Although it is generally accepted that cellular differentiation requires changes to transcriptional networks, dynamic regulation of promoters and enhancers at specific sets of genes has not been previously studied en masse. Exploiting the fact that active promoters and enhancers are transcribed, we simultaneously measured their activity in 19 human and 14 mouse time courses covering a wide range of cell types and biological stimuli. Enhancer RNAs, then messenger RNAs encoding transcription factors, dominated the earliest responses. Binding sites for key lineage transcription factors were simultaneously overrepresented in enhancers and promoters active in each cellular system. Our data support a highly generalizable model in which enhancer transcription is the earliest event in successive waves of transcriptional change during cellular differentiation or activation.

The immediate-early response mediates cell fate in response to a variety of extracellular stimuli and is dysregulated in many cancers. However, the specificity of the response across stimuli and cell types, and the roles of non-coding RNAs are not well understood. Using a large collection of densely-sampled time series expression data we have examined the induction of the immediate-early response in unparalleled detail, across cell types and stimuli. We exploit cap analysis of gene expression (CAGE) time series datasets to directly measure promoter activities over time. Using a novel analysis method for time series data we identify transcripts with expression patterns that closely resemble the dynamics of known immediate-early genes (IEGs) and this enables a comprehensive comparative study of these genes and their chromatin state. Surprisingly, these data suggest that the earliest transcriptional responses often involve promoters generating non-coding RNAs, many of which are produced in advance of canonical protein-coding IEGs. IEGs are known to be capable of induction without de novo protein synthesis. Consistent with this, we find that the response of both protein-coding and non-coding RNA IEGs can be explained by their transcriptionally poised, permissive chromatin state prior to stimulation. We also explore the function of non-coding RNAs in the attenuation of the immediate early response in a small RNA sequencing dataset matched to the CAGE data: We identify a novel set of microRNAs responsible for the attenuation of the IEG response in an estrogen receptor positive cancer cell line. Our computational statistical method is well suited to meta-analyses as there is no requirement for transcripts to pass thresholds for significant differential expression between time points, and it is agnostic to the number of time points per dataset.

Vascular permeability and angiogenesis underpin neovascular age-related macular degeneration and diabetic retinopathy. While anti-VEGF therapies are widely used clinically, many patients do not respond optimally, or at all, and small-molecule therapies are lacking. Here, we identified a dibenzoxazepinone BT2 that inhibits endothelial cell proliferation, migration, wound repair in vitro, network formation, and angiogenesis in mice bearing Matrigel plugs. BT2 interacts with MEK1 and inhibits ERK phosphorylation and the expression of FosB/ΔFosB, VCAM-1, and many genes involved in proliferation, migration, angiogenesis, and inflammation. BT2 reduced retinal vascular leakage following rat choroidal laser trauma and rabbit intravitreal VEGF-A165 administration. BT2 suppressed retinal CD31, pERK, VCAM-1, and VEGF-A165 expression. BT2 reduced retinal leakage in rats at least as effectively as aflibercept, a first-line therapy for nAMD/DR. BT2 withstands boiling or autoclaving and several months’ storage at 22°C. BT2 is a new small-molecule inhibitor of vascular permeability and angiogenesis.

Smooth muscle cells (SMC) in blood vessels are normally growth quiescent and transcriptionally inactive. Our objective was to understand promoter usage and dynamics in SMC acutely exposed to a prototypic growth factor or pro-inflammatory cytokine. Using cap analysis gene expression (FANTOM5 project) we report differences in promoter dynamics for immediate-early genes (IEG) and other genes when SMC are exposed to fibroblast growth factor-2 or interleukin-1β. Of the 1871 promoters responding to FGF2 or IL-1β considerably more responded to FGF2 (68.4%) than IL-1β (18.5%) and 13.2% responded to both. Expression clustering reveals sets of genes induced, repressed or unchanged. Among IEG responding rapidly to FGF2 or IL-1β were FOS, FOSB and EGR-1, which mediates human SMC migration. Motif activity response analysis (MARA) indicates most transcription factor binding motifs in response to FGF2 were associated with a sharp induction at 1 h, whereas in response to IL-1β, most motifs were associated with a biphasic change peaking generally later. MARA revealed motifs for FOS_FOS{B,L1}_JUN{B,D} and EGR-1..3 in the cluster peaking 1 h after FGF2 exposure whereas these motifs were in clusters peaking 1 h or later in response to IL-1β. Our findings interrogating CAGE data demonstrate important differences in promoter usage and dynamics in SMC exposed to FGF2 or IL-1β.

Background and Aim: MicroRNAs (miRNAs or miRs) are an abundant class of short (17 to 25 nt) non-coding single-stranded RNAs that are significant controllers of cellular gene expression. Mature miRNA is created from long primary genomic transcripts (pri-miRNA), which are processed in the nucleus. The resulting 60-80-nucleotide precursor miRNAs (pre-miRNAs) are transferred to the cytoplasm and are treated to produce a short RNA duplex. One strand of the duplex is degraded, the remaining single-stranded miRNA molecule associates with members of the Argonaute protein family, to form the RNA-induced silencing complex (RISC). RISC plus the guide miRNA bind to the 3′untranslated region (3′-UTR) of the targeted mRNA and induce gene down-regulation by mRNA cleavage or translational repression. Serum stimulation of cancer cells has been shown to prompt positive regulatory transcription factors, such as c-Jun, c-Fos and Egr-1, as well as increasing transcriptional repressors such as YY1, NAB2 and GCF2. These transcription factors play critical parts in the early stimulation of cancer disease phenotypes and are prime targets for clarifying disease pathways and as therapeutic candidates. The identification of microRNAs regulating the mRNAs of transcription factors will improve our understanding of the mechanism of disease gene stimulation and subsequent pathway initiation leading to cancer disease phenotypes. MicroRNAs that ablate disease phenotypes are possible pharmaceutical therapies. Our aim was to identify and profile microRNAs that interact with the mRNA of transcription factors such as c-Jun and c-Fos. Results: We measured the expresion of c-Jun and c-Fos at the mRNA and protein levels (time course 0 to 6 hrs) in MM200 (human melanoma). We measured the expression of two microRNAs, miR155 and miR125b, which have been reported as playing a role in regulating c-Jun and c-Fos in Dermal Fibroblasts and melanoma cells (Song, Liu et al. Cellular Physiology and Biochemistry 2012 and Kappelmann, Kuphal et al. Oncogene 2013). We show that miR-155 and miR-125b expression is repressed following serum activation in these two cell lines (HEK293 and MM200). Loss of function and gain-of-function studies in these cancer cell lines, by supressing and overexpressing miR155/125b were performed. Subsquently, a target 3′UTR vs microRNA study was conducted that demonstrated that the miR-155 and miR-125b have the ability to recognize and repress c-Jun and c-Fos. Moreover, phenotypic studies such as migration, proliferation, cell cycle were performed. Conclusions: miR-155 and miR-125b are able to regulate c-Jun and c-Fos mRNAs and to inhibit their expression at the protein level. Citation Format: Ahmad M.N Alhendi, Leonel Prado-Lourenço, Noel Whitaker. Targeting c-Jun and c-Fos using microRNA's has a potential in contesting melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2114. doi:10.1158/1538-7445.AM2015-2114