Methamphetamine-associated memory is regulated by a writer and an eraser of permissive histone methylation

Abstract

Background: Memories associated with drugs of abuse, such as methamphetamine (METH), increase relapse vulnerability to substance use disorder by triggering craving. The nucleus accumbens (NAc) is essential to these drug-associated memories, but underlying mechanisms are poorly understood. Posttranslational chromatin modifications, such as histone methylation, modulate gene transcription; thus, we investigated the role of the associated epigenetic modifiers in METH-associated memory.

Methods: Conditioned place preference was used to assess the epigenetic landscape in the NAc supporting METH-associated memory (n = 79). The impact of histone methylation (H3K4me2/3) on the formation and expression of METH-associated memory was determined by focal, intra-NAc knockdown (KD) of a writer, the methyltransferase mixed-lineage leukemia 1 (Mll1) (n = 26), and an eraser, the histone lysine (K)-specific demethylase 5C (Kdm5c) (n = 38), of H3K4me2/3.

Results: A survey of chromatin modifications in the NAc of animals forming a METH-associated memory revealed the global induction of several modifications associated with active transcription. This correlated with a pattern of gene activation, as revealed by microarray analysis, including upregulation of oxytocin receptor (Oxtr) and FBJ osteosarcoma oncogene (Fos), the promoters of which also had increased H3K4me3. KD of Mll1 reduced H3K4me3, Fos and Oxtr levels and disrupted METH-associated memory. KD of Kdm5c resulted in hypermethylation of H3K4 and prevented the expression of METH-associated memory.

Conclusions: The development and expression of METH-associated memory are supported by regulation of H3K4me2/3 levels by MLL1 and KDM5C, respectively, in the NAc. These data indicate that permissive histone methylation, and the associated epigenetic writers and erasers, represent potential targets for the treatment of substance abuse relapse, a psychiatric condition perpetuated by unwanted associative memories.

Keywords: Demethylase; KDM5C; MLL; epigenetics; methyltransferase; nucleus accumbens.

Figure 1

Induction of posttranslational histone modifications associated with active gene expression in the NAc during the formation of a METH-associated memory. (A) Schematic of experimental design. (B) Histone modifications associated with transcriptional activation (H3K4me2, me3, pan-AcK H2-3, pan-AcK H4) or repression (H3K9me2, H3K27me2) were measured in the NAc by Western blot and normalized to total H4. (C) Representative images of Western blots. * P<0.05, error bars represent SEM.

Figure 2

Formation of a METH-associated memory is associated with transcriptional activation in the NAc. (A) Using the same experimental design depicted in Fig. 1A, microarray ananalysis in the NAc revealed the upregulation of 91 genes, relative to control groups (Sal CPP, METH HC). (B) From the group of 91 genes selectively upregulated in the METH CPP group, Cebpd, Fos and Oxtr were selected for qRT-PCR validation. (C) Schematic of the region of the Fos and Oxtr promoters amplified from genomic DNA samples obtained from H3K4me3 chromatin immunoprecipitation (upper panel). Promoter-specific H3K4me3 at Fos and Oxtr was measured by ChIP-qRT-PCR in the NAc of METH CPP and control mice (lower panel). * P<0.05, error bars represent SEM.

Figure 3

The H3K4 histone methyltransferase, MLL1, is increased during METH CPP acquisition and its loss results in H3K4 demethylation. (A) Using the same experimental design depicted in Fig. 1A, Mll1 expression was measured by qRT-PCR. (B) Representative coronal section of a mouse brain injected in the NAc core with a adeno-associate virus (AAV1) expressing the enhanced Green Fluorescent Protein (eGFP). The same brain coordinates were used for subsequent siRNA delivery. (C) Mll1 levels in the NAc two and five days after intra-NAc siRNA injection. (D) Intra-NAc KD of Mll1 resulted in demethylation of H3K4 in the NAc. (E) Effect of Mll1 KD on Fos and Oxtr mRNA levels. * P<0.05, error bars represent SEM.

Figure 4

NAc MLL1 is required for METH-associated memory. (A) Schematic of experimental design. (B) Effect of focal KD of Mll1 in the NAc on METH-associated memory. (C) Mll1 KD in the NAc was confirmed after behavioral testing. * P<0.05, error bars represent SEM.

Figure 5

Kdm5c is the most abundant H3K4 demethylase in the NAc. (A) Expression of the KDM5 (Kdm5a, Kdm5b, Kdm5c and Kdm5d) and KDM1 (Kdm1a) families of H3K4 demethylases was assayed in the NAc by qRT-PCR and expressed relative to Gapdh expression. (B) Kdm5c mRNA expression was also measured across several brain regions relevant to psychostimulants and memory (NAc, dStr, mPFC, AMY and anterior HPC). Error bars represent SEM.

Figure 6

Loss of Kdm5c in the NAc results in H3K4 hypermethylation. (A) Using the same experimental design depicted in Fig. 1A, Mll1 expression was measured by qRT-PCR. (B) Kdm5c levels in the NAc two and five days after intra-NAc siRNA injection. (C) KDM5C protein levels in the NAc two days after intra-NAc siRNA injection. (D) Intra-NAc KD of Kdm5c resulted in hypermethylation of H3K4 in the NAc. * P<0.05, error bars represent SEM.

Figure 7

Focal knockdown of Kdm5c in the NAc after consolidation disrupts METH-associated contextual memory. (A) Schematic of experimental design for B-C. (B) Pre-training intra-NAc Kdm5c siRNA had no effect on METH-associated memory. (C) KDM5C protein and H3K4me3 levels in the NAc were measured after behavioral testing. Lower panel contains representative images of Western blots for KDM5C (~170kDa), H3K4me3 (~17kDa) and GAPDH (~36kDa). (D) Schematic of experimental design for E-F. (E) METH-associated memory was assayed before (Test 1) and after (Test 2) Kdm5c siRNA infusion. (F) KDM5C protein and H3K4me3 levels in the NAc were measured after behavioral testing. Lower panels contain representative Western blot images of KDM5C, H3K4me3 and GAPDH. * P<0.05, error bars indicate SEM.