Pioneer factors, genetic competence, and inductive signaling: programming liver and pancreas progenitors from the endoderm.

The endoderm is a multipotent progenitor cell population in the embryo that gives rise to the liver, pancreas, and other cell types and provides paradigms for understanding cell-type specification. Studies of isolated embryo tissue cells and genetic approaches in vivo have defined fibroblast growth factor/mitogen-activated protein kinase (FGF/MAPK) and bone morphogenetic protein (BMP) signaling pathways that induce liver and pancreatic fates in the endoderm. In undifferentiated endoderm cells, the FoxA and GATA transcription factors are among the first to engage silent genes, helping to endow competence for cell-type specification. FoxA proteins can bind their target sites in highly compacted chromatin and open up the local region for other factors to bind; hence, they have been termed "pioneer factors." We recently found that FoxA proteins remain bound to chromatin in mitosis, as an epigenetic mark. In embryonic stem cells, which lack FoxA, FoxA target sites can be occupied by FoxD3, which in turn helps to maintain a local demethylation of chromatin. By these means, a cascade of Fox factors helps to endow progenitor cells with the competence to activate genes in response to tissue-inductive signals. Understanding such epigenetic mechanisms for transcriptional competence coupled with knowledge of the relevant signals for cell-type specification should greatly facilitate efforts to predictably differentiate stem cells to liver and pancreatic fates.

Ikaros-family proteins: in search of molecular functions during lymphocyte development.

The regulatory steps that lead to the differentiation of hematopoietic cells from a multipotential stem cell remain largely unknown. A beginning to the understanding of these steps has come from the study of DNA-binding proteins that are thought to regulate the expression of genes required for specific developmental events. Ikaros is the founding member of a small family of DNA-binding proteins required for lymphocyte development, but the members of this family differ from other key regulators of lymphopoiesis in that direct target genes have not been conclusively identified, and reasonable support has been presented for only a few potential targets. Therefore, the molecular mechanisms that Ikaros uses for regulating lymphocyte development remain largely unknown. Current data suggest that, in some instances, Ikaros may function as a typical transcription factor. However, recent results suggest that it may function more broadly, perhaps in the formation of silent and active chromatin structures. In this review, our current knowledge of the molecular functions of Ikaros will be discussed.

Initiator recognition in a primitive eukaryote: IBP39, an initiator-binding protein from Trichomonas vaginalis.

While considerable progress has been made in understanding the mechanisms of transcription in higher eukaryotes, transcription in single-celled, primitive eukaryotes remains poorly understood. Promoters of protein-encoding genes in the parasitic protist Trichomonas vaginalis, which represents one of the deepest-branching eukaryotic lineages, have a bipartite structure with gene-specific regulatory elements and a conserved core promoter encompassing the transcription start site. Core promoters in T. vaginalis appear to consist solely of a highly conserved initiator (Inr) element that is both a structural and a functional homologue of its metazoan counterpart. Using DNA affinity chromatography, we have isolated an Inr-binding protein from T. vaginalis. Cloning of the gene encoding the Inr binding protein identified a novel 39-kDa protein (IBP39). We show that IBP39 binds to both double and single Inr motifs found in T. vaginalis genes and that binding requires the conserved nucleotides necessary for Inr function in vivo. Analyses of the cloned IBP39 gene revealed no homology at the protein sequence level with identified proteins in other organisms or the presence of known DNA-binding domains. The relationship between IBP39 and Inr-binding proteins in metazoa presents interesting evolutionary questions.

Down-regulation of TDT transcription in CD4(+)CD8(+) thymocytes by Ikaros proteins in direct competition with an Ets activator.

Ikaros is a unique regulator of lymphopoiesis that associates with pericentromeric heterochromatin and has been implicated in heritable gene inactivation. Binding and competition experiments demonstrate that Ikaros dimers compete with an Ets activator for occupancy of the lymphocyte-specific TdT promoter. Mutations that selectively disrupt Ikaros binding to an integrated TdT promoter had no effect on promoter function in a CD4(+)CD8(+) thymocyte line. However, these mutations abolished down-regulation on differentiation, providing evidence that Ikaros plays a direct role in repression. Reduced access to restriction enzyme cleavage suggested that chromatin alterations accompany down-regulation. The Ikaros-dependent down-regulation event and the observed chromatin alterations appear to precede pericentromeric repositioning. Current models propose that the functions of Ikaros should be disrupted by a small isoform that retains the dimerization domain and lacks the DNA-binding domain. Surprisingly, in the CD4(+)CD8(+) thymocyte line, overexpression of a small Ikaros isoform had no effect on differentiation or on the pericentromeric targeting and DNA-binding properties of Ikaros. Rather, the small isoform assembled into multimeric complexes with DNA-bound Ikaros at the pericentromeric foci. The capacity for in vivo multimer formation suggests that interactions between Ikaros dimers bound to the TdT promoter and those bound to pericentromeric repeat sequences may contribute to the pericentromeric repositioning of the inactive gene.

Nucleosome remodeling at the IL-12 p40 promoter is a TLR-dependent, Rel-independent event.

Lipopolysaccharide (LPS) induction of the gene encoding interleukin 12 p40 requires remodeling of a promoter-encompassing nucleosome and the Toll-like receptor (TLR)-mediated activation of a c-Rel-containing complex. Analysis of TLR4-mutant mice revealed that remodeling requires TLR signaling. However, Rel proteins and other proteins required for transcription of an integrated p40 promoter were insufficient for remodeling. c-Rel was also unnecessary for remodeling, as remodeling was observed in c-Rel-/- macrophages, which lack p40 transcripts. These results suggest that remodeling requires TLR signaling pathways that diverge from the c-Rel activation pathways. The factors that stimulate remodeling may represent, therefore, newly identified targets of TLR signaling and of agents that regulate inflammatory responses and TH1 development.

Selective requirement for c-Rel during IL-12 P40 gene induction in macrophages.

A major challenge in the study of gene regulation by NF-kappaB/Rel transcription factors is to understand, at the biological and mechanistic levels, the selective functions of individual Rel family members. To study selectivity, we have examined the NF-kappaB/Rel protein binding site (Rel site) within the IL-12 p40 promoter. IL-12 is a proinflammatory cytokine expressed by activated macrophages that serves as an essential inducer of T helper 1 cell development. In nuclear extracts from lipopolysaccharideactivated macrophages, the predominant Rel dimers capable of binding the IL-12 p40 Rel site were the p50/p65 and p50/c-Rel heterodimers and p50/p50 homodimer. The two heterodimers bound the site with comparable affinities and exhibited comparable transactivation activities. In striking contrast, p40 mRNA and protein concentrations were reduced dramatically in c-Rel(-/-) macrophages and only modestly in p65(-/-) macrophages. Other proinflammatory cytokine mRNAs and proteins were not significantly reduced in c-Rel(-/-) macrophages. These results reveal that a c-Rel-containing complex is an essential and selective activator of p40 transcription, which may reflect unique regulatory mechanisms or biological functions of IL-12. Furthermore, because selectivity was not observed in vitro or in transient transactivation experiments, these findings suggest that an understanding of the selectivity mechanism may require an analysis of the endogenous p40 locus.

Targeting of Ikaros to pericentromeric heterochromatin by direct DNA binding.

Ikaros is a sequence-specific DNA-binding protein that is essential for lymphocyte development. Little is known about the molecular function of Ikaros, although recent results have led to the hypothesis that it recruits genes destined for heritable inactivation to foci containing pericentromeric heterochromatin. To gain further insight into the functions of Ikaros, we have examined the mechanism by which it is targeted to centromeric foci. Efficient targeting of Ikaros was observed upon ectopic expression in 3T3 fibroblasts, demonstrating that lymphocyte-specific proteins and a lymphoid nuclear architecture are not required. Pericentromeric targeting did not result from an interaction with the Mi-2 remodeling factor, as only a small percentage of Mi-2 localized to centromeric foci in 3T3 cells. Rather, targeting was dependent on the amino-terminal DNA-binding zinc finger domain and carboxy-terminal dimerization domain of Ikaros. The carboxy-terminal domain was required only for homodimerization, as targeting was restored when this domain was replaced with a leucine zipper. Surprisingly, a detailed substitution mutant analysis of the amino-terminal domain revealed a close correlation between DNA-binding and pericentromeric targeting. These results show that DNA binding is essential for the pericentromeric localization of Ikaros, perhaps consistent with the presence of Ikaros binding sites within centromeric DNA repeats. Models for the function of Ikaros that are consistent with this targeting mechanism are discussed.

A prominent role for Sp1 during lipopolysaccharide-mediated induction of the IL-10 promoter in macrophages.

IL-10 is an antiinflammatory cytokine secreted by activated macrophages and Th2 cells. IL-10 secretion promotes the down-regulation of proinflammatory cytokine synthesis and the development of Th2 responses. In macrophages, proinflammatory cytokines appear to be induced by similar mechanisms, but the IL-10 induction mechanisms have not been examined. We have analyzed the murine IL-10 promoter in the RAW264.7 macrophage line activated with LPS. A comprehensive mutant analysis revealed only one element upstream of the core promoter that was essential for promoter induction. A refined mutant analysis localized this element to nucleotides -89 to -78, and gel shift experiments revealed that it represents a nonconsensus binding site for Sp1. The functional relevance of Sp1 was supported by the high affinity of the interaction, the close correlation between the nucleotides required for Sp1 binding and promoter function, and the ability of an Sp1 consensus sequence to substitute for the -89/-78 promoter sequence. Evidence that Sp1 may be a target of signaling pathways involved in IL-10 induction was provided by the exclusive requirement for the Sp1 binding site, by the ability of the Sp1 site to confer induction to a heterologous promoter, and by the delineation of an Sp1 domain that can mediate induction. No relevant contribution from Rel, C/EBP (CCAAT/enhancer-binding protein), or AP-1 binding sites, which regulate most proinflammatory cytokine promoters, was observed. Together, these results demonstrate that IL-10 gene regulation is distinct from the regulation of proinflammatory cytokine genes, and suggest that Sp1 may be a central mediator of IL-10 induction.

Host defense mechanisms triggered by microbial lipoproteins through toll-like receptors.

The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.

Rapid and selective remodeling of a positioned nucleosome during the induction of IL-12 p40 transcription.

Nucleosomes are important for gene regulation, but comprehensive studies of nucleosome positioning, remodeling, and transcription factor binding at inducible mammalian promoters have not been reported. We have analyzed the IL-12 p40 promoter, which is induced in macrophages by bacterial products. High-resolution micrococcal nuclease analyses revealed that a positioned nucleosome, nucleosome 1, spans the promoter, with three positioned nucleosomes further upstream. Upon activation, nucleosome 1 was rapidly and selectively remodeled in a protein synthesis-dependent manner. In primary macrophages, IFNgamma synergistically enhanced p40 expression, but little effect on remodeling or promoter occupancy was observed. These results suggest that remodeling complexes are selectively targeted to a single, promoter-encompassing nucleosome and that IFNgamma influences an event that is independent or downstream of remodeling.

Helios, a T cell-restricted Ikaros family member that quantitatively associates with Ikaros at centromeric heterochromatin.

The Ikaros gene encodes multiple protein isoforms that contribute critical functions during the development of lymphocytes and other hematopoietic cell types. The intracellular functions of Ikaros are not known, although recent studies have shown that Ikaros proteins colocalize with inactive genes and centromeric heterochromatin. In this study, Ikaros proteins were found to be components of highly stable complexes. The complexes from an immature T cell line were purified, revealing associated proteins of 70 and 30 kD. The p70 gene, named Helios, encodes two protein isoforms with zinc finger domains exhibiting considerable homology to those within Ikaros proteins. Helios and Ikaros recognize similar DNA sequences and, when overexpressed, Helios associates indiscriminately with the various Ikaros isoforms. Although Ikaros is present in most hematopoietic cells, Helios was found primarily in T cells. The relevance of the Ikaros-Helios interaction in T cells is supported by the quantitative association of Helios with a fraction of the Ikaros. Interestingly, the Ikaros-Helios complexes localize to the centromeric regions of T cell nuclei, similar to the Ikaros localization previously observed in B cells. Unlike the B cell results, however, only a fraction of the Ikaros, presumably the fraction associated with Helios, exhibited centromeric localization in T cells. These results establish immunoaffinity chromatography as a useful method for identifying Ikaros partners and suggest that Helios is a limiting regulatory subunit for Ikaros within centromeric heterochromatin.

Sp1 activation of a TATA-less promoter requires a species-specific interaction involving transcription factor IID.

Sp1 is a ubiquitous activator of numerous TATA-containing and TATA-less promoters within the human genome. This transcription factor is distinct from several other mammalian activators because it cannot stimulate transcription of reporter genes when ectopically expressed in Saccharomyces cerevisiae . Here we report that in cultured cells from Drosophila melanogaster human Sp1 efficiently activates transcription from synthetic promoters containing TATA boxes, but not from promoters that contain an initiator instead of a TATA box. The inability of Sp1 to activate initiator-mediated transcription did not result from inactivity of the consensus initiator element used for the experiments, as other initiator functions were conserved in Drosophila cells. Interestingly, a difference between the Drosophila and human TFIID complexes was found to be responsible for the selective inability of Sp1 to activate initiator-mediated transcription in Drosophila; in a complementation assay with a TFIID-depleted HeLa cell extract both the Drosophila and human TFIID complexes supported TATA-mediated transcription, but only the human complex supported initiator-mediated transcription. These results suggest that a species-specific interaction is required for activation of TATA-less promoters by Sp1, revealing a difference in transcriptional activation mechanisms between vertebrates and invertebrates.

CIF150, a human cofactor for transcription factor IID-dependent initiator function.

The transcription factor IID (TFIID) complex is highly conserved between the Drosophila and mammalian systems. A mammalian homolog has been described for all the Drosophila TATA box-binding protein-associated factors (TAFs), with the exception of dTAF(II)150. We previously reported the identification of CIF, an essential cofactor for TFIID-dependent transcription from promoters containing initiator (Inr) elements. Here we describe the molecular cloning of CIF150, the human homolog of dTAF(II)150, and present biochemical evidence that this factor is involved in Inr activity. CIF150 is capable of mediating TFIID-dependent Inr activity in a complementation assay, and a protein fraction lacking Inr activity lacks detectable amounts of CIF150. Despite the striking similarity to dTAF(II)150, CIF150 does not appear to be associated with human TFIID. However, in vitro binding assays revealed a specific and direct interaction between CIF150 and hTAF(II)135. This interaction might be structurally important for the functional interaction between CIF150 and human TFIID, since CIF150 stabilizes TFIID binding to a core promoter.

Hematopoietic stem cells and lymphoid progenitors express different Ikaros isoforms, and Ikaros is localized to heterochromatin in immature lymphocytes.

The generation of lymphoid cells in mice depends on the function of the Ikaros protein. Ikaros has been characterized as a lymphoid-restricted, zinc-finger transcription factor that is derived from an alternatively spliced message. Ikaros knockout mice have defects in multiple cell lineages, raising the question of whether the protein regulates multiple committed progenitors and/or multipotent stem cells. To address this issue, we examined Ikaros expression in purified populations of multipotent cells and more committed progenitors. We found that the DNA-binding isoforms of Ikaros were localized in the nucleus of the most primitive hematopoietic stem cell subset. Changes in the RNA splicing pattern of Ikaros occurred at two stages: (i) as long-term self-renewing stem cells differentiated into short-term self-renewing stem cells and (ii) as non-self-renewing multipotent progenitors differentiated into lymphoid-committed progenitors. Unexpectedly, we found Ikaros localized to heterochromatin in Abelson-transformed pre-B lymphocytes by using immunogold electron microscopy. These observations suggest a complex role for Ikaros in lymphoid development.

Mechanism of synergy between TATA and initiator: synergistic binding of TFIID following a putative TFIIA-induced isomerization.

The TFIID complex interacts with at least three types of core promoter elements within protein-coding genes, including TATA, initiator (Inr), and downstream promoter elements. We have begun to explore the mechanism by which the TFIID-Inr interaction leads to functional synergy between TATA and Inr elements during both basal and activated transcription. In DNase I footprinting assays, GAL4-VP16 recruited TFIID-TFIIA to core promoters containing either a TATA box, an Inr, or both TATA and Inr elements, with synergistic interactions apparent on the TATA-Inr promoter. Appropriate spacing between the two elements was essential for the synergistic binding. Despite the sequence-specific TFIID-Inr interactions, gel shift experiments revealed that TFIID alone possesses similar affinities for the TATA-Inr and TATA promoters. Interestingly, however, recombinant TFIIA strongly and selectively enhanced TFIID binding to the TATA-Inr promoter, with little effect on binding to the TATA promoter. Studies of the natural adenovirus major late promoter confirmed these findings, despite the existence of specific but nonfunctional TFIID interactions downstream of the Inr in that promoter. These results suggest that a TFIIA-induced conformational change is essential for the sequence-specific TFIID-Inr interaction to occur with sufficient affinity to support the functional synergism between TATA and Inr elements.

Association of transcriptionally silent genes with Ikaros complexes at centromeric heterochromatin.

Ikaros proteins are required for normal T, B, and NK cell development and are postulated to activate lymphocyte-specific gene expression. Here we examined Ikaros distribution in the nucleus of B lymphocytes using confocal microscopy and a novel immunofluorescence in situ hybridization (immuno-FISH) approach. Unexpectedly, Ikaros localized to discrete heterochromatin-containing foci in interphase nuclei, which comprise clusters of centromeric DNA as defined by gamma-satellite sequences and the abundance of heterochromatin protein-1 (HP-1). Using locus-specific probes for CD2, CD4, CD8alpha, CD19, CD45, and lambda5 genes, we show that transcriptionally inactive but not transcriptionally active genes associate with Ikaros-heterochromatin foci. These findings support a model of organization of the nucleus in which repressed genes are selectively recruited into centromeric domains.

Multiple control elements mediate activation of the murine and human interleukin 12 p40 promoters: evidence of functional synergy between C/EBP and Rel proteins.

Interleukin 12 (IL-12) is a heterodimeric cytokine whose activity is critical for T-helper 1 responses. The gene for the IL-12 p40 subunit is expressed in macrophages following induction by bacterial products, and its expression is augmented by gamma interferon. In this study, we performed a functional analysis of the murine and human p40 promoters in the murine macrophage cell line RAW 264.7. Transcription from the murine p40 promoter was strongly induced by lipopolysaccharide and heat-killed Listeria monocytogenes (HKLM), but promoter activity was not enhanced by gamma interferon. Multiple cis-acting elements involved in activated transcription were identified through an extensive mutant analysis. The most critical element, whose activity is conserved in mice and humans, is located between positions -96 and -88 relative to the murine transcription start site. This element exhibits functional synergy with a previously described NF-kappaB half-site which interacts with Rel proteins. DNase I footprinting and electrophoretic mobility shift assays demonstrated that C/EBP proteins interact with the critical element, but in nuclear extracts, cooperative binding of C/EBP and Rel proteins to their respective sites was not observed. Interestingly, promoter activity was induced by HKLM in the presence of cycloheximide, consistent with induction by posttranslational mechanisms. The results suggest that C/EBP and Rel proteins play important roles in the activation of IL-12 p40 transcription by bacteria. However, many complex interactions will need to be clarified to fully understand p40 regulation.