FGRS: research description & training
 

Stream Research Summary


The genome of an organism is most simply the complete set of all genes contained on a single (haploid) set of chromosomes.  The advent of DNA sequencing provided a more complete view of the genome.  Thus, the term genome often now refers to the ordered DNA sequence across all chromosomes of an organism.


Functional genomics is the intersection of experimental molecular biology with sequence-based genomics.  The field takes advantage of various experimental techniques and sources of complementary data in order to better characterize the roles of genes, proteins, and other components of an entire genome as it operates and interacts.


The Functional Genomics Research Stream is focused upon using high-throughput next-generation technologies in order to characterize the molecular interactions that define cell cycle transcriptional regulation and progression.  These include the analysis of nucleosomes, transcription factors, and transcriptional abundance in such a way so as to produce novel understanding of how and when each of these regulatory components either affects or is affected by a regulatory interaction.




Specific techniques utilized by the research stream are capable of measuring nucleosome position (A) and modification (D).  Mapping of nucleosome occupation is possible through specific purification of nucleosome-bound DNA fragments (ChIP-seq).  Expression profiling

(B) is accomplished by quantifying absolute transcript copies for all actively transcribed regions of a genome (RNA-seq).  Finally, transcription factors (C) can be mapped to genomic loci thus facilitating the characterization of regulatory DNA sequence motifs (ChIP-seq).


Research Methods and Technologies

The study my colleagues and I published in 2007 focused on the characterization of discrete deletions of transcription factors in S. cerevisiae.  By performing whole-genome expression profiling on individual deletion strains we were able to establish target genes for hundreds of transcription factors.  Computational analysis characterized a functional network of transcription factor interaction and described the depth of cascading regulation.  Biological pathway enrichment analysis enabled the functional characterization of many unannotated regulators.

The Functional Genomics Research Stream is now engaged in the characterization of dozens of these transcription factors across four species of Saccharomyces sensu stricto including Saccharomyces bayanus, cerevisiae, mikatae and paradoxus. While molecular evolution within these species has beens studied, several of the publications are computational in their methodology and predictive in their findings.  Other publications engaged experimental methods but focused on only a few key regulators and binding regions.  Most of our experiments will be performed using treatments that will allow us to characterize the function of unannotated regulators and elucidate the extent to which molecular evolution has affected their specific gene targets in individual species of yeast.  We will profile transcription factors under conditions most similar to those in which they are cellularly active.  For example, when investigating unannotated transcription factors implicated by our biological pathway analysis to be involved in DNA damage repair, we will use methyl methanesulfonate (MMS) treatments to induce double-stranded DNA breaks.  We will then measure whole genome gene expression across discrete deletion mutants of each species using next generation sequencing (RNA-seq).


Relevant Literature


Saccharomyces cerevisiae

Simon et al. Serial regulation of transcriptional regulators in the yeast cell cycle. Cell (2001) vol. 106 (6) pp. 697-708


Spellman et al. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization.

Mol. Biol. Cell (1998) vol. 9 (12) pp. 3273-97


Gasch et al. The genomics of yeast responses to environmental stress and starvation.

Funct Integr Genomics (2002) vol. 2 (4-5) pp. 181-92


Harbison et al. Transcriptional regulatory code of a eukaryotic genome.

Nature (2004) vol. 431 (7004) pp. 99-104


Hu et al. Genetic reconstruction of a functional transcriptional regulatory network.

Nat. Genet. (2007) pp.


Next Generation Sequencing

Farnham. Insights from genomic profiling of transcription factors. Nat Rev Genet (2009) vol. 10 (9) pp. 605-16


Ansorge. Next-generation DNA sequencing techniques. N Biotechnol (2009) vol. 25 (4) pp. 195-203


Park. ChIP-seq: advantages and challenges of a maturing technology. Nat Rev Genet (2009)


Lefrançois et al. Efficient yeast ChIP-Seq using multiplex short-read DNA sequencing. BMC Genomics (2009) vol. 10 pp. 37


Shendure et al. Next-generation DNA sequencing.

Nat Biotechnol (2008) vol. 26 (10) pp. 1135-45


Mardis. Next-generation DNA sequencing methods.

Annual review of genomics and human genetics (2008) vol. 9 pp. 387-402


Morozova et al. Applications of next-generation sequencing technologies in functional genomics.

Genomics (2008) pp.


Kharchenko et al. Design and analysis of ChIP-seq experiments for DNA-binding proteins. Nat Biotechnol (2008)

 

Introduction

The FRI is a ground-breaking and highly special opportunity for undergraduate students interested in research.  Typically, undergraduates do not have the chance to get involved with research until their junior or senior years.  Once they do they are slow to start projects due to lack of comprehensive training.

Thus, most undergraduate research experiences are delayed, shallow and rarely lead to real results for the students and the professors involved.  Your experience with the FRI has the opportunity change all of that - are you ready?


Stream Philosophy

I am primarily interested in achieving two outcomes for you per your membership in the research stream:

  1. 1.You will experience your education at the University of Texas in a fundamentally different way through exposure to research concepts, techniques, technologies and mind-sets.

    It is my goal that during the year you spend with me your educational process is transformed.  Everyone knows that classes can seem monotonous, pointless and exercises in checking off boxes.  You wonder endlessly why you are learning concepts and wonder when, where and why you may ever use them.  We have all been there (we have all been students).

    The FRI and your membership in my stream has the ability to change your experience.  Everything that I teach you will be backed up by an understanding of why.  I will not just be training your hands in the name of research productivity;  I will be training you to seek understanding of processes and procedures as they relate to modern biology, chemistry and bioinformatics.  You will come to understand a great deal about modern genetics, genomics, technologies, techniques and eukaryotic regulation of gene expression.  We will discuss fairly advanced concepts such as DNA microarrays, qPCR, next-generation sequencing methods, ChIP-seq and RNA-seq.  Through exposure to this material many of your other classes will begin to come “in focus”.  You will see and understand why your are learning what you are learning elsewhere.   Trust me.  The stepwise initiation of transcription, for example, can seem quite inconsequential .... but when you come to
    want to understand it because it will aid you in understanding a research problem - at that moment your education is transformed.  It is my goal that you experience that event and feeling many times under my umbrella.

  2. 2.You will leave my research stream with a packed tool belt - you will be well equipped to pursue and engage an amazing array of research opportunities that may interest you.

    The FRI was started on the idea of both
    exposing undergraduates to research and achieving research results with this student population.  Great.  Sounds good.  I believe these should continue to be the goals of our program.  There can be a conflict of interest at times, however, between those two goals.  The FRI should achieve research results with students - but not at the cost of turning them into glorified laboratory technicians.  I believe that your time with the FRI is more about building your potential in research than building your list of immediate accomplishments.  In this manner I will expose you to a wide breadth of training and development (see below).  I would rather have you learn twenty different techniques with me than two - even if it might cost us a bit of research productivity.  That productivity will be earned back many times over when you move one to research in one of the many labs on campus or around the country (summer programs).  It is my goal that these other researchers that work with you are amazed by the skill you have ready to bring to bear on new problems.


Research

I encourage you to take some time to explore this website.  On it you can learn a great deal about our research agenda, our methods used and the curricula that I use to teach the spring and fall semesters.  You should take a look at some of my training and propaganda videos.  Finally, I have a blog-like environment that my researchers use to track current research issues.  It is the main way in which my research stream communicates on a day-to-day basis.  This environment is called Results Central - be sure to check it out!


Training and Development


Much of this information is lifted from the research page.  I thought that it would interest you to read it here, however.  This is a somewhat formal but accurate enumeration of the main areas in which all of my students are trained.  Many more sub-projects and techniques are also engaged during the course of research.


Training Philosophy

The Functional Genomics Research Stream focuses a significant portion of time in the spring semester on the delivery of a hierarchical and continually building training regimen.  Training is considered paramount at this stage of student development for two primary reasons.


First, the collaborative and individual research engaged by each student during the summer and fall portions of stream membership is quite simply: complicated.  Questions posed by the research stream require the execution of sophisticated in vivo experimentation and subsequent purification methodologies.  In order to engage a single experiment characterizing the in vivo regulatory binding behavior of a transcription factor, for example, a student would be required to aseptically grow a cell culture of a previously PCR-verified epitope-tagged strain of Saccharomyces cerevisiae.  They must manage sterile culture growth to a specific point of log-phase growth, chemically treat the culture, and then engage a multi-day procedure resulting in the purification of genomic DNA fragments correlated with regulatory binding.  The skills engaged in this line of experimentation are diverse and required of every student in the research stream.


The second reason training is significantly emphasized in the initial months of stream membership is one of research portability.  The Functional Genomics Research Stream curricula is specifically designed to achieve short-term (one year) research productivity and deliverables. 


Additionally, it is considered important by the stream leaders that the students leave the research stream with a broadly applicable set of skills that will serve them well in their future research endeavors.  In this manner, meticulous training pays off both now and later for everyone involved in the process; both the immediate beneficiaries in the research stream and the research colleagues who will later accept these trained students into their laboratory environments.


Training Regimen and Skills Delivered

The training regimen begins the first week of the spring semester and is aggregative in nature - each new step utilizes the majority of skills attained in the previous steps.


Professional Scientific Communication

The training hierarchy was purposefully initiated outside the laboratory environment in order to set a continuity of expectation across all students and all research activities - clear, precise and professional communication.


Specifically focused upon is written communication and rigorous maintenance of a laboratory notebook.  These skills are iteratively revisited throughout the spring semester through formal reports, notebook evaluations and interpersonal communications between research staff and students.  We utilize peer review in order to encourage an environment of high expectations and teach the process of collegial review and communication.


Basic Equipment Familiarity

Functional genomics is a cross-disciplinary laboratory field that requires operational familiarity with the equipment that is now typical to both chemistry and molecular biology laboratories.  In the beginning weeks of training students are trained to be capable of accurate and precise mass and volume manipulations (analytical balances, micropipetting).  The researched understanding of both safe and clean handing of reagents is emphasized through familiarity with online resources such as chemical and MSDS databases.


Reagents and Buffer Preparation

With basic equipment familiarity in tow, students next engage a process of learning to produce reagents and solutions that will be used throughout experimentation.  Emphasis is placed upon the understanding of reagent manipulation rather than the simple production of immediately needed reagents so as to empower students to be capable of producing any solution they may need in future research scenarios.  Also emphasized are typical laboratory practices such as the production of concentrated stock solutions, stock dilution, serial dilutions, and multi-component solution production.  All typical methods of solution concentration nomenclature are taught (molarity, weight by mass, weight by volume, etc).


After basic reagent production and dilution is understood students engage the process of understanding the biological and laboratory need for buffered solutions and reagents.  Basic skills in pH measurement are taught with emphasis upon clean and accurate manipulation of buffers to appropriate pH levels.  Students engage the process of characterizing several buffered solutions through titration in order to better understand how buffers are designed and selected for biological experimentation.


Aseptic Cell Culture and Differential Growth

Students have now acquired the ability to use all laboratory equipment and can produce each needed reagent.  We now begin a process of shifting them to focus upon the attainment of biological laboratory skills in order to compliment the chemical skills they have previously mastered.  The Functional Genomics Research Stream utilizes the eukaryotic model organism Saccharomyces cerevisiae (budding yeast) in order to perform large-scale experiments characterizing cell cycle regulation and control.  In order to engage any experiment performed by the research stream, students must now learn the techniques of aseptic cell culture.


Aseptic technique is emphasized strongly.  Briefly, aseptic technique is the set of procedures and measures taken that prevent the unwanted contamination of cell cultures and other related resources (cell stocks, common reagents and media).  Such measures are often referred to as sterile technique.  Aseptic technique is employed by students such that subsequent experimentation involves the manipulation of specific and desired strains of Saccharomyces cerevisiae - not eukaryotic or prokaryotic organisms that might contaminate such environments.


Finally, in their initial education of cellular growth and culture maintenance, students learn several methods by which they can quantitatively characterize strain-by-strain growth relative to some appropriate control.  Specifically they learn this methodology through growth curve execution and analysis as well as a complementary assay that is based upon the use of solid media.  This technique is important in that it demonstrates how rapidly cell cultures biologically deteriorate once nutrient saturation has occurred. Secondarily, it provides mechanisms by which students can evaluate tagged and mutant strains used in their independent research projects.


Molecular Preparation and Purification (DNA, RNA, Protein), Gel Electrophoresis

The hierarchy of training continues:  students not have the ability to use all lab equipment, manufacture and pH all reagents and aseptically growth and characterize various strains of the eukaryotic model organism Saccharomyces cerevisiae.  At this stage of training we teach them how to take the cell cultures they have maintained, lyse the cells and specifically purify biological molecules of interest.  The two techniques focused upon in the spring semester are purification of genomic DNA and total RNA.  The former assay (genomic DNA purification) provides a nucleic substrate that can be used to teach enzyme mediated reactions (in the next stage of training). 


The latter assay (total RNA preparation) provides for a lesson in the consequences of less-than-aseptic technique.  Total RNA is easily degraded by a number of common laboratory contaminations and thus the purification of total RNA is a valuable lesson in stepwise, consistent and focused attention to detailed protocols and methods.  Gel electrophoresis is presented at this stage of training as an assay by which purified nucleic acids can be either analyzed or further separated from other species of molecules.


Enzyme Mediated Reactions (PCR, Reverse Transcription)

Following the growth and experimental perturbation of a cell culture as well as the purification of nucleic acids is the stage of training where we shepherd students toward the final peak of functional capability in a modern molecular biology laboratory - enzyme mediated reactions and manipulations of biological and chemical substrates.  We specifically use the substrates purified in the previous stage of training (genomic DNA, total RNA) in order to teach polymerase chain reaction (PCR) and reverse transcription (RT).  This stage of training further emphasizes the new for sterile technique, precise volume manipulation and the use of advanced equipment in (thermal cyclers and NanoDrop-mediated quantification of nucleic acids).


Complex Techniques Measuring Whole-Genome Regulation (ChIP)

The completion of training in enzyme mediated reactions marks the completion of student training in our research stream.  The summer fellows immediately begin work on independent research through the cumulative use of all of the above areas of training in one complex multi-day experiment: chromatin immunoprecipitation characterizing in vivo transcription factor binding to genomic DNA.  In order to even begin this experiment students must PCR-verify an epitope-tagged strain of Saccharomyces cerevisiae.  Next, the rigors of clean reagent production and aseptic cell culture are tested through the controlled execution of cellular growth and end-testing for contamination.  Finally, a multi-day procedure of stage-by-stage molecular preparation is engaged.  This process results in the purification of tiny fragments of genomic DNA representing the regulatory sites engaged by a transcription factor of interest.  This entire process demands significant skill, attention to detail and problem-solving capacity of the researcher.  These requirements we deliver through the training phases of stream operations.


Computational Biology & Bioinformatics

The completion of a ChIP-seq experiment marks the culmination of an amazing amount of bench-education for our students.  The field of Functional Genomics, however, demands that their education has only begun at this point.  Each ChIP-seq experiment has the potential to generate several gigabytes (GB) of primary data.  This data is meaningless without the use and development of sophisticated analysis tools.  Thus, the students are given the opportunity to engage and conquer a hurdle that blocks many on the biology/chemistry side of the Natural Sciences from using cutting-edge technologies - the ability to handle vast amounts of multi-format data.  Students learn to install and navigate a UNIX-based operating environment.  They learn to program in the interpretive language Perl and solve common bioinformatic problems.  We work specifically on next-generation specific tasks such as sequence alignment, peak detection and gene-target determination.  These tasks involve an enormous amount of custom programming to handle constantly evolving file formats, analytical processes and biological questions.  I assume no previous experience and train all students from step zero.