Mouse Gene Promoters Research Articles

Adjuvant effect of diphtheria toxin after mucosal administration in both wild type and diphtheria toxin receptor engineered mouse strains

The finding that murine and simian cells have differential susceptibility to diphtheria toxin (DTx) led to the development of genetically engineered mouse strains that express the simian or human diphtheria toxin receptor (DTR) under the control of various mouse gene promoters. Injection of DTx into DTR engineered mice allows for rapid and transient depletion of various cell populations. There are several advantages to this approach over global knockout mice, including normal mouse development and temporal control over when cell depletion occurs. As a result, many DTR engineered mouse strains have been developed, resulting in significant insights into the cell biology of various disease states. We used Foxp3DTR mice to attempt local depletion of Foxp3+ cells in the lung in a model of tolerance breakdown. Intratracheal administration of DTx resulted in robust depletion of lung Foxp3+ cells. However, DTx administration was accompanied by significant local inflammation, even in control C57Bl/6 mice. These data suggest that DTx administration to non-transgenic mice is not always an immunologically inert event, and proper controls must be used to assess various DTx-mediated depletion regimens.

Abstract 2791: SINE retrotransposons cause epigenetic reprogramming of adjacent gene promoters

Abstract Retrotransposons of SINE and LINE classes have been extremely successful in colonizing mammalian genomes. Almost half of the human genome and as much as 40% of the mouse genome is composed of repetitive DNA sequences. The comparison of human and mouse genomes revealed a spatial concordance in positioning of SINE but not LINE repeats, indicating evolutionary pressure to maintain and/or exclude these repeats from orthologous regions. We hypothesized that this phenomenon is due to a negative influence of SINE repeats on gene expression and counter selection over evolution, resulting in exclusion of these repeats from vulnerable genomic environments. To test the capacity of SINE B1 elements in promoting gene silencing, we generated a system where the luciferase gene is under the control of three different mouse gene promoters (cdkn2a, p14arf and mlh1), and we inserted two and four copies of SINE B1 elements upstream to these promoters. Using this in vitro system, we found that SINE B1 elements can influence the activity of downstream gene promoters, with acquisition of DNA methylation and loss of activating histone marks, thus resulting in a repressed state. Interestingly, SINE sequences themselves do not immediately acquired DNA methylation, but became marked by H3K9me2 and H3K27me3. Moreover, gain of DNA methylation depended on cryptic genomic methylation centers rather than spreading from SINE B1 repeats. Since mouse SINE B1 elements caused transcriptional repression, we sought to investigate whether human SINE Alu repeats could have the same effect. The human E-cadherin gene has three SINE Alus adjacent to the 3’ region of its CpG island, and in reporter assays the removal of these repeats resulted in higher, stable promoter activity. Genome-wide analysis of SINE repeats distribution in the human and mouse genomes showed that they are excluded from proximal promoters and their enrichment is directly correlated with the presence of USF1, USF2 and CTCF binding, proteins with insulator function. In conclusion, our work supports the concept that SINE repeats interfere negatively with gene expression and that their presence near gene promoters is dependent on, and co-evolved with the presence of insulator elements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2791. doi:10.1158/1538-7445.AM2011-2791

Annotation of gene promoters by integrative data-mining of ChIP-seq Pol-II enrichment data

BackgroundUse of alternative gene promoters that drive widespread cell-type, tissue-type or developmental gene regulation in mammalian genomes is a common phenomenon. Chromatin immunoprecipitation methods coupled with DNA microarray (ChIP-chip) or massive parallel sequencing (ChIP-seq) are enabling genome-wide identification of active promoters in different cellular conditions using antibodies against Pol-II. However, these methods produce enrichment not only near the gene promoters but also inside the genes and other genomic regions due to the non-specificity of the antibodies used in ChIP. Further, the use of these methods is limited by their high cost and strong dependence on cellular type and context.MethodsWe trained and tested different state-of-art ensemble and meta classification methods for identification of Pol-II enriched promoter and Pol-II enriched non-promoter sequences, each of length 500 bp. The classification models were trained and tested on a bench-mark dataset, using a set of 39 different feature variables that are based on chromatin modification signatures and various DNA sequence features. The best performing model was applied on seven published ChIP-seq Pol-II datasets to provide genome wide annotation of mouse gene promoters.ResultsWe present a novel algorithm based on supervised learning methods to discriminate promoter associated Pol-II enrichment from enrichment elsewhere in the genome in ChIP-chip/seq profiles. We accumulated a dataset of 11,773 promoter and 46,167 non-promoter sequences, each of length 500 bp, generated from RNA Pol-II ChIP-seq data of five tissues (Brain, Kidney, Liver, Lung and Spleen). We evaluated the classification models in building the best predictor and found that Bagging and Random Forest based approaches give the best accuracy. We implemented the algorithm on seven different published ChIP-seq datasets to provide a comprehensive set of promoter annotations for both protein-coding and non-coding genes in the mouse genome. The resulting annotations contain 13,413 (4,747) protein-coding (non-coding) genes with single promoters and 9,929 (1,858) protein-coding (non-coding) genes with two or more alternative promoters, and a significant number of unassigned novel promoters.ConclusionOur new algorithm can successfully predict the promoters from the genome wide profile of Pol-II bound regions. In addition, our algorithm performs significantly better than existing promoter prediction methods and can be applied for genome-wide predictions of Pol-II promoters.

Temporal Induction Pattern of STAT4 Target Genes Defines Potential for Th1 Lineage-Specific Programming

STAT4 is a critical component in the development of inflammatory adaptive immune responses. It has been extensively characterized as a lineage-determining factor in Th1 development. However, the genetic program activated by STAT4 that results in an inflammatory cell type is not well defined. In this report, we use DNA isolated from STAT4-chromatin immunoprecipitation to perform chromatin immunoprecipitation-on-chip analysis of over 28,000 mouse gene promoters to identify STAT4 targets. We demonstrate that STAT4 binds multiple gene-sets that program distinct components of the Th1 lineage. Although many STAT4 target genes display STAT4-dependent IL-12-inducible expression, other genes displayed IL-12-induced histone modifications but lack induction, possibly due to high relative basal expression. In the subset of genes that STAT4 programs for expression in Th1 cells, IL-12-induced mRNA levels remain increased for a longer time than mRNA from genes that are not programmed. This suggests that STAT4 binding to target genes, while critical, is not the only determinant for STAT4-dependent gene programming during Th1 differentiation.

Positional bias of general and tissue-specific regulatory motifs in mouse gene promoters

BackgroundThe arrangement of regulatory motifs in gene promoters, or promoter architecture, is the result of mutation and selection processes that have operated over many millions of years. In mammals, tissue-specific transcriptional regulation is related to the presence of specific protein-interacting DNA motifs in gene promoters. However, little is known about the relative location and spacing of these motifs. To fill this gap, we have performed a systematic search for motifs that show significant bias at specific promoter locations in a large collection of housekeeping and tissue-specific genes.ResultsWe observe that promoters driving housekeeping gene expression are enriched in particular motifs with strong positional bias, such as YY1, which are of little relevance in promoters driving tissue-specific expression. We also identify a large number of motifs that show positional bias in genes expressed in a highly tissue-specific manner. They include well-known tissue-specific motifs, such as HNF1 and HNF4 motifs in liver, kidney and small intestine, or RFX motifs in testis, as well as many potentially novel regulatory motifs. Based on this analysis, we provide predictions for 559 tissue-specific motifs in mouse gene promoters.ConclusionThe study shows that motif positional bias is an important feature of mammalian proximal promoters and that it affects both general and tissue-specific motifs. Motif positional constraints define very distinct promoter architectures depending on breadth of expression and type of tissue.

Cis-Acting Human ApoE Tissue Expression Element is Associated with Human Pattern of Intraneuronal ApoE in Transgenic Mice

Apolipoprotein E polymorphic variants (ApoE-ϵ2, ϵ3, and ϵ4) are associated with the age of onset distribution and risk of Alzheimer disease. The question of whether ApoE is expressed at a comparatively low level in human neurons compared to astrocytes, or whether ApoE enters neuronal cytoplasm via altered endosomal metabolism is important to understanding potential pathogenic roles for ApoE as a susceptibility gene in Alzheimer disease. ApoE deficient (“knock-out”) mice received large human genomic DNA fragment transgenes for each of the three common apoE alleles. All transgenic mice demonstrated glial/astrocytic (normal rodent pattern), as well as cortical intraneuronal ApoE immunoreactivity with all three human isoforms and at multiple ApoE human allele doses (Xu et al. [32]). To test whether ApoE intraneuronal immunoreactivity was due to ApoE gene sequences between mouse and human, we examined another set of mice constructed using targeted replacement so that the human ApoE gene was placed under mouse gene promoters. Current analyses show that targeted replacement recombinant mice show normal rodent glial expression pattern, but no ApoE neuronal immunoreactivity through six months of age compared to large human genomic DNA fragment transgenic mice, which show neuronal content of ApoE throughout adult life. We conclude that cis-acting DNA sequences, rather than the specific sequence of the ApoE gene, may be responsible for low levels of transcription activity in cortical neurons.

Coordinate Expression and Distinct DNA-Binding Characteristics of the Four EGR-Zinc Finger Proteins in Jurkat T lymphocytes

The Early Growth Response Genes (EGR-1 to AT133/EGR-4) encode a family of proteins that are composed of three homologous consecutive zinc fingers of the Cys 2-His, type and different flanking sequences. Upon growth stimulation of resting cells the four EGR-genes are simultaneously transcribed. We have analyzed the expression of the four EGR-proteins in Jurkat T cells and show by Western blot analysis that the four EGR-proteins are coordinately induced upon treatment with a combination of PHA and PMA. As the individual proteins are reported to bind to identical target sequences, we have analyzed the DNA-binding of the native proteins. Using nuclear extract in which we have demonstrated expression of all four EGR-proteins, only EGR-1, but no other member of this protein family is found to bind to the EGR-consensus site (GCG GGG GCG). In addition, DNA-binding of both native EGR-1 and of recombinant EGR-1 and AT133/EGR-4 proteins expressed in insect cells was analyzed. This comparison revealed distinct binding properties of recombinant EGR-1 and AT133/EGR-4 to oligonucleotides that include the EGR-consensus sites. The distinct binding affinities suggest that in vivo EGR-proteins bind to different target sequences and that each EGR-protein regulates distinct target genes. This is underlined by demonstrating that EGR-1 but not AT133/EGR-4 binds to a related G-rich promoter element with the sequence GGG GTG GGG. This G-rich sequence serves as an overlapping binding site for the two zinc finger proteins EGR-1 and Sp1. As similar overlapping binding sites for EGR-1 and Sp1 have been identified in several human and mouse gene promoters, we raise the question whether the Sp1 binding sites described in a large number of eukaryotic gene promoters also represent binding sites for EGR-1.