GCAT-SEEKquence
The Genome Consortium for Active Teaching
NextGen Sequencing Group
NextGen Sequencing Request Form
Complete fields below, save file with your last name at the beginning of
the filename (e.g. newman-GCAT-SEEK Sequence request form.pdf) and
email to Vincent Buonaccorsi <BUONACCORSI@juniata.edu>
A. Contact Information
1. Name:
Emily Humphrey Dixon
2. Department: Biology and Chemistry (Joint appointment)
3. Institution: St. Lawrence University
4. Phone Number: (315) 229-5671
5. Email Address: edixon@stlawu.edu
B. Project Information
1. Title: Determining the roles of the yeast 14-3-3 proteins in rapamycin-mediated transcription
2. Category: RNA-seq and ChIP-seq
3. Total amount of sequence requested: Help! For the RNA-seq, it would be 12 yeast RNA extractions
that would need to be sequenced, probably in triplicate if possible. For the ChIP-seq, it would be 6
IPs plus 6 whole cell DNA extract controls that would need to be sequenced, probably in triplicate if
possible.
4. Preferred technology: No idea!
5. Do you have funds for a partial run next Spring? No.
Describe the background, hypotheses and specific aims (500 words max)
14-3-3 proteins are highly conserved and have been found in all eukaryotic organisms investigated. They are
involved in many varied cellular processes, and interact with hundreds of other proteins. Among many other
roles in cells, yeast 14-3-3 proteins have been implicated in rapamycin-mediated cell signaling. Preliminary
microarray data (unpublished) suggests that BMH1 and BMH2 are required for rapamycin-induced regulation of
distinct, but overlapping sets of genes. In addition, preliminary data (unpublished) using chromatin
immunoprecipitation followed by quantitative PCR suggests that both Bmh1p and Bmh2p associate with the
promoters of at least some of the genes that require BMH1 and BMH2 for rapamycin-induced regulation.
Preliminary data (unpublished) suggests that H3S28 phosphorylation may be involved in recruiting Bmh1 and
Bmh2 to the promoters of some of these genes and in mediating some of the transcriptional effects we observe.
Hypothesis: H3S28 phosphorylation recruits BMH1 and BMH2 to yeast genes upon rapamycin treatment and is
required for mediating some of the transcriptional changes that are observed following rapamycin treatment.
Specific Aims:
1. Determine the roles of BMH1, BMH2, and histone H3 S28 phosphorylation in mediating the
transcriptional response to rapamycin
To do this, we will determine the transcriptional effects of deleting BMH1 and BMH2 in the presence of
both wild-type histone H3 (serine 28 can be phosphorylated) and histone H3 with serine 28 mutated to
alanine (serine 28 cannot be phosphorylated).
2. Determine the role of histone H3 S28 phosphorylation in recruiting Bmh1p and Bmh2p to DNA
To do this, we will use chromatin immunoprecipitation to determine the genome-wide locations of
Bmh1p and Bmh2p in the presence of both wild-type histone H3 and histone H3 with serine 28 mutated
to alanine. We will also use chromatin immunoprecipitation to determine the genome-wide distribution
of phosphorylated H3S28 both in the presence and absence of rapamycin.
C. Describe the methods [sample prep, calculation of amount of sequence required, analysis plan]
bmh1, bmh2, and an otherwise isogenic wild-type strain with unmutated histone H3 as well as bmh1
H3S28A, bmh2 H3S28A and H3S28A yeast will be grown and either treated with rapamycin or mock-treated.
RNA will be extracted and used for sequencing. [I’m not exactly sure of the methods beyond this. I hope
that with training we would be able to prepare the samples properly- perhaps bar coding to use fewer lanesand analyze the resulting data.]
We will use chromatin immunoprecipitation to isolated DNA associated with Bmh1-HA and Bmh2-HA in
yeast with wild-type histone H3 and in yeast with the H3S28A mutation, and in the presence and absence of
rapamycin. We will also use chromatin immunoprecipitation to isolate DNA associated with
phosphorylation H3S28 both in the presence and absence of rapamycin. This DNA as well as DNA isolated
from samples of corresponding whole cell extract will be used for sequencing. [Again, beyond this I’m not
really sure of the methods.]
D. Describe the role and number of undergraduates involved in the project, and how they would benefit.
Undergraduates will be involved in all stages of the project. Students in my Research Methods in Molecular
Biology class (about 12 per year) as well as independent research students (2-3 new students per year) will help
with sample preparation and, with supervision, will take a lead role in analyzing the data. Approximately 50
students will be involved in the project over three years. This will likely be the only experience available to these
students for collecting and analyzing genome-wide data.
E. I agree to administer the GCAT-SEEK pre- and post-activity assessment test for students and to complete the
faculty post-utilization survey. __X__ yes, ____ no
F. Describe any other broader impact or intellectual merit considerations.
G. References