INTRODUCTION
Telomeres are highly conserved, non-coding DNA sequences that form
protective caps at the end of eukaryotic chromosomes (Blackburn,1991,
Blackburn & Epel 2012). In the absence of telomerase (a reverse
transcriptase that adds telomeric repeats de novo after each cell
division), telomeres shorten with each round of cell division (Harley,
Futcher, & Greider, 1990). When a critical length is lost, telomeres
become dysfunctional and cells enter a state of replicative senescence
(Hornsby, 2003; Verdun & Karlseder, 2007, see also Victorelli &
Passos, (2017) for length independent damage to telomeres triggering
cell senescence). The accumulation of senescent cells is known to
contribute to age-related declines in tissue and organ function (Wong et
al., 2003). Accordingly, within species, relatively short telomeres and
accelerated rates of telomere shortening have been associated with
fitness costs, predominantly via reduced lifespan, at the individual
level (Monaghan, 2010; Eastwood et al., 2019; Boonekamp, Mulder,
Salomons, Dijkstra, & Verhulst, 2014, Wilbourn et al., 2018). Given the
relationship with lifespan, telomeres have been widely used as
biomarkers of biological age (Jylhava, Pedersen, & Hagg, 2017). This
has also led to a recent focus in understanding how inter-individual
variation in telomere length arises. Early life telomere length is
partly determined by genetic factors (Olsson et al., 2011; Dugdale &
Richardson 2018), however, accumulating evidence suggests that
environmental cues also impact telomere length dynamics across the life
course (Kotrschal, Ilmonen, Penn, 2007; Vedder, Verhulst, Zuidersma, &
Bouwhuis, 2018; Dupoue et al., 2017; Boonekamp, et al., 2014).
Information on the relationship between environmental and telomere
length variation is steadily increasing, yet surprisingly little is
known about whether other DNA modifications mirror telomere dynamics.
Insight on how telomere length can vary in concert with other genetic
traits can expand our understanding of DNA based biological markers of
aging, and of physiological reactions to environmental change.
DNA methylation (DNAm) is an epigenetic mechanism that appears to be an
important component of telomere length regulation (Blasco, 2007), and is
also considered a promising biological clock (Horvath & Raj 2018). Yet,
the relationship between DNAm and telomere length has not yet been
explored in an ecological context in any species. DNAm usually refers to
the addition of a methyl group to a cytosine base at a CG dinucleotide
(a ‘CpG’ site) on the DNA sequence, but can occur at other sites in
different taxa (Anglers et al., 2010). When DNAm occurs at a CpG site
close to a gene regulatory region, it can modulate phenotypic variation
through its effects on gene expression. Evidence suggests that DNAm
could be involved in two, key telomere regulatory processes; those
involving telomerase (Buxton et al., 2014), and ‘alternative mechanisms’
relying on homologous recombination between telomeric sequences such as
alternative lengthening of telomeres (Gonzalo et al., 2006). Indeed,
while telomeres themselves do not contain CpG sites, decreases in global
and subtelomeric DNAm are known to be concomitant with increased
homologous recombination between telomeric sequences and dramatically
elongated telomeres in mouse cells (Gonzalo et al., 2006). Additionally,
a negative relationship between telomere length and genome-wide (Lee et
al., 2019) and gene-specific (Lee et al., 2019; Buxton et al., 2014)
DNAm has been described in humans (however, this relationship may be
complicated as a positive relationship has been detected in a different
correlative study (Dong et al., 2018)). Evidence also suggests that
short telomeres have specific epigenetic marks that may facilitate their
preferential elongation (Hemann, Strong, Hao, & Greider, 2001; de
Lange, 2005). These studies suggest the existence of a direct
relationship between DNAm and telomere length. However, there is also
plenty of scope for indirect relationships between DNAm and telomere
length to occur given that they are associated with a range of the same
biological and ecological factors (Figure 1) and are also both
independently identified as molecular measures of age and ageing (Lu et
al., 2019; Banszerus, Vetter, Salewsky, König & Demuth, 2019).
To date, surprisingly little work has explored the potential links
between telomere length and DNAm, although a few studies have indicated
some conceptual connections (Horvath et al 2018; Blasco, 2007; Figure
1). DNAm and telomere length are highly responsive to environmental cues
(Feil & Fraga, 2012; Monaghan, 2014), particularly during early life
(Watson, Bolton & Monaghan, 2019; Boonekamp et al., 2014). For example,
studies have detected associations between DNAm or telomere length and
clutch/brood size (Noguera & Velando, 2020; Jimeno, Hau, Gomez-Diaz, &
Verhulst, 2019; Sheldon, Schrey, Ragsdale & Griffith 2018; Nettle et
al., 2016; Reichert et al., 2014; Costanzo et al., 2016; Boonekamp et
al., 2014); ambient temperature (Stier, Metcalfe & Monaghan, 2020;
Sheldon, Schrey, Hurley, & Griffith, 2020; Yan et al., 2015); and body
size/growth rate (Young et al., 2017; Vedder et al., 2018). It is
therefore important to account for these influences when testing the
association between DNAm and telomere length. The present study is the
first examination, to date, of the relationship between DNAm and
telomere length dynamics in a species in the wild in which individuals
are exposed to natural variation in early life conditions. Specifically,
we collected longitudinal measures of telomere length (using qPCR) and
genome-wide levels of DNAm (using methylation sensitive- amplification
fragment length polymorphisms (MS-AFLP)) across the early life of wild
zebra finches (Taeniopygia guttata ). Although MS-AFLP analyses do
not provide inference on the functional, gene regulatory consequences of
DNAm differences, they do provide a useful tool to compare the
relationship between telomere length dynamics and changes in the
percentage of a consistent subset of CpG loci that are methylated among
individuals (Schrey et al., 2013). Our study aims to provide an initial
exploration into the relationship between early life DNAm changes and
telomere length changes in wild animals as well as potential
associations with ambient temperature and life history effects. This can
establish a base for future research to link and/or disentangle the
relationship between early life conditions, DNA based biomarkers of age,
and individual fitness parameters.
In addition to considering the links between telomere length and DNAm,
our study is also of value in providing the first investigation of
telomere length dynamics in wild zebra finches Taenopygia
guttata . Characterisations of telomere dynamics in the wild have become
increasingly common for a wide range of bird species (Supplementary
Table 1a and 1b). In contrast, laboratory studies on birds are largely
performed using the zebra finch, in which paradoxically, telomere
dynamics have yet to be explored in the wild (Supplementary Table 1a).
Studies of ecologically relevant populations of wild zebra finches are
thus of value in helping to contextualize and interpret the controlled
laboratory tests that have made such a significant contribution to our
understanding of telomere biology in birds.