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Title

Key Factors in Drosophila Melanogaster HP1a Partner Binding

Date of Award

Summer 8-15-2012

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Computational & Molecular Biophysics)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Drosophila melanogaster heterochromatin protein 1a (HP1a) is an essential protein that plays a central role in heterochromatin formation and concomitant gene silencing. Structurally, HP1a is defined by the presence of a chromodomain (CD), which binds H3K9me2/3, and a chromo shadow domain (CSD), which generates a homodimer with a hydrophobic surface that interacts with more than 12 different binding partners. These partners are involved in different processes, ranging from gene silencing to chromosome architecture, so correct discrimination among partners is of central importance. Several partners have been found to have a consensus sequence PXVXL which is utilized for binding. Due to the multiplicity of partners described to interact with the CSD, we wondered whether an alternative binding surface was being used for protein partners such as Heterochromatin Protein 2 (HP2), which does not have an easily identifiable PXVXL sequence. I found however that the CSD PXVXL binding surface is being used, interacting with the HP2 sequence LCVKI centered on V-2468 (position 0). Despite the non-canonical motif, I found that HP2 binds 66-fold more tightly to the HP1a CSD than does PIWI, which interacts with the HP1a CSD using the same surface via a PRVKV sequence centered on V-30. Further investigation showed that HP2 uses an extended binding surface on HP1a, contacting amino acids such as L165, outside of the pentamer binding site. This additional binding contact imparts a substantial portion of the enhanced affinity. In addition, HP2 binding is more dependent on the residues C-terminal to the chromo shadow domain. The difference in affinity is also partly attributed to the PXVXL pentamer sequence in HP2 itself. This sequence, LCVKI, has two large hydrophobic groups in the plus and minus two positions, whereas PIWI does not. Phosphorylation of the HP1a CSD at residues S199 and S202 improves the affinity of both HP2 and PIWI. This HP1a modification enhances partner binding where positively charged residues are found in the plus one and minus one positions, as in the case for both HP2 and PIWI. Thus we find that there is a fine-tuned interplay between the HP1a amino acids in the chromo shadow domain, the C-terminal extension, post translational modifications, and the binding partner's pentamer sequence together with residues outside of that sequence; the combination of these effects allows HP1a to discriminate among its partners.

Language

English (en)

Chair and Committee

Sarah C.R. Elgin

Committee Members

Robert E. Blankensip, Douglas L. Chalker, Kathleen B. Hall, Joseph M. Jez, John E. Majors

Comments

Permanent URL: https://doi.org/10.7936/K7ZW1HVP

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