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  • Single-nucleotide Polymorphisms that Create Transcription Factor Binding Sites

    Pranavi Aradhyula, William Law, Anthony AntonellisDepartment of Human Genetics, University of Michigan

    IntroductionCharcot-Marie-Tooth (CMT) disease is the most common inherited peripheral neuropathy in the United States. One form of CMT is caused by demyelination of the Schwann cells which are responsible for wrapping around a peripheral nerve and keeping electrical impulses within the axon. Patients with CMT have a wide range of severity of disease, even among patients with identical coding mutations. The cause of this variability is unknown, however gene regulatory regions and transcription factors may modify the severity of disease. Because this form of CMT affects both motor and sensory neurons, weakness and muscle atrophy may occur, resulting in difficulty in carrying out fine motor skills. The onset of symptoms can vary from adolescence to early adulthood or even mid adulthood. Although CMT is not a fatal disease, pain may vary from mild to severe, where some may even have to use orthopedic devices or wheelchairs for mobility [Reference National Institute of Neurological disorders and Stroke].

    While there are many transcriptional factors that regulate Schwann cells, our lab is interested in SOX10 (SRY- box containing gene 10). SOX10 is a transcription factor gene that is critical for the development of neural crest, which are stem cells that for atop of the future spinal cord and develop into peripheral nerves. A transcription factor gene codes for a protein that in turn binds to specific regions of the DNA (enhancers for other genes) and controlling the transcription rate of that gene. Mutations in the SOX10 gene can cause up regulation or down regulation of the genes regulated by SOX10, thus resulting in peripheral nerve diseases including demyelinating peripheral neuropathy. The goal of this lab is to define transcriptional hierarchies for SOX10.

    The DSCS (Dimeric SOX10 binding sites Created by SNPs) project explores the single-nucleotide polymorphism (SNP) alleles that create SOX10 binding sites (as shown in Figure 1 which will be replaced with better image) to identify potential modifiers of peripheral nerve disease. A SNP is basically a common mutation (in a population) where a single nucleotide is altered to a different nucleotide. For this project, we defined a dimeric SOX10 site as those consisting of two monomeric SOX10 sites that are each 5 bp in length with a 5-10 bp gap in between the two sites. Dimeric SOX10 binding sites were selected for functional analysis based on: (1) the evolutionary conservation (amongst mouse, chicken, and rat) of one monomeric site; and (2) the presence of a SNP where the minor allele creates a dimeric SOX10 site. We utilized computational analysis to narrow down the regions of interest to 20 dimeric SOX10 binding sites, of which 13 did not overlap a gene; we do not want the dimeric sites to overlap a gene because we want to use regions that have a predicted role in transcriptional regulation. Of these, 12 dimeric SOX10 sites were amplified around the genomic DNA surrounding the 12 minor alleles. The regions are currently being assessed by luciferase assays to identify active (greater than 5 fold) regions in cultured Schwann cells. Active regions will then be mutagenized to the major allele (ablating the dimeric site) and reassessed in luciferase assays to compare the activity between the major and minor alleles. Thus far, we have tested 5 regions and identified one active region with 100 fold activity. We are currently assessing the major allele for altered activity. Any SNP identified that causes a change in activity, represents an excellent candidate modifier of peripheral nerve disease.

    The long term goal of this research is to identify potential drugs that ameliorate the symptoms of patients suffering from this disease.

    Methods

    Start

    Computational Analysis

    Predicting Transcription Factor Binding via Genome Browser

    and desired criteria

    PCR Amplify

    Design primers to amplify Region of Interest and PCR

    amplify

    Gateway Clone - BP Reaction

    Insert Region of Interest into pDonor vector via BP cloning

    Sequence Verication

    Verify via BSRGI digest and Sangers Sequencing that we have the correct DNA region

    Freeze Down

    Freeze cells in -80 degrees for Backup

    Sequence Verication

    Verify via BSRGI digest and Sangers Sequencing that we have the correct DNA region

    Luciferase Assay

    Test Luciferase activity of the Region of Interest

    Analyze Data

    Normalize Luciferase activity to Renilla and pE1B control;

    Compare amongst regions

    Mutagenesis

    Changing one SNP to create dimeric SOX10 binding site

    (minor allele)

    minor allele

    major allele

    A T A A T A A A G A A T T G T

    A T A A T A A A G A A T T G T

    A C A A T A A A G A A T T G T

    Gateway Clone - LR Reaction

    Insert Region of Interest into pE1B vector via LR cloning in Forward and Reverse oreintations

    The Flow chart above shows the various methods we used to analyze the regions of interest. The methods section could be divided into two parts: 1) computational analysis and 2) experimental analysis. Computation analysis in-volves narrowing down our regions of interest to match the desired critieria (i.e. conservation, SNP change creates dimeric site). Experimental analysis involves experimentally testing the regions to see if they are actually functional SOX10 binding sites. This is done by Luciferase assay, where we insert our region of interest into the pE1B vector and test for luciferase activity.

    Results

    Of the 12 regions that were amplified, 5 of the regions were placed into vector pE1B in forward and reverse orientations and tested for luciferase activity. As can be seen in Figure 3, DSCS 8 placed in the reverse orientation showed a 100 fold increase in activity compared to the normalized pE1B empty vector.

    DSCS 8 (chr12:24078888-24078903) minor allele has a SNP change from a C (major allele in humans) to T (minor allele in humans) that creates the Dimeric SOX10 site. The minor allele frequency rate is 5.97% of the 299 people tested. This region was found just downstream of SOX5 gene, suggesting the potential for it to be an enhancer. This theory is further supported because only DSCS 8 in the reverse orientation showed luciferase activity while the same region placed in the forward orientation showed low levels of activity. SOX5 is also oriented in the reverse direction with respect to the genome browser, and thereby is in the same orientation as the DSCS 8-rev. This suggests that DSCS 8 may indeed play some role in SOX5 regulation.

    Future Directions

    The next immediate step in this project is to test the major allele of DSCS 8-rev with luciferase assay. We would like to observe diminished luciferase activity to support our claim that DSCS 8 is indeed a SOX10 binding site that enhances activity of SOX5 gene. Another immediate step is to test the remaining 7 minor alleles for luciferase activity above 5 fold increase.

    References

    Scalechr12:

    DSCS 8

    MouseChicken

    200 kb hg1823,700,000 23,800,000 23,900,000 24,000,000 24,100,000 24,200,000 24,300,000 24,400,000 24,500,000

    PCR Product

    SOX5SOX5SOX5SOX5SOX5

    L-SOX5

    Figure 2. DSCS 8 is just downstream of SOX5, suggesting potential to be an enhancer0

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    pE1B

    Empty

    DSCS

    3 Fo

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    3 Re

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    8 Fo

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    Figure 3. DSCS 8-Rev shows 100 fold increase in activity

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