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Background: Major transcription factors controlling TH1 and TH2 development, such as T-box transcription factor and GATA3, might be centrally involved in asthma and atopic diseases. Only recently, the homeobox transcription factor H.20-like homeobox 1 (HLX1), interacting closely with T-box transcription factor, has been identified as an important regulator of TH1 differentiation and suppressor of TH2 commitment. Objective: We investigated whether genetic variations in the HLX1 gene exist and whether these could affect the development of childhood asthma. Methods: The HLX1 gene was resequenced in 80 chromosomes. Associations between identified polymorphisms, asthma, and atopic diseases were investigated in German children (total n = 3099) from the cross-sectional International Study of Asthma and Allergy in Childhood phase II. Functional properties of polymorphisms were studied by using luciferase reporter gene assays and electrophoretic mobility shift assays in T cells. All statistical analyses were performed with SAS/Genetics software (SAS Institute, Inc, Cary, NC). Results: Nineteen polymorphisms were identified in the HLX1 gene, and 2 tagging single nucleotide polymorphisms representing 7 polymorphisms were associated with childhood asthma in our study population. Two promoter polymorphisms, C−1407T and C−742G, contained in 1 tagging block were associated with asthma (odds ratio, 1.44; 95% CI, 1.11-1.86; P = .0061), significantly decrease promoter transactivation, and disrupt specificity protein–transcription factor binding in in vitro experiments. Conclusions: Our data suggest that polymorphisms in the HLX1 gene increase the risk for childhood asthma. On the cellular level, altered binding of specificity protein–transcription factors to the HLX1 promoter and subsequent changes in HLX1 gene expression might contribute to these effects. [Copyright &y& Elsevier]

Abstract: The cytokine colony stimulating factor-1 (CSF-1) is a key regulator of the proliferation, differentiation and activation of mononuclear phagocytes. CSF-1 also plays an important role in reproduction. CSF-1 is produced in the placenta and activates signal transduction pathways that significantly increase the proliferation of placental trophoblast cells in culture. The target genes activated by CSF-1 mediated signal transduction in the nucleus are not well understood. Here, we use placental trophoblast cells to investigate potential downstream effector genes of CSF-1. HLX1 is a homeobox gene that controls proliferation in embryonic cell types and haematopoietic cell lineages. We have shown HLX1 is expressed in placental trophoblast cells but its functional role in the placenta is unknown. Following CSF-1 stimulation, HLX1 mRNA expression was significantly increased in SGHPL-4 and HTR-8/SVNeo cultured trophoblast cells (p <0.001, n =3). siRNA-mediated reduction of HLX1 mRNA levels with four independent oligonucleotides (siRNAs) resulted in significantly decreased cell proliferation in both cell lines (p <0.001, n =4). When HLX1 mRNA levels were reduced in the presence of CSF-1 stimulation, proliferation remained significantly decreased (p <0.001, n =4) in both the cell lines. We have shown for the first time that a homeobox gene, HLX1, is a downstream effector gene of CSF-1, that HLX1 regulates placental cell proliferation and that CSF-1 acts, at least in part, through HLX1 to control cell proliferation. [Copyright &y& Elsevier]

Abstract: Sonic Hedgehog (Shh) signaling helps pattern the vertebrate neural tube, in part by regulating the dorsal/ventral expression of a number of homeodomain containing transcription factors. These Hh responsive genes have been divided into two classes, with Class II genes being activated by Hh signaling and Class I genes being repressed by Hh signaling. While the transcriptional response to varying Hh levels is well defined in chick and mouse, it is only partially described in zebrafish, despite the fact that zebrafish has emerged as a powerful genetic system for the study of neural patterning. To better characterize the Hh response in the zebrafish neural tube, we cloned the zebrafish Class II Hh target genes nkx2.9 and nkx6.2. We then analyzed the expression of a number of Class I and Class II Hh responsive genes in wild type, Hh mutant, and Hh over-expressing zebrafish embryos. We show that expression of Class I and Class II genes is highly conserved in the vertebrate neural tube. Further, ventral-most Class II gene expression was completely lost in all Hh pathway mutants analyzed, indicating high levels of Hh signaling are blocked in all of these mutants. In contrast, more dorsally expressed genes were variably affected in different Hh pathway mutants, indicating mid-levels of Hh signaling are differentially affected. This comprehensive expression study provides an important tool for the characterization of Hh signaling in zebrafish and provides a sensitive assay for determining the degree to which newly identified zebrafish mutants affect Hh signaling. [Copyright &y& Elsevier]

Background: The T cell–specific T-box transcription factor (TBX21) plays a crucial role in the regulation of the immune system because this factor induces the differentiation of TH1 and blocks TH2 commitment together with the homeobox transcription factor HLX1. Objective: The role of genetic variants in TBX21 alone and in combination with HLX1 polymorphisms was investigated in the development of TH2-associated atopy and asthma. Methods: The TBX21 gene was resequenced in 37 adult volunteers. Polymorphisms identified were genotyped in a cross-sectional (N = 3099) and nested asthma case-control population (N = 1872) using mainly matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Effects of promoter polymorphisms on TBX21 gene expression were studied by reporter gene assays. Furthermore, the impact of combinations of TBX21 and HLX1 polymorphisms on the development of asthma was assessed by using a risk score model. Statistical analyses were performed by using SAS/Genetics. Results: Forty-three polymorphisms were identified in the TBX21 gene. Considering a minor allele frequency of at least 10%, single nucleotide polymorphisms were assigned to 7 linkage disequilibrium blocks. Three tagging single nucleotide polymorphisms increased childhood asthma risk significantly (odds ratio [OR], 2.60, 95% CI, 1.34-5.03, P = .003; OR, 1.39, 95% CI, 1.02-1.90, P = .039; and OR, 1.97, 95% CI, 1.18-3.30, P = .009). TBX21 promoter polymorphisms contained in 2 blocks significantly influenced TBX21 promoter activity. In a risk score model, the combination of TBX21 and HLX1 polymorphisms increased the asthma risk by more than 3-fold. Conclusions: These data suggest that TBX21 polymorphisms contribute to the development of asthma, potentially by altering TBX21 promoter activity. A risk score model indicates that TBX21 and HLX1 polymorphisms may have synergistic effects on asthma risk. [Copyright &y& Elsevier]