Editing BioMicroCenter:Research (section)

== 2012 ==
=== Genomics of Toxoplasma gondii - [http://web.mit.edu/biology/www/facultyareas/facresearch/saeij.html SAEIJ LAB] - [http://web.mit.edu/biology Biology]===
The overall goal of the Saeij lab is to understand the molecular basis for individual differences in outcome of infection. We believe that these differences are either due to host differences in resistance/susceptibility to infection or to genetic differences in the pathogen that causes the infection. As a model we use infection with the obligate intracellular pathogen Toxoplasma gondii. Toxoplasma can infect all warm-blooded animals and a third of the world’s population is estimated to be infected. There are many different Toxoplasma strains that differ hugely in virulence in both mice and humans. Mouse and rat strain differences in resistance/susceptibility to Toxoplasma have also been described.<BR><BR>
To characterize Toxoplasma genetic diversity we sequenced (Illumina HiSeq), with the help of the BioMicro Center, the whole genome of 10 Toxoplasma strains and the transcriptome of 32 different strains, representing global diversity. This data allowed us to construct the first Toxoplasma haplotype map and to propose a new model explaining current global diversity. The results were published in PNAS (Minot et al, 2012). We also determined the transcriptome of murine macrophages infected with these 32 different strains. This approach allowed us to correlate genotype with phenotype and have led to the identification of Toxoplasma loci and genes that affect fitness, clonality, virulence and modulation of host signaling pathways. Some of these results were published in PLoS Pathogens (Niedelman et al, 2012).<BR><BR>
To determine mouse genetic differences in resistance/susceptibility to Toxoplasma we determined the transcriptome and toxoplasmacidal activity of naïve or IFNγ+TNF-stimulated macrophages isolated from 29 recombinant inbred mice, derived from A/J (Toxoplasma resistant) and C57BL/6 (susceptible) mice. We then identified mouse genomic loci affecting mouse gene expression and toxoplasmacidal activity using quantitative trait locus (QTL) analysis. We are now using genetic approaches to confirm mouse candidate genes involved in macrophage differences in Toxoplasma killing ability. The results of this study have helped us secure four years of grant support from the New England Regional Center of Excellence Biodefense and Emerging Infectious Diseases.<BE><BE>
We have received excellent help from the BioMicro Center at every step of these analyses, their many suggestions on how to analyze the data have been instrumental in getting high quality data.<BR><BR>

===Sequencing of tRNAs - DEDON/NILES LABS - [http://web.mit.edu/be BE] and [http://web.mit.edu/cehs CEHS] ===
Emerging evidence points to selective translation of critical proteins as a major facet of the cellular stress response.  The Dedon lab has shown that while one contribution to this translational control of cell response involves changes in the relative quantities of dozens of modified ribonucleosides in tRNA, there is also evidence for control of the number of copies of individual tRNA molecules.  This dual control problem makes it difficult to distinguish changes observed in the level of tRNA modifications caused by altered activity of ribonucleoside-modifying enzymes from changes in the number of tRNA copies.  To solve this problem, the Dedon lab has undertaken the development of a high-throughput deep-sequencing method to identify and quantify individual species of tRNA in a bulk population of tRNAs.  This approach exploits the paucity of ribonucleoside modifications at the 3’-end of tRNA molecules, modifications that can interfere with RNA sequencing reactions. Following ligation of a custom primer to the 3’-ends of tRNA molecules, reverse transcription is performed to create cDNA that is subsequently subjected to linear amplification off of the custom primer.  The amplified products are then identified and quantified by Illumina sequencing. This method allows for the simultaneous quantification of individual tRNA species in total tRNA isolated from cells subjected to different stresses, with application to study controlled degradation of tRNA during cell responses and to differentiate between changes in tRNA copy numbers from changes in RNA modifications due to enzymatic activity. <BR><BR>
The BioMicro Center has been very instrumental in providing technical expertise towards optimization of experimental design for the conversion of tRNA to cDNA for sequencing, as well as assisting with bioinformatics analysis of the output data from the Illumina HiSeq 2000. The Dedon lab was able to barcode six different experimental samples so that multiple sequencing libraries could be run in a single lane of an Illumina flow cell. Using the Illumina HiSeq 2000, we were able to identify every tRNA species found in S.cerevisiae and are in the process of optimizing the sequencing method in order to reliably quantify relative changes in the levels of specific tRNAs isoacceptors from different populations of tRNAs. <BR><BR>
The Niles lab has partnered with the Dedon Lab and the BioMicro Center in their efforts to develop a protocol for sequencing bulk tRNA.  While the Dedon lab is investigating the effect of different stresses on tRNA expression, the Niles lab is interested in the basic endogenous biology of the malaria parasite.  Currently, very little is known in the field about tRNA dynamics across the complicated life cycle of Plasmodium falciparum.  While many published studies have tracked RNA expression using microarrays and deep sequencing, these protocols enrich for protein coding RNAs, excluding an important component of host biology.  Additionally, while the mitochondria does not have any genes to produce tRNA, the apicoplast organelle has a full complement of tRNA genes.  As the apicoplast organelle is required for successful parasite infection, having a better understanding of the expression of tRNA from the reduced but necessary apicoplast plastid will assist in the downstream search for both vaccines and pharmacological treatments.<BR><BR>
Using the method described above, all tRNAs found in both the genome and apicoplast were detected from extracts of Plasmodium falciparum.  However, there were several species of tRNA that jackpotted, appearing at concentrations several orders of magnitude above the remaining species.  We are continuing to work with the BioMicro Center and the Dedon Lab to troubleshoot the protocol to determine a more quantitative picture of tRNA dynamics in the parasite.<BR><BR>   

=== Elucidating Brn targeting of Sox2 in embryonic stem cells - [http://web.mit.edu/biology/www/facultyareas/facresearch/jaenisch.html JAENISCH LAB] - [http://web.mit.edu/biology Biology] and [http://web.mit.edu/ki KI]===
In mammals, a few thousand transcription factors regulate the differential expression of greater than 20,000 genes to specify ~200 cell types during development.  How this is accomplished has been a major focus of biology for many years.  Transcription factors bind sequence-specific regulatory elements, including proximal promoters and distal enhancers, to control gene expression.  Emerging evidence indicates that transcription factor binding at distal enhancers plays important roles in the establishment of tissue-specific gene expression programs during development.  Combinatorial binding among groups of transcription factors can further increase the diversity and specificity of regulatory modules governed by a particular factor.   The identification of regulatory modules comprised of groups of transcription factors which occupy important regulatory regions of genes which govern cell fate determination would shed light on how developmental decisions are made.<BR><BR>
Work from the Jaenisch and Young labs elucidated the role of  a group of three transcription factors, Oct4, Sox2, and Nanog, in regulating cell identity in embryonic stem cells (ESCs) using ChIP-on-Chip technology in 2005.  Recently, Michael Lodato from the Jaenisch lab, in collaboration with the Boyer lab, has studied the genome-wide role of Sox2 in neural progenitor cells (NPCs).   Their work showed that Sox2 occupied a distinct set of targets in NPCs relative to it targets in ESCs, and further that Sox2 switched partner factors during this transition: form Oct4 and Nanog in ESCs to Brn2, and Oct4 family member, in NPCs. <BR><BR>  
Taking advantage of the expertise in the BioMicro Center, they were able to examine the effect of the NPC-specific Sox2 partner factor, Brn2, on Sox2 binding in ESCs, where Sox2 normally partners with Oct4.  The BioMicro Center Staff utilized the IP-Star Automated ChIP System to facilitate the automation of a large number of ChIP-Seq experiments, successfully querying the genome-wide occupancy of Sox2, Brn2, Histone H3 Lysine 4 monomethylation, Histone H3 Lysine 27 acetylation, total Histone H3, and p300 in both control and Brn2-induced ESCs in a rapid and controlled manner.  Thus, not only could the binding of Sox2 and Brn2 be investigated, but due to the availability of the IP-STAR automated ChIP system the Jaenisch Lab could investigate changes to the epigenome as well.  The BioMicro Center then prepared Illumina libraries from these samples ran them in a single lane of an Illumina flow cell, saving time and immensely reducing the cost to the Jaenisch Lab.  Finally, the BioMicro Center implanted quality control metrics on both the samples before loading on the sequencer and on the quality of the data generated during the run. <BR><BR>
Using this data, the Jaenisch Lab was able to define a set of regions where ectopic Brn2 could recruit endogenous Sox2 (Figure x), and they are currently investigating the effects of this binding on the chromatin status of these regions.   The technical expertise of the staff of the BioMicro Center contributed significantly to the successful completion of this project during both the planning and execution of these experiments.  <BR><BR>