1. HypothesisAbstract
Investigating the loss of Chd1 gene expression through
position effect variegation in Drosophila melanogaster
Background
Future Studies
Acknowledgements
References
Results
Discussion
• DNA exists in both as euchromatin, a
loosely package form associated with
active genes, and heterochromatin, a
tightly package form associated with
silenced genes.1
• Chromatin remodeling proteins alter
nucleosomal structure, and by in large
exposing the gene segment to be
accessed by transcriptional factors.2
CHD1 is implicated in maintenance of active chromatin
I propose that the homozygous loss of Chd1 will result in more silencing of the
white gene when positioned next to heterochromatin. Therefore, the phenotype
of the PEV eyes will be more white.
Tram Bui and Jennifer A. Armstrong
I would like to extend my deepest thanks to members of the Armstrong
lab. Special thanks goes to Professor Armstrong for her guidance,
enthusiasm, and patience throughout the years. I would also like to
acknowledge Lakshmi Bugga for her knowledge of everything lab
related and constant support in lab. Thank you NSF for their generous
funding. Lastly, my deepest gratitude goes out to my parents for their
unending sacrifices.
Loss of CHD1 can modify PEV differently according
to the location of white gene insertions
Chromodomain helicase DNA binding protein 1 (CHD1) is a conserved chromatin
remodeling and assembly factor implicated in the regulation of nucleosome dynamics
at transcriptionally active genes. We observed that increased levels of CHD1 result in
loss of HP1a binding on chromosomes, despite different localization patterns of the
two proteins. Given the role of CHD1 in chromatin structure at active genes, and its
ability to antagonize binding of heterochromatin proteins, we wondered whether loss
of function Chd1 alleles could function as enhancers of variegation [E(var)] of the
mosaic expression of euchromatic genes placed near or within heterochromatin.
Given that Chd1 is not an essential gene, we examined the effect of the recessive loss
of Chd1 on variegation of In(1)wm4h as well as lines carrying the white gene inserted
in various locations on the fourth chromosome. When homozygous, the Chd1 mutant
allele acted as an E(var) of white inserted into the telomere of the fourth chromosome
and a Su(var) of wm4h. Thus, we observed that a loss of function Chd1 allele can
modify PEV in a manner that varies depending upon the chromosomal location of the
white gene.
• CHD1 is a chromatin remodeler that is recruited to highly transcribed gene and
co-localizes with early elongation form of RNA Polymerase II.3
• CHD1 expression is inversely proportional to Heterochromatic Protein 1 (HP1a)
and H3K9me2, both are marks of heterochromatin and repressed genes.4 No change of
enhancement or
suppression was
observed in the
pericentric region.
An enhancement of
variegation was
observed in the
telomeric region.
white+
white+
A suppression of
variegation was
observed in the white
mottled 4h line.
white+
Wildtype
chd1
Heterozygous
chd1 mutant
Homozygous
chd1 mutant
A subtle
enhancement of
variegation was
observed in the
medial region.
white+
+
⇁
;
𝑎𝑙 𝑏 𝑐 𝑠𝑝
+
;
+
+
;
118 𝐸−15
+
+
⇁
;
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
𝑎𝑙 𝑏 𝑐 𝑠𝑝
;
+
+
;
118 𝐸−15
+
+
⇁
;
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
𝑎𝑙 𝑏 𝑐 𝑠𝑝
;
+
+
;
118 𝐸−15
+
+
⇁
;
𝑎𝑙 𝑏 𝑐 𝑠𝑝
+
;
+
+
;
118 𝐸−10
+
+
⇁
;
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
𝑎𝑙 𝑏 𝑐 𝑠𝑝
;
+
+
;
118 𝐸−10
+
+
⇁
;
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
𝑎𝑙 𝑏 𝑐 𝑠𝑝
;
+
+
;
39 𝐶−12
+
+
⇁
;
𝑎𝑙 𝑏 𝑐 𝑠𝑝
+
;
+
+
;
39 𝐶−12
+
+
⇁
;
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
𝑎𝑙 𝑏 𝑐 𝑠𝑝
;
+
+
;
118 𝐸−10
+
+
⇁
;
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
𝑎𝑙 𝑏 𝑐 𝑠𝑝
;
+
+
;
39 𝐶−12
+
𝐼𝑛(1)𝑤 𝑚4ℎ
⇁
;
𝐶𝑦𝑂
𝑎𝑙 𝑏 𝑐 𝑠𝑝
;
+
+
;
+
+
𝐼𝑛(1)𝑤 𝑚4ℎ
⇁
;
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
𝑎𝑙 𝑏 𝑐 𝑠𝑝
;
+
+
;
+
+
𝐼𝑛(1)𝑤 𝑚4ℎ
⇁
;
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
𝑐ℎ𝑑15 𝑏 𝑐 𝑠𝑝
;
+
+
;
+
+
Position Effect Variegation and Suppressors and
Enhancers of PEV
• Stochastic silencing of the white gene
(normally found in euchromatin), after
it is placed near heterochromatin.5
• Gene is silenced in some cells
causing a mosaic expression
phenotypically that can be use to
study and identify modifiers of gene
expression.
• Alleles of genes are categorized into
enhancers of variegation [E(var)s] or
suppressors of variegation [Su(var)s]
based on how they modify PEV.
• E(var) alleles enhance the silencing
effect of the white gene, making the
eyes more white.
• Su(var) alleles suppress the silencing
effect of the white gene, thus making
the eyes more red.
• The loss of function Chd1 allele can modify PEV in a manner that
varies depending of the chromosomal location of the white gene.
• When homozygous, the Chd1 mutant allele acted as an E(var) of
white inserted into the telomere of the fourth chromosome and a
Su(var) of wm4h.
• Results deviated from the findings by Elgin and Wallrath done on
some of the same PEV lines as they did not witness any modifying
effect at the telomeric region.6
• In the wm4h line, similar suppression of variegation was observed
for the heterozygous Chd1 mutant.7
• Conduct spectrophotometry analyzes in order to quantify the results and
determine if there are any statistical differences between the genotypes.
• Redo the Wildtype Chd1 controls in order to account for the
homozygous loss of black, curved, and specked marker genes on PEV
gene expression for the 118 E-15 line.
1. Li, B., Carey, M., & Workman, J. L. (2007). The Role of Chromatin during Transcription. Cell, 128(4),
707-719. doi:10.1016/j.cell.2007.01.015
2. Phillips, T. & Shaw, K. (2008) Chromatin Remodeling in Eukaryotes. Nature Education 1(1):209
3. Park, D., Shivram, H., Iyer, V.R. 2014. Chd1 co-localizes with early transcription elongation factors
independently of H3K36 methylation and releases stalled RNA polymerase II at introns.
Epigenetics Chromatin. 7(1):32.
4. Bugga, Lakshmi et al. “The Drosophila Melanogaster CHD1 Chromatin Remodeling Factor
Modulates Global Chromosome Structure and Counteracts HP1a and H3K9me2.” PloS one 8.3
(2013): e59496. Web.
5. Elgin, S. C. R., and G.Reuter. "Position-Effect Variegation, Heterochromatin Formation, and Gene
Silencing in Drosophila." Cold Spring Harbor Perspectives in Biology 5.8 (2013): n. pag. Web.
6.Wallrath, L. L., and S. C R Elgin. “Position Effect Variegation in Drosophila Is Associated with an
Altered Chromatin Structure.” Genes and Development 9.10 (1995): 1263–1277. Web.
7. Wang, Frances. "Investigation of CHD1 Function in Heterochromatic Gene Silencing through Position
Effect Variegation in Drosophila Melanogaster." Thesis. Claremont McKenna College, 2014. Print.
A
D
G
J
B C
E F
H I
K L
Each row corresponds to the white gene insert at different location on the
chromosome. The left column (A,D,G,J) displays the wildtype Chd1 condition.
The middle column (B,E,H,K) displays the heterozygous Chd1 mutants. The
right column (C,F,I,L) shows the homozygous Chd1 mutants. The columns were
compared to each other in order to identify Chd1’s role in gene expression of
the white gene.