Enhancers are highly sensitive to DNase I attact and have specific histone modifications, H3K4me1 and H3K27ac. However, it is unclear the reciprocal roles of these modifications for enhancer activity. To study the distinct roles of both histone modifications in it, catalytic domain of histone methyltransferases MLL3 and MLL4 and acetyltransferase p300, respectively, was deleted by CRISPR/Cas9 system in erythroid K562 cells. The loss of H3K4me1 decreased H3K27ac at the β-globin enhancer LCR HSs but the H3K27ac reduction did not affect H3K4me1. Notably, histone H3 depletion was reduced at putative enhancers by the loss of H3K4me1 but not by the loss of H3K27ac. Histone depletion in the putative enhancers was decreased in △Mll3/4 cells, but it was not any affected in △p300 cells. Furthermore, chromatin remodeling complexes were recruited at β-globin LCR HSs in only H3K4me1-dependent manner. △Mll3/4 cells but not △p300 cells. However, the transcription of eRNAs was decreased at the active enhancers in both loss of H3K4me1 and loss of H3K27ac. Hyperacetylation of H3K27ac by histone deacetylase inhibitor TSA treatment increased eRNA transcription without affect H3K4me1 level. These results indicate that H3K4me1 is required for histone depletion at enhancers but H3K27ac is required for eRNAs transcription even H3K4me1 was not enough for it. Thus, this study imply that H3K4me1 and H3K27ac affect each other differently at enhancers and play distinct roles in enhancer activity.