AbstractHistone ubiquitylation/deubiquitylation plays a major role in the epigenetic regulation of gene expression. In plants, OTLD1, a member of the ovarian tumor (OTU) deubiquitinase family, deubiquitylates monoubiquitylated histone 2B and represses the expression of genes involved in growth, cell expansion, and hormone signaling. Like many other histone-modifying enzymes, OTLD1 lacks the intrinsic ability to bind DNA. How OTLD1, as well as most other known plant histone deubiquitinases, is recruited specifically to the promoters of its target genes remains unknown. Here, we show that Arabidopsis transcription factor LSH10, a member of the ALOG protein family, interacts with OTLD1 in living plant cells. Loss-of-function LSH10 mutations relieve the OTLD1-promoted transcriptional repression of the target genes, resulting in their elevated expression, whereas recovery of the LSH10 function results in down-regulated transcription of the same genes. We then show that LSH10 associates directly with the target gene chromatin as well as with the specific DNA sequence motifs in the promoter regions of the target genes. Furthermore, in the absence of LSH10, the degree of H2B monoubiquitylation in the target promoter chromatin increases. Hence, our data suggest that OTLD1-LSH10 acts as a co-repressor complex, in which LSH1 recruits OTLD1 to the target gene promoters, potentially representing a general mechanism for recruitment of plant histone deubiquitinases to the target chromatin.