Detecting differential activation of transcription factors (TFs) in response to perturbation provides insight into cellular processes. Transcription Factor Enrichment Analysis (TFEA) is a robust and reliable computational method that detects differential activity of hundreds of TFs given any set of perturbation data. TFEA draws inspiration from GSEA and detects positional motif enrichment within a list of ranked regions of interest (ROIs). As ROIs are typically inferred from the data, we also introduce muMerge, a statistically principled method of generating a consensus list of ROIs from multiple replicates and conditions. TFEA is broadly applicable to data that informs on transcriptional regulation including nascent (eg. PRO-Seq), CAGE, ChIP-Seq, and accessibility (e.g. ATAC-Seq). TFEA not only identifies the key regulators responding to a perturbation, but also temporally unravels regulatory networks with time series data. Consequently, TFEA serves as a hypothesis-generating tool that provides an easy, rigorous, and cost-effective means to broadly assess TF activity yielding new biological insights.

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Zinc is the second most abundant transition metal in humans and an essential nutrient required for growth and development of newborns. During lactation, mammary epithelial cells differentiate into a secretory phenotype, uptake zinc from blood circulation, and export it into mother’s milk. At the cellular level, many zinc-dependent cellular processes, such as transcription, metabolism of nutrients, and proliferation are involved in the differentiation of mammary epithelial cells. Using mouse mammary epithelial cells as a model system, we investigated the remodeling of zinc homeostasis during differentiation induced by treatment with the lactogenic hormones cortisol and prolactin. RNA-Seq at different stages of differentiation revealed changes in global gene expression, including genes encoding zinc-dependent proteins and regulators of zinc homeostasis. Increases in mRNA levels of three zinc homeostasis genes, Slc39a14 (ZIP14) and metallothioneins (MTs) I and II were induced by cortisol but not by prolactin. The cortisol-induced increase was partially mediated by the nuclear glucocorticoid receptor signaling pathway. An increase in the cytosolic labile Zn2+ pool was also detected in lactating mammary cells, consistent with upregulation of MTs. We found that the zinc transporter ZIP14 was important for the expression of a major milk protein, whey acid protein (WAP), as knockdown of ZIP14 dramatically decreased WAP mRNA levels. In summary, our study demonstrated remodeling of zinc homeostasis upon differentiation of mammary epithelial cells resulting in changes in cytosolic Zn2+ and differential expression of zinc homeostasis genes, and these changes are important for establishing the lactation phenotype.

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Flaviviruses such as dengue encode a protease that is essential for viral replication. The protease functions by cleaving well-conserved positions in the viral polyprotein. In addition to the viral polyprotein, the dengue protease cleaves at least one host protein involved in immune response. This raises the question, what other host proteins are targeted and cleaved? Here we present a new computational method for identifying putative host protein targets of the dengue virus protease. Our method relies on biochemical and secondary structure features at the known cleavage sites in the viral polyprotein in a two-stage classification process to identify putative cleavage targets. The accuracy of our predictions scaled inversely with evolutionary distance when we applied it to the known cleavage sites of several other flaviviruses—a good indication of the validity of our predictions. Ultimately, our classifier identified 257 human protein sites possessing both a similar target motif and accessible local structure. These proteins are promising candidates for further investigation. As the number of viral sequences expands, our method could be adopted to predict host targets of other flaviviruses.

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Nascent transcription assays, such as global run-on sequencing (GRO-seq) and precision run-on sequencing (PRO-seq), have uncovered a myriad of unstable RNAs being actively produced from numerous sites genome-wide. These transcripts provide a more complete and immediate picture of the impact of regulatory events. Transcription factors recruit RNA polymerase II, effectively initiating the process of transcription; repressors inhibit polymerase recruitment. Efficiency of recruitment is dictated by sequence elements in and around the RNA polymerase loading zone. A combination of sequence elements and RNA binding proteins subsequently influence the ultimate stability of the resulting transcript. Some of these transcripts are capable of providing feedback on the process, influencing subsequent transcription. By monitoring RNA polymerase activity, nascent assays provide insights into every step of the regulated process of transcription.

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Transcriptional responses to external stimuli remain poorly understood. Using global nuclear run-on followed by sequencing (GRO-seq) and precision nuclear run-on sequencing (PRO-seq), we show that CDK8 kinase activity promotes RNA polymerase II pause release in response to interferon-γ (IFN-γ), a universal cytokine involved in immunity and tumor surveillance. The Mediator kinase module contains CDK8 or CDK19, which are presumed to be functionally redundant. We implemented cortistatin A, chemical genetics, transcriptomics, and other methods to decouple their function while assessing enzymatic versus structural roles. Unexpectedly, CDK8 and CDK19 regulated different gene sets via distinct mechanisms. CDK8-dependent regulation required its kinase activity, whereas CDK19 governed IFN-γ responses through its scaffolding function (i.e., it was kinase independent). Accordingly, CDK8, not CDK19, phosphorylates the STAT1 transcription factor (TF) during IFN-γ stimulation, and CDK8 kinase inhibition blocked activation of JAK-STAT pathway TFs. Cytokines such as IFN-γ rapidly mobilize TFs to “reprogram” cellular transcription; our results implicate CDK8 and CDK19 as essential for this transcriptional reprogramming.

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The glucocorticoid receptor (NR3C1, also known as GR) binds to specific DNA sequences and directly induces transcription of anti-inflammatory genes that contribute to cytokine repression, frequently in cooperation with NF-kB. Whether inflammatory repression also occurs through local interactions between GR and inflammatory gene regulatory elements has been controversial. Here, using global run-on sequencing (GRO-seq) in human airway epithelial cells, we show that glucocorticoid signaling represses transcription within 10 min. Many repressed regulatory regions reside within "hyper-ChIPable" genomic regions that are subject to dynamic, yet nonspecific, interactions with some antibodies. When this artifact was accounted for, we determined that transcriptional repression does not require local GR occupancy. Instead, widespread transcriptional induction through canonical GR binding sites is associated with reciprocal repression of distal TNF-regulated enhancers through a chromatin-dependent process, as evidenced by chromatin accessibility and motif displacement analysis. Simultaneously, transcriptional induction of key anti-inflammatory effectors is decoupled from primary repression through cooperation between GR and NF-kB at a subset of regulatory regions. Thus, glucocorticoids exert bimodal restraints on inflammation characterized by rapid primary transcriptional repression without local GR occupancy and secondary anti-inflammatory effects resulting from transcriptional cooperation between GR and NF-kB.

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Mycobacterium vaccae (NCTC 11659) is an environmental saprophytic bacterium with anti-inflammatory, immunoregulatory, and stress resilience properties. Previous studies have shown that whole, heat-killed preparations of M. vaccae prevent allergic airway inflammation in a murine model of allergic asthma. Recent studies also demonstrate that immunization with M. vaccae prevents stress-induced exaggeration of proinflammatory cytokine secretion from mesenteric lymph node cells stimulated ex vivo, prevents stress-induced exaggeration of chemically induced colitis in a model of inflammatory bowel disease, and prevents stress-induced anxiety-like defensive behavioral responses. Furthermore, immunization with M. vaccae induces anti-inflammatory responses in the brain and prevents stress-induced exaggeration of microglial priming. However, the molecular mechanisms underlying anti-inflammatory effects of M. vaccae are not known.

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Bioinformatics answers questions of cancer and career path

Phil Richardson, an author on a paper recently published in Nature, developed a love for bioinformatics in BioFrontiers' Robin Dowell's lab. His next move: pursuing a graduate degree in medical genomics.

At some point in school, we were taught that humans are a diploid species, made of cells with two sets of chromosomes – one set contributed by each parent. This idea neatly packaged the way we believed cells carried on with dividing themselves and creating more cells, but it isn’t the only way. Polyploidy occurs when cells have more than two complete sets of chromosomes. Polyploids are common: Plants, some fish and amphibians are polyploid. Aneuploidy is yet another chromosomal mix – one where there is an abnormal number of chromosomes in a cell due to extra or missing chromosomes. While being diploid looks much simpler on paper, recent research points to the chromosomal flexibility of the ancestors of many diploid species, because as it turns out, polyploidy and aneuploidy appear to be pretty helpful in smoothing out the evolutionary ride.

In a recent paper in , BioFrontiers Institute faculty member, Robin Dowell, an assistant professor of Molecular, Cellular and Developmental Biology, describes the influence that polyploidy has on accelerating evolutionary adaptation. By studying Saccharomyces cerevisiae, a helpful species of yeast well-known in winemaking, baking and beer brewing, Dowell was able to show that many individual strains can switch between polyploidy and aneuploidy, and they do so to adapt to evolutionary and environmental changes.

“To me, the interesting thing that came out of this study is that being aneuploid can help polyploids,” says Dowell. “When faced with a lot of evolutionary or environmental pressure, polyploid cells can switch quickly to being aneuploid. It seems like it becomes a short-term solution for these cells.”

Dowell’s lab is, in part, focused on studying how chromosomes influence adaptation and how these adaptations affect cellular processes. These processes are poorly studied because genetic data creates huge datasets and many labs lack the computational tools and abilities to dig through them. Dowell’s lab, in the Jennie Smoly Caruthers biotechnology building, blends a biological wet lab with a computational dry lab resulting in research that has the capability to look deeply at bioinformatics.

Phil Richmond is part of Dowell’s lab and a co-author on the Nature paper. He did much of the groundwork for this research as an undergraduate in the Department of Molecular, Cellular and Developmental Biology at CU-�鶹ӰԺ. His job was to comb through the data of 130 genome-sequenced strains, narrowing the dataset to 78 that were of acceptable quality to study.

“One of my biggest contributions on this project was benchmarking a way to track mutations in the genes,” says Richmond. “Everything was built for humans with a diploid assumption and the current tools couldn’t find mutations in polyploid yeast. This was my first really cool experience in digging into genomics.”

Polyploidy and aneuploidy become interesting study subjects when it comes to cancer. From a cancer standpoint, polyploidy is a mistake in replication of chromosomes, and the odds of a polyploid cell becoming cancerous are increased. Some cancers appear to start when the cell divides and extra chromosomes go into one cell instead of splitting evenly between two. As this happens over and over, a cell can quickly become what scientists call “self-interested” and become a soft tumor.

Genetic sequencing is showing promise as a powerful tool for learning about different types of cancers. Richmond started at CU-�鶹ӰԺ as a pre-medical student and quickly fell in love with biology. Dowell hired him into her lab as a sophomore where he filed papers and washed glassware, but it wasn’t long before Dowell recruited him for a programming project.

“I had never programmed anything,” says Richmond. “But since then, the number of tools available and people that are interested in bioinformatics has grown exponentially, and I’ve been riding that wave ever since. I’m no expert in any of the fields I’ve collaborated in, but my bioinformatics skillset has allowed me to be useful in a lot of different projects. I chose a path that will just keep growing.”

Richmond would love to see cancerous tumors undergo genetic sequencing so that doctors will know what they are treating and how best to treat it. He believes that, although sequencing costs are still relatively high, it might be more cost effective in the long run to sequence a tumor rather than have a patient take the wrong drugs without results.

“This study really brought out one big outlying question,” says Richmond. “When a tumor becomes polyploid, is it the result of some random system, or is it providing an advantage to the cell? We’ve shown that yeast—a eukaryote—is able to adapt faster in the polyploid state, so what we learned is helping us to think of the chromosomal copy number profile in cancer as more of an evolutionary advantage rather than the result of a chaotic and unstable system operating in the cell. This may change the way we approach the treatment of this disease in the future.”

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I recently attended the 2014 Association for Computing Machinery Conference on Bioinformatics, Computational Biology, and Health Informatics (ACM BCB) with fellow IQ Biology student Joey Azofeifa and our advisor Robin Dowell. The conference had many interesting talks, ranging from theory-heavy explanations of algorithm improvements to very applied talks on using computational analysis for medical procedures. Joey presented his work on FStitch, a tool for measuring RNA transcription with GRO-seq data, which is soon to be published in the conference journal. His talk went very well and he fielded many good questions from interested attendees. In addition, Robin was a panelist for the “Women in Bioinformatics Panel” which addressed specific issues women might face in the field of bioinformatics.

I presented my poster titled “Inferring Ancestry in Mouse Genomes using a Hidden Markov Model”, where I showed my work on determining haplotype block inheritance using single-nucleotide polymorphism data from two selectively bred mouse strains and six of the eight ancestor strains that they were bred from (the other two ancestor strains haven’t been sequenced). To infer ancestry, I used a hidden Markov model (HMM)- a probabilistic model used to find the maximum likelihood path through a state machine. The poster session was great and I ended up having many visitors over during the two-hour timeframe. Some were simply intrigued by the pretty pictures and wanted to know what an HMM was, while others had worked on similar inheritance problems and had good questions about my process. I even spoke with a group that works with the mice strains we used and have imputated the genomes of the two unsequenced ancestor mouse strains, so I’m now looking into incorporating this data in my model.

The conference was held in Newport Beach, CA and while it was hot and sunny the entire weekend, I unfortunately never got a chance to visit the beach. I was lucky enough, however, to have a good friend who lives in the area and whom I hadn’t seen in over a year, so I got to spend some quality time with her. We were trying to plan a trip to see each other soon anyways, so it was really lucky that the conference happened to be in her area!

Currently, other members of the Dowell Lab and I are in the process of writing a paper on the sequencing of the two selectively bred mice strains, which will include my ancestor inference piece as a section. We then hope to extend my work by refining our methods, running simulations, and including the imputed genomes of the missing ancestors. This can hopefully be published as a conference paper later this year.

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Mary Allen holds up a valentine sent to her from a childhood friend. It sits in her cubicle where she is hard at work tearing apart genomic data looking for patterns. This friend, who has Down syndrome, is part of the reason that Allen, a postdoctoral researcher in  at the , became interested in studying aneuploidy. Aneuploidy means that cells have too many, or too few, of one or more chromosomes. In the case of Down syndrome, there is an extra copy of chromosome 21. Allen is exploring what makes people with this extra chromosome survivors.

“Down syndrome is actually not all that survivable,” says Allen. “Only 25 percent of embryos with three copies of chromosome 21 survive to live birth. These people who are surviving and living long lives have something in their DNA—from their genetic background—that is helping them.”

Down syndrome is the most commonly occurring chromosomal condition and more than 400,000 people in the United States are currently living with it. Allen is right about them being survivors. According to the , life expectancy for people with the syndrome has increased dramatically from 25 years in 1983 to 60 years now, due in part to better educational programs, health care and support from families and communities.

Allen is taking genetic sequencing data from people with Down syndrome and their parents to understand how that extra copy of chromosome 21 puts this population at higher risk for health issues such as heart defects, thyroid conditions, leukemia, , and respiratory and hearing problems. She is also trying to understand why they are at lower risk for heart attack, stroke, and solid tumor cancers. Allen isn’t out to find a cure for Down syndrome. Her goal is to find what in their DNA is helping these survivors, and how can we design targeted molecular therapy to help them have better lives.

“Once you have had a friend with Down syndrome, stopping the occurrence of the syndrome isn’t on the table,” says Allen. “They are just such great people.”

Allen recently was awarded a Sie Foundation Postdoctoral Fellowship to continue her Down syndrome research for the next two years. This fellowship was created under the Anna and John J. Sie Endowment Fund for the BioFrontiers Institute, which is targeted specifically at funding research to prevent the cognitive and medical ill effects associated with the extra chromosome 21. The fellowship is offered as a collaboration between BioFrontiers and the  at the University of Colorado, Anschutz Medical Campus.

The BioFrontiers Institute also awarded Sie Fellowships to Geertruida Josien Levenga of CU-�鶹ӰԺ’s Institute of Behavioral Genetics and to  of CU-�鶹ӰԺ’s Department of Molecular, Cellular and Developmental Biology. Dr. Levenga is a neuroscientist whose research holds promise for ameliorating the seizures that afflict so many individuals with Down syndrome. Dr. Garrido-Lecca will test the hypothesis that alteration of microRNA levels in individuals with Down syndrome contributes to some of their health challenges.

Dr. Allen sees the new fellowship as welcome news for her work. Research funding for Down syndrome has always been extremely low. The National Institutes of Health in 2012 allocated only $50 in research funding per person living with the condition, versus $270 for Fragile X research, $329 for multiple sclerosis research and $2,867 for cystic fibrosis research. Individuals with Down syndrome have special health needs, like heart conditions and decreased immunity, which can be helped by further research. In addition, since Alzheimer’s disease, leukemia, low muscle tone and weight gain are seen at a high incidence in people with Down syndrome, researching the syndrome may lead to treatments for these associated disorders in the broader population.

“Research on the smaller ear canals of people with Down syndrome is now helping people who suffer from deafness and other auditory disorders,” says Allen. “Unlocking the cellular processes behind one disorder can help us with so many others.”

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