Jean-Bosco Tagne

Transcriptional regulatory code of a eukaryotic genome

DNA-binding transcriptional regulators interpret the genomes regulatory code by binding to specific sequences to induce or repress gene expression. Comparative genomics has recently been used to identify potential cis-regulatory sequences within the yeast genome on the basis of phylogenetic conservation, but this information alone does not reveal if or when transcriptional regulators occupy these binding sites. We have constructed an initial map of yeasts transcriptional regulatory code by identifying the sequence elements that are bound by regulators under various conditions and that are conserved among Saccharomyces species. The organization of regulatory elements in promoters and the environment-dependent use of these elements by regulators are discussed. We find that environment-specific use of regulatory elements predicts mechanistic models for the function of a large population of yeasts transcriptional regulators.

A Systems Approach to Mapping DNA Damage Response Pathways

Failure of cells to respond to DNA damage is a primary event associated with mutagenesis and environmental toxicity. To map the transcriptional network controlling the damage response, we measured genomewide binding locations for 30 damage-related transcription factors (TFs) after exposure of yeast to methyl-methanesulfonate (MMS). The resulting 5272 TF-target interactions revealed extensive changes in the pattern of promoter binding and identified damage-specific binding motifs. As systematic functional validation, we identified interactions for which the target changed expression in wild-type cells in response to MMS but was nonresponsive in cells lacking the TF. Validated interactions were assembled into causal pathway models that provide global hypotheses of how signaling, transcription, and phenotype are integrated after damage.

A nanoemulsion formulation of tamoxifen increases its efficacy in a breast cancer cell line.

This paper reports on the preparation of a water-soluble nanoemulsion of the highly lipid-soluble drug tamoxifen (TAM). In addition, relative to a suspension of TAM, the nanoemulsion preparation demonstrated a greater zeta potential (increased negative charge) which has previously been associated with increasing drug/membrane permeability. This study also reports that relative to suspensions of TAM with particle sizes greater than 6000 nm, nanoemulsions of TAM, having mean particle sizes of 47 nm, inhibited cell proliferation 20-fold greater and increased cell apoptosis 4-fold greater in the HTB-20 breast cancer cell line. Thus, this work suggests that a nanoemulsion compared to a suspension preparation of TAM increases its anticancer properties relative to breast cancer.

The transcription factors Grainyhead-like 2 and NK2-homeobox 1 form a regulatory loop that coordinates lung epithelial cell morphogenesis and differentiation.

Background: Grhl2 regulates cell-junction gene transcription in several epithelia but has not been fully characterized in lungs. Results: In lung epithelial cells GRHL2 regulates cell-cell interaction genes, collective cell migration, and Nkx2-1 transcription. Conversely, NKX2-1 regulates transcription of Grhl2. Conclusion: A Grhl2- and Nkx2-1-positive transcriptional loop coordinates morphogenesis and differentiation of lung epithelial cells. Significance: This regulatory loop reinforces normal lung epithelial cell identity. The Grainyhead family of transcription factors controls morphogenesis and differentiation of epithelial cell layers in multicellular organisms by regulating cell junction- and proliferation-related genes. Grainyhead-like 2 (Grhl2) is expressed in developing mouse lung epithelium and is required for normal lung organogenesis. The specific epithelial cells expressing Grhl2 and the genes regulated by Grhl2 in normal lungs are mostly unknown. In these studies we identified the NK2-homeobox 1 transcription factor (Nkx2-1) as a direct transcriptional target of Grhl2. By binding and transcriptional assays and by confocal microscopy we showed that these two transcription factors form a positive feedback loop in vivo and in cell lines and are co-expressed in lung bronchiolar and alveolar type II cells. The morphological changes observed in flattening lung alveolar type II cells in culture are associated with down-regulation of Grhl2 and Nkx2-1. Reduction of Grhl2 in lung epithelial cell lines results in lower expression levels of Nkx2-1 and of known Grhl2 target genes. By microarray analysis we identified that in addition to Cadherin1 and Claudin4, Grhl2 regulates other cell interaction genes such as semaphorins and their receptors, which also play a functional role in developing lung epithelium. Impaired collective cell migration observed in Grhl2 knockdown cell monolayers is associated with reduced expression of these genes and may contribute to the altered epithelial phenotype reported in Grhl2 mutant mice. Thus, Grhl2 functions at the nexus of a novel regulatory network, connecting lung epithelial cell identity, migration, and cell-cell interactions.

Nanoemulsion preparations of the anticancer drug dacarbazine significantly increase its efficacy in a xenograft mouse melanoma model.

This article reports on the preparation of a water-soluble nanoemulsion of the highly lipid-soluble drug Dacarbazine (DAC). In addition, relative to suspensions of DAC, the nanoemulsion preparation demonstrated a lower zeta-potential (decreased negative charge, less anionic and more cationic) which has previously been associated with influencing drug membrane permeability. This study also reports that, relative to suspensions of DAC with a mean particle size of 5470 nm, nanoemulsions of DAC having mean particle sizes of 131 nm were more efficacious. For example, in a mouse xenograft model using a human melanoma cell line, a topical application of nanoemulsions of DAC compared to the suspension preparation of DAC produced up to 10-fold greater percent (%) reductions of tumor size. The reduction in tumor size by the intramuscular (IM) injection (-61%) and topical application of the nanoemulsion preparations of DAC (-49%) appeared to be comparable in efficacy, although the former was statistically greater (p < 0.05). In addition, 12 weeks after DAC treatment cessation, 98% of the animals given the IM application of the nanoemulsion of DAC remained tumor-free compared to the control or untreated animals. During this drug cessation period, and compared to the suspension preparations, nanoemulsions of DAC showed 5-fold greater efficacies (73% versus 14%) in preventing tumor growth. In conclusion, in this xenograft mouse model of melanoma, nanoemulsion suspensions of DAC are more efficacious in the treatment and prevention of tumor growth.

A nanoemulsion formulation of dacarbazine reduces tumor size in a xenograft mouse epidermoid carcinoma model compared to dacarbazine suspension

UNLABELLED Dacarbazine (DAC) is an anticancer drug that has been used to treat various types of cancers. The aim of the current study was to test whether there is an increased efficacy of DAC as a nanoemulsion on reducing tumor size in an epidermoid carcinoma xenograft mouse model. Tumors were induced in 5-week-old nude mice by subcutaneous injection. The mice were untreated or treated with a suspension of DAC (0.1 mg/kg), a nanoemulsion of DAC (0.1 mg/kg), or Nano-Control (same composition as the suspension and nanoemulsion but no DAC), every 2 days by either intramuscular injection (IM) or topical application. After 40 days, the final tumor size of mice receiving the nanoemulsion of DAC IM (0.83 ± 0.55 mm(3)) was significantly reduced compared to the suspension of DAC IM (4.75 ± 0.49 mm(3)), Nano-Control IM (7.63 ± 0.91 mm(3)), and untreated (10.46 ± 0.06 mm(3)). The final tumor size of mice receiving the nanoemulsion of DAC topically (3.33 ± 0.63 mm3) was also significantly reduced compared to the suspension of DAC topically (7.64 ± 0.68 mm(3)). This increased efficacy maybe partially attributed to: 1) the reduced particle size of the nanoemulsion in comparison with suspension (111 versus > 6000 nm), 2) reduction in zeta potential of the nanoemulsion compared to suspension (-3.2 versus -89.1 mV), 3) production of a stable water dispersion relative to unstable suspension, 4) decreased polydispersity index of the nanoemulsion compared to suspension, and 5) greater stability of drug with the nanoemulsion in comparison with the suspension. FROM THE CLINICAL EDITOR In this clinically relevant study, the anti-tumor efficacy of dacarbazine was found to be significantly increased as a nanoemulsion in epidermoid carcinoma xenograft mice, both with IM and topical administration.

Epigenetic Mechanisms Modulate Thyroid Transcription Factor 1-mediated Transcription of the Surfactant Protein B Gene

Epigenetic regulation of transcription plays an important role in cell-specific gene expression by altering chromatin structure and access of transcriptional regulators to DNA binding sites. Surfactant protein B (Sftpb) is a developmentally regulated lung epithelial gene critical for lung function. Thyroid transcription factor 1 (Nkx2-1) regulates Sftpb gene expression in various species. We show that Nkx2-1 binds to the mouse Sftpb (mSftpb) promoter in the lung. In a mouse lung epithelial cell line (MLE-15), Nkx2-1 knockdown reduces Sftpb expression, and mutation of Nkx2-1 cis-elements significantly reduces mSftpb promoter activity. Whether chromatin structure modulates Nkx2-1 regulation of Sftpb transcription is unknown. We found that DNA methylation of the mSftpb promoter inversely correlates with known patterns of Sftpb expression in vivo. The mSftpb promoter activity can be manipulated by altering its cytosine methylation status in vitro. Nkx2-1 activation of the mSftpb promoter is impaired by DNA methylation. The unmethylated Sftpb promoter shows an active chromatin structure enriched in the histone modification H3K4me3 (histone 3-lysine 4 trimethylated). The ATP-dependent chromatin remodeling protein Brg1 is recruited to the Sftpb promoter in Sftpb-expressing, but not in non-expressing tissues and cell lines. Brg1 knockdown in MLE-15 cells greatly decreases H3K4me3 levels at the Sftpb promoter region and expression of the Sftpb gene. Brg1 can be co-immunoprecipitated with Nkx2-1 protein. Last, Nkx2-1 and Brg1 with intact ATPase activity are required for mSftpb promoter activation in vitro. Our findings suggest that DNA methylation and chromatin modifications cooperate with Nkx2-1 to regulate Sftpb gene cell specific expression.

Genome-Wide Analyses of Nkx2-1 Binding to Transcriptional Target Genes Uncover Novel Regulatory Patterns Conserved in Lung Development and Tumors

The homeodomain transcription factor Nkx2-1 is essential for normal lung development and homeostasis. In lung tumors, it is considered a lineage survival oncogene and prognostic factor depending on its expression levels. The target genes directly bound by Nkx2-1, that could be the primary effectors of its functions in the different cellular contexts where it is expressed, are mostly unknown. In embryonic day 11.5 (E11.5) mouse lung, epithelial cells expressing Nkx2-1 are predominantly expanding, and in E19.5 prenatal lungs, Nkx2-1-expressing cells are predominantly differentiating in preparation for birth. To evaluate Nkx2-1 regulated networks in these two cell contexts, we analyzed genome-wide binding of Nkx2-1 to DNA regulatory regions by chromatin immunoprecipitation followed by tiling array analysis, and intersected these data to expression data sets. We further determined expression patterns of Nkx2-1 developmental target genes in human lung tumors and correlated their expression levels to that of endogenous NKX2-1. In these studies we uncovered differential Nkx2-1 regulated networks in early and late lung development, and a direct function of Nkx2-1 in regulation of the cell cycle by controlling the expression of proliferation-related genes. New targets, validated in Nkx2-1 shRNA transduced cell lines, include E2f3, Cyclin B1, Cyclin B2, and c-Met. Expression levels of Nkx2-1 direct target genes identified in mouse development significantly correlate or anti-correlate to the levels of endogenous NKX2-1 in a dosage-dependent manner in multiple human lung tumor expression data sets, supporting alternative roles for Nkx2-1 as a transcriptional activator or repressor, and direct regulator of cell cycle progression in development and tumors.

Transcription factor and microRNA interactions in lung cells: an inhibitory link between NK2 homeobox 1, miR-200c and the developmental and oncogenic factors Nfib and Myb

BackgroundThe transcription factor NK2 homeobox 1 (Nkx2-1) plays essential roles in epithelial cell proliferation and differentiation in mouse and human lung development and tumorigenesis. A better understanding of genes and pathways downstream of Nkx2-1 will clarify the multiple roles of this critical lung factor. Nkx2-1 regulates directly or indirectly numerous protein-coding genes; however, there is a paucity of information about Nkx2-1-regulated microRNAs (miRNAs).Methods and resultsBy miRNA array analyses of mouse epithelial cell lines in which endogenous Nkx2-1 was knocked-down, we revealed that 29 miRNAs were negatively regulated including miR-200c, and 39 miRNAs were positively regulated by Nkx2-1 including miR-1195. Mouse lungs lacking functional phosphorylated Nkx2-1 showed increased expression of miR-200c and alterations in the expression of other top regulated miRNAs. Moreover, chromatin immunoprecipitation assays showed binding of NKX2-1 protein to regulatory regions of these miRNAs. Promoter reporter assays indicated that 1kb of the miR-200c 5′ flanking region was transcriptionally active but did not mediate Nkx2-1- repression of miR-200c expression. 3′UTR reporter assays support a direct regulation of the predicted targets Nfib and Myb by miR-200c.ConclusionsThese studies suggest that Nkx2-1 controls the expression of specific miRNAs in lung epithelial cells. In particular, we identified a regulatory link between Nkx2-1, the known tumor suppressor miR-200c, and the developmental and oncogenic transcription factors Nfib and Myb, adding new players to the regulatory mechanisms driven by Nkx2-1 in lung epithelial cells that may have implications in lung development and tumorigenesis.

NKX2-1-AS1 negatively regulates CD274/PD-L1, cell-cell interaction genes, and limits human lung carcinoma cell migration.

The function of most long noncoding RNAs (lncRNAs) is unknown. However, recent studies reveal important roles of lncRNAs in regulating cancer-related pathways. Human antisense lncRNA-NKX2-1-AS1 partially overlaps the NKX2-1/TTF1 gene within chromosomal region 14q13.3. Amplification of this region and/or differential expression of genes therein are associated with cancer progression. Herein we show higher levels of NKX2-AS1 and NKX2-1 in lung adenocarcinomas relative to non-tumor controls but no correlation between NKX2-1-AS1 and NKX2-1 levels across specimens, or with amplification of the 14q13.3 region, suggesting that NKX2-1-AS1 and NKX2-1 are independently regulated. Loss-and-gain of function experiments showed that NKX2-1-AS1 does not regulate NKX2-1 expression, or nearby genes, but controls genes in trans. Genes up-regulated by NKX2-1-AS1-knockdown belong to cell adhesion and PD-L1/PD-1 checkpoint pathways. NKX2-1-AS1 negatively regulates endogenous CD274/PD-L1, a known target of NKX2-1, and the transcriptional activity of -1kb-CD274 promoter-reporter construct. Furthermore, NKX2-1-AS1 interferes with NKX2-1 protein binding to the CD274-promoter, likely by NKX2-1 protein-NKX2-1-AS1 interactions. Finally, NKX2-1-AS1 negatively regulates cell migration and wound healing, but not proliferation or apoptosis. These findings support potential roles of NKX2-1-AS1 in limiting motility and immune system evasion of lung carcinoma cells, highlighting a novel mechanism that may influence tumorigenic capabilities of lung epithelial cells.