Detection of histone acetylation levels in the dorsal hippocampus reveals early tagging on specific residues of H2B and H4 histones in response to learning

Abstract

The recent literature provides evidence that epigenetic mechanisms such as DNA methylation and histone modification are crucial to gene transcription linked to synaptic plasticity in the mammalian brain--notably in the hippocampus--and memory formation. We measured global histone acetylation levels in the rat hippocampus at an early stage of spatial or fear memory formation. We found that H3, H4 and H2B underwent differential acetylation at specific sites depending on whether rats had been exposed to the context of a task without having to learn or had to learn about a place or fear therein: H3K9K14 acetylation was mostly responsive to any experimental conditions compared to naive animals, whereas H2B N-terminus and H4K12 acetylations were mostly associated with memory for either spatial or fear learning. Altogether, these data suggest that behavior/experience-dependent changes differently regulate specific acetylation modifications of histones in the hippocampus, depending on whether a memory trace is established or not: tagging of H3K9K14 could be associated with perception/processing of testing-related manipulations and context, thereby enhancing chromatin accessibility, while tagging of H2B N-terminus tail and H4K12 could be more closely associated with the formation of memories requiring an engagement of the hippocampus.

Figure 1. Short spatial memory training differentially modulates histone acetylation in the rat hippocampus.

(A) Performance of rats trained in the Morris water maze task during one or three consecutive days in the Morris Water Maze (left panel) and probe trial performance after 1 or 3 days of training (right panel). During training, rats had to search for the location of a platform hidden at a constant location (HPf); their controls swam to a visible platform (VPf) whose location was changed from trial to trial. Probe trial performances of the HPf groups are presented after 1- or 3 days of training (right panel) as the mean time (+ SEM) spent in the target quadrant. After 3 days of training, the rats trained with the hidden platform performed significantly above chance (i.e., 15 s), *p<0.05, an effect not observed after only 1 day of training. (B) Comparison of acetylated and total histone levels between home cage rats (HC, n = 5), rats trained to swim to a visible platform (VPf, n = 5) and rats trained to learn the location of a hidden platform (HPf, n = 5) in a single daily session (4 trials). Acetylation levels were measured by western blot performed on total extracts from dorsal hippocampus with specific antibodies (Tetra Ac: H2BK5K12K15K20, K5Ac: H2BK5, H4K12 and H3K9K14). Typical western blots are presented in duplicates on the left. Corresponding quantifications are shown on the right. Ratios of acetylated/total histone corresponding to the home cage rats (HC) were arbitrarily set at 100% and other values normalized accordingly. Newman-Keuls multiple comparisons test: ***p<0.001, **p<0.01, *p<0.05, for comparisons with the HC group or as indicated. Both H2B and H4 histones showed hyperacetylation in the group trained to find the hidden platform (HPf) compared to either control (VPf or HC), while H3 was hyperacetylated in the VPf and HPf groups, thus more reflecting task-related context processing.

Figure 2. Impact of contextual fear conditioning on histone acetylation in the rat hippocampus.

(A) Experimental design. Three groups of rats (n = 16/group) were used. In one group, rats were kept in the context but received no shock (CX). Others received three immediate and consecutive shocks and were subsequently left in the context for 8 min (IS). In the last group, rats received three randomly-distributed shocks while being kept in the context as noted (CS). Animals (n = 10/group) were then either tested for freezing behavior after 24 h (probe) (B; n = 10/group) or euthanized after 1 h for tissue collection (dorsal hippocampus) and western blot analyses of acetylated histones (C; n = 6/group). (B) Freezing levels at 24 h. Notice that marked freezing was observed only in the Context-shock group (CS), demonstrating that rats of this group were the only ones to have associated the shock with the context and memorized this association. (C) Comparison of acetylated and total histone levels in the three groups relative to their counterparts taken from the home cage (HC, n = 6). Lysine acetylations measured were H2BK5 (K5Ac, plain histograms), H2BK5K12K15K20 (Tetra Ac, stripped histograms), H4K12 (K12Ac) and H3K9K14 (K9K14Ac). Typical western blots are shown in duplicates. Quantified results are represented as % induction of the Acetylated/total ratio for each histone. The ratio obtained in the HC condition was arbitrarily set at 100%. Newman-Keuls multiple comparisons test: ***p<0.001**p<0.01, *p<0.05, as compared to HC group. Global H2B and H4 histone acetylation levels were clearly increased in the group exhibiting fear towards the context (CS) as compared to the other situations, while H3 acetylation levels were increased in CS and both controls (CX and IS ) as compared to rats completely naive to the test situation (HC).