Abstract: Objective: To elucidate the regulatory impact of Setd2 within osteocytes on bone formation. Methods: Setd2^flox/flox mice were crossed with the Dmp1^Cre strain to generate Dmp1^Cre, Setd2^flox/flox mice. All mice analyzed were maintained on the C57BL/6 background. Immunohistochemical staining was used to confirm the specific deletion of Setd2 in osteocytes. Micro-CT and three-dimensional reconstruction were used to analyze cortical and trabecular bone mass variations in bone tissues from same-litter mice at 6 months of age. Differences in bone morphology parameters, such as trabecular number, thickness, and separation, were conducted using CTAn software. Additionally, safranine O/fast green staining was utilized to observe changes in the blue-stained bone tissue regions in Setd2 cKO and control mice. Immunohistochemical staining was used to observe trimethylation of lysine 36 on histone 3 (H3K36me3). Results: With successful Setd2 deletion in osteocytes, Setd2 cKO mice exhibited smaller body size compared to littermate controls. Micro-CT and bone morphometry analysis revealed that, at 6 months of age, Setd2 cKO mice displayed thinner cortical bone, decreased bone density, reduced trabecular thickness, lower trabecular number, and increased trabecular separation compared to control mice (P<0.05). Safranine O/fast green staining further indicated a reduced blue-stained bone tissue area in Setd2 cKO mice compared to controls. Compared to the control group mice, there was a loss of H3K36me3 modification in Setd2 cKO mice. Conclusion: The loss of Setd2 in osteocytes affected the trimethylation modification of H3K36, resulting in a decrease in mouse bone mass.

Key words: Setd2, gene knockout, osteocytes, bone mass