DOI: 10.5061/dryad.9zw3r22rw
title: A potential role for epigenetic mechanisms enabling appropriate seasonal reproductive transitions of liver yolk-precursor production

description: Animals utilize mechanisms that enable breeding at times that ensure
 offspring production and growth during periods of abundant resources. DNA
 methylation is one mechanism by which the expression of genes necessary
 for reproduction may be regulated so that they are expressed only at
 appropriate times of the year. To date, much work on seasonal breeding in
 vertebrates has focused on the neuroendocrine system; however, oviparous
 vertebrates, including birds, must also rely on the liver for the
 production of yolk precursor hormones that will provide the nutrients
 necessary for developing young in ovo. We hypothesized that
 changes in DNA methylation in the liver may be one mechanism ensuring
 appropriately timed seasonal breeding. We observed that liver expression
 of (VTG2) was upregulated in birds sampled during the early breeding
 compared with those sampled during the pre-breeding period, and also
 observed changes in liver expression of DNMT1 and DNMT3a between these two
 periods. Contrary to our predictions, we observed an increase in
 methylation from pre-breeding to breeding at one of the three CpG sites in
 the promoter region of the VTG2 gene, with no differences at the other two
 CpG sites. Finally, we asked if patterns of methylation of VTG2 are
 similar between the liver and the blood. We observed strong correlations
 between blood and liver in two sites that did not change between
 pre-breeding and breeding, while there was only a trend for a significant
 association between blood and liver DNA methylation at the site that
 displayed a significant increase in liver CpG methylation between sampling
 time periods. Together, these findings suggest that changes in DNA
 methylation in a tissue important for reproduction outside of the
 reproductive endocrine axis (liver) may play a critical role in
 appropriate timing of seasonal clutch initiation.
description: # A potential role for epigenetic mechanisms enabling appropriate seasonal
 reproductive transitions of liver yolk-precursor production Dataset DOI:
 [10.5061/dryad.9zw3r22rw](10.5061/dryad.9zw3r22rw) ## Description of the
 data and file structure Each individual was sacrificed during one of two
 sampling periods as described in the text. All individuals had their DNA
 bisulfite converted and the percent of methylation at the targeted sites
 denoted in the text were analyzed using pyrosequencing.  Pyrosequencing
 results that did not meet quality control criteria described in the text
 were not included in the analysis. ### Files and variables #### File:
 methylation.junco.combined.DRYAD.withage.csv **Description:**  #####
 Variables * Female No.: Female number was assigned to each bird captured
 and sampled sequentially. Cells marked 'null' had no age
 information recorded on the datasheet.  * age: AY are birds determined as
 young adults (after hatch year) in their first breeding year using plumage
 to classify age; AO are adult birds determined to be 2 or more years old
 using plumage characteristics. Cells marked 'null' had no age
 information recorded on the datasheet.  * age.yr: Converted AY and AO into
 a numerical variable. AY birds were assigned a 1, and AO birds were
 assigned a 2. * Date: this was the calendar day that birds were sacrificed
 for sampling. * Sample.tim: This is a categorical variable to denote the
 two distinct sampling time periods described in the text. * Pre-breeding
 were from the sampling dates from 4/25/16 through 4/27/16. *
 Early-breeding were from the sampling period of 5/12/16 through 5/14/16
 and corresponded to the time period when the earliest females in the
 population were initiating clutches. * Sample.time: Converted sample.tim
 to a numeric value. Birds sampled in the pre-breeding period were assigned
 a 0, and birds sampled during the early breeding period were assigned a 1.
 * Time of Capture: This variable denotes the hour and minute that birds
 were sacrificed. * Liver_site1_methylation: The percent of methylated
 cytosinse at CpG site 1 in liver as measured by pyrosequencing. Cells
 marked 'null' did not pass the QC check.  *
 Liver_site2_methylation: The percent of methylated cytosinse at CpG site 2
 in liver as measured by pyrosequencing. Cells marked 'null' did
 not pass the QC check.  * Liver_site3_methylation: The percent of
 methylated cytosinse at CpG site 3 in liver as measured by pyrosequencing.
 Cells marked 'null' did not pass the QC check.   *
 Blood_site1_methylation: The percent of methylated cytosinse at CpG site 1
 in blood as measured by pyrosequencing. Cells marked 'null' did
 not pass the QC check.   * Blood_site2_methylation: The percent of
 methylated cytosinse at CpG site 2 in blood as measured by
 pyrosequencing. Cells marked 'null' did not pass the QC check.  
 * Blood_site3_methylation: The percent of methylated cytosinse at CpG site
 3 in blood as measured by pyrosequencing. Cells marked 'null'
 did not pass the QC check.   * Liver_VTG2_expression: Relative expression
 of VTG2  in liver sample from qPCR.  * LOG10_Liver_VTG2_expression:
 log~10~ relative expression of VTG2  in liver sample from qPCR. *
 Liver_DNMT1_expressoin: Relative expression of DNMT1  in liver sample from
 qPCR. Cells marked 'null' did not have enough sample remaining
 to run.  * Liver_DNMT3A_expressoin: Relative expression of DNMT3a in liver
 sample from qPCR. Cells marked 'null' did not have enough sample
 remaining to run.  * F1_size_mm: Size of the largest follicle in the ovary
 sample.  Follicles were only measured during the early breeding period, as
 no bird sampled during the pre-breeding period displayed developed
 follicles.