BMC Molecular biology - Latest articles

Background: The ETS transcription factor Elf5 (also known as ESE-2) is highly expressed in the mammary gland and plays an important role in its development and differentiation. Indeed studies in mice have illustrated an essential role for Elf5 in directing alveologenesis during pregnancy. Although the molecular mechanisms that underlie the developmental block in Elf5 null mammary glands are beginning to be unraveled, this investigation has been hampered by limited information about the identity of Elf5-target genes. To address this shortcoming, in this study we have performed ChIP-cloning experiments to identify the specific genomic segments that are occupied by Elf5 in pregnant mouse mammary glands. Results: Sequencing and genomic localization of cis-regulatory regions bound by Elf5 in vivo has identified several potential target genes covering broad functional categories. A subset of these target genes demonstrates higher expression levels in Elf5-null mammary glands suggesting a repressive functional role for this transcription factor. Here we focus on one putative target of Elf5, the Ccnd2 gene that appeared in our screen. We identify a novel Elf5-binding segment upstream of the Ccnd2 gene and demonstrate that Elf5 can transcriptionally repress Ccnd2 by directly binding to the proximal promoter region. Finally, using Elf5-null mammary epithelial cells and mammary glands, we show that loss of Elf5 in vivo leads to up regulation of Ccnd2 and an altered expression pattern in luminal cells. Conclusions: Identification of Elf5-targets is an essential first step in elucidating the transcriptional landscape that is shaped by this important regulator. Our studies offer new toolbox in examining the biological role of Elf5 in mammary gland development and differentiation.

Background: The c-myb proto-oncogene is the founding member of a family of transcription factors involved principally in haematopoiesis, in diverse organisms, from zebrafish to mammals. Its deregulation has been implicated in human leukaemogenesis and other cancers. The expression of c-myb is tightly regulated by post-transcriptional mechanisms involving microRNAs. MicroRNAs are small, highly conserved non-coding RNAs that inhibit translation and decrease mRNA stability by binding to regulatory motifs mostly located in the 3'UTR of target mRNAs conserved throughout evolution. MYB is an evolutionarily conserved miR-150 target experimentally validated in mice, humans and zebrafish. However, the functional miR-150 sites of humans and mice are orthologous, whereas that of zebrafish is different. Results: We identified the avian mature miRNA-150-5P, Gallus gallus gga-miR-150 from chicken leukocyte small-RNA libraries and showed that, as expected, the gga-miR-150 sequence was highly conserved, including the seed region sequence present in the other miR-150 sequences listed in miRBase. Reporter assays showed that gga-miR-150 acted on the avian MYB 3'UTR and identified the avian MYB target site involved in gga-miR-150 binding. A comparative in silico analysis of the miR-150 target sites of MYB 3'UTRs from different species led to the identification of a single set of putative target sites in amphibians and zebrafish, whereas two sets of putative target sites were identified in chicken and mammals. However, only the target site present in the chicken MYB 3'UTR that was identical to that in zebrafish was functional, despite the additional presence of mammalian target sites in chicken. This specific miR-150 site usage was not cell-type specific and persisted when the chicken c-myb 3'UTR was used in the cell system to identify mammalian target sites, showing that this miR-150 target site usage was intrinsic to the chicken c-myb 3'UTR. Conclusion: Our study of the avian MYB/gga-miR-150 interaction shows a conservation of miR-150 target site functionality between chicken and zebrafish that does not extend to mammals.

Background: Postnatal expansion of the pancreatic beta-cell mass is required to maintain glucose homeostasis immediately after birth. This beta-cell expansion is regulated by multiple growth factors, including glucose, insulin, insulin-like growth factor (IGF-1) and epidermal growth factor (EGF). These mitogens signal through several downstream pathways (AKT, ERK, STAT3, and JNK) to regulate the survival and proliferation of betacells. Survivin, an oncofetal protein with both pro-proliferative and anti-apoptotic properties, is a known transcriptional target of both IGF-1 and EGF in cancer cells. Here, we analyzed the effects of the beta-cell mitogens IGF-1 and EGF on survivin regulation in the established pancreatic beta-cell model cell lines, MIN6 and INS-1 and in primary mouse islets. Results: In pancreatic beta-cells, treatment with glucose, insulin, or EGF increased survivin protein levels at early time points. By contrast, no significant effects on survivin were observed following IGF-1 treatment. EGF-stimulated increases in survivin protein were abrogated in the presence of downstream inhibitors of the Raf-1/MEK/ERK pathway. EGF had no significant effect on survivin transcription however it prolonged the half-life of the survivin protein and stabilized survivin protein levels by inhibiting survivin ubiquitination. Conclusions: This study defines a novel mechanism of survivin regulation by EGF through the Raf-1/MEK/ERK pathway in pancreatic beta-cells, via prolongation of survivin protein half-life and inhibition of the ubiquitin-mediated proteasomal degradation pathway. This mechanism may be important for regulating beta-cell expansion after birth.

Background: In insects, hemocyanin superfamily proteins accumulate apparently to serve as sources of amino acids during metamorphosis, reproduction and development. Storage hexamerins are important members of the hemocyanin superfamily. Although insects possess storage hexamerins, very little is known about the character and specific functions of hexamerin 1 and storage protein 1 in insect development. Results: To gain insight into the function of storage proteins in insects, cDNAs for two storage proteins were cloned from the fat body of Spodoptera exigua. S. exigua hexamerin 1 (SeHex) cDNA contained an open reading frame of 2124 nucleotides encoding a protein of 707 amino acids with a predicted molecular weight of 82.12 kDa. S. exigua storage protein 1 (SeSP1) cDNA contained an open reading frame of 2256 bp encoding a protein of 751 amino acids with a predicted molecular weight of ~88.84 kDa. Northern blotting analyses revealed that SeHex mRNA is expressed in the fat body, cuticle, midgut and Malpighian tubules and SeSP1 in fat body, Malpighian tubules and tracheae. SeHex and SeSP1 mRNAs were expressed in fat body at different levels from first instar larvae to pupae, with expression was much lower from first instar larvae to first-day fifth instar larvae. SeHex transcript expression was high in fat body of wandering larvae (pre-pupae) and steadily decreased to the seventh pupal day. SeSP1 transcript expression was high in fat body of wandering larvae, 2-day-old fifth instar larvae and 2-, 4- and 7-day-old pupae. SeHex and SeSP1 mRNAs levels were expressed lower than control on the condition of starvation at 12 h . Of insects injected with SeHex and SeSP1 dsRNA, 38.7% and 24.3% survived to 204 h after treatment, respectively. This was significantly lower than in the controls groups. Conclusions: These findings provide new data on the tissue distribution, expression patterns and the function in starvation of storage proteins. RNA interference results revealed that storage protein genes are key in metamorphosis, reproduction and insect development. The results for SeHex and SeSP1 interference reveal that a potential method to control this pest is to disrupt the regulation of storage proteins.

Background: The L1 cell adhesion molecule (L1CAM) was originally identified as a neural adhesion molecule involved in axon guidance. In many human epithelial carcinomas L1CAM is overexpressed and thereby augments cell motility, invasion and metastasis formation. L1CAM positive carcinomas are associated with bad prognosis. Recent data point out that L1CAM is regulated in a fashion similar to epithelial-mesenchymal transition (EMT). Previous studies have implied the transcription factors Slug and/or beta-catenin in L1CAM transcriptional regulation. However, the regulation of human L1CAM expression at the transcriptional level is not well understood. Results: To better understand the molecular basis of L1CAM transcriptional regulation, we carried out a detailed characterization of the human L1CAM promoter. We identified two transcription start sites, the first in front of a non-translated exon 0 (promoter 1) and the other next to the first protein-coding exon 1 (promoter 2). Both sites could be verified in endometrial carcinoma (EC) cell lines and appear to be used in a cell-type specific manner. The two identified promoter regions showed activity in luciferase reporter assays. Chromatin-IP analyses confirmed the in silico predicted E-boxes, binding sites for transcription factors Snail and Slug, as well as Lef-1 sites, which are related to beta-catenin-mediated transcriptional regulation, in both promoters. Overexpression of beta-catenin exclusively augmented activity of promoter 1 whereas Slug enhanced promoter 1 and 2 activity suggesting that both promoters can be active. Overexpression of beta-catenin or Slug could upregulate L1CAM expression in a cell-type specific manner. Conclusions: Our results, for the first time, provide evidence that the L1CAM gene has two functionally active promoter sites that are used in a cell-type specific manner. Slug and beta-catenin are involved L1CAM transcriptional regulation. Nevertheless, Slug rather than beta-catenin levels are correlated with L1CAM expression in EC cell lines. Our findings suggest that the L1CAM transcriptional regulation is more complex than anticipated and this study provides the basis for a better understanding of L1CAM regulation in non-neuronal/tumor cells.

Background: Smith-Magenis Syndrome is a contiguous gene syndrome in which the dosage sensitive gene has been identified: the Retinoic Acid Induced 1 (RAI1). Little is known about the function of human RAI1. Results: We generated the full-length cDNA of the wild type protein and five mutated forms: RAI1-HA 2687delC, RAI1-HA 3103delC, RAI1 R960X, RAI1-HA Q1562R, and RAI1-HA S1808N. Four of them have been previously associated with SMS clinical phenotype. Molecular weight, subcellular localization and transcription factor activity of the wild type and mutant forms were studied by western blot, immunofluorescence and luciferase assays respectively. The wild type protein and the two missense mutations presented a higher molecular weight than expected, localized to the nucleus and activated transcription of a reporter gene. The frameshift mutations generated a truncated polypeptide with transcription factor activity but abnormal subcellular localization, and the same was true for the 1-960aa N-terminal half of RAI1.Two different C-terminal halves of the RAI1 protein (1038aa-end and 1229aa-end) were able to localize into the nucleus but had no transactivation activity. Conclusion: Our results indicate that transcription factor activity and subcellular localization signals reside in two separate domains of the protein and both are essential for the correct functionality of RAI1. The pathogenic outcome of some of the mutated forms can be explained by the dissociation of these two domains.

Background: The mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket. Results: In order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity. Conclusions: These results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.

Background: RT-qPCR analysis is a widely used method for the analysis of mRNA expression throughout the field of mesenchymal stromal cell (MSC) research. Comparison between MSC studies, both in vitro and in vivo, are challenging due to the varied methods of RT-qPCR data normalization and analysis. Therefore, this study focuses on putative housekeeping genes for the normalization of RT-qPCR data between heterogeneous commercially available human MSC, compared with more homogeneous populations of MSC such as MIAMI and RS-1 cells. Results: Eight genes including; ACTB, B2M, EF1α, GAPDH, RPL13a, YWHAZ, UBC and HPRT1 were tested as possible housekeeping genes based on their expression level and variability. EF1α and RPL13a were validated for RT-qPCR analysis of MIAMI cells during expansion in varied oxygen tensions, endothelial differentiation, neural precursor enrichment, and during the comparison with RS-1 cells and commercially available MSC. RPL13a and YWHAZ were validated as normalization genes for the cross-species analysis of MIAMI cells in an animal model of focal ischemia. GAPDH, which is one of the most common housekeeping genes used for the normalization of RT-qPCR data in the field of MSC research, was found to have the highest variability and deemed not suitable for normalization of RT-qPCR data. Conclusions: In order to make comparisons between heterogeneous MSC populations, as well as adult stem cell like MSC which are used in different laboratories throughout the world, it is important to have a standardized, reproducible set of housekeeping genes for RT-qPCR analysis. In this study we demonstrate that EF1α, RPL13a and YWHAZ are suitable genes for the RT-qPCR analysis and comparison of several sources of human MSC during in vitro characterization and differentiation as well as in an ex vivo animal model of global cerebral ischemia. This will allow for the comparative RT-qPCR analysis of multiple MSC populations with the goal of future use in animal models of disease as well as tissue repair.

Background: Circadian rhythms have a profound effect on human health. Their disruption can lead to serious pathologies, such as cancer and obesity. Gene expression studies in these pathologies are often studied in different mouse strains by quantitative real time polymerase chain reaction (qPCR). Selection of reference genes is a crucial step of qPCR experiments. Recent studies show that reference gene stability can vary between species and tissues, but none has taken circadian experiments into consideration. Results: In the present study the expression of ten candidate reference genes (Actb, Eif2a, Gapdh, Hmbs, Hprt1, Ppib, Rn18s, Rplp0, Tbcc and Utp6c) was measured in 131 liver and 97 adrenal gland samples taken from three mouse strains (C57BL/6JOlaHsd, 129Pas plus C57BL/6J and Crem KO on 129Pas plus C57BL/6J background) every 4 h in a 24 h period. Expression stability was evaluated by geNorm and NormFinder programs. Differences in ranking of the most stable reference genes were observed both between individual mouse strains as well as between tissues within each mouse strain. We show that selection of reference gene (Actb) that is often used for analyses in individual mouse strains leads to errors if used for normalization when different mouse strains are compared. We identified alternative reference genes that are stable in these comparisons. Conclusions: Genetic background and circadian time influence the expression stability of reference genes. Differences between mouse strains and tissues should be taken into consideration to avoid false interpretations. We show that the use of a single reference gene can lead to false biological conclusions. This manuscript provides a useful reference point for researchers that search for stable reference genes in the field of circadian biology.

Background: Several genes have been used as housekeeping genes and choosing an appropriate reference gene is important for accurate quantitative RNA expression in real time RT-PCR technique. The expression levels of reference genes should remain constant between the cells of different tissues and under different experimental conditions. The purpose of this study was to determine the effect of different experimental treatments on the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA so that the reliability of GAPDH as reference gene for quantitative real time RT-PCR in human diploid fibroblasts (HDFs) can be validated. HDFs in 4 different treatment groups viz; young (passage 4), senescent (passage 30), H2O2-induced oxidative stress and γ-tocotrienol (GTT)-treated groups were harvested for total RNA extraction. Total RNA concentration and purity were determined prior to GAPDH mRNA quantification. Standard curve of GAPDH expression in serial diluted total RNA, melting curve analysis and agarose gel electrophoresis were used to determine the reliability of GAPDH as reference gene. Results: HDFs with different experimental treatments exhibited diverse cell morphology with different expression of senescence-associated β-galactosidase (SA β-gal) activity. However the expression level of GAPDH was consistent in all treatment groups. Conclusion: The study demonstrated that GAPDH is reliable as reference gene for quantitative gene expression analysis in HDFs. Therefore it can be used as housekeeping gene for quantitative real time RT-PCR technique in human diploid fibroblasts particularly in studying cellular senescence.