Transcript Profiling of Elf5+/− Mammary Glands during Pregnancy Identifies Novel Targets of Elf5

Background: Elf5, an epithelial specific Ets transcription factor, plays a crucial role in the pregnancy-associated development of the mouse mammary gland. Elf5 2/2 embryos do not survive, however the Elf5 +/2 mammary gland displays a severe pregnancy-associated developmental defect. While it is known that Elf5 is crucial for correct mammary development and lactation, the molecular mechanisms employed by Elf5 to exert its effects on the mammary gland are largely unknown. Principal Findings: Transcript profiling was used to investigate the transcriptional changes that occur as a result of Elf5 haploinsufficiency in the Elf5 +/2 mouse model. We show that the development of the mouse Elf5 +/2 mammary gland is delayed at a transcriptional and morphological level, due to the delayed increase in Elf5 protein in these glands. We also identify a number of potential Elf5 target genes, including Mucin 4, whose expression, is directly regulated by the binding of Elf5 to an Ets binding site within its promoter. Conclusion: We identify novel transcriptional targets of Elf5 and show that Muc4 is a direct target of Elf5, further elucidatin

Mitochondrial DNA haplotypes induce differential patterns of DNA methylation that result in differential chromosomal gene expression patterns

Mitochondrial DNA copy number is strictly regulated during development as naive cells differentiate into mature cells to ensure that specific cell types have sufficient copies of mitochondrial DNA to perform their specialised functions. Mitochondrial DNA haplotypes are defined as specific regions of mitochondrial DNA that cluster with other mitochondrial sequences to show the phylogenetic origins of maternal lineages. Mitochondrial DNA haplotypes are associated with a range of phenotypes and disease. To understand how mitochondrial DNA haplotypes induce these characteristics, we used four embryonic stem cell lines that have the same set of chromosomes but possess different mitochondrial DNA haplotypes. We show that mitochondrial DNA haplotypes influence changes in chromosomal gene expression and affinity for nuclear-encoded mitochondrial DNA replication factors to modulate mitochondrial DNA copy number, two events that act synchronously during differentiation. Global DNA methylation analysis showed that each haplotype induces distinct DNA methylation patterns, which, when modulated by DNA demethylation agents, resulted in skewed gene expression patterns that highlight the effectiveness of the new DNA methylation patterns established by each haplotype. The haplotypes differentially regulate α-ketoglutarate, a metabolite from the TCA cycle that modulates the TET family of proteins, which catalyse the transition from 5-methylcytosine, indicative of DNA methylation, to 5-hydroxymethylcytosine, indicative of DNA demethylation. Our outcomes show that mitochondrial DNA haplotypes differentially modulate chromosomal gene expression patterns of naive and differentiating cells by establishing mitochondrial DNA haplotype-specific DNA methylation patterns

Functional Crosstalk between Type I and II Interferon through the Regulated Expression of STAT1

Small "priming" quantities of type I interferon enhance cellular responses to type II interferon by maintaining basal levels of STAT1, explaining the observed crosstalk between these two cytokines

RNA-eXpress annotates novel transcript features in RNA-seq data

Summary: Next-generation sequencing is rapidly becoming the approach of choice for transcriptional analysis experiments. Substantial advances have been achieved in computational approaches to support these technologies. These approaches typically rely on existing transcript annotations, introducing a bias towards known genes, require specific experimental design and computational resources, or focus only on identification of splice variants (ignoring other biologically relevant transcribed features contained within the data that may be important for downstream analysis). Biologically relevant transcribed features also include large and small non-coding RNA, new transcription start sites, alternative promoters, RNA editing and processing of coding transcripts. Also, many existing solutions lack accessible interfaces required for wide scale adoption. We present a user-friendly, rapid and computation-efficient feature annotation framework (RNA-eXpress) that enables identification of transcripts and other genomic and transcriptional features independently of current annotations. RNA-eXpress accepts mapped reads in the standard binary alignment (BAM) format and produces a study-specific feature annotation in GTF format, comparison statistics, sequence extraction and feature counts. The framework is designed to be easily accessible while allowing advanced users to integrate new feature-identification algorithms through simple class extension, thus facilitating expansion to novel feature types or identification of study-specific feature types. Availability and implementation: RNA-eXpress software, source code, user manuals, supporting tutorials, developer guides and example data are available at http://www.rnaexpress.org. Contact: [email protected] Supplementary information: Supplementary data are available at Bioinformatics online

Microarray profiling to analyse adult cardiac fibroblast identity

Heart failure is one of the leading causes of death worldwide [1–4]. Current therapeutic strategies are inefficient and cannot cure this chronic and debilitating condition [5]. Ultimately, heart transplants are required for patient survival, but donor organs are scarce in availability and only prolong the life-span of patients for a limited time. Fibrosis is one of the main pathological features of heart failure [6,7], caused by inappropriate stimulation of fibroblasts and excessive extracellular matrix production. Therefore, an in-depth understanding of the cardiac fibroblast is essential to underpin effective therapeutic treatments for heart failure [5]. Fibroblasts in general have been an underappreciated cell type, regarded as relatively inert and providing only basic functionality; they are usually referred to as the ‘biological glue’ of all tissues in the body. However, more recent literature suggests that they actively participate in organ homeostasis and disease [7,8]. We have recently uncovered a unique molecular identity for fibroblasts isolated from the heart [9], expressing a set of cardiogenic transcription factors that have been previously associated with cardiomyocyte ontogenesis. This signature suggests that cardiac fibroblasts may be ideal for use in stem cell replacement therapies, as they may retain the memory of where they derive from embryologically. Our data also revealed that about 90% of fibroblasts from both tail and heart origins share a cell surface signature that has previously been described for mesenchymal stem cells (MSCs), raising the possibility that fibroblasts and MSCs may in fact be the same cell type. Thus, our findings carry profound implications for the field of regenerative medicine. Here, we describe detailed methodology and quality controls related to the gene expression profiling of cardiac fibroblasts, deposited at the Gene Expression Omnibus (GEO) under the accession number GSE50531. We also provide the R code to easily reproduce the data quantification and analysis processes

Purification and biological characterization of soluble, recombinant mouse IFNβ expressed in insect cells

Interferon β (IFNβ) is a member of the type I interferon family of cytokines widely recognised for their anti-viral, anti-proliferative and immunomodulatory properties. Recombinant, biologically active forms of this cytokine are used clinically for the treatment of multiple sclerosis and in laboratories to study the role of this cytokine in health and disease. Established methods for expression of IFNβ utilise either bacterial systems from which the insoluble recombinant proteins must be refolded, or mammalian expression systems in which large volumes of cell culture are required for recovery of acceptable yields. Utilising the baculovirus expression system and Trichoplusia ni (Cabbage Looper) BTI-TN-5B1-4 cell line, we report a reproducible method for production and purification of milligram/litre quantities of biologically active murine IFNβ. Due to the design of our construct and the eukaryotic nature of insect cells, the resulting soluble protein is secreted allowing purification of the Histidine-tagged natively-folded protein from the culture supernatant. The IFNβ purification method described is a two-step process employing immobilised metal-ion affinity chromatography (IMAC) and reverse-phase high performance liquid chromatography (RP-HPLC) that results in production of significantly more purified IFNβ than any other reported eukaryotic-based expression system. Recombinant murine IFNβ produced by this method was natively folded and demonstrated hallmark type I interferon biological effects including antiviral and anti-proliferative activities, and induced genes characteristic of IFNβ activity in vivo. Recombinant IFNβ also had specific activity levels exceeding that of the commercially available equivalent. Together, our findings provide a method for production of highly pure, biologically active murine IFNβ

Suppressor of cytokine signaling (SOCS) 1 inhibits type I interferon (IFN) signaling via the interferon alpha receptor (IFNAR1)-associated tyrosine kinase Tyk2

Type I IFNs are critical players in host innate and adaptive immunity. IFN signaling is tightly controlled to ensure appropriate immune responses as imbalance could result in uncontrolled inflammation or inadequate responses to infection. It is therefore important to understand how type I IFN signaling is regulated. Here we have investigated the mechanism by which suppressor of cytokine signaling 1 (SOCS1) inhibits type I IFN signaling. We have found that SOCS1 inhibits type I IFN signaling not via a direct interaction with the IFN ? receptor 1 (IFNAR1) receptor component but through an interaction with the IFNAR1-associated kinase Tyk2. We have characterized the residues/regions involved in the interaction between SOCS1 and Tyk2 and found that SOCS1 associates via its SH2 domain with conserved phosphotyrosines 1054 and 1055 of Tyk2. The kinase inhibitory region of SOCS1 is also essential for its interaction with Tyk2 and inhibition of IFN signaling. We also found that Tyk2 is preferentially Lys-63 polyubiquitinated and that this activation reaction is inhibited by SOCS1. The consequent effect of SOCS1 inhibition of Tyk2 not only results in a reduced IFN response because of inhibition of Tyk2 kinase-mediated STAT signaling but also negatively impacts IFNAR1 surface expression, which is stabilized by Tyk2

CiIIder:A tool for predicting and analysing transcription factor binding sites

The availability of large amounts of high-throughput genomic, transcriptomic and epigenomic data has provided opportunity to understand regulation of the cellular transcriptome with an unprecedented level of detail. As a result, research has advanced from identifying gene expression patterns associated with particular conditions to elucidating signalling pathways that regulate expression. There are over 1,000 transcription factors (TFs) in vertebrates that play a role in this regulation. Determining which of these are likely to be controlling a set of genes can be assisted by computational prediction, utilising experimentally verified binding site motifs. Here we present CiiiDER, an integrated computational toolkit for transcription factor binding analysis, written in the Java programming language, to make it independent of computer operating system. It is operated through an intuitive graphical user interface with interactive, high-quality visual outputs, making it accessible to all researchers. CiiiDER predicts transcription factor binding sites (TFBSs) across regulatory regions of interest, such as promoters and enhancers derived from any species. It can perform an enrichment analysis to identify TFs that are significantly over- or under-represented in comparison to a bespoke background set and thereby elucidate pathways regulating sets of genes of pathophysiological importance

Restoration of normal embryogenesis by mitochondrial supplementation in pig oocytes exhibiting mitochondrial DNA deficiency

An increasing number of women fail to achieve pregnancy due to either failed fertilization or embryo arrest during preimplantation development. This often results from decreased oocyte quality. Indeed, reduced mitochondrial DNA copy number (mitochondrial DNA deficiency) may disrupt oocyte quality in some women. To overcome mitochondrial DNA deficiency, whilst maintaining genetic identity, we supplemented pig oocytes selected for mitochondrial DNA deficiency, reduced cytoplasmic maturation and lower developmental competence, with autologous populations of mitochondrial isolate at fertilization. Supplementation increased development to blastocyst, the final stage of preimplantation development, and promoted mitochondrial DNA replication prior to embryonic genome activation in mitochondrial DNA deficient oocytes but not in oocytes with normal levels of mitochondrial DNA. Blastocysts exhibited transcriptome profiles more closely resembling those of blastocysts from developmentally competent oocytes. Furthermore, mitochondrial supplementation reduced gene expression patterns associated with metabolic disorders that were identified in blastocysts from mitochondrial DNA deficient oocytes. These results demonstrate the importance of the oocyte’s mitochondrial DNA investment in fertilization outcome and subsequent embryo development to mitochondrial DNA deficient oocytes