Identification of Novel Transcription Factors in Osteoclast Differentiation Using Genome-wide Analysis of Open Chromatin Determined by DNase-seq

Abstract

Clarification of the mechanisms underlying osteoclast differentiation enables us to understand the physiology of bone metabolism as well as the pathophysiology of bone diseases such as osteoporosis. Recently, it has been reported that epigenetics can determine cell fate and regulate cell type-specific gene expression. However, little is known about epigenetics during osteoclastogenesis. To reveal a part of epigenetics, especially focused on chromatin dynamics, during early osteoclastogenesis and to identify novel transcription factors involved in osteoclastogenesis, we performed a genome-wide analysis of open chromatin during receptor activator of NF-kB ligand (RANKL)-induced osteoclastogenesis using DNase I hypersensitive sites sequencing (DNase-seq). DNase-seq was performed using the extracted nuclei from RAW264 cells treated with or without RANKL for 24 hours, followed by several bioinformatic analyses. DNase I hypersensitive sites (DHSs) were dynamically changed during RANKL-induced osteoclastogenesis and they accumulated in promoter regions. The distributions of DHSs among cis-regulatory DNA regions were identical regardless of RANKL stimulation. Motif discovery analysis successfully identified well-known osteoclastogenic transcription factors including Jun, CREB1, FOS, ATF2, and ATF4, but also novel transcription factors for osteoclastogenesis such as Zscan10, Atf1, Nrf1, and Srebf2. siRNA knockdown of these identified novel transcription factors impaired osteoclastogenesis. Taken together, DNase-seq is a useful tool for comprehension of epigenetics, especially chromatin dynamics during osteoclastogenesis and for identification of novel transcription factors involved in osteoclastogenesis. This study may reveal underlying mechanisms that determine cell type-specific differentiation of bone cells and may lead to investigation of novel therapeutic targets for osteoporosis.

Reprogramming human A375 amelanotic melanoma cells by catalase overexpression: Upregulation of antioxidant genes correlates with regression of melanoma malignancy and with malignant progression when downregulated

“…In human skin tumors NFE2L1 was lower than in normal skin [70]. The involvement of NF2L1 has also been described in osteoblast differentiation [71, 72]. Thus, GCLC and NFE2L1 upregulation in A7 may contribute to both the increased ROS scavenging capacity compared with G10 and controls and its more differentiated and less aggressive melanoma.…”

Uhrf1 is indispensable for normal limb growth by regulating chondrocyte differentiation through specific gene expression

“…Each experiment was biologically replicated at least three times. siRNA siRNA was purchased from Sigma-Aldrich and transfected into primary chondrocytes obtained from cKO mice using an electroporation apparatus (Neon, Invitrogen) as previously described (Inoue and Imai, 2014). After siRNA transfection, cells were treated with 100 ng/ml of rhBMP2 for 3 days and harvested for RNA extraction.…”

Epigenetic regulation of bone cells

“…Although these interactions are detected in cells in which RUNX2 is silent, the interaction frequency between RUNX2-Supt3h is significantly increased during osteoblastogenesis as shown by chromosome conformation capture (3C) analysis, suggesting Supt3h as a potential regulator of bone specific RUNX2 P1 promoter. DNase-seq analysis also detected that RANKL facilitates the dynamic changes of chromatin accessibility in RAW 264.7 cells, a murine monocytic cell line that is capable of differentiating into osteoclasts upon RANKL stimulation [107]. Using motif discovery analysis, Inoue et al further profiled the transcription motifs in open chromatin regions and identified novel players for osteoclastogenesis in addition to other well-defined osteoclastogenic transcription factors.…”