Several traits associated with asthma phenotypes, such as high total serum immunoglobulin E and bronchial hyperresponsiveness, have been linked by numerous genome-screen studies and linkage analyses to markers on human chromosome 5q31-q34. In the present article, we describe UGRP1 (encoding uteroglobin-related protein 1) as one of asthma-susceptibility genes that is located on chromosome 5q31-q32. UGRP1 is a homodimeric secretory protein of 17 kDa and is expressed only in lung and trachea. The G --> A polymorphism was identified at -112 bp in the human UGRP1 gene promoter. The -112A allele is responsible for a 24% reduction in the promoter activity in relation to the -112G allele, as examined by transfection analysis. Electrophoretic mobility-shift analysis revealed that an unknown nuclear factor binds to the region around -112 bp. The binding affinity with the -112A oligonucleotide was reduced by approximately one half, as compared with the -112G oligonucleotide. In a case-control study using 169 Japanese individuals (84 patients with asthma and 85 healthy control individuals), those with a -112A allele (G/A or A/A) were 4.1 times more likely to have asthma than were those with the wild-type allele (G/G).
When establishing the most appropriate cells from the huge numbers of a cell library for practical use of cells in regenerative medicine and production of various biopharmaceuticals, cell heterogeneity often found in an isogenic cell population limits the refinement of clonal cell culture. Here, we demonstrated high-throughput screening of the most suitable cells in a cell library by an automated undisruptive single-cell analysis and isolation system, followed by expansion of isolated single cells. This system enabled establishment of the most suitable cells, such as embryonic stem cells with the highest expression of the pluripotency marker Rex1 and hybridomas with the highest antibody secretion, which could not be achieved by conventional high-throughput cell screening systems (e.g., a fluorescence-activated cell sorter). This single cell-based breeding system may be a powerful tool to analyze stochastic fluctuations and delineate their molecular mechanisms.
Laminin-1 is the major component of embryonic basement membrane and consists of ␣1, 1, and ␥1 chains. The expression of laminin-1 is induced in mouse F9 embryonal carcinoma cells upon differentiation into parietal endoderm cells. We recently identified a parietal endoderm-specific enhancer in the mouse laminin ␣1 (Lama1) gene and showed that Sp1/Sp3 and YY1 transcription factors were involved in the enhancer activity. Although here we identified that NF-Y binds to the enhancer sequence between Sp1/Sp3-and YY1-binding sites, all these transcription factors are ubiquitously expressed and thus are not sufficient to explain parietal endoderm-specific enhancer activity. In the present study, we further showed that SOX7 and SOX17 are involved in the regulation of parietal endoderm-specific enhancer activity of the mouse Lama1 gene. Northern blot analysis revealed that the steady-state levels of mouse Sox7 and Sox17 mRNAs increased in parallel with that of Lama1 mRNA during the differentiation of F9 cells. Both SOX7 and SOX17 markedly trans-activated the transcription of the Lama1 enhancer-reporter construct in undifferentiated F9 cells in a manner dependent on high mobility group box-mediated DNA binding. Electrophoretic mobility shift assays and mutational analyses revealed that SOX7 and SOX17 bound specifically to two SOX-binding sites within the Lama1 enhancer, and that these SOX-binding sites functioned synergistically to confer the trans-activation by SOX7 and SOX17. Furthermore, this trans-activation was dependent on the integrity of the binding sites for Sp1/Sp3 and NF-Y located at upstream of the two SOX-binding sites. These results indicate that the transcription of the mouse Lama1 gene during the differentiation of F9 cells is controlled by a combination of the actions of the ubiquitous factors, Sp1/Sp3 and NF-Y, and the parietal endoderm-specific factors, SOX7 and SOX17.Mouse F9 embryonal carcinoma cells are widely used as an in vitro model to study early embryonic development and cell differentiation (1, 2). When treated with retinoic acid (RA), 1 F9 cells differentiate into primitive endoderm-like cells, and then further differentiate into parietal endoderm-like cells by the addition of reagents elevating intracellular cAMP concentration. Parietal endoderm cells are major fetal components of the yolk sac, synthesizing large amounts of basement membrane components including collagen IV and laminin-1. They are organized into a monolayer by a thick sheet of basement membrane, Reichert's membrane, in the early stage embryo (3, 4). Differentiated F9 cells also synthesize and secrete large amounts of basement membrane components, resembling parietal endoderm cells in many of their biochemical properties (5). In recent years, retinoid signaling during this RA-induced differentiation has been well characterized; the action of RA is mediated through nuclear receptors that in turn modulate gene expression. Some of the direct transcriptional targets of RA are known, such as GATA transcription factors (6) and lamini...
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