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Attenuation of Forkhead signaling by DACH1

The Drosophila Dachshund (Dac) gene, cloned as a dominant inhibitor of the hyperactive growth factor mutant ellipse, encodes a key component of the retinal determination gene network that governs cell fate. In this study cyclic amplification and selection of targets identified a DACH1 DNA-binding sequence that resembles the FOX (Forkhead box-containing protein) binding site. Genome-wide in silico promoter analysis of DACH1 binding sites identified gene clusters populating cellular pathways associated with the cell cycle and growth factor signaling. ChIP coupled with high-throughput sequencing mapped DACH1 binding sites to corresponding gene clusters predicted in silico and identified as weight matrix resembling the cyclic amplification and selection of targets-defined sequence. DACH1 antagonized FOXM1 target gene expression, promoter occupancy in the context of local chromatin, and contact-independent growth. Attenuation of FOX function by the cell fate determination pathway has broad implications given the diverse role of FOX proteins in cellular biology and tumorigenesis (Zhou, 2010).

This study provides evidence that the RDGN and Forkhead pathways integrate to control the expression of genes that associate with contact-independent growth. The key RDGN protein, DACH1, directly binds DNA and competes in the context of local chromatin with FOXM1, thereby attenuating key Forkhead regulatory gene networks. The cell fate determination factor, DACH1, is thus a DNA sequence-specific inhibitor of Forkhead signaling (Zhou, 2010).

The FOX proteins are a family of evolutionarily conserved transcription regulators involved in diverse biological processes. FOX protein function can either promote or inhibit tumorigenesis, and deregulation of FOX protein function in human tumorigenesis may occur by alteration in upstream regulators or genetic events such as mutations of the DBD, or translocations, which often disrupt the DBD. FOXM1 is overexpressed in human tumors and promotes tumor growth. Inhibition of FOXM1 expression reduces growth of murine tumors in response to carcinogens, and diminishes DNA replication and mitosis of tumor cells. FOXC2, associated with aggressive basal-like breast cancer, enhances tumor metastasis and invasion (Zhou, 2010).

In this study DACH1 was shown to inhibit FOXM1-mediated contact-independent growth of U2OS cells, antagonize FOXM1-mediated gene expression, and reduce occupancy of FOXM1 at target genes known to regulate the G2/M phase progression. Through competitive inhibition of FOXM1 occupancy at target genes, DACH1 could be essential for transcription of F-Box protein S-phase kinase-associated protein 2 (Skp2) and the Cdk subunit 1 (Cks1), which are substrate-specific subunits of the Skp1-Cullin-F-box protein (SCF) ubiquitin-ligase complex, that regulate p21CIP1 and p27KIP1. FOXM1 protein activates gene transcription and DACH1 competes with FOXM1 in the context of local chromatin to repress gene expression. Loss of DACH1 expression and increased oncogenic FOX protein expression could lead to deregulation of a subset of genes required for tumorigenesis. It has been shown that loss of DACH1 expression in human breast and endometrial cancers predicts poor outcome. Future studies will address whether loss of DACH1 correlates with increased FOXM1 expression during the progression of breast and endometrial cancers as well as other type of cancers (Zhou, 2010).

Identification of DACH1 DNA binding sequences by CAST, together with genome-wide in silico screening for putative target genes and ChIP-Seq for genes whose promoters were engaged by DACH1 through direct DNA binding, provided an alternative approach to establish the regulatory networks or individual genes that are governed by DACH1. In silico screening identified 2,887 genes whose promoter regions contain a potential DACH1 binding site. Many FOXM1-targeted genes including Aurora A (STK6), Aurora B (AURKB), CDC25A, CDC25C, E-cadherin (CDH1), and CENPB have DACH1 binding sequences within their 2-kb promoter region. A subset of these genes, chosen based on gene expression change as a result of DACH1 induction into MDA-MB-231 cells, were also enriched in their promoter region for the Forkhead family of transcription factors, supporting the competition/cooperation model of DACH1 and Forkhead transcription factors in gene regulation. Genes with DACH1 binding sites in their promoters populate cellular pathways associated with cancers such as the cell cycle pathway and the glioma pathway. Taken together, these results provide a rationale for further investigations into DACH1-mediated gene regulation in tumorigenesis. Given the diverse roles of Forkhead family proteins in cellular differentiation, survival, and DNA repair, the finding that the RDGN network protein DACH1 intersects FOX signaling may have broad implications for the understanding of cellular biology and tumorigenesis (Zhou, 2010).

Mammalian Forkhead homologs: Wing-helix nude (Hfh 11) is a product of the nude locus

Mutations at the nude locus of mice and rats disrupt normal hair growth and thymus development, causing nude mice and rats to be immune-deficient. The mouse nude locus has been localized on chromosome 11 within a region of < 1 megabase. One of the genes from this critical region, designated whn, encodes a new member of the winged-helix domain family of transcription factors, and it is disrupted on mouse nu and rat rnuN alleles. Mutant transcripts do not encode the characteristic DNA-binding domain, strongly suggesting that the whn gene is the nude gene. Mutations in winged-helix domain genes cause homeotic transformations in Drosophila and distort cell-fate decisions during vulval development in Caenorhabditis elegans. The whn gene is thus the first member of this class of genes to be implicated in a specific developmental defect in vertebrates (Nehls, 1994).

The development of the thymus depends initially on epithelial-mesenchymal and subsequently on reciprocal lympho-stromal interactions. The genetic steps governing development and differentiation of the thymic microenvironment are unknown. By a targeted disruption of the whn gene, which recapitulates the phenotype of the athymic nude mouse, the Whn transcription factor has been shown to be the product of the nude locus. Formation of the thymic epithelial primordium before the entry of lymphocyte progenitors does not require the activity of Whn. However, subsequent differentiation of primitive precursor cells into subcapsular, cortical, and medullary epithelial cells of the postnatal thymus depends on activity of the whn gene. These results define the first genetically separable steps during thymic epithelial differentiation (Nehls, 1996).

Mutations in the winged-helix nude (whn) gene are associated with the phenotype of congenital athymia and hairlessness in mouse and rat. The whn gene encodes a presumptive transcription factor with a DNA binding domain of the forkhead/winged-helix class. Two previously described null alleles encode truncated whn proteins lacking the characteristic DNA binding domain. In the rat rnu allele described here, a nonsense mutation in exon 8 of the whn gene was identified. The truncated whnrnu protein contains the DNA binding domain but lacks the 175 C-terminal amino acids of the wild-type protein. To facilitate the identification of functionally important regions in this region, a whn homolog from the pufferfish Fugu rubripes was isolated. Comparison of derived protein sequences with the mouse whn gene reveals the presence of a conserved acidic protein domain in the C terminus, in addition to the highly conserved DNA binding domain. Using fusions with a heterologous DNA binding domain, a strong transcriptional activation domain was localized to the C-terminal cluster of acidic amino acids. Since the whnrnu mutant protein lacks this domain, these results indicate that a transactivation function is essential for the activity of the whn transcription factor (Schuddekopf, 1996).

Mutations in the winged-helix nude (whn) gene result in the nude mouse and rat phenotypes. The pleiotropic nude phenotype, which affects the hair, skin, and thymus, suggests that whn plays a pivotal role in the development and/or maintenance of these organs. However, little is known about whn function in these organs. In skin whn is specifically expressed in epithelial cells and not the mesenchymal cells: using a hair reconstitution assay, it has been demonstrated that the abnormal nude mouse hair development is attributable to a functional defect of the epithelial cells. Examination of nude mouse primary keratinocytes in culture reveals that these cells have an increased propensity to differentiate in an abnormal fashion, even under conditions that promote proliferation. Furthermore, nude mouse keratinocytes display a 100-fold increased sensitivity to the growth-inhibitory/differentiation effects of the phorbol ester TPA. In parallel with these findings, it has been directly shown that Whn functions as a transcription factor that can specifically suppress expression of differentiation/TPA-responsive genes. The region of Whn responsible for these effects was mapped to the carboxy-terminal transactivating domain. These results establish whn as a key regulatory factor involved in maintaining the balance between keratinocyte growth and differentiation. The general implications of these findings for an epithelial self-renewal model are discussed (Brissette, 1996).

In the mouse, the product of the nude locus, Whn, is required for the keratinization of the hair shaft and the differentiation of epithelial progenitor cells in the thymus. A bacterially expressed peptide representing the presumptive DNA binding domain of the mouse whn gene in vitro specifically binds to an 11-bp consensus sequence containing the invariant tetranucleotide 5'-ACGC. In transient transfection assays, such binding sites stimulate reporter gene expression about 30- to 40-fold, when positioned upstream of a minimal promotor. Whn homologs from humans, bony fish (Danio rerio), cartilaginous fish (Scyliorhinus caniculus), agnathans (Lampetra planeri), and cephalochordates (Branchiostoma lanceolatum) share at least 80% of amino acids in the DNA binding domain. In agreement with this remarkable structural conservation, the DNA binding domains from zebrafish, which possesses a thymus but no hair, and amphioxus, which possesses neither thymus nor hair, recognize the same target sequence as the mouse DNA binding domain in vitro and in vivo. The genomes of vertebrates and cephalochordates contain only a single whn-like gene, suggesting that the primordial whn gene was not subject to gene-duplication events. Although the role of whn in cephalochordates and agnathans is unknown, its requirement in the development of the thymus gland and the differentiation of skin appendages in the mouse suggests that changes in the transcriptional control regions of whn genes accompanied their functional reassignments during evolution (Schlake, 1997).

Loss-of-function mutations in Whn (wing-helix nude; Hfh 11), a winged-helix/forkhead transcription factor, result in the nude mouse phenotype. To determine the whn expression pattern during development, mice were used in which a beta-galactosidase reporter gene was placed under the control of the wild-type whn promoter by homologous recombination. Sites of reporter expression were confirmed by immunohistochemical staining for Whn protein or by in situ hybridization for whn mRNA. At all developmental stages, whn expression is restricted to epithelial cells. In addition to the skin and thymus, whn is expressed in the developing nails, nasal passages, tongue, palate, and teeth. In embryonic epidermis, suprabasal cells induce whn expression at the same time that terminal differentiation markers first appear. As the epidermis matures, whn promoter activity is found primarily in the first suprabasal layer, which contains keratinocytes in the early stages of terminal differentiation. In developing and mature anagen hair follicles, whn is expressed at high levels in the postmitotic precursor cells of the hair shaft and inner root sheath. Though principally associated with terminal differentiation, whn expression is also detected in progenitor cell compartments; in the hair bulb matrix and basal epidermal layer, a small subclass of cells expresses whn, while in the outer root sheath, whn promoter activity is induced as the follicle completes its elongation. Within these compartments, rare cells exhibit both whn expression and the nuclear proliferation marker Ki-67. The results suggest that whn expression encompasses the transition from a proliferative to a postmitotic state and that whn regulates the initiation of terminal differentiation. During thymus development, whn expression first appears in epithelial cells of the thymic primordium, and in the mature thymus, whn expressing epithelial cells are present throughout the medulla, cortex, and subcapsular region. Given the sites of whn expression in the mouse, as well as the presence of homologs in lower vertebrates and cephalochordates, the whn gene may influence a fundamental or common features of epithelial cell differentiation (Lee, 1999).

Table of contents

forkhead: Biological Overview | Regulation | Targets of Activity | Developmental Biology | Effects of Mutation | References

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