HDAC3 ensures stepwise epidermal stratification via NCoR/SMRT-reliant mechanisms independent of its histone deacetylase activity (HDAC3 NCoRSMRT microarray)

Chromatin modifiers play critical roles in epidermal development, but the functions of histone deacetylases in this context are poorly understood. We find that the Class I HDAC, HDAC3, is expressed broadly in embryonic epidermis, and is required for its orderly stepwise stratification. Stability of HDAC3 protein in vivo is reliant on NCoR and SMRT, which function redundantly in epidermal development. However, point mutations in the NCoR and SMRT Deacetylase Activating Domains, which are required for HDAC3's enzymatic function, permit normal stratification, indicating that HDAC3's roles in this context are independent of its histone deacetylase activity. HDAC3 functions both in conjunction with, and independent of, KLF4 to repress premature expression of different sets of terminal differentiation genes and suppresses expression of inflammatory cytokines through a RelA-dependent mechanism. These data identify HDAC3 as a hub coordinating multiple aspects of epidermal barrier acquisition. We used microarrays to determine transcriptional changes in Hdac3 deleted epidermis compared to control and Ncor1/Ncor2 deleted epidermis compared to control. For the Hdac3 experiment, epidermis was collected from 5 E18.5 controls and 5 E18.5 mutants. For the Ncor1/Ncor2 experiment, epidermis was collected from 4 E18.5 controls and 4 E18.5 mutants.染色质修饰因子在表皮发育中发挥关键作用，但目前对组蛋白去乙酰化酶（histone deacetylases, HDACs）在此过程中的功能尚不清楚。本研究发现，I类组蛋白去乙酰化酶HDAC3（Class I HDAC, HDAC3）在胚胎表皮中广泛表达，且对于表皮有序的渐进式分层发育必不可少。体内HDAC3蛋白的稳定性依赖于NCoR与SMRT，二者在表皮发育过程中功能冗余。然而，NCoR与SMRT的去乙酰化酶激活结构域（Deacetylase Activating Domains）是HDAC3发挥酶学功能所必需的，若在此区域引入点突变，表皮仍可正常分层，这表明HDAC3在此过程中的功能不依赖于其组蛋白去乙酰化酶活性。HDAC3既可与KLF4协同发挥作用，也可独立于KLF4行使功能：一方面抑制多组终末分化基因的过早表达，另一方面通过RelA依赖的机制抑制炎性细胞因子的表达。上述研究结果表明，HDAC3是协调表皮屏障建立多个环节的核心调控枢纽。本研究通过基因芯片（microarrays）技术，分别检测了Hdac3敲除表皮与对照表皮、Ncor1/Ncor2双敲除表皮与对照表皮之间的转录组差异。在Hdac3敲除实验中，研究样本采集自5只E18.5期野生型对照小鼠与5只E18.5期突变型小鼠的表皮组织；在Ncor1/Ncor2双敲除实验中，研究样本采集自4只E18.5期野生型对照小鼠与4只E18.5期突变型小鼠的表皮组织。