Macronuclear nanochromosomes studied and DNA methylation pattern of developmentally expressed genes during vegetative growth and sexual reproduction. (A) Actin, tubulin and histone H4 are constitutively expressed genes; mdp1 and mdp2 are only expressed during sexual reproduction in exconjugant cells. Red shaded areas show the open reading frames of the nanochromosomes. Cytosines are methylated at positions 28, 35 and 38 in mdp1 and at position 44 in mdp2 (red stars). Black dashed lines: amplicons analyzed for cytosine methylation; blue lines: amplicons used for ChIP analyses. (B, C) DNA methylation pattern in mdp1 (B) and mdp2 (C) during vegetative growth (mdp1/mdp2 repressed) and in exconjugant cells (mdp1/mdp2 expressed).