Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 167-187). / Posttranscriptional regulation of mRNA by RNA-binding proteins plays key roles in regulating the transcriptome over the course of development, between tissues and in disease states. The specific interactions between mRNA and protein are controlled by the proteins' inherent affinities for different RNA sequences as well as other features such as translation and RNA structure which affect the accessibility of mRNA. The stabilities of mRNA transcripts are regulated by nonsense-mediated mRNA decay (NMD), a quality control degradation pathway. In this thesis, I present a novel method for high throughput characterization of the binding affinities of proteins for mRNA sequences and an integrative analysis of NMD using deep sequencing data. This thesis describes RNA Bind-n-Seq (RBNS), which comprehensively characterizes the sequence and structural specificity of RNA binding proteins (RBPs), and application to the developmentally-regulated splicing factors RBFOX2, MBNL1 and CELF1/CUGBP1. For each factor, the canonical motifs are recovered as well as additional near-optimal binding motifs. RNA secondary structure inhibits binding of RBFOX2 and CELF1, while MBNL1 favors unpaired Us but tolerates C/G pairing in UGC-containing motifs. In a project investigating how NMD shapes the embryonic transcriptome, this thesis presents integrated genome-wide analyses of UPF1 binding locations, NMD-regulated gene expression, and translation in murine embryonic stem cells (mESCs). Over 200 direct UPF1 binding targets are identified using crosslinking/immunoprecipitation-sequencing (CLIP-seq). Results from ribosome foot printing show that actively translated upstream open reading frames (uORFs) are enriched in transcription factor mRNAs and predict mRNA repression by NMD, while poorly translated mRNAs escape repression. / by Alexander De Jong Robertson. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Transmission of information from DNA to RNA to protein underlies the core of modem life forms. The advance in sequencing and genetic technologies has revolutionized the study of molecular biology, genetics and developmental biology enabling delineation of biological processes in unprecedented details. Through the study of epigenetics and posttranscriptional regulation of gene expression by high-throughput sequencing technologies in several biological processes, namely embryonic stem cells, somatic reprogramming, erythroid differentiation, epithelial-mesenchymal transition and cancer metastasis, this thesis work has identified novel players and regulatory mechanisms underlying these developmental processes and diseases. Furthermore, an attempt to engineer CRISPRzymes - protein fusions of RNA-guided DNA binding dCas9 - will enable experiments to directly test biological processes at defined genomic loci and expands the toolbox for synthetic biology and potentially opens up opportunities for novel therapeutics. / by Wu Albert Cheng. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Mammalian spermatogenesis includes two types of cell divisions. First, germ cells undergo transit-amplifying mitotic divisions, which enable prodigious output of mature spermatozoa. Second, they undergo reductive meiotic divisions to produce haploid gametes. In this thesis, I examine gene expression and regulation during the mitotic and meiotic phases of spermatogenesis. Chapter 2 describes how RA-STRA8 signaling regulates two key transitions: spermatogonial differentiation, which begins the transit-amplifying mitotic divisions, and meiotic initiation, which ends them. First, in mice lacking the RA (retinoic acid) target gene Stra8, undifferentiated spermatogonia accumulated; thus, Stra8 promotes spermatogonial differentiation as well as meiotic initiation. Second, injection of RA into wild-type males induced precocious spermatogonial differentiation and meiotic initiation; thus, RA acts instructively on germ cells at both transitions. Finally, competencies of germ cells to undergo spermatogonial differentiation or meiotic initiation in response to RA were found to be distinct and periodic. Chapter 3 describes a novel method for isolating precise populations of mitotic and meiotic germ cells from the mouse testis. We first synchronize germ cell development in vivo, and perform histological staging to verify synchronization. We then separate these germ cells from contaminating somatic and stem cells by FACS, to achieve ~90% purity of each distinct germ cell type, from the stem cell pool through mid/late meiotic prophase. Utilizing this "3S" method (synchronize, stage, and sort), we can robustly and efficiently separate germ cell types that were previously challenging or impossible to distinguish, with sufficient yield for transcriptomic and epigenetic studies. Chapter 4 presents a systematic comparison of the male and female gene expression programs of meiotic prophase. We performed transcriptional profiling of postnatal testes synchronized in precise stages of meiotic prophase, and compared to the same stages in the fetal ovary. We identified 260 genes up-regulated during both male and female prophase; this shared gene set represents a core meiotic program, composed of known and potential novel meiotic players. We also identified over two thousand genes that are up-regulated during meiotic prophase specifically in the male. These comprise both a male-specific meiotic program, and a preparatory program for cellular differentiation of spermatozoa. / by Katherine A. Romer. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2014. / Cataloged from PDF version of thesis. / Includes bibliographical references / The vast majority of the mammalian genome does not encode proteins. Only 2% of the genome is exonic, yet recent deep survey of human transcripitome suggested that 75% of the genome is transcribed, including half of the intergenic regions. Such pervasive transcription typically leads to short-lived, low-copy number noncoding RNAs (ncRNAs). We are starting to understand the biogenesis and mechanisms regulating the noncoding transcription. However, it is still unclear what's the functional impact of pervasive transcription and the ncRNAs at the level of the'genome, the cell, and the organism. A large fraction of ncRNAs in cells is generated by divergent transcription that occurs at the majority of mammalian gene promoters. RNA polymerases transcribe divergently on opposite strands, producing precursor mRNAs (pre-mRNAs) on one side and promoter upstream antisense RNAs (uaRNAs) on the other side. Like typical products of pervasive transcription, uaRNAs are relatively short and unstable as compared to pre-mRNAs, suggesting there are mechanisms suppressing uaRNA transcription and enforcing promoter directionality. We describe the Ul-PAS axis, a mechanism that enhances gene transcription but suppresses noncoding transcription. Two RNA processing signals, the Ul signal, or 5' splice site sequences recognized by Ul snRNP during splicing, and polyadenylation signal (PAS), differentially mark the two sides of gene transcription start site (TSS), ensuring the generation of full-length mRNA but inducing early termination of uaRNAs. The Ul-PAS axis also suppresses pervasive transcription on the antisense strand of genes, as well as intergenic transcription. Transcription is a mutagenic process that could accelerate evolution. We uncover a link between pervasive transcription and genome evolution. Specifically, transcription-induced mutational bias in germ cells could strengthen the Ul-PAS axis, which in turn enhances transcription, thus forming a positive feedback loop, which eventually drives new gene origination, and facilitates genome rearrangements. Tools to directly interfere with transcription with specificity are necessary to understand the function of noncoding transcription, especially when the RNA product is rapidly degraded or nonfunctional. The newly emerged CRISPR-Cas9 system provides the opportunity to target any desired locus. We comprehensively characterize the binding specificity of Cas9 in the mouse genome. We find that Cas9 specificity varies dramatically but in a predictable manner, depending on the seed sequence and chromatin accessibility. Our results will facilitate Cas9 target design and enable genome manipulation with high precision. / by Xuebing Wu. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2010. / Cataloged from PDF version of thesis. / Includes bibliographical references (p. 157-168). / The epidermal growth factor receptor (EGFR, also known as ErbB 1) is a prototypical receptor tyrosine kinase (RTK) that activates multi-kinase phosphorylation cascades to regulate diverse cellular processes, including proliferation, migration and differentiation. ErbB 1 heterooligomerizes with three close homologues: ErbB2, ErbB3 and ErbB4. ErbB1-3 receptors are frequently mutated, overexpressed or activated by autocrine or paracrine ligand production in solid tumors and have been the target of extensive drug discovery efforts. Multiple small molecule kinase inhibitors and therapeutic antibodies against ErbB receptors are in clinical use or development. Despite their importance as RTKs, oncogenes and drug targets, regulation of ErbB receptors by the interplay of conformational change, phosphorylation, phosphatases and receptor trafficking remains poorly understood, and the impact of these dynamics on physiological activity and cellular responses to anti-ErbB drugs is largely unknown. This thesis investigates the dynamic opposition of kinases and phosphatases within the ErbB pathway. By standard biochemical analysis, ErbB receptors and downstream proteins appear to become phosphorylated and then dephosphorylated in approximately 30 minutes. However, pulse chase experiments where cells are exposed to ligand and then to small molecule kinase inhibitors reveal that individual proteins must in fact cycle rapidly between being phosphorylated and dephosphorylated in seconds. We construct a succession of differential equation-based models of varying biochemical resolution, each model appropriate for analyzing a different aspect of ErbB regulation, to help interpret the data and gain quantitative insight into receptor and drug biology. Rapid phosphorylation and dephosphorylation of receptors has important implications for the assembly dynamics of signalosomes. We find that signals are rapidly propagated through some downstream pathways but slowly through others, resulting in prolonged activation in the absence of upstream signal. We show that fast phosphorylation/dephosphorylation may provide cells with the flexibility necessary to rapidly detect and respond to changes in their extracellular environment. These fast dynamics also play a crucial role in determining the response to ErbB 1-targeting cancer therapies, which we find to vary significantly between drugs with different mechanisms of action. We show that treatment with one class of these drugs results in sustained signaling, instead of inhibition, and thus may actually promote tumor proliferation or invasion. Our work may help explain why certain drugs have been more effective in patients than others and suggests new approaches for evaluating biochemical signaling networks and targeted therapeutics. / By Laura B. Kleiman. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2012. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student submitted PDF version of thesis. / Includes bibliographical references. / Understanding the effectiveness of a drug therapy on halting disease progression is an essential aspect of cancer biology. Conventional assays that study cell behavior after a drug intervention report the average response of a cell population which can mask the heterogeneity and dynamics of seemingly identical cells. Recently, many single-cell techniques have been developed, but there are currently no methods that can fully characterize the long-term effects of drug treatment on cancer cell growth. To accomplish such, we developed an instrument to measure single-cell growth before and after drug treatment. In order to achieve femtogram-level mass resolution, we employed the suspended microchannel resonator (SMR), a vacuum-packaged cantilever with an embedded channel. Here, we describe three implementations that involve different technologies (optical trap, mechanical trap, and dynamic ow trapping) to capture a cell for repeated measurements and to perform drug delivery. Applying the technique we developed based on the dynamic ow trapping, we were able to monitor one or more generations of a cancer cell before and after drug treatment. We investigated the growth of mouse leukemia cells in response to drugs that inhibit the mammalian target of rapamycin (mTOR) pathway, induce apoptosis, or prevent translational activity directly at the ribosome. Our method was able to discern a particular growth signature for each drug investigated and to discover a new phenotype in cells following mTOR inhibition. Furthermore, our data demonstrates that the instantaneous growth rate changes following a drug treatment could potentially predict the long-term inhibitory effect on cellular biogenesis and mass accumulation. / by Yaochung Weng. / Ph.D.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2016. / Cataloged from PDF version of thesis. / Includes bibliographical references. / A protein's function in the cell depends on its structure, which in turn depends on the intracellular environment. Stress like heat shock or nutrient starvation can alter intracellular conditions, leading to protein misfolding - i.e. the inability of a protein to reach or maintain its native conformation. Since many proteins interact with each other, protein misfolding and cellular stress response must be examined both on the scale of individual protein conformational changes and on a more global level, where interaction patterns can reveal larger-scale protein responses to cellular stress. On the individual scale, one example of a protein particularly susceptible to misfolding is the human von Hippel-Lindau (VHL) tumor suppressor. When expressed in the absence of its cofactors, VHL cannot fold correctly and is quickly degraded by the cell's quality control machinery. Here, I present a biophysical characterization of a VHL mutation that confers increased resistance to misfolding. Mathematical modeling provides an explanation for this mutant's increased stability in the cell by predicting how its cofactor and chaperone interaction sites are buried or exposed in the protein's predicted conformation. On a more global level, a budding yeast cell undergoing glucose deprivation both acidifies its cytosol and exhibits widespread protein clustering. By employing a proteome-wide computational assay, I examine how this drop in pH could lead to the formation of higher order protein structures. This modeling framework also provides a rationale for why these two related phenotypes might be beneficial, since protein clustering can help regulate relevant metabolic pathways and provide protection from protein misfolding and/or degradation. / by Kelly Paige Brock. / Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Computational and Systems Biology Program, 2018. / This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. / Cataloged from student-submitted PDF version of thesis. / Includes bibliographical references (pages 205-226). / RNA not only shuttles information between DNA and proteins but also carries out many other essential cellular functions. Nearly all steps of an RNA's life cycle are controlled by approximately one thousand RNA binding proteins (RBPs) that direct RNA splicing, cleavage and polyadenylation, localization, translation, and degradation. Despite the central role of RBPs in RNA processing and gene expression, they have been less well studied than DNA binding proteins, in part due to the historical dearth of technologies to probe RBP binding and activity in a high-throughput, comprehensive manner. In this thesis, I describe the affinity landscapes of the largest set of human RBPs to date elucidated through a highthroughput version of RNA Bind-N-Seq (RBNS), an unbiased in vitro assay that determines the primary sequence, secondary structure, and contextual preferences of an RBP. The 78 RBPs bound an unexpectedly low diversity of RNA motifs, implying convergence of binding specificity toward a small set of RNA motifs characterized by low compositional complexity. Offsetting the low diversity of sequence motifs, extensive preferences for contextual features beyond short linear motifs were observed, including bipartite motifs, flanking nucleotide content, and preference for or against RNA structure. These features likely refine which motif occurrences are selected in cells, enabling RBPs that bind the same linear motif to act on distinct subsets of transcripts. Additionally, RBNS data is integrated with complementary in vivo binding sites from enhanced crosslinking and immunoprecipitation (eCLIP) and functional (RNAi/RNA-seq) data produced through collaborative efforts with the ENCODE consortium. These data enable creation of "RNA maps" of RBP activity in pre-mRNA splicing and gene expression levels, either with (eCLIP) or without (RBNS) crosslinking-based assays. The mapping and characterization of RNA elements recognized by over 200 human RBPs is also presented in two human cell lines, K562 and HepG2 cells. Together, these novel data augment the catalog of functional elements encoded in the human genome to include those that act at the RNA level and provide a basis for how RBPs select their RNA targets, a fundamental requirement in more fully understanding RNA processing mechanisms and outcomes. / by Peter Freese. / Ph. D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / Cellular signaling and regulatory networks underlie fundamental biological processes such as growth, differentiation, and response to the environment. Although there are now various high-throughput methods for studying these processes, knowledge of them remains fragmentary. Typically, the majority of hits identified by transcriptional, proteomic, and genetic assays lie outside of the expected pathways. In addition, not all components in the regulatory networks can be exposed in one experiment because of systematic biases in the assays. These unexpected and hidden components of the cellular response are often the most interesting, because they can provide new insights into biological processes and potentially reveal new therapeutic approaches. However, they are also the most difficult to interpret. We present a technique, based on the Steiner tree problem, that uses a probabilistic protein-protein interaction network and high confidence measurement and prediction of protein-DNA interactions, to determine how these hits are organized into functionally coherent pathways, revealing many components of the cellular response that are not readily apparent in the original data. We report the results of applying this method to (1) phosphoproteomic and transcriptional data from the pheromone response in yeast, and (2) phosphoproteomic, DNaseI hypersensitivity sequencing and mRNA profiling data from the U87MG glioblastoma cell lines over-expressing the variant III mutant of the epidermal growth factor receptor (EGFRvIII). In both cases the method identifies changes in diverse cellular processes that extend far beyond the expected pathways. Analysis of the EGFRVIII network connectivity property and transcriptional regulators that link observed changes in protein phosphorylation and differential expression suggest a few intriguing hypotheses that may lead to improved therapeutic strategy for glioblastoma. / by Shao-shan Carol Huang. / Ph.D.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Computational and Systems Biology Program, 2011. / Cataloged from PDF version of thesis. / Includes bibliographical references. / An ability to control the degree of heterogeneity in cellular phenotypes may be important for cell populations to survive uncertain and ever-changing environments or make cell-fate decisions in response to external stimuli. Cells may control the degree of gene expression heterogeneity and ultimately levels of phenotypic heterogeneity by modulating promoter switching dynamics. In this thesis, I investigated various mechanisms by which heterogeneity in the expression of FLO 11 in S. cerevisiae could be generated and controlled. First, we show that two copies of the FLOJ1 locus in S. cerevisiae switch between a silenced and competent promoter state in a random and independent fashion, implying that the molecular event leading to the transition occurs in cis. Through further quantification of the effect of trans regulators on both the slow epigenetic transitions between a silenced and competent promoter state and the fast promoter transitions associated with conventional regulation of FLO11, we found different classes of regulators affect epigenetic, conventional, or both forms of regulation. Distributing kinetic control of epigenetic silencing and conventional gene activation offers cells flexibility in shaping the distribution of gene expression and phenotype within a population. Next, we demonstrate how multiple molecular events occurring at a gene's promoter could lead to an overall slow step in cis. At the FLO] 1 promoter, we show that at least two pathways that recruit histone deacetylases to the promoter and in vivo association between the region -1.2 kb from the ATG start site of the FLO11 ORF and the core promoter region are all required for a stable silenced state. To generate bimodal gene expression, the activator Msnlp forms an alternate looped conformation, where the core promoter associates with the non-coding RNA PWR1's promoter and terminator regions, located at -2.1 kb and -3.0 kb from the ATG start site of the FLO]1 ORF respectively. Formation of the active looped conformation is required for Msnlp's ability to stabilize the competent state without destabilizing the silenced state and generate a bimodal response. Our results support a model where multiple stochastic steps at the promoter are required to transition between the silenced and active states, leading to an overall slow step in cis. Finally, preliminary investigations of heterozygous diploids revealed possible transvection occurring at FLO] 1, where a silenced allele of FLO 11 appeared to transfer silencing factors to a desilenced FLO11 allele on the homologous chromosome. These observations suggest a new mechanism through which heterogeneity in FL011 expression could be further controlled, in addition to the molecular events at the FL011 promoter we elucidated previously. / by Leah M. Octavio. / Ph.D.