Forkhead transcription factor complex with special ...

Bull Vet Inst Pulawy 57, 455-459, 2013 DOI: 10.2478/bvip-2013-0079

Forkhead transcription factor complex with special focus on Foxp3, Foxn1, and Foxj1 genes determining immunological parameters - a review Paweł Wojtaszczyk, Krzysztof Kostro, Krzysztof Niemczuk1, Urszula Lisiecka, Krzysztof Stojecki2, Andrzej Żmuda Department of Epizootiology and Clinic of Infectious Diseases, Faculty of Veterinary Medicine, University of Life Sciences in Lublin, 20-033 Lublin, Poland 1 National Veterinary Research Institute, 24-100 Pulawy, Poland 2 Department of Parasitology and Invasive Diseases, National Veterinary Research Institute, 24-100 Pulawy, Poland [email protected] Received: September 5, 2013

Accepted: December 4, 2013

Abstract The Forkhead genes (transcription complex Fox) play many important roles in the maintenance and determination of biological processes underlying carbon-based life. The expression of Fox genes occurring as a result of reciprocal interactions at the transcriptional level influences the correct function of the immune system as regards the context of general activity of immunological parameters, as well as exposure to aetiological agents. In the case of model organisms and species, in which the knowledge about genomic sequences is incomplete, understanding the above-mentioned transcriptional complex is still insufficient, despite the existence of numerous scientific publications. It is worth noting that Fox genes exist in amphioxus Branchiostoma lanceolatum and in the majority of species they were not characterised. Among many coding sequences included in the transcriptional complex Forkhead, Foxp3, Foxn1, and Foxj1 genes are of a great importance from immunological point of view, being partially jointly responsible for determining features related to productivity. The paper provides general characterisation of selected Fox genes and is an introduction to the presentation of new data, which can be applied at the level of biological differentiation of the populations of domestic and wild animals.

Key words: Forkhead box, Fox genes, immune system. General characteristics. The Forkhead transcription complex containing Fox genes is a conservative set of genetic factors, and their expression is connected with important physiological processes existing at different functional levels. The name “Forkhead” is derived from the forkhead gene discovered in 1989 by Weigel et al. (19) in a mutant of Drosophila melanogaster. In 1990, Weigel et al. (19) demonstrated a close relationship of the forkhead gene with the mammalian HNF-3 gene, thus proving that they exist in living organisms belonging to different taxonomic groups. The current knowledge is restricted to sequences of Fox genes only in animal models, although this complex occurs in amphioxus Branchiostoma lanceolatum (21). The distribution of Forkhead gene complex throughout the genomes does not form physically linked clusters (4).

Currently the Forkhead genes are described as Fox genes and this nomenclature is used in different species. Fox genes are divided into subfamilies and each subfamily is identified by a number, e.g. Foxf2 (4). The identification of a given Fox gene in different animal species and humans is enabled by including a species name, but in literature and databases the synonyms are used, e.g. HNF3α for FoxA1, or FKHR for Foxo1. According to Tuteja and Kaestner (16, 17), the role of at least 40 genes of the Forkhead transcription complex is currently known. From an evolutionary point of view, genes belonging to forkhead box are strongly conservative factors. It is attributed to certain regularities in their locations despite species-specific structure of chromosomes. Foxp3 gene is located on the X chromosome in the majority of species. On the other

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hand, Foxj1 gene shows similarities in Homo sapiens and Pan troglodytes species, in which it is located on chromosome 17. This location probably explains its functional relatedness with Foxn1 gene, also located on this chromosome. In the case of Canis lupus familiaris, Foxn1 and Foxj1 genes are found on chromosome 9. The transcriptional factor Foxo3 in Rattus norvegicus and its variant „b” in Danio rerio are located on chromosome 20. In the case of Homo sapiens and Pan troglotydes, Foxq1 gene is located on chromosome 6. The forkhead box genes are responsible for a series of life functions in different species, including the correct performance of cellular and humoral immune response, in which the genes Foxp3, Foxn1, and Foxj1 are mostly involved. The immune system is homeostatic, and it classifies antigens to one of two categories: „self” and “non-self” (13). An intrusion of the foreign antigens induces an immune response and initiates the mechanisms of non-specific immunity (innate, natural). This type of immunity, phylogenetically the oldest, is species specific, transferred to offspring, and is revealed after the first contact with pathogen. Among systemic factors, genetic background and immunological competence play the most important role (6, 7, 11). If the innate immunity is not efficient, viruses, bacteria, and fungi that invade an organism (or more precisely–antigens of these pathogens) can accumulate in the peripheral lymphatic organs, and cells of the immune system begin to come in contact with these antigens (not belonging to the „self” category), and then the acquired immunity, directed towards the destruction of particular antigens, develops. The most common features of the acquired immunity, different from the innate immunity, include: recognition of antigen, specificity of reaction, and immunological memory (1, 2, 11). Mechanisms of non-specific and specific immunity are divided into two major functional areas. The first one is the exposure conditions comprising routes of entry, as well as the presence and type of pathogen. The second is the quality of genetic material of which the expression is responsible not only for effectiveness of immunological mechanisms but also for manifestation of particular diseases that have genetic roots. Another issue is connected to the polymorphism of loci in the regions such as MHC, responsible for the synthesis of proteins linked to class I MHC, and represented by several dozens of alleles in each locus, and this phenomenon is responsible for intraspecies variation in the activity of major histocompatibility complex. Therefore, the genetic background, apart from its influence on immunological mechanisms and genetic diseases, is responsible for measurable gradation of activity of immunological factors. The analysis of functional basis of genetic factors determining the quality of immunological parameters shows unambiguously that genomes of vertebrates are characterised by structural dynamism, especially

marked in the process of expression of nucleotide sequences. Additionally, the genome of each organism is constantly exposed to factors causing genetic instability, expressed by the emergence of mutations. The effectiveness of DNA repairing mechanisms, taking into consideration the complexity of their regulatory factors, is not perfect, resulting in errors with frequency in conformity with the value of statistical error, and thus not causing any significant changes at the whole genome level, but introducing substitutions at the level sufficient to induce functional modifications of the specific coding sequences, which are revealed in the process of expression (5). Genetic instability is characteristic for cancer cells and can be expressed by a so called microsatellite instability (MSI), resulting in the appearance of new fragments of tandem sequences characterised by a high degree of polymorphism at the species level but highly conservative at the individual level. Proteins produced as a result of translation of nucleotide sequence into amino acids (based on genetic code) undergo the process of post-translational modification, which is the source of functional variability of the primary linear shape of coding complexes. Variability between individuals, caused by these factors, in the efficiency of physiological mechanisms in terms of switching-off the influence of fluctuating factors should encourage the contemporary scientists to search for the causes in the genome dynamics. It should be stressed that, from the point of view of the above mentioned mechanisms, the structural variability of nucleotide sequences and functional fluidity of peptides produced as a result of translation concerns also the regions of genetic material known as highly conservative; whereas, these phenomena are characteristic for polymorphic loci. The current scope of immunogenetics does not seem to explicitly include the knowledge existing beyond the major histocompatibility complex, antigenic complexes of the blood cells, or inherited immune system disorders, such as severe combined immunodeficiency (SCID), and related to the control of natural immunological mechanisms. This article describes the function of Foxp3, Foxn1, and Foxj1 genes in relation to immune system and also other factors of exogenic and endogenic origin, which, with varying degrees, guarantee the proper functioning of immunological mechanisms. Fox genes and basic metabolic genes. Basic metabolic genes (described as housekeeping genes HGK) are coding complexes, their expression level is constant for all cells and includes sequences responsible for processes essential for the survival of cells. However, as described by Romanowski et al. (12), the expression level of these genes can be diverse for different tissues, and thus evaluation of their reference values should be performed in each case individually. The role of genetic regions included in the HGK group is usually known, and the assessment of the expression level of genes responsible for, e.g.



immunological parameters, should be performed in relation to quantity of mRNA complementary to their linear sequences. In view of existing results of the studies on the expression of genes Foxp3, Foxj1, and Foxn1, a high usefulness of the following basic metabolism genes has been shown: GADPH (glyceraldehyde-3-phosphate dehydrogenase), HPRT (hypoxanthine phosphoribosyltransferase), ACTB (β-actin) determining the production of structural proteins of the cytoskeleton, and B2M encoding β2-microglobulin (the protein of the major histocompatibility complex). It should be stressed that incorrect expression level of some of HGK genes is linked to oncogenesis. As shown by Romanowski et al. (12), the use of quantitive real-time RT-PCR (Q-RT-PCR) technique enabled to demonstrate that esophageal, stomach, colon and liver cancer are related to incorrect expression of HPRT or GAPDH genes. It should be also underlined that in the case of these cancers, statistical differences in the expression levels of Fox genes measured by real-time PCR exist. This method combined with reverse transcriptase (RT-PCR) can be used to measure gene expression based on standardised copy numbers of the studied sequence against nanogram quantity of the specific nucleic acid or, as a relative method, based on the internal control represented by HGK genes, like HPRT or GAPDH. Forkhead complex and immunological parameters. Among numerous Fox genes responsible for the regulation of cell cycle, development of hair follicles, and silkiness of hair (Foxn1 and Foxq1) (8, 16, 17), development of the heart (Foxp4), skeleton (Foxc2), and eyes (Fox3) (16, 17), there are also transcriptional factors responsible for immunoregulation. The Foxj1, which inhibits spontaneous activation of T lymphocytes, or Foxn1 responsible for proliferation of epithelial cells, differentiation of keratinocytes, and development of the thymus, belong to the group of genes determining immunological function in animal species studied so far (5, 16, 17). On the other hand, erythropoiesis, inhibition of spontaneous activation of T lymphocytes, and proliferation and/or induction of apoptosis, as well as autoimmunological phenomena are related to the expression of Foxo3 gene. Foxq1 gene is responsible for the modulation of NK cells function, while Foxp3 gene regulates proliferation and differentiation of T lymphocytes, as well as the function of regulatory cells of the phenotype TCD4+CD25+TReg induced by dendritic cells CD11c+CD11b+DCs (9). Expression of genes of the Foxo family is probably linked to the production of proapoptotic protein Bim in haematopoietic cells, which (together with proteins Nur77) take part in the negative selection of T lymphocytes (14). Sunters et al. (15) and Lin et al. (10) showed that the Foxo3a gene inhibits the activity of the transcriptional factor NK-κB, which indirectly regulates oncogenic processes.

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Foxp3 gene. The gene belongs to the family of transcriptional complex Fox, and was described in the Homo sapiens - accession number NC000023, Pan troglodytes - NC006491, Canis lupus familiaris NC006621, Bos taurus - NC007331, Mus musculus NC000086, and Rattus norvegicus- NC005120. The region Foxp3 is characterised by the length ranging from 6358 bp (Bos taurus) to 15 542 bp (Mus musculus) (Homologene), and is responsible for differentiation and T lymphocyte activity of the phenotype CD4+CD25+TReg (7, 13). The analysis of expression of this gene has shown its highest level in lymphatic organs, particularly in TCD4+ cells (5). In autoimmunological disorders in humans and mice, a lack or low level of the expression was found (5, 6). In turn, in vitro stimulation of mouse T lymphocytes of the phenotype CD4+CD25 has shown that - as opposed to humans - expression of Foxp3 gene is not enhanced due to lymphocyte activation. Similarly, mouse lymphocytes of the phenotype CD8+ are also not connected with the expression of this gene (18). Expression of Foxp3 gene is induced by oestrogens through a direct interaction of oestrogen receptor with the promoter of this gene (5, 7, 13). It can be connected to an increase in the percentage of regulatory lymphocytes CD4+CD25+TReg during pregnancy. Other factors correlating with an increase in the Foxp3 gene expression are the serum level of prostaglandin E2, and expression of CD3 particles and TCR receptors on T lymphocytes (16, 17). Expression of Foxp3 gene is also responsible for the regulation of activity of certain cytokines (IL4, IL2, IL5, IL2RA) and immunoglobulins (IgG1, IgG2a, IgM). The major role of the transcription factor Foxp3, i.e. regulation of lymphocyte differentiation, can be disturbed, which was demonstrated in the mouse mutants in which improper expression of this gene was manifested by loss of stability of cytokine production and eosinophilia (16, 17). The cooperation of T lymphocytes and dendritic cells, as well as a specific microenvironment of regulatory lymphocytes are the reason why under certain conditions, determined by the presence of appropriate cytokines, modulation of the transcription factor Foxp3 can take place, e.g. in the case of interleukine 6, which has the ability to inhibit the expression of this gene causing the loss of suppressive effect on proliferating T lymphocytes (20). The changes in the expression level of Foxp3 gene are linked to the development of tumours. Biller et al. (3) investigated populations of healthy dogs, and dogs with tumours in the context of identification of regulatory lymphocytes. These studies demonstrated that the total number of TReg cells in diseased dogs was much higher, which was reflected in the expression level of the Foxp3 transcription factor. Foxj gene. Foxj is the next gene of the forkhead box family regulating immunological parameters. This gene was described in the Homo sapiens–accession number NC000017, Pan troglotydes - NC006484,


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Canis lupus familiaris - NC006591, Bos taurus NC007317, Mus musculus - NC000077, Gallus gallus NC006105, and Danio rerio (FOXJ1a) - NC007114. The length of this gene varies from 3040 bp (Canis lupus familiaris) to 16 220 bp (Foxj1 variant in Danio rerio, Homologene). The Foxj1 gene is mostly responsible for the regulation of lymphocyte proliferation. The factor, regulating its expression is tFoxd1 gene, which belongs to the same complex, and is responsible for development of the kidneys. Tuteja and Kaestner (16, 17) have demonstrated that the Foxj1 gene regulates the activity of IL-2 and IFN-γ, as well as transcription factor NF-κB, responsible for the induction of the expression of pro-inflammatory mediators. According to Coffer and Burgering (5), Foxj1 is also responsible for the inhibition of T lymphocyte activation, and development of autoimmune reaction. Therefore, the consequence of Foxj1 mutations in mice is the development of systemic autoimmune inflammation. Moreover, mutations in Foxj1 gene in mice are responsible for random leftright asymmetry, defective ciliogenesis, impaired growth, and hydrocephalus. Deletions in gene Foxj1 can result in abnormal laterality of the heart and other internal organs (8). However, no relationship was found between the intensity of expression of Foxj1 gene and the occurrence of tumours (16, 17). Regulation of proliferation and differentiation of helper TCD4+ lymphocytes by Foxj1 gene appears through the influence on the group of genes involved in the abovementioned process, or modulation of function of the key transcription factors, such as Foxo3a (5). Foxn1 gene. Foxn1 is a transcription factor, which expression and activity determine not only the function of the immune system, but regulate many other important physiological processes. The gene has been described in the Homo sapiens–accession number NC000017, Pan troglotydes - NC006484, Canis lupus familiaris - NC006591, Bos taurus - NC007317, Mus musculus - NC000077, Rattus norvegicus NC005109, Gallus gallus - NC006106, and Danio rerio - NC007126. The length of the gene ranges from 12 977 bp (Canis lupus familiaris) to 35 972 (Gallus gallus). The major role of this gene is encoding the proliferation factors of epithelial cells, differentiation of keratinocytes, and development of the thymus. Regulation of the Foxn1 expression takes place through factors such as WNT5B i WNT4 (genes of the family WNT encoding signal proteins engaged in the process of oncogenesis and embryogenesis), BMP4 (bone morphogenetic protein) and Noggin (signal protein also responsible for the embryo development). Foxn1 gene is functionally responsible for the regulation of biological properties of IL-7, playing a significant role in lymphopoiesis, and expression of the CD274 particles, as well as other factors, such as: FAM57A (epithelial-like lung adenocarcinoma), SERPINB1 (leukocyte elastase inhibitor gene), GZMA (granzyme A gene), MREG (melanoregulin protein; protein

involved in the differentiation of cell organelles and in the process of pigmentation), PPP1R16B (protein of the phosphatase family), C19ORF28 (ORF- open reading frame 28 on the chromosome 19), and genes TRG responsible for the expression of γ-receptors on T lymphocytes. In mice, mutations within Foxn1 gene cause alopecia, improper development of the thymus and T lymphocytes, and premature death (16, 17). Alopecia is caused by a recessive mutation in Foxn1 gene, also responsible for athymia (5). Genes Foxe1 and Foxq1 are jointly responsible for the described disorders in the hair development. These types of mutations are also manifested by the serious deficiency of T lymphocytes (8). In humans, an improper expression level of Foxn1 gene causes hair loss and nail dystrophy. There are assumptions that the level of Foxn1 expression is related to cancerogenesis or immunodeficiency. Summary. The current state of knowledge about Fox genes in domestic animals is insufficient. Therefore, there is a need for closer investigation of their functions at the molecular level, and their role in the regulation of expression of some immunological phenotypes. The complexity of the described genes’ functions and multidimensional regulation of their expression, with a possible contribution of environmental factors, make them a promising area of research. Acknowledgments: The research was supported by the National Centre for Research and Development (project ZKB\PB-R\1). References 1.

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