H3K4me1 facilitates promoter-enhancer interactions and gene activation during embryonic stem cell differentiation. Histone H3 lysine 4 mono-methylation (H3K4me1) marks poised or active enhancers. KMT2C (MLL3) and KMT2D (MLL4) catalyze H3K4me1, but their histone methyltransferase activities are largely dispensable for transcription during early embryogenesis in mammals. To better understand the role of H3K4me1 in enhancer function, the study analyzed dynamic enhancer-promoter (E-P) interactions and gene expression during neural differentiation of mouse embryonic stem cells. The results showed that KMT2C/D catalytic activities were only required for H3K4me1 and E-P contacts at a subset of candidate enhancers induced upon neural differentiation. In contrast, a majority of enhancers retained H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, H3K4me1 signals at these KMT2C/D-independent sites were reduced after acute depletion of KMT2B, resulting in aggravated transcriptional defects. These findings suggest that KMT2B contributes to H3K4me1 at enhancers, and H3K4me1 actively promotes E-P interactions and enhancer-driven transcription during differentiation. The study also found that KMT2C/D catalytic activity is required for new E-P contacts in NPC, and loss of KMT2C/D catalytic activity delayed NPC differentiation and activation of hundreds of genes. Additionally, KMT2B contributes to H3K4me1 at KMT2C/D-independent enhancers, and its loss significantly affected gene induction. The study highlights the role of H3K4me1 in enhancer-promoter interactions and transcription in mammalian cells.H3K4me1 facilitates promoter-enhancer interactions and gene activation during embryonic stem cell differentiation. Histone H3 lysine 4 mono-methylation (H3K4me1) marks poised or active enhancers. KMT2C (MLL3) and KMT2D (MLL4) catalyze H3K4me1, but their histone methyltransferase activities are largely dispensable for transcription during early embryogenesis in mammals. To better understand the role of H3K4me1 in enhancer function, the study analyzed dynamic enhancer-promoter (E-P) interactions and gene expression during neural differentiation of mouse embryonic stem cells. The results showed that KMT2C/D catalytic activities were only required for H3K4me1 and E-P contacts at a subset of candidate enhancers induced upon neural differentiation. In contrast, a majority of enhancers retained H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, H3K4me1 signals at these KMT2C/D-independent sites were reduced after acute depletion of KMT2B, resulting in aggravated transcriptional defects. These findings suggest that KMT2B contributes to H3K4me1 at enhancers, and H3K4me1 actively promotes E-P interactions and enhancer-driven transcription during differentiation. The study also found that KMT2C/D catalytic activity is required for new E-P contacts in NPC, and loss of KMT2C/D catalytic activity delayed NPC differentiation and activation of hundreds of genes. Additionally, KMT2B contributes to H3K4me1 at KMT2C/D-independent enhancers, and its loss significantly affected gene induction. The study highlights the role of H3K4me1 in enhancer-promoter interactions and transcription in mammalian cells.