Higher expression levels of MSL2 in D. virilis lead to histone locus binding that is not seen in other Drosophila species. Open Access

Abstract

Nuclear bodies are membraneless structures containing concentrated regulatory factors that coordinate nuclear processes such as gene expression. The histone locus body (HLB) is a nuclear body that is the main site of histone mRNA production. While many factors of the HLB are known, there are likely many unknown factors that contribute to histone gene regulation. In addition, while HLB function is highly conserved, it is also unknown how the HLB may function differently in different species. The histone locus (HL) of the model organism Drosophila melanogaster contains ~100 tandem arrays of the five histone genes. While D. melanogaster has one HL, the related species, Drosophila virilis, has two HL. We observe localization of male-specific dosage compensation proteins MSL2 and MSL3 to the major D. virilis histone locus using polytene chromosome immunofluorescence, which we do not observe in other Drosophila species. To confirm our immunofluorescence observations, we mapped existing MSL2 ChIP-seq data and discovered that when MSL2 from either species is overexpressed, it targets the H2a-H2b promoter. Finally, we performed RT-qPCR analysis on D. melanogaster and D. virilis to compare expression levels of msl2, observing higher levels of msl2 in D. virilis, compared to D. melanogaster. Our results indicate that increased MSL2 expression leads to artificial HL targeting in D. melanogaster and natural targeting in D. virilis. In addition, our results caution against using D. melanogaster MSL2 overexpression systems to infer the role of this protein in dosage compensation.

Table of Contents

Introduction. 1

Figure 1: CLAMP targets GA-rich sequences across the genome. 2

Figure 2: CLAMP targets GA-repeats at the histone3/4 promoter, which promotes histone locus body formation. 2

Figure 3: Conservation of Mxc, CLAMP, and MSL2. 3

Figure 4: Arrangement of the dosage compensation complex (DCC) in Drosophila. 5

Figure 5: The D. melanogaster histone locus and D. virilis histone loci. 7

Figure 6: Localization of MSL2. (A) In D. melanogaster, the protein MSL2 targets the X-chromosome. 8

Methods. 10

Results. 13

Figure 7: The DCC complex targets the major histone locus (HL) in D. virilis. 14

Figure 8: The DCC does not localize to the histone locus in D. melanogaster. 15

Figure 9: Immunostaining of MSL2 on polytene chromosomes from D. pseudoobscura and D. willistoni. 16

Table 1: Table summary of polytene immunostaining results. 17

Figure 10: Overexpression of MSL2 in D. melanogaster leads to peaks at the histone2a-histone2b promoter. 19

Figure 11: No MSL2 peaks are observed in the histone array upon normal MSL2 expression in D. melanogaster. 20

Figure 12: RT-qPCR results on the relative msl2 RNA expression levels in D. melanogaster (mel) and D. virilis (vir). 21

Discussion. 22

Figure 13: Phylogenetic tree of Drosophila species. 23

References. 26

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Genetics Biology, Molecular
Keyword	Drosophila virilis Dosage compensation MSL3 Histone locus body Drosophila melanogaster MSL2
Committee Chair / Thesis Advisor	Leila Rieder, Emory University
Committee Members	Rachelle M. Spell, Emory University Kenneth H. Moberg, Emory University

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