عنوان

Visualization of transcriptional dynamics in the early Drosophila embryo

Number

TL9cm913xj

.Language of Text, Soundtrack etc

انگلیسی

Title Proper

Visualization of transcriptional dynamics in the early Drosophila embryo

General Material Designation

[Thesis]

First Statement of Responsibility

Esposito, Emilia Esposito

Subsequent Statement of Responsibility

Levine, Michael S

Name of Publisher, Distributor, etc.

UC Berkeley

Date of Publication, Distribution, etc.

2016

Body granting the degree

UC Berkeley

Text preceding or following the note

2016

Text of Note

To the observer it is immediately visible that an elephant and an ant are verydifferent animals. However, when observed at a molecular level, we realize thatthey share a comparable basic machinery of development. This concept isessentially true for all the animals, either vertebrates or invertebrates.Over the years, different biologists tried to explain how phenotypic diversity iscreated, to conclude that living beings are made of similar genetic material.However, the instructions that drive their development may vary considerably.To make a simple comparison, we can combine the same ingredients to formmultiple types of food. For example, we can mix flour, salt, water and a bit ofyeast to make pizza dough. The same ingredients can be used to make bread orsavory cookies. The final products depend on the quantity ratio among theseingredients, the preparation, cooking strategy and the time we allow for each ofthese products to develop consistency and flavor.Similarly, the same genes may lead to the formation of various cell fates justbecause they are expressed (or not) at different levels or different time in thatspecific cell. But what does regulate when, where and how much a particulargene is expressed? In other words, where can we find the manual that instructson how organism form and develop?When in the sixties, Jacob and Monod described that in bacteria the Lac operontranscription is controlled by non-coding elements positioned right upstream thegene (Jacob & Monod 1961), no one would imagine that a similar process wouldregulate gene expression in eukaryotic cells. It took about two extra decades todiscover that also in eukaryotes some non-coding DNA elements were able toenhance the activity of a gene (for a complete review on enhancer discovery, seeSchaffner 2015). Contrarily to bacteria, these elements, called enhancers, werefunctioning in any orientation and also on a distance. At the same time, it wasdiscovered that enhancers contain binding sites for activator and repressorproteins (Lewis 1978). Thus, a gene could be either active or inactive dependingon whether activators (or repressors) were bound to its enhancer. When all thegenes of a multicellular organism are expressed at the right place, time and in theright quantity, development proceeds normally.Regulation of gene expression is far from a static process; on the contrary it ischaracterized by a series of dynamic events. The amount of regulatory proteinschanges constantly among cells and even within the same cell. To complicatethings further, enhancers can be found everywhere in the genome, often, faraway from the gene they regulate. More over, DNA is a very mobile molecule andacquires multiple conformations that potentially could prevent, or favor, proteinaccessibility to regulatory domains and interaction between enhancers andgenes.Until recently, most of the information about gene expression derived fromexperimental analysis in fixed biological samples, using for example in situhybridization assays that allow detection of nuclear and cytoplasmic mRNA. Thistechnique has been useful to understand the spatial information of geneexpression. Yet, in fixed samples it is very challenging to deduce the dynamicchanges that occur in a developing embryo.The continuous advancement in the molecular biology field, improvements on thevisualization and computational techniques, allow now seeing and analyzingbiological processes occurring in real time at increasingly higher resolution. This,together with collaborations among people with different expertise, represents amulti-disciplinary task force to understand the rules that govern gene regulationand, to a large extent, how multicellular organisms are produced.In this thesis I intend to discuss how newly developed imaging techniques allowto push our knowledge forward about gene regulation dynamics. I will presentthree instances in which we reveal subtle mechanisms that fine tune mRNAproduction in Drosophila melanogaster embryos. Gene regulation is a pervasivefeature that underlies the formation of all living beings and I believe that by beingable to observe and analyze how biological processes occur in real-time in vivowe will, to some extent, be able to comprehend how life is generated.This thesis consists of four chapters. In the first introductory chapter I present thebackground on gene regulation and imaging techniques. In particular I focus onthe bacteriophage MCP-MS2 system since this has been my elective way tovisualize transcription. In the following chapters I discuss my work done incollaboration with several bright people to visualize transcriptional dynamicsduring the hours that precede gastrulation in the Drosophila embryo. I presenthow transcription occurs in burst of expression in a pair-rule gene (chapter 2),describe the existence of post-mitotic transcriptional memory (chapter 3) and, inthe forth chapter, I report the analysis done to understand how transcriptionalrepression regulates gene activity during the formation of the mesoderm ectodermboundary. In this work we provide evidence that mitotic silencingfacilitates repression and we suggest a model whereby repressor exploit pausesin transcriptional activity to ensure rapid gene inactivation.

Esposito, Emilia Esposito

Levine, Michael S

UC Berkeley

Electronic name

p

[Thesis]

276903

a

Y