The protein biotin ligase, holocarboxylase synthetase (HLCS), is a chromatin protein that interacts physically with the DNA methyltransferase DNMT1, the methylated cytosine-binding protein MeCP2 and the histone H3 K9-methyltransferase EHMT1, all of which participate in folate-dependent gene repression. Here we tested the hypothesis that biotin and folate synergize in the repression of pro-inflammatory cytokines and long-terminal repeats (LTRs), mediated by interactions between HLCS and other chromatin proteins. Biotin and folate supplementation could compensate for each other's deficiency in the repression of LTRs in Jurkat and U937 cells. For example, when biotin-deficient Jurkat cells were supplemented with folate, the expression of LTRs decreased by >70%. Epigenetic synergies were more complex in the regulation of cytokines compared with LTRs. For example, the abundance of TNF-α was 100% greater in folate- and biotin-supplemented U937 cells compared with biotin-deficient and folate-supplemented cells. The NF-κB inhibitor curcumin abrogated the effects of folate and biotin in cytokine regulation, suggesting that transcription factor signalling adds an extra layer of complexity to the regulation of cytokine genes by epigenetic phenomena. We conclude that biotin and folate synergize in the repression of LTRs and that these interactions are probably mediated by HLCS-dependent epigenetic mechanisms. In contrast, synergies between biotin and folate in the regulation of cytokines need to be interpreted in the context of transcription factor signalling.