitin known to block transcription by RNA polymerase III interfered with mRNA decay suggesting the involvement of polymerase III transcripts in the degradation pathway.

Regulation of transferrin receptor mRNA expression. Distinct regulatory features in erythroid cells.

In proliferating non-erythroid cells, the expression of transferrin receptors (TfR) is negatively regulated by the amount of intracellular iron. Fe-dependent regulation of TfR occurs post-transcriptionally and is mediated by iron-responsive elements (IRE) located in the 3' untranslated region of the TfR mRNA. IREs are recognized by a specific cytoplasmic binding protein (IRE-BP) that, in the absence of Fe, binds with high affinity to TfR mRNA, preventing its degradation. While TfR numbers are positively correlated with proliferation in non-erythroid cells, in hemoglobin-synthesizing cells, their numbers increase during differentiation and are, therefore, negatively correlated with proliferation. This suggests a distinct regulation of erythroid TfR expression and evidence, as follows, for this was found in the present study. (a) With nuclear run-on assays, our experiments show increased TfR mRNA transcription following induction of erythroid differentiation of murine erythroleukemia (MEL) with Me2SO. (b) Me2SO treatment of MEL cells does not increase IRE-BP activity which is, however, increased in uninduced MEL cells by Fe chelators. (c) Following induction of MEL cells, there is an increase in the stability of TfR mRNA, whose level is only slightly affected by iron excess. (d) Heme-synthesis inhibitors, such as succinylacetone and isonicotinic acid hydrazide, which inhibit numerous aspects of erythroid differentiation, also inhibit TfR mRNA expression in induced MEL cells. However, heme-synthesis inhibition does not lead to a decrease in TfR mRNA levels in uninduced MEL cells. Thus, these studies indicate that TfR gene expression is regulated differently in hemoglobin synthesizing as compared to uninduced MEL cells.

Characterization of a second RNA-binding protein in rodents with specificity for iron-responsive elements.

Iron regulatory factor (IRF) is a cytoplasmic RNA-binding protein involved in regulating iron homeostasis. IRF controls expression of ferritin and transferrin receptor post-transcriptionally via specific binding to stem-loop iron-responsive elements (IREs) located in the untranslated regions of the respective mRNAs. We have confirmed by RNA band-shift analysis that a second IRE-protein complex observed in different rodent cell extracts is, like IRF, regulated by intracellular iron levels. This faster migrating complex appears to represent a specific interaction between the ferritin IRE and an iron-regulated protein that is distinct from IRF, as concluded from the following lines of evidence. First, UV cross-linking and partial digestion with different proteases revealed different peptide patterns for the two IRE-protein complexes. Second, antiserum raised against IRF peptides immunoprecipitated only authentic IRF and not the protein of the faster migrating complex, as determined by band-shift analysis. Following separation of the two IRE-binding proteins by ion-exchange chromatography, only the IRF-containing fraction reacted with the antibodies on Western blots. The second protein binds IREs with an affinity similar to that of IRF as demonstrated by competition with a ferritin IRE and related stem-loop RNAs. UV cross-linking experiments indicate that this second protein, tentatively named IRFB, has a molecular mass of approximately 105 kDa. Analysis of mouse tissues revealed differences in the distribution of IRF and IRFB. Whereas IRF protein and IRE binding activity were predominant in liver, intestine, and kidney, the IRFB protein(s) revealed highest binding activity in intestine and brain. Our data support the existence of two distinct iron-regulated IRE-binding proteins in rodents.

Interleukin-2-dependent transcriptional and post-transcriptional regulation of transferrin receptor mRNA.

Interleukin-2 (IL-2) controls the proliferation of the murine T cell line B6.1 and induces transferrin receptor (TfR) mRNA steady-state levels 50-fold when added to arrested, IL-2-deprived cells. In addition, TfR mRNA is post-transcriptionally regulated by intracellular iron. Low iron levels activate a cytoplasmic RNA-binding protein, called iron regulatory factor (IRF) or iron-responsive element-binding protein, which coordinately stabilizes TfR mRNA and inhibits ferritin mRNA translation. Since ferritin expression is known to be modulated by cytokines, we decided to investigate the mechanism by which IL-2 activates TfR gene expression in B6.1 cells. Induction by IL-2 of both nuclear and cytoplasmic TfR RNA was compared with run-on transcription rates in isolated nuclei. The results revealed a 3-fold increase in TfR gene transcription and a 6-fold rise in nuclear TfR RNA reaching its steady-state level within 2 h. The main accumulation of mature mRNA in the cytoplasm occurred after 6 h in parallel with the activation of IRF. However, stimulation of IRF binding activity by the iron chelator desferrioxamine, in the absence of IL-2, failed to induce TfR mRNA. Moreover, deprivation of growing B6.1 cells of IL-2 resulted in cell arrest and a rapid decay of TfR mRNA, which was not prevented by the activation of IRF with desferrioxamine. TfR mRNA stabilization appears, therefore, to depend on IL-2. We conclude that TfR mRNA expression is controlled by at least three steps at the onset of cell proliferation: (i) the growth factor-dependent activation of transcription; (ii) mRNA stabilization by IRF in the cytoplasm; and (iii) an additional IL-2-dependent activity which prevents TfR mRNA degradation. Our results indicate that expression of TfR, like ferritin, is controlled by both iron and cytokines.

Expression of active iron regulatory factor from a full-length human cDNA by in vitro transcription/translation.

Iron regulatory factor (IRF), also called iron responsive element-binding protein (IRE-BP), is a cytoplasmic RNA-binding protein which regulates post-transcriptionally transferrin receptor mRNA stability and ferritin mRNA translation. By using the polymerase chain reaction (PCR) and the sequence published by Rouault et al. (1990) a probe was derived which permitted the isolation of three human IRF cDNA clones. Hybridization to genomic DNA and mRNA, as well as sequencing data indicated a single copy gene of about 40 kb specifying a 4.0 kb mRNA that translates into a protein of 98,400 dalton. By in vitro transcription of a assembled IRF cDNA coupled to in vitro translation in a wheat germ extract, we obtained full sized IRF that bound specifically to a human ferritin IRE. In vitro translated IRF retained sensitivity to sulfhydryl oxidation by diamide and could be reactivated by beta-mercaptoethanol in the same way as native placental IRF. An IRF deletion mutant shortened by 132 amino acids at the COOH-terminus was no longer able to bind to an IRE, indicating that this region of the protein plays a role in RNA recognition. Placental IRF has previously been shown to migrate as a doublet on SDS-polyacrylamide gels. After V8 protease digestion the heterogeneity was located in a 65/70 kDa NH2-terminal doublet. The liberated 31 kDa COOH-terminal polypeptide was found to be homogeneous by amino acid sequencing supporting the conclusion of a single IRF gene.