Verna & Marrs McLean Department of Biochemistry & Molecular Biology, Baylor College of Medicine, One Baylor Plaza, MS-125, Houston, TX, 77030-3411, USA.
Department of Molecular and Cellular Biology, Baylor College of Medicine, Houston, TX, USA.
Anal Bioanal Chem. 2023 Apr;415(9):1627-1639. doi: 10.1007/s00216-023-04555-4. Epub 2023 Feb 8.
Histone proteins are essential to the regulation of the eukaryotic genome. Histone post-translational modifications (PTMs) and single-molecule combinations of these modifications (proteoforms) allow for the regulation of many DNA-templated processes, most notably transcription. Histone H4 is a part of the core histone octamer, which packages DNA into nucleosomes. Top-down proteomics allows for the inquiry of the epigenetic landscape with proteoform-level specificity. Although these approaches are well-demonstrated ex vivo, our knowledge of in vivo histone proteoform biology remains sparse. Here, we demonstrate the first in vivo quantitative top-down analysis of histone H4 and analyze the forebrains and hindbrains of differently aged mice. This reveals novel differences between the mouse forebrain and hindbrain and region-specific changes during adolescence in histone H4 PTMs and proteoforms. At 25 days of age (P25), histone H4 of the hindbrain is more acetylated than the forebrain. At 47 days of age (P47), there are fewer significant differences in histone H4 PTMs and their combinations between regions. Histone H4 of the forebrain is more acetylated in P47 than in P25 forebrains. Hindbrains exhibit the opposite difference with histone H4 of the P25 hindbrain being more acetylated than that of P47 hindbrains. These differences are mainly driven by less abundant hyperacetylated proteoforms. Transcription of histone acetyltransferases such as p300, CBP, and HAT1 is known to be higher in cortical neurons, consistent with the observed acetylation levels. Lysine 20 methylation (K20me1, K20me2, and K20me3) is notably invariant with brain region and age difference.
组蛋白蛋白对于真核基因组的调控至关重要。组蛋白翻译后修饰（PTMs）和这些修饰的单分子组合（蛋白形式）允许调控许多 DNA 模板过程，尤其是转录。组蛋白 H4 是核心组蛋白八聚体的一部分，将 DNA 包装成核小体。自上而下的蛋白质组学允许在蛋白形式水平特异性上研究表观遗传景观。虽然这些方法在体外得到了很好的证明，但我们对体内组蛋白蛋白形式生物学的了解仍然很少。在这里，我们展示了第一个体内定量自上而下的组蛋白 H4 分析，并分析了不同年龄的小鼠的前脑和后脑。这揭示了小鼠前脑和后脑之间的新的差异，以及青春期组蛋白 H4 PTM 和蛋白形式的区域特异性变化。在 25 天龄（P25）时，后脑的组蛋白 H4 乙酰化程度高于前脑。在 47 天龄（P47）时，不同区域之间组蛋白 H4 PTM 及其组合的差异较少。P47 前脑中的组蛋白 H4 比 P25 前脑中的组蛋白 H4 更乙酰化。相反，P25 后脑中的组蛋白 H4 比 P47 后脑中的组蛋白 H4 更乙酰化。这些差异主要是由丰度较低的高乙酰化蛋白形式驱动的。已知 p300、CBP 和 HAT1 等组蛋白乙酰转移酶的转录在皮质神经元中更高，与观察到的乙酰化水平一致。赖氨酸 20 甲基化（K20me1、K20me2 和 K20me3）在脑区和年龄差异上明显不变。
