Extensive Variation in Chromatin States Across Humans

The majority of disease-associated variants lie outside protein-coding regions, suggesting a link between variation in regulatory regions and disease predisposition. We studied differences in chromatin states using five histone modifications, cohesin, and CTCF in lymphoblastoid lines from 19 individuals of diverse ancestry. We found extensive signal variation in regulatory regions, which often switch between active and repressed states across individuals. Enhancer activity is particularly diverse among individuals, whereas gene expression remains relatively stable. Chromatin variability shows genetic inheritance in trios, correlates with genetic variation and population divergence, and is associated with disruptions of transcription factor binding motifs. Overall, our results provide insights into chromatin variation among humans

Functional annotation of microRNAs in signal transduction pathways via high-throughput cell based screening

MicroRNAs (miRNAs) are a class of regulatory genes whose role in cell biology is increasingly appreciated [1]. In metazoans, miRNAs mediate post-transcriptional regulation by base-pairing with partially complementary sequences in their target transcripts. This interaction with mRNA frequently inhibits its translation but can also result in its deadenylation and consequent degradation [2]. Although estimates vary, miRNAs are thought to target as much as 60% of human genes, highlighting their potential as master regulators of gene expression [3, 4]. However, despite the identification of hundreds of human miRNAs and computational predictions of thousands of their target genes, many lack experimentally validated functions. Functional genomics methodologies such as cell-based screening (CBS) offer a high-throughput approach to close the gap between the identification of miRNAs and understanding their function. To this end, this thesis examines the role of miRNAs in the regulation of signal transduction pathways using CBS. I identified and characterized novel regulators of NF-κB and CREB signaling. Notably, I found miR-517a and miR-517c were potent activators of NF-κdemonstrated that miR-132 could act as a feedback repressor or activator of CREB signaling depending on the pathway stimulus and the promoter makeup of CREB target genes. Lastly, I developed a novel computational pipeline to deduce functional coding components of a pathway by integrating CBS data and miRNA target predictions. Together these studies extend our knowledge of the general function of miRNAs and serve as a resource for their functional output in signal transduction pathways

Functional annotation of microRNAs in signal transduction pathways via high-throughput cell based screening

MicroRNAs (miRNAs) are a class of regulatory genes whose role in cell biology is increasingly appreciated [1]. In metazoans, miRNAs mediate post-transcriptional regulation by base-pairing with partially complementary sequences in their target transcripts. This interaction with mRNA frequently inhibits its translation but can also result in its deadenylation and consequent degradation [2]. Although estimates vary, miRNAs are thought to target as much as 60% of human genes, highlighting their potential as master regulators of gene expression [3, 4]. However, despite the identification of hundreds of human miRNAs and computational predictions of thousands of their target genes, many lack experimentally validated functions. Functional genomics methodologies such as cell-based screening (CBS) offer a high-throughput approach to close the gap between the identification of miRNAs and understanding their function. To this end, this thesis examines the role of miRNAs in the regulation of signal transduction pathways using CBS. I identified and characterized novel regulators of NF-κB and CREB signaling. Notably, I found miR-517a and miR-517c were potent activators of NF-κdemonstrated that miR-132 could act as a feedback repressor or activator of CREB signaling depending on the pathway stimulus and the promoter makeup of CREB target genes. Lastly, I developed a novel computational pipeline to deduce functional coding components of a pathway by integrating CBS data and miRNA target predictions. Together these studies extend our knowledge of the general function of miRNAs and serve as a resource for their functional output in signal transduction pathways

Placental expression of miR-517a/b and miR-517c contributes to trophoblast dysfunction and preeclampsia.

Preeclampsia is a pregnancy specific hypertensive disease that confers significant maternal and fetal risks. While the exact pathophysiology of preeclampsia is unknown, it is widely accepted that placental dysfunction is mechanistically involved. Recent studies reported aberrant expression of placenta-specific microRNAs (miRNAs) in preeclampsia including miR-517a/b and miR-517c. Using placental biopsies from a preeclampsia case-control study, we found increased expression of miR-517a/b in term and preterm preeclampsia vs controls, while, miR-517c was increased only in preterm preeclampsia vs controls. To determine if miR-517a/b and miR-517c are regulated by hypoxia, we treated first trimester primary extravillous trophoblast cells (EVTs) with a hypoxia mimetic and found both were induced. To test for a mechanistic role in placental function, we overexpressed miR-517a/b or miR-517c in EVTs which resulted in decreased trophoblast invasion. Additionally, we found that miR-517a/b and miR-517c overexpression increased expression of the anti-angiogenic protein, sFLT1. The regulation of sFLT1 is mostly unknown, however, TNFSF15, a cytokine involved in FLT1 splicing, was also increased by miR-517a/b and miR-517c in EVTs. In summary, we demonstrate that miR-517a/b and miR-517c contribute to the development of preeclampsia and suggest that these miRNAs play a critical role in regulating trophoblast and placental function

miR-517a/b and miR-517c regulates trophoblast invasion.

<p>Bar graph results from invasion assays of EVT cells transfected with (<b>A</b>) miR-517a/b/c mimics or or (<b>B</b>) miR-517a/b/c inhibitors. Associated light microscopy photographs of invaded cells transfected with (<b>C</b>) miR-517a/b/c mimics or (<b>D</b>) miR-517a/b/c inhibitors. Values in (<b>A) and (B</b>) are expressed as a percent of control (miR-negative or anti-miR-negative transfected cells). n = 3. *p<0.001</p

miR-517a/b and miR-517c increases TNFSF15 expression in trophoblast cells.

<p>TNFSF15 expression in extravillous trophoblast cells transfected with miR-negative control, miR-517a/b and miR-517c. Values are mean ± SEM. n = 5 *p<0.001.</p

mRNA expression levels of TNFSF15 associated pathway genes involved in sFLT1 regulation in first trimester extravillious trophoblast cells transfected with miR-517a/b and miR-517c.

<p>mRNA expression levels of TNFSF15 associated pathway genes involved in sFLT1 regulation in first trimester extravillious trophoblast cells transfected with miR-517a/b and miR-517c.</p

Placental miR-517a/b and miR-517c expression is upregulated in preeclampsia.

<p>Boxplots of miR-517a/b (<b>A</b>) and miR-517c (<b>B</b>) expression in human placentas from a preeclampsia case-control study. Case cohort (case-all) samples were divided into preterm (case-preterm) and term (case-term) subgroups. All comparisons are relative to the control group. *p<0.05</p

miR-517a/b and miR-517c increases the release of sFLT1 from trophoblast cells.

<p>sFtl1 (<b>A</b>), VEGF (<b>B</b>) and PLGF (<b>C</b>) levels in cell culture media after extravillous trophoblast cell transfection with miR-negative control, miR-517a/b and miR-517c. VEGF and PLGF levels were unchanged by miR-517a and miR-517c transfection. Values are mean ± SEM. n = 4 *p<0.001.</p

Schematic of hypothesized molecular pathways contributing to preeclampsia.

<p>Based on the results of this study, we provide evidence that abnormal pathologic placental hypoxia early in pregnancy could lead to increased miR-517a/b and miR-517c expression resulting in alterations in trophoblast function including 1) decreased trophoblast invasion and 2) increased TNFSF15 expression. Consequently, TNFSF15 is able to mediate FLT1 splicing and increase sFLT1 expression leading to a decrease in placental angiogenesis. Therefore, miR-517 mediated alterations in trophoblast function could propagate the placental hypoxia and contribute to the development of preeclampsia.</p