Transcriptional Regulation of the Human IL5RA Gene through Alternative Promoter Usage during Eosinophil Development

Regulation of the IL-5 receptor alpha (IL5RA) gene is complicated, with two known promoters (P1 and P2) driving transcription, and two known isoforms (transmembrane and soluble) dichotomously affecting the signaling potential of the protein products. Here, we sought to determine the patterns of P1 and P2 promoter usage and transcription factor occupancy during primary human eosinophil development from CD34+ hematopoietic stem cell progenitors. We found that during eosinophilopoiesis, both promoters were active but subject to distinct temporal regulation, coincident with combinatorial interactions of transcription factors, including GATA-1, PU.1, and C/EBP family members. P1 displayed a relatively constant level of activity throughout eosinophil development, while P2 activity peaked early and waned thereafter. The soluble IL-5Rα mRNA peaked early and showed the greatest magnitude fold-induction, while the signaling-competent transmembrane isoform peaked moderately. Two human eosinophilic cell lines whose relative use of P1 and P2 were similar to eosinophils differentiated in culture were used to functionally test putative transcription factor binding sites. Transcription factor occupancy was then validated in primary cultures by ChIP. We conclude that IL-5-dependent generation of eosinophils from CD34+ precursors involves complex and dynamic activity including both promoters, several interacting transcription factors, and both signaling and antagonistic protein products.

Validation of a Novel Fgf10Cre–ERT2 Knock-in Mouse Line Targeting FGF10Pos Cells Postnatally

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a knock-in Fgf10Cre–ERT2 line (with the Cre-ERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki–v1, to target FGF10Pos cells. While this line allowed fairly efficient and specific labeling of FGF10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus of our research. We report here the generation and validation of a new knock-in Fgf10Cre–ERT2 line (called thereafter Fgf10Ki–v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. Fgf10Ki−v2/+ heterozygous mice exhibited comparable Fgf10 expression levels to wild type animals. However, a mismatch between Fgf10 and Cre expression levels was observed in Fgf10Ki–v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki–v2 mice. Bioinformatic analysis shows that the 3′UTR, where the Cre-ERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Importantly, Fgf10Ki–v2 mouse is capable of significantly targeting FGF10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki–v2 line opens the way for future mechanistic experiments involving the postnatal lung.

Priming Maritime Pine Megagametophytes during Somatic Embryogenesis Improved Plant Adaptation to Heat Stress

In the context of global climate change, forest tree research should be addressed to provide genotypes with increased resilience to high temperature events. These improved plants can be obtained by heat priming during somatic embryogenesis (SE), which would produce an epigenetic-mediated transgenerational memory. Thereby, we applied 37 °C or 50 °C to maritime pine (Pinus pinaster) megagametophytes and the obtained embryogenic masses went through the subsequent SE phases to produce plants that were further subjected to heat stress conditions. A putative transcription factor WRKY11 was upregulated in priming-derived embryonal masses, and also in the regenerated P37 and P50 plants, suggesting its role in establishing an epigenetic memory in this plant species. In vitro-grown P50 plants also showed higher cytokinin content and SOD upregulation, which points to a better responsiveness to heat stress. Heat exposure of two-year-old maritime pine plants induced upregulation of HSP70 in those derived from primed embryogenic masses, that also showed better osmotic adjustment and higher increases in chlorophyll, soluble sugars and starch contents. Moreover, ɸPSII of P50 plants was less affected by heat exposure. Thus, our results suggest that priming at 50 °C at the SE induction phase is a promising strategy to improve heat resilience in maritime pine.

Characterization of a novel Fgf10CreERT2 knock-in mouse line targeting postnatal lung Fgf10 lineages

Fgf10 is a key gene during development, homeostasis and repair after injury. We previously reported a Fgf10CreERT2 line (with the CreERT2 cassette inserted in frame with the start codon of exon 1), called thereafter Fgf10Ki-v1, to target Fgf10Pos cells. While this line allowed fairly efficient and specific labeling of Fgf10Pos cells during the embryonic stage, it failed to target these cells after birth, particularly in the postnatal lung, which has been the focus on our research. We report here the generation and validation of a new Fgf10CreERT2 (called thereafter Fgf10Ki-v2) with the insertion of the expression cassette in frame with the stop codon of exon 3. This new Fgf10Ki-v2 line exhibited comparable Fgf10 expression level to their wild type counterpart. However, a disconnection between the Fgf10 and the Cre expression was observed in Fgf10Ki-v2/+ lungs. In addition, lung and limb agenesis were observed in homozygous embryos suggesting a loss of Fgf10 functional allele in Fgf10Ki-v2 mice. Bio-informatics analysis shows that the 3’UTR, where the CreERT2 cassette is inserted, contains numerous putative transcription factor binding sites. By crossing this line with tdTomato reporter line, we demonstrated that tdTomato expression faithfully recapitulated Fgf10 expression during development. Significantly, Fgf10Ki-v2 mouse is capable of significantly targeting Fgf10Pos cells in the adult lung. Therefore, despite the aforementioned limitations, this new Fgf10Ki-v2 line opens the way for future mechanistic experiments involving the postnatal lung.

Cis-regulatory elements in conserved non-coding sequences of nuclear receptor genes indicate for crosstalk between endocrine systems

Nuclear receptors (NRs) are ligand-activated transcription factors that regulate gene expression when bound to specific DNA sequences. Crosstalk between steroid NR systems has been studied for understanding the development of hormone-driven cancers but not to an extent at a genetic level. This study aimed to investigate crosstalk between steroid NRs in conserved intron and exon sequences, with a focus on steroid NRs involved in prostate cancer etiology. For this purpose, we evaluated conserved intron and exon sequences among all 49 members of the NR Superfamily (NRS) and their relevance as regulatory sequences and NR-binding sequences. Sequence conservation was found to be higher in the first intron (35%), when compared with downstream introns. Seventy-nine percent of the conserved regions in the NRS contained putative transcription factor binding sites (TFBS) and a large fraction of these sequences contained splicing sites (SS). Analysis of transcription factors binding to putative intronic and exonic TFBS revealed that 5 and 16%, respectively, were NRs. The present study suggests crosstalk between steroid NRs, e.g., vitamin D, estrogen, progesterone, and retinoic acid endocrine systems, through cis-regulatory elements in conserved sequences of introns and exons. This investigation gives evidence for crosstalk between steroid hormones and contributes to novel targets for steroid NR regulation.

Genetic Diversity of the Noncoding Control Region of the Novel Human Polyomaviruses

The genomes of polyomaviruses are characterized by their tripartite organization with an early region, a late region and a noncoding control region (NCCR). The early region encodes proteins involved in replication and transcription of the viral genome, while expression of the late region generates the capsid proteins. Transcription regulatory sequences for expression of the early and late genes, as well as the origin of replication are encompassed in the NCCR. Cell tropism of polyomaviruses not only depends on the appropriate receptors on the host cell, but cell-specific expression of the viral genes is also governed by the NCCR. Thus far, 15 polyomaviruses have been isolated from humans, though it remains to be established whether all of them are genuine human polyomaviruses (HPyVs). The sequences of the NCCR of these HPyVs show high genetic variability and have been best studied in the human polyomaviruses BK and JC. Rearranged NCCRs in BKPyV and JCPyV, the first HPyVs to be discovered approximately 30 years ago, have been associated with the pathogenic properties of these viruses in nephropathy and progressive multifocal leukoencephalopathy, respectively. Since 2007, thirteen novel PyVs have been isolated from humans: KIPyV, WUPyV, MCPyV, HPyV6, HPyV7, TSPyV, HPyV9, HPyV10, STLPyV, HPyV12, NJPyV, LIPyV and QPyV. This review describes all NCCR variants of the new HPyVs that have been reported in the literature and discusses the possible consequences of NCCR diversity in terms of promoter strength, putative transcription factor binding sites and possible association with diseases.

Genome-wide identification and characterization of functionally relevant microsatellites from putative transcription factor genes of tea for efficient genotyping.

Background: SSR markers derived from coding region and non-coding un-translated regions (UTRs) act as potential “functional genetic markers”. Tea, one among the most popular herbal drink with multiple medicinal properties is greatly suffered due to several bottlenecks of self-incompatibility, genome heterozygosity and long gestation period. Despite the huge genomic resources, experimentally validated functionally relevant SSR markers are still scarce in tea. Transcription factors (TFs) accounts for larger proportion of plant genomes regulating expression of various desirable traits have not been explored for development of functional marker resource in tea.Results: In current study, putative 2776 transcripts encoding TFs were identified harbouring 3687 SSR loci. High abundance of repeats in ORF (44.37%), UTRs (5'UTR: 32.57%; 3'UTR: 23.0%) suggests significant functional and regulatory relevance of novel SSR loci in trait dissection. 589 polymorphic novel experimentally validated SSR markers (ORF: 297; 5’UTR: 211; 3'UTR: 81) amplified 2864 alleles with allele numbers (Na) of 2 to 17 per locus. Interestingly, repetitive pattern of tri/hexa-repeats in coding region revealed single amino acid repeats reiteration of serine, glycine and proline, leucine in TFs of tea. Moreover, high representation of TF derived polymorphic SSR markers in bHLH (53), WRKY (37), C3H (32), and Myb-related (31) can be utilised for candidate-gene based mapping in tea. Successful utilization of polymorphic SSR makers for genetic diversity characterization of 115 tea cultivars suggests their wider utility in arge-scale fingerprinting studies. Furthermore, 185 SSR markers representing hk x hk, lm x ll, nn x np, ab x cd and ef x eg segregating patterns in selective genotyping of bi-parental mapping population can be potentially employed in genome mapping studies in tea. Conclusion: SSR repeats in regulatory genes influencing normal activity and function of the gene due to the repeat length (expansion and contraction) causing phenotypic variation in plants. Therefore, identification of first set of 1843 novel putative TFs derived microsatellite markers resource with experimentally validated potential polymorphic markers stipulates their importance in various genotyping studies in tea. Interestingly, SSRs retrieved in important TFs may have direct implications for molecular dissection desirable in tea. Genic functional markers developed in the study are powerful tools and can be potentially utilized to accelerate molecular breeding and trait dissection efforts for implantation of marker-asserted selection in tea.

Evolutionarily conserved transcription factors drive the oxidative stress response in Drosophila

ABSTRACTAs organisms are constantly exposed to the damaging effects of oxidative stress through both environmental exposure and internal metabolic processes, they have evolved a variety of mechanisms to cope with this stress. One such mechanism is the highly conserved p38 MAPK (p38K) pathway, which is known to be post-translationally activated in response to oxidative stress, resulting in the activation of downstream antioxidant targets. However, little is known about the role of p38K transcriptional regulation in response to oxidative stress. Therefore, we analyzed the p38K gene family across the genus Drosophila to identify conserved regulatory elements. We found that oxidative stress exposure results in increased p38K protein levels in multiple Drosophila species and is associated with increased oxidative stress resistance. We also found that the p38Kb genomic locus includes conserved AP-1 and lola-PT transcription factor consensus binding sites. Accordingly, over-expression of these transcription factors in D. melanogaster is sufficient to induce transcription of p38Kb and enhances resistance to oxidative stress. We further found that the presence of a putative lola-PT binding site in the p38Kb locus of a given species is predictive of the species' survival in response to oxidative stress. Through our comparative genomics approach, we have identified biologically relevant putative transcription factor binding sites that regulate the expression of p38Kb and are associated with resistance to oxidative stress. These findings reveal a novel mode of regulation for p38K genes and suggest that transcription may play as important a role in p38K-mediated stress responses as post-translational modifications.

Gene regulation gravitates towards either addition or multiplication when combining the effects of two signals

Signals often ultimately affect the transcription of genes, and often, two different signals can affect the transcription of the same gene. In such cases, it is natural to ask how the combined transcriptional response compares to the individual responses. Mechanistic models can predict a range of combined responses, with the most commonly applied models predicting additive or multiplicative responses, but systematic genome-wide evaluation of these predictions are not available. Here, we performed a comprehensive analysis of the transcriptional response of human MCF-7 cells to two different signals (retinoic acid and TGF-β), applied individually and in combination. We found that the combined responses exhibited a range of behaviors, but clearly favored both additive and multiplicative combined transcriptional responses. We also performed paired chromatin accessibility measurements to measure putative transcription factor occupancy at regulatory elements near these genes. We found that increases in chromatin accessibility were largely additive, meaning that the combined accessibility response was the sum of the accessibility responses to each signal individually. We found some association between super-additivity of accessibility and multiplicative or super-multiplicative combined transcriptional responses, while sub-additivity of accessibility associated with additive transcriptional responses. Our findings suggest that mechanistic models of combined transcriptional regulation must be able to reproduce a range of behaviors.