THE ROLE OF CHROMODOMAIN PROTEIN 1 (CDP-1) AT NEUROSPORA CRASSA HETEROCHROMATIN
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Date
2016-06
Authors
Kim, Carissa
Journal Title
Journal ISSN
Volume Title
Publisher
University of Oregon
Abstract
Proper heterochromatin I formation is a significant factor in chromosome
organization and gene regulation. Therefore, it is vital to study the role of all proteins
that contribute to proper heterochromatin formation in order to form a comprehensive
understanding of the importance of heterochromatin. In Neuro~pora crassa2,
heterochromatin formation depends on a number of well-studied proteins. However, we
still have an incomplete understanding of this process.
In order to improve our understanding of heterochromatin formation in N.
crassa, the Selker lab has identified many novel proteins involved in this process. One
novel protein is chromodomain protein I (CDP-I) that specifically localizes to
heterochromatin. We hypothesized that CDP-I is involved in proper heterochromatin
formation. To begin understanding the role of CDP-I at heterochromatin, we asked:
1 Heterochromatin- generally, it is a tightly packed fonn of chromatin (organizes DNA) that can (though
not always) prevent certain proteins from accessing certain regions of DNA
2 Neurospora crassa (N. crassa)-fungal species used in the Selker Jab, as well as other labs, as a genetic
model
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1) Is CDP-1 required for proper heterochromatin formation at specific regions,
such as the centromeres, telomeres, and interspersed heterochromatic
regions?
2) Which domains3 of CDP-1 are required for proper protein function?
3) Do changes in gene expression occur when the cdp-1 gene is deleted?
4) Do proteins or protein complexes associate with CDP-1? If so, do they play
a role in CDP-1 function?
To test if CDP-1 is required for proper silencing at heterochromatic regions, my
mentor knocked out4 the cdp-1 gene and inserted antibiotic resistance genes at various
heterochromatic regions (centromere on linkage group5 I and VI, a telomere on linkage
group VII, and in interspersed heterochromatin). I screened strains for the antibiotic
resistance genes and the knockouts of certain genes of interest (e.g. cdp-1, hpo6) using
Southern blots7. I tested the growth of the strains on antibiotic media to determine if
heterochromatin was still forming properly at these regions with cdp-1 deleted.
Once I determined where CDP-1 is required for proper heterochromatin
formation, I tested which domains of CDP-1 are required for CDP-1 function. I
transformed8 a cdp-1 knockout strain with plasmids9 that contained the cdp-1 gene with
3 Domains—proteins contain amino acid sequences which form a three-dimensional structure that carries
out specific functions for the protein (catalytic, binding). CDP-1 has 2 types of domains: a chromodomain
that allows CDP-1 to recognize and bind to chromatin and AT hooks that help CDP-1 bind to AT-rich
DNA regions.
4 Knocked out/knockout—deletion (or replacement) of gene from genome
5 Linkage group—a chromosome in N. crassa
6 hpo—a gene encoding the protein, HP1, in N. crassa that is essential for heterochromatin formation. It
is a highly conserved protein in higher organisms.
7 Southern blot—assay to detect the placement of restriction sites around a specific DNA sequence. See
methods.
8 Transformed/Transformation—genetic technique to insert a specific gene into the genome of an
organism. See methods.
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specific point mutations (chromodomain (CD), AT hook 1, AT hook 2, and AT hook
1/2 double mutant)10. These strains also had antibiotic resistance genes inserted into the
genome (CenVIR::bar or CenIL::nat-1). I will test the CDP-1 mutant strains for growth
on antibiotic media and antibiotic resistance gene expression using qRT-PCR.
RNA sequencing11 (RNA-seq) showed that there were some genes whose
expression levels changed when cdp-1 was deleted. Based on the RNA-seq results, I
focused on the expression of six different genes to confirm the data from the RNA-seq.
We confirmed that one gene, NCU1671812, had significantly higher expression levels
when cdp-1 was deleted compared with wild type. This gene is located in the subtelomeric
region of linkage group III, indicating that CDP-1 is required for silencing of
this gene at telomere IIIR.
Finally, to determine if CDP-1 interacts with other proteins or protein complexes
in vivo, I examined four different proteins that immunoprecipitated with CDP-1 in a
large scale pull-down assay performed by a former member of the Selker lab. The four
proteins (referred to as interacting partners from here on) that I am interested in are
CRF8-1, SPT-16 and POB-3 (FACT complex), and HIR-1 (HIRA complex). Each
interacting partner has a role at chromatin and potentially associates with CDP-1 in N.
crassa. I tagged13 all four interacting partners with 3xHA::hph14 and crossed15 them to
9 Plasmid—circular bacterial DNA that can be manipulated for genetic experiments, such as
transformations.
10 Point mutations—mutations in the DNA sequence that alters one nucleotide into another nucleotide
changing the amino acid sequence of the protein encoded by the DNA.
11 RNA sequencing—technique to measure RNA levels
12 NCU16718—a hypothetical gene in N. crassa genome whose function has yet to be defined.
13 Epitope tag—a nucleotide sequence genetically inserted at the end of a gene that is translated with the
gene and can be detected with antibodies
14 3xHA::hph—3 sequence repeats of HA tag that can be detected with HA antibodies. The hph antibiotic
resistance gene is used for the selection of the tag [11]
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another strain with 3xflag::hph16 tagged CDP-1. I screened for strains with both CDP-1
and interacting partners tagged as well as strains with only interacting partners tagged. I
then performed a Co-immunoprecipitation (Co-IP)17 to determine if each interacting
partner is pulled down with CDP-1 when CDP-1 is immunoprecipitated.
My results demonstrate that CDP-1 is required for silencing at centromere I and
VI, as well as telomere IIIR. However, CDP-1 is not required for silencing at telomere
VIIL or at interspersed heterochromatic regions. In addition, my results demonstrate
that CDP-1 may interact with other proteins in order to carry out its function at
heterochromatin. Further testing of the association between CDP-1 and the interacting
partners, as well as, further efforts to produce stable mutant CDP-1 proteins will expand
our understanding of CDP-1 function at heterochromatin. However, the preliminary
findings that CDP-1 is required for proper heterochromatin formation at only certain
heterochromatic regions in N. crassa contribute to our understanding of
heterochromatin.
15 Crossed—2 N. crassa strains were mated, see methods
16 3xflag::hph—similar to 3xHA::hph, repeated sequence for flag tag that can be detected with flag
antibodies[11]
17 Co-immunoprecipitation—target protein co-precipitates a protein partner, see methods.
Description
36 pages. A thesis presented to the Department of Biology and the Clark Honors College of the University of Oregon in partial fulfillment of the requirements for degree of Bachelor of Science, Spring 2016.
Keywords
Biology, Genetics, Neurospora crassa, Epigenetics, Heterochromatin, Chromodomain Protein, Molecurlar Biology