Changes in melanocyte RNA and DNA methylation favour pheomelanin synthesis and may avoid systemic oxidative stress after dietary cysteine supplementation in birds

Abstract

Cysteine plays essential biological roles, but excessive amounts produce cellular oxidative stress. Cysteine metabolism is mainly mediated by the enzymes cysteine dioxygenase and γ‐glutamylcysteine synthetase, respectively coded by the genes CDO1 and GCLC. Here we test a new hypothesis posing that the synthesis of the pigment pheomelanin also contributes to cysteine homeostasis in melanocytes, where cysteine can enter the pheomelanogenesis pathway. We conducted an experiment with the Eurasian nuthatch Sitta europaea, a bird producing large amounts of pheomelanin for feather pigmentation, to investigate if melanocytes show epigenetic lability under exposure to excess cysteine. We increased systemic cysteine levels in nuthatches by supplementing them with dietary cysteine during growth. In feather melanocytes this led to the downregulation of genes involved in intracellular cysteine metabolism (GCLC), cysteine transport to the cytosol from the extracellular medium (Slc7a11) and from melanosomes (CTNS), and regulation of tyrosinase activity (MC1R and ASIP). These changes were mediated by increases in DNA m5C in all genes except Slc7a11, which experienced RNA m6A depletion. Birds supplemented with cysteine synthesized more pheomelanin than controls, but did not suffer higher systemic oxidative stress. These results suggest that excess cysteine activates an epigenetic mechanism that favours pheomelanin synthesis and may protect against oxidative stress.

Exposure to a competitive social environment activates an epigenetic mechanism that limits pheomelanin synthesis in zebra finches

“…An absence of changes in CTNS expression in feather melanocytes has also been reported in the lesser kestrel ( Falco naumanni ) under exposure to a different natural source of environmental stress such as breeding colony size (Galván, Moraleda, et al, ). However, when dietary cysteine intake was experimentally increased in developing Eurasian nuthatches, a downregulation of CTNS that favours pheomelanin synthesis was detected (Rodríguez‐Martínez et al, ). Indeed, CTNS expression has been suggested to influence pheomelanin synthesis only when systemic cysteine levels are above a certain threshold (Chiaverini et al, ).…”

“…On the other hand, Slc45a2 has also been proposed to participate in the transport of cysteine in bird melanocytes (Galván, Inácio, & Nielsen, ), but a previous experimental increment of systemic cysteine did not affect its expression in nuthatches (Rodríguez‐Martínez et al, ). Here, the proportion of m 5 C DNA in Slc45a2 decreased in male zebra finches kept in groups as compared to solitary males, but no changes in its expression were found.…”

“…DNA was extracted using a Quick‐DNA Plus kit (Zymo Research) from follicular melanin units. We quantified 5‐methylcytosine (m 5 C) at the target genes by immunoprecipitation and real‐time qPCR following a procedure previously used with the same type of samples (Rodríguez‐Martínez et al, ). The proportion of DNA with m 5 C was calculated by dividing the C t of the input DNA control by that of the immunoprecipitated test sample (Rodríguez‐Martínez et al, ).…”

“…After 15 days under treatment, the growing flank feathers were collected to measure gene expression in melanocytes and pheomelanin-based colour intensity (Galván, Inácio, Romero-Haro, et al, 2017;Rodríguez-Martínez et al, 2019). We also quantified DNA methylation on these samples.…”

“…Rodríguez-Martínez et al, 2019). The proportion of DNA with m 5 C was calculated by dividing the C t of the input DNA control by that of the immunoprecipitated test sample.…”