Data Sheet 4_SIRT1 retention in elongating spermatids interferes with histone displacement by counteracting MOF-dependent H4K16 acetylation.pdf

IntroductionThe histone H4 hyperacetylation (i.e., acetylation of H4 at lysines -K5, -K8, -K12, and -K16, here reported as H4tetraAc) occurs in elongating spermatids (eSPTs) during spermiogenesis. Although it is critically involved in histone displacement, the mechanistic involvement of histone -acetyltransferases (HATs) and -deacetylases (HDACs) in the pathway underlying H4 hyperacetylation is poorly defined. Here, we investigate the involvement of SIRT1 deacetylase, and its functional interaction with the histone acetyltransferase MOF, in regulating H4 hyperacetylation underlying histone-to-protamine exchange.
MethodsExploiting the cannabinoid receptor 1 (Cb1) null mice (Cb1−/−) as a model of impaired histone displacement, we assessed in eSPTs the expression and the localization of SIRT1 in combination with the enrichment of H4tetraAc and the relative monoacetylated forms (H4-K5ac, -K8ac, -K12ac and -K16ac), by Western Blot and immunohistochemistry analyses. Then, focusing on SIRT1 interaction with MOF HAT by protein immunoprecipitation experiments, we verified the H4K16ac and H4TetraAc enrichment in eSPTs in response to ex vivo SIRT1 inhibition by using the selective EX-527 inhibitor.
ResultsWe show that the hyperacetylation of histone H4 occurs progressively in steps 8–9 eSPTs and bursts in step 10 eSPTs, appearing inversely correlated to the expression pattern of SIRT1, being SIRT1 present in step 8, detectable in step 9 and absent in step 10 eSPTs. The abnormal SIRT1 retention in step 10 eSPTs of Cb1−/− mice, despite the observed enrichment of H4-K5ac, -K8ac, and -K12ac, counteracts the H4 hyperacetylation burst by limiting the H4 acetylation at lysine K16. Mechanistically, SIRT1 directly or indirectly interacts with and negatively regulates MOF acetyltransferase, specifically affecting its acetylation status and protein content, thereby interfering with H4K16 acetylation. Counteracting the MOF/SIRT1 interaction by SIRT1 inhibition in ex vivo Cb1−/− testis, both MOF protein content and acetylation status increase, downstream promoting recovery of H4K16ac and H4tetraAc in step 10 eSPT, and full rescue of histone displacement.
ConclusionThese results underscore the key involvement of SIRT1–MOF axis in modulating H4K16 acetylation. Our findings provide mechanistic insights into H4K16 acetylation pathway in eSPTs and support the key role of H4K16ac in chromatin remodeling underlying histone displacement.

引言
组蛋白H4高度乙酰化（histone H4 hyperacetylation，即H4在赖氨酸残基K5、K8、K12及K16位点的乙酰化，本文中记为H4四乙酰化（H4tetraAc)）发生于精子发生（spermiogenesis）过程中的长形精子细胞（elongating spermatids, eSPTs）。尽管其在组蛋白替换过程中发挥关键作用，但组蛋白乙酰转移酶（histone acetyltransferases, HATs）与组蛋白去乙酰化酶（histone deacetylases, HDACs）在H4高度乙酰化相关通路中的具体调控机制仍有待阐明。本研究探究了去乙酰化酶SIRT1（SIRT1 deacetylase）以及其与组蛋白乙酰转移酶MOF（histone acetyltransferase MOF）的功能互作在调控组蛋白-鱼精蛋白替换相关H4高度乙酰化过程中的作用。

方法
本研究以大麻素受体1（cannabinoid receptor 1, Cb1）敲除小鼠（Cb1−/−，一种组蛋白替换缺陷模型）为研究对象，通过蛋白质免疫印迹（Western Blot）与免疫组织化学（immunohistochemistry）分析，检测了长形精子细胞中SIRT1的表达与定位，同时分析了H4tetraAc以及相对应的单乙酰化形式（H4-K5ac、-K8ac、-K12ac及-K16ac）的富集水平。随后，通过蛋白质免疫沉淀（protein immunoprecipitation）实验验证SIRT1与MOF HAT的相互作用，并利用选择性EX-527抑制剂（EX-527 inhibitor）进行离体SIRT1抑制实验，检测长形精子细胞中H4K16ac与H4tetraAc的富集变化。

结果
本研究发现，组蛋白H4的高度乙酰化在第8-9期长形精子细胞中逐步发生，并在第10期长形精子细胞中达到峰值，且其动态变化与SIRT1的表达模式呈负相关：SIRT1在第8期长形精子细胞中存在，第9期仍可检测到，而第10期则完全缺失。在Cb1−/−小鼠的第10期长形精子细胞中，尽管H4-K5ac、-K8ac及-K12ac的富集水平升高，但SIRT1的异常滞留会通过限制赖氨酸K16位点的H4乙酰化，抑制H4高度乙酰化的爆发。从机制上来说，SIRT1可直接或间接与MOF乙酰转移酶相互作用并对其产生负调控，特异性影响其乙酰化状态与蛋白含量，进而干扰H4K16位点的乙酰化。在离体Cb1−/−睾丸中通过抑制SIRT1以阻断MOF/SIRT1相互作用后，MOF的蛋白含量与乙酰化状态均得以提升，下游可促进第10期长形精子细胞中H4K16ac与H4tetraAc的恢复，并完全恢复组蛋白替换过程。

结论
本研究结果突显了SIRT1-MOF轴（SIRT1–MOF axis）在调控H4K16乙酰化过程中的关键作用。我们的研究为长形精子细胞中H4K16乙酰化通路提供了机制层面的新见解，并证实了H4K16ac在组蛋白替换相关染色质重塑（chromatin remodeling）过程中的核心功能。