A critical directive of the BioMicro Center is to provide a cutting-edge research core for members of the MIT community. Creating and maintaining the BioMicro Center at the forefront of technology improves our ability to support MIT faculty in grant applications, manuscript publishing and in the recruitment of new faculty members. In part, this goal is achieved through ongoing collaborations with many labs at MIT. A selection of these collaborations taken from the annual reports of the BioMicro Center is presented below.
Large-scale discovery and functional analysis of distal enhancer elements - BOYER LAB - Biology and KI
The overall goal of the Boyer lab is to understand how a single cell can ultimately specify the diversity of cell types during mammalian development. An exciting and emerging area of biology in the post-genomics era has been the genome-wide identification of non-coding regulatory elements in what was once known as “junk DNA”. Enhancers are key cis-regulatory elements that can affect gene transcription independent of their orientation or distance that are required for tissue specific patterning of gene expression during development, though only few examples had been known. Global identification of these regions as well as their contribution to target gene expression has been challenging because enhancers can often reside thousands of base pairs away from their target of regulation.
The Boyer lab has recently discovered that specific histone modification patterns could identify enhancers by genome-wide ChIP-Seq in embryonic stem cells (ESCs) as well as in a range of differentiated cell types and moreover, that these patterns distinguish enhancers as either active or poised (or inactive). Remarkably, genes connected to active enhancers code for genes with cell type specific functions and more importantly, poised enhancers could predict future developmental potential of that cell by marking genes that have the potential to become activated. However, it had been unclear how enhancer states were correlated during lineage commitment. Using cutting edge high-throughput sequencing methods, the Boyer lab has now defined a large set (~80,000) of both poised and active enhancers throughout the genome based on chromatin modification patterns derived from four key time points during cardiomyocyte differentiation. The differentiation system provides a unique opportunity to study enhancer state transitions during embryonic patterning of cardiomyocytes, which ultimately comprise the majority of the cell types in the developing heart.
The BioMicro Center was instrumental in providing the technical expertise necessary for the generation of the large number of high quality sequencing libraries from chromatin immunoprecipitated material. The BioMicro Center adapted the use of the IP-Star automated ChIP system (currently under evaluation) to facilitate automation of ChIP followed by library generation on the SPRI-TE. Additionally, the Boyer lab was able to barcode each experimental sample so that multiple sequencing libraries could be run in a single lane of an Illumina flow cell. Barcoded libraries were then analyzed by a number of quality control measures developed by the BioMicro Center to ensure the highest quality of sequence. These steps represented substantial improvements over previous protocols and allowed us to perform many experiments in a cost and time-efficient manner.
Together with the BioMicro Center, the Boyer lab analyzed the substantial amount of sequencing data and developed new algorithms to identify and to functionally dissect the role of distal enhancer elements in regulating gene expression patterns during lineage commitment. As a result of this study, they found that enhancer utilization is highly cell type specific and that enhancer state transitions are dynamic and non-random and likely occur during short windows of developmental time. These exciting findings have provided new details about how tissue specific expression patterns are established early in development and how mutations in these elements may contribute to cardiac diseases.
A major challenge in bacterial genetics is the identification of the molecular targets and pathways affected by newly discovered genes. Toward this end, one powerful technique involves the unbiased selection for mutations that are able to suppress the deleterious effects of gain- or loss-of-function mutations in the gene of interest. However, finding the genomic locations of these suppressor mutations by traditional mapping methods can be time and labor intensive. The BioMicro Center has worked with the Laub lab to bypass the need for genetic mapping by sequencing the entire genomes of mutant bacterial strains.
The Laub lab, working in the bacterium Caulobacter crescentus, has recently characterized a novel gene, sidA, which inhibits cell division in response to DNA damage. To identify the protein targets of sidA, the Laub lab performed a suppressor screen for mutations allowing cells to form colonies despite sidA overproduction. By directed sequencing of candidate genes, most of these mutations were mapped. However, one suppressor strain did not contain mutations in any of the known cell division genes and was directly sequenced to find the mutation.
In order to generate libraries from this strain, the BioMicro Center piloted a new protocol using the Nextera tagmentation system. Standard approaches using fragmentation had appeared to be unsuccessful, possibly due to the high GC percentage of Caulobacter. The tagmentation system uses a Tn5 transposase to insert sequence tags into intact genomes, both tagging them and fragmenting them at the same time. This reduces the number of operator steps and avoids the need for sonication. With the suppressor samples, the Nextera system was directly compared to sonicated DNA prepared with the SPRI-TE. The sequencing data showed that the Nextera system was able to produce very even and consistent coverage of the genome and is now being offered as a service through the BioMicro Center.
Screening yeast libraries for genes involved in DNA damage response - SAMSON LAB - Biology-BE-KI-CEHS
A myriad of new chemicals have been introduced into our environment and exposure to these agents can have detrimental effects on biological systems. Many of these chemicals are thought to have mutagenic activity. Analysis of the cellular response to these potential toxins using S. cerevisiae can provide a description of systems level responses to environmental stress and identify new pathways of DNA damage response. High-throughput techniques have become important tools to establish and clarify toxicity-modulating pathways of potential environmental carcinogens.
The BioMicro Center has worked closely with Laia Quiros-Pesudo from the Samson lab in multiple complimentary screening methods to identify the cellular systems that respond to DNA damage. The initial screening method involved performing barcode-sequencing (Bar-Seq) described by Smith et al. (Genome Res. 2009. 19: 1836-1842) on a haploid yeast knockout library. In Bar-Seq, each knockout strain is identified by two unique barcode sequences (“uptag” and “downtag” barcodes) that can be amplified from its genome and identified using Illumina sequencing, allowing the whole library to be grown together in a single vessel. The importance of each knocked out gene is then measured by comparing the frequency of the strain in the initial knockdown library pool to the frequency after subjecting the pool to an environmental stress which is summarized as the fitness defect ratio. Multiple conditions can be tested simultaneously by using a second molecular barcode added to the library that identifies the experiment.
Using the Bar-Seq approach, the Samson lab was able to simultaneously analyze the frequencies of ~4,800 strains of S. cerevisiae in up to 19 treatments and doses in a single Illumina sequencing lane. The Bar-Seq method was able to reproduce previous results using a solid agar assay and the alkylating agent MMS (Begley et al, 2004). In addition, new groups of sensitive strains have been identified and analysis of these new pathways is currently underway.
In addition to Bar-Seq approaches, the Samson lab is directly screening GFP-fusion libraries to identify proteins that respond to environmental stress. GFP-fusion libraries monitor both changes in protein expression and localization as a result of chemical exposure instead of survival. In these experiments, each strain is individually screened across a number of conditions, requiring significant automation to make the experiment feasible.
In order to perform these screens, the Samson lab has relied on the Tecan EVO 150 liquid handler in the BioMicro Center and the Cellomics ArrayScan VTI HCS reader available through the CEHS Genomics and Imaging Core (similar instruments are also available through the Whitehead Institute and will be available through the Koch Institute). The Tecan EVO150 performed the cellular treatment, fixation and staining of multiple GFP tagged library plates simultaneously and significantly improved the throughput of the library screen. Initial results have been promising.