Cis-regulatory methylation of the VIP gene in the white-throated sparrow (Zonotrichia albicollis) Open Access

Prichard, Mackenzie R. (Spring 2021)

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Pathways between genotype and phenotype, especially a behavioral phenotype, are complicated by many levels of biological organization, making direct connections difficult. Animal models with clear connections between genotype and behavioral phenotype are needed. One such model is the white-throated sparrow. In this species, a rearrangement of the second chromosome is linked with territorial aggression. Birds with a copy of the rearrangement are more aggressive than those without it. The rearrangement has captured the gene VIP, which encodes vasoactive intestinal peptide, a neuromodulator known to be causal for aggression in other songbirds. VIP expression is higher in the anterior hypothalamus of birds with the rearrangement than those without it and expression of VIP in this region predicts the level of territorial aggression regardless of genotype. Thus, hypothalamic VIP is a strong candidate to mediate this behavioral polymorphism. For example, due to differentiation between the standard and rearranged chromosomes, there are two distinct alleles of VIP. The two alleles may be transcribed at different levels, leading to morph differences in expression. Here, we aimed to identify epigenetic mechanisms that could contribute to such allelic imbalance. We extracted and bisulfite-converted DNA from samples of the hypothalamus in wild-caught adults and nestlings and used high-throughput sequencing to measure DNA methylation of a region 1.2kb upstream of the VIP start site. We found that the methylation of this region was lower on the allele inside the rearrangement than on the alternative allele. In addition, methylation differed between the alleles at specific sites within this putative cis-regulatory element. This work represents an initial step toward understanding how epigenetic differentiation inside chromosomal inversions leads to the development of alternative behavioral phenotypes.

Table of Contents

Introduction 1

Methods 6

Tissues and Extractions 6

RNA Sequencing 7

Measurements of Methylation 8

Bisulfite Conversion and Polymerase Chain Reaction 8

Targeted Bisulfite Sequencing 9

Library Preparation 9

Data Analysis 10

Differences in Methylation 10

Regressions of Methylation on Expression 11

Results 12

Differences in Methylation 12

Regressions of Methylation on Expression 13

Discussion 13

References 19

Tables 28

Figures 50

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