N⁶-methyladenosine (m⁶A) is the most abundant modification in linear RNA molecules. Over the last few years, interestingly, many circRNA molecules are also found to have extensive m⁶A modification sites with temporal and spatial specific expression patterns. To date, however, little information is available concerning the expression profiling and functional regulatory characteristics of m⁶A modified circRNAs (m⁶A-circRNAs) in secondary hair follicles (SHFs) of cashmere goats. In this study, a total of fifteen m⁶A-circRNAs were identified and characterized in the skin tissue of cashmere goats. Of these, six m⁶A-circRNAs were revealed to have significantly higher expression in skin at anagen compared with those at telogen. The constructed ceRNA network indicated a complicated regulatory relationship of the six anagen up-regulated m⁶A-circRNAs through miRNA mediated pathways. Several signaling pathways implicated in the physiological processes of hair follicles were enriched based on the potential regulatory genes of the six anagen up-regulated m⁶A-circRNAs, such as TGF-beta, axon guidance, ribosome, and stem cell pluripotency regulatory pathways, suggesting the analyzed m⁶A-circRNAs might be essentially involved in SHF development and cashmere growth in cashmere goats. Further, we showed that four m⁶A-circRNAs had highly similar expression trends to their host genes in SHFs of cashmere goats including m⁶A-circRNA-ZNF638, -TULP4, -DNAJB6, and -CAT. However, the expression patterns of two m⁶A-circRNAs (m⁶A-circRNA-STAM2 and -CAAP1) were inconsistent with the linear RNAs from their host genes in the SHFs of cashmere goats. These results provide novel information for eluci-dating the biological function and regulatory characteristics of the m⁶A-circRNAs in SHF development and cashmere growth in goats.

The cashmere, a kind of nature protein fiber, is one of the main use of cashmere goats. The induced activation of secondary hair follicle (SHF) stem cells by the dermal papilla cell-derived signals is a key biological process for the morphogenesis and growth of cashmere fiber in cashmere goats. Previously, the circRNA-ERCC6 (circERCC6) was identified from cashmere goat SHFs; however, its biological significance is unclear in the SHF physiology process of cashmere goats. In this study, we found that circERCC6 exhibited significantly higher expression at anagen SHF bulge compared with the counterpart of telogen and harbored three m⁶A modified sites (named m⁶A-685, m⁶A-862, and m⁶A-995) through methylation immunoprecipitation using a real-time quantitative polymerase chain reaction (Me-RIP-qPCR) technique. The knockdown experiments of circERCC6 in SHF stem cells showed that circERCC6 positively regulates the induced activation of SHF stem cells in cashmere goats. Through a dual-luciferase reporter assay, we demonstrated that m⁶A-modified circERCC6 (m⁶A-circERCC6) sponged miR-412-3p to upregulate the expression of BNC2 mRNA in SHFstem cells. Through m⁶A-deficient mutant assay in circERCC6 knockdown SHF stem cells, we further showed that m⁶A modification within circERCC6 is required to mediate the miR-412-3p/BNC2 axis to finally promote the proper induced activation of SHF stem cells in cashmere goats.

Cashmere goats are widely distributed in the cold, arid and semi-arid remote regions of northern China. Their main economic value is the production of precious cashmere fibers. The differentiation of second hair follicle (SHF) stem cells into hair follicle lineages plays a crucial role in SHF regeneration as well as in the morphogenesis and growth of cashmere fibers; however, its precise molecular mechanism is still unclear. In this study, we found that N⁶-methyladenosine (m⁶A)-circHECA recruiting FUS promoted the differentiation of SHF stem cells into hair follicle lineages through stabilizing FOXM1 mRNA in SHF stem cells, thereby activating the NOTCH pathway in cashmere goats. Furthermore, we confirmed that the m⁶A modification of circHECA is required for the FUS/FOXM1-mediated NOTCH signaling pathway to facilitate the differentiation of SHF stem cells into hair follicle lineages via transfecting circHECA m⁶A-deficient mutants. Our results contribute to elucidating the functional mechanism of m⁶A-circHECA in the differentiation of SHF stem cells into hair follicle lineages in cashmere goats.