Transcription-associated recombination is dependent on replication in Mammalian cells.

Transcription can enhance recombination; this is a ubiquitous phenomenon from prokaryotes to higher eukaryotes. However, the mechanism of transcription-associated recombination in mammalian cells is poorly understood. Here we have developed a construct with a recombination substrate in which levels of recombination can be studied in the presence or absence of transcription. We observed a direct enhancement in recombination when transcription levels through the substrate were increased. This increase in homologous recombination following transcription is locus specific, since homologous recombination at the unrelated hprt gene is unaffected. In addition, we have shown that transcription-associated recombination involves both short-tract and long-tract gene conversions in mammalian cells, which are different from double-strand-break-induced recombination events caused by endonucleases. Transcription fails to enhance recombination in cells that are not in the S phase of the cell cycle. Furthermore, inhibition of transcription suppresses induction of recombination at stalled replication forks, suggesting that recombination may be involved in bypassing transcription during replication.

Spontaneous homologous recombination is induced by collapsed replication forks that are caused by endogenous DNA single-strand breaks.

Homologous recombination is vital to repair fatal DNA damage during DNA replication. However, very little is known about the substrates or repair pathways for homologous recombination in mammalian cells. Here, we have compared the recombination products produced spontaneously with those produced following induction of DNA double-strand breaks (DSBs) with the I-SceI restriction endonuclease or after stalling or collapsing replication forks following treatment with thymidine or camptothecin, respectively. We show that each lesion produces different spectra of recombinants, suggesting differential use of homologous recombination pathways in repair of these lesions. The spontaneous spectrum most resembled the spectra produced at collapsed replication forks formed when a replication fork runs into camptothecin-stabilized DNA single-strand breaks (SSBs) within the topoisomerase I cleavage complex. We found that camptothecin-induced DSBs and the resulting recombination repair require replication, showing that a collapsed fork is the substrate for camptothecin-induced recombination. An SSB repair-defective cell line, EM9 with an XRCC1 mutation, has an increased number of spontaneous gammaH2Ax and RAD51 foci, suggesting that endogenous SSBs collapse replication forks, triggering recombination repair. Furthermore, we show that gammaH2Ax, DSBs, and RAD51 foci are synergistically induced in EM9 cells with camptothecin, suggesting that lack of SSB repair in EM9 causes more collapsed forks and more recombination repair. Furthermore, our results suggest that two-ended DSBs are rare substrates for spontaneous homologous recombination in a mammalian fibroblast cell line. Interestingly, all spectra showed evidence of multiple homologous recombination events in 8 to 16% of clones. However, there was no increase in homologous recombination genomewide in these clones nor were the events dependent on each other; rather, we suggest that a first homologous recombination event frequently triggers a second event at the same locus in mammalian cells.

Regulation of postnatal lung development and homeostasis by estrogen receptor beta.

Estrogens have well-documented effects on lung development and physiology. However, the classical estrogen receptor alpha (ERalpha) is undetectable in the lung, and this has left many unanswered questions about the mechanism of estrogen action in this organ. Here we show, both in vivo and in vitro, that ERbeta is abundantly expressed and biologically active in the lung. Comparisons of lungs from wild-type mice and mice with an inactivated ERbeta gene (ERbeta(-/-)) revealed decreased numbers of alveoli in adult female ERbeta(-/-) mice and findings suggesting deficient alveolar formation as well as evidence of surfactant accumulation. Platelet-derived growth factor A (PDGF-A) and granulocyte-macrophage colony-stimulating factor (GM-CSF), key regulators of alveolar formation and surfactant homeostasis, respectively, were decreased in lungs of adult female ERbeta(-/-) mice, and direct transcriptional regulation of these genes by ERbeta was demonstrated. This suggests that estrogens act via ERbeta in the lung to modify PDGF-A and GM-CSF expression. These results provide a potential molecular mechanism for the gender differences in alveolar structure observed in the adult lung and establish ERbeta as a previously unknown regulator of postnatal lung development and homeostasis.

C/EBP transcription factors in the lung epithelium.

During recent years, the biological roles of CCAAT/enhancer binding proteins (C/EBPs) in the lung have started to be uncovered. C/EBPs form a family within the basic region-leucine zipper class of transcription factors. In the lung epithelium C/EBPalpha, -beta, and -delta are expressed. Lung-specific target genes for these transcription factors include the surfactant proteins A and D, the Clara cell secretory protein, and the P450 enzyme CYP2B1. As more information is gathered, a picture is emerging in which C/EBPalpha has a role in regulating proliferation as well as differentiation-dependent gene expression, whereas C/EBPbeta and -delta, in addition to a partly overlapping role in regulating expression of differentiation markers, also seem to be involved in responses to injury and hormones.

Evidence that peroxisome proliferator-activated receptor delta influences cholesterol metabolism in men.

OBJECTIVE: The objective of this work was to explore the role of peroxisome proliferator-activated receptor delta (PPARD) in lipid metabolism in humans. METHODS AND RESULTS: PPARD is a nuclear receptor involved in lipid metabolism in primates and mice. We screened the 5'-region of the human gene for polymorphisms to be used as tools in association studies. Four polymorphisms were detected: -409C/T in the promoter region, +73C/T in exon 1, +255A/G in exon 3, and +294T/C in exon 4. The frequencies of the rare alleles were 4.2%, 4.2%, 1.2% and 15.6%, respectively, in a population-based group of 543 healthy men. Only the +294T/C polymorphism showed significant association with a metabolic trait. Homozygotes for the rare C allele had a higher plasma LDL-cholesterol concentration than homozygotes for the common T allele, which was verified in an independent cohort consisting of 282 healthy men. Transfection studies showed that the rare C allele had higher transcriptional activity than the common T allele. Electrophoretic mobility shift assays demonstrated that the +294T/C polymorphism influenced binding of Sp-1. An interaction with the PPAR alpha L162V polymorphism was also detected for several lipid parameters. CONCLUSIONS: These findings suggest that PPARD plays a role in cholesterol metabolism in humans.

Synergistic transactivation of the differentiation-dependent lung gene Clara cell secretory protein (secretoglobin 1a1) by the basic region leucine zipper factor CCAAT/enhancer-binding protein alpha and the homeodomain factor Nkx2.1/thyroid transcription factor-1.

The basic region-leucine zipper transcription factor CCAAT/enhancer-binding protein alpha (C/EBPalpha) and the homeodomain transcription factor Nkx2.1/thyroid transcription factor-1 are essential for normal lung morphogenesis. Nkx2.1 is expressed from the onset of lung development, whereas C/EBPalpha expression is turned on at later stages. The expression of C/EBPalpha correlates to the appearance of lung-specific proteins with differentiation-dependent expression patterns, such as the Clara cell secretory protein (secretoglobin 1a1 (Scgb1a1), CCSP). In this study, we demonstrate synergistic transactivation by C/EBPalpha and Nkx2.1 in the regulation of the CCSP gene. We show that the synergistic activity of C/EBPalpha and Nkx2.1 originates from cis-acting elements in the proximal promoter of CCSP and that the synergism is dependent on NH(2)-terminal transactivation domains of C/EBPalpha and Nkx2.1. Our results suggest that the cooperation of C/EBPalpha and Nkx2.1 is a major determinant for the high level, lung epithelial-specific expression of CCSP during the later stages of lung development and in the adult lung.

Glucocorticoids regulate the CCSP and CYP2B1 promoters via C/EBPbeta and delta in lung cells.

Glucocorticoids have several important roles in the lung and play a key role in lung development and maturation. However, the specific molecular mechanisms of glucocorticoid action in lung are unclear. In this study, we have investigated two glucocorticoid-regulated genes expressed in the lung epithelium, the secretory protein CCSP, and the P450-enzyme CYP2B1. In transient transfections of lung epithelial cells, glucocorticoids increased expression from the CCSP and CYP2B1 promoters and we demonstrated that induction was dependent on the integrity of C/EBP-binding sites in both promoters. Electrophoretic mobility shift assays revealed increased DNA-binding of C/EBPbeta and C/EBPdelta after glucocorticoid treatment, which was not correlated to altered protein levels. The results of this study indicate a previously unknown role for C/EBP transcription factors in glucocorticoid signaling in the lung epithelium.

Decreased serum and bronchoalveolar lavage levels of Clara cell secretory protein (CC16) is associated with bronchiolitis obliterans syndrome and airway neutrophilia in lung transplant recipients.

BACKGROUND: The major hinderance for long-term survival after lung transplantation is chronic rejection in the form of bronchiolitis obliterans syndrome (BOS). BOS is a fibrosing process in the small airways causing irreversible airway obstruction. BOS is associated with increased oxidative burden and activation of inflammatory and growth-stimulating mediators. The Clara cell secretory protein (CCSP or CC16) is a secreted differentiation marker for the bronchiolar epithelium with both antioxidative and antiinflammatory/immmunomodulatory properties. We asked whether this molecule could have a role in the development of BOS. METHODS: Serum and bronchoalveolar lavage (BAL) fluid samples were collected from 22 consecutive lung transplant recipients, the majority (19) was followed for 2 years. Six patients developed BOS. CCSP in serum was measured in 162 samples from 19 patients with an ELISA method, and CCSP in 191 BAL samples from 22 patients with quantitative Western blot. RESULTS: CCSP in both serum and BAL was significantly lower in BOS compared with acute rejection or no rejection. After the first postoperative month, serum and BAL CCSP levels were consistently lower in the patients who developed BOS than in those who did not. The percentage of neutrophils in BAL correlated negatively with CCSP in BAL. CONCLUSIONS: Levels of CCSP in serum and BAL is lowered in BOS. Serum CCSP could have a potential as an early marker for BOS. The correlation between decreased CCSP and increased neutrophils in BAL suggests a loss of local airway defense capacity in BOS.