tram thesis poster

1
Hypothesis Abstract 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 w m4h . 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 + ;;; ;;; ;;; ;;; ;;; ;;; ;;; ;;; ;;; ;;; ;;; ;;; 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 • 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 w m4h . • 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 w m4h 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.

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Page 1: TRAM THESIS POSTER

HypothesisAbstract

Investigating the loss of Chd1 gene expression throughposition effect variegation in Drosophila melanogaster

BackgroundFuture 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

Heterozygouschd1 mutant

Homozygouschd1 mutant

A subtle enhancement of variegation was

observed in the medial region.

white+

;;; ;;; ;;;

;;; ;;;

;;; ;;;

;;;

;;;

;;; ;;; ;;;

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.2007.01.0152. Phillips, T. & Shaw, K. (2008) Chromatin Remodeling in Eukaryotes. Nature Education 1(1):2093. 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.

Bui, Phuong (Tram)
need to add figure legends
Bui, Phuong (Tram)
Q: Don't know if I should add the dark-colored eyes also? Afraid that it'll take up too much space
Bui, Phuong (Tram)
More on discussion?
Bui, Phuong (Tram)
More on future studies?