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Kdm3b Haploinsufficiency Impairs the Consolidation of Cerebellum

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Kdm3b Haploinsufficiency Impairs the Consolidation of Cerebellum Kim et al. Mol Brain (2021) 14:106 https://doi.org/10.1186/s13041-021-00815-5 RESEARCH Open Access Kdm3b haploinsufciency impairs the consolidation of cerebellum-dependent motor memory in mice Yong Gyu Kim1,2†, Myeong Seong Bak1,2†, Ahbin Kim1,2†, Yujin Kim3,4, Yun‑Cheol Chae5, Ye Lee Kim2,6, Yang‑Sook Chun1,2,6, Joon‑Yong An3,4, Sang‑Beom Seo5, Sang Jeong Kim1,2,7* and Yong‑Seok Lee1,2,7* Abstract Histone modifcations are a key mechanism underlying the epigenetic regulation of gene expression, which is critically involved in the consolidation of multiple forms of memory. However, the roles of histone modifcations in cerebellum‑dependent motor learning and memory are not well understood. To test whether changes in histone / methylation are involved in cerebellar learning, we used heterozygous Kdm3b knockout (Kdm3b+ −) mice, which show reduced lysine 9 on histone 3 (H3K9) demethylase activity. H3K9 di‑methylation is signifcantly increased / selectively in the granule cell layer of the cerebellum of Kdm3b+ − mice. In the cerebellum‑dependent optokinetic / response (OKR) learning, Kdm3b+ − mice show defcits in memory consolidation, whereas they are normal in basal oculomotor performance and OKR acquisition. In addition, RNA‑seq analyses revealed that the expression levels of several plasticity‑related genes were altered in the mutant cerebellum. Our study suggests that active regulation of histone methylation is critical for the consolidation of cerebellar motor memory. Keywords: Kdm3b, Optokinetic response (OKR), Histone modifcation, Cerebellum Introduction Post-translational modifcations of histone pro- One of the hallmarks of memory consolidation is that it teins, such as acetylation and methylation, determine requires changes in gene expression [1, 2]. Since DNA is whether the gene is in a transcriptionally permissive tightly packaged with histone proteins to form chroma- or repressive state [6, 7]. Methylated histones are gen- tin in the nucleus, chromatin structure should be remod- erally considered codes for gene repression, but their eled to induce gene activation or repression by epigenetic efects on transcription are very complex [8]. For exam- mechanisms, such as modifcations of histone proteins ple, tri-methylation of histone 3 lysine 9 (H3K9me3) and DNA [3]. It has been shown that epigenetic regula- represents a repressive code, whereas H3K4me3 is con- tion, such as histone modifcation and DNA methyla- sidered an activating code [9]. Histone methylation is tion, is a key mechanism for memory consolidation from dynamically regulated by many histone methyltrans- invertebrates to mammals [4, 5]. ferases and demethylases [10]. Lysine demethylase 3b (KDM3B) encodes a demethylase that targets mono- and di-methylated lysine 9 on histone 3 (H3K9me1/2) *Correspondence: [email protected]; [email protected] [11]. KDM3B has been shown to regulate many cellular †Yong Gyu Kim, Myeong Seong Bak, and Ahbin Kim have contributed processes, including gene expression in leukemia, sper- equally to this work matogenesis, and autophagy [11–13]. Recent studies 1 Department of Physiology, Seoul National University College of Medicine, Seoul 03080, Korea have shown that KDM3B mutations are associated with Full list of author information is available at the end of the article cognitive defcits, including intellectual disability and © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Kim et al. Mol Brain (2021) 14:106 Page 2 of 11 schizophrenia [14, 15]. However, the functional role of Histone purifcation KDM3B in the nervous system remains unclear. In order to extract purifed histone protein from tissues, Te cerebellum plays a central role in adaptive tissues were homogenized with TEB bufer (0.5 × Triton motor control. For newly formed motor memory to be X-100, 2 mM PMSF, 1 × Protease inhibitor cocktail) at expressed in an appropriate circumstance, consolida- 4 °C for 30 min. Homogenized tissues were centrifuged tion of motor memory by the cerebellum is essential at 7,000 rpm for 2 min at 4 °C. After removing the super- [16]. Similar to other types of learning, the consoli- natant, the pellet was washed with additional TEB bufer dation of cerebellum-dependent motor memory also and centrifuged under the same conditions. After remov- requires de novo transcription and translation [17]. ing the supernatant, 0.5 M HCl was added to the pellet. However, the epigenetic mechanisms underlying cer- Te tube was rotated overnight at 4 °C. After centrifu- ebellar learning and memory have rarely been inves- gation at 13,500 rpm for 10 min at 4 °C, the supernatant tigated. In this study, we investigated how changes (~ 90% of the total volume) was transferred to a fresh in histone methylation afect cerebellum-dependent tube. Subsequently, 1/3 volume of 100% TCA was added motor behavior and gene expression in the cerebel- to the tube, which was inverted and placed on ice for 2 h. lum. Using Kdm3b heterozygous knockout (Kdm3b+/−) Occasionally, the tube was vortexed. Te tube was cen- mice, we found defcits in retention of cerebellum- trifuged at 13,500 rpm for 10 min at 4 °C. Te superna- dependent motor memory in the mutant mice, dem- tant was removed and acetone was added. Te tube was onstrating that regulation of histone methylation in the centrifuged at 13,500 rpm for 5 min at 4 °C. Acetone was cerebellum is critical for cerebellum-dependent mem- air-dried and distilled water was added. Te tube was ory consolidation. agitated overnight at 4 °C. Te tube was centrifuged at 13,500 rpm for 5 min at 4 °C. Te supernatant containing the histone extract was then saved. Materials and methods Animals Western blot analyses / Adult (> 7-week-old) male Kdm3b+ − mice and their Western blot analysis was performed as described pre- wild-type (WT) littermates were used. Kdm3b-targetted viously [19]. Briefy, tissues were homogenized and ES cell line (YTC406) was obtained from International sonicated with RIPA bufer (25 mM Tris–HCl [pH 7.6], Gene Trap Consortium (https:// igtc. org/ cgi- bin/ annot 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% ation. py? celll ine= YTC406). Genomic sequencing was SDS) with a phosphatase inhibitor cocktail and a protease performed to identify the targeting vector (pGT0lxf) inhibitor cocktail. Proteins were quantifed using bicin- insertion site in the ES cell line. Te heterozygous knock- choninic acid (BCA) analysis according to the manu- out mice were generated by injecting the ES cells and facturer’s instructions (Pierce). Either 3 μg of histone or backcrossed to C57Bl/6N background (Macrogen, Inc). 20 μg of total tissue extract were used for Western blot Genotypes of the mice were identifed by PCR using the analyses. Te membranes were imaged with a digital ′ following primers: common forward 5 -GGC ACC AGA imager (Amersham Imager 600, GE Life Science). Pro- ′ ′ CCC TGG GAG CTA G-3 , WT reverse 5 -CAC CCA tein quantifcation was performed using the AI600 soft- ′ ′ CGA CCT GGC TTA CAC C-3 , KO reverse 5 -CAC ware (GE Healthcare), and each band was normalized ′ CCA CGA CCT GGC TTA CAC C-3 . Te mutant mice to α-tubulin or β-actin in the same gel. Te following were crossed to C57Bl/6J 3–4 times before the experi- primary antibodies and dilution ratios were used: anti- ments. Animals were housed with food and water avail- KDM3B antibody (2621S, 1:500; Cell Signaling Tech- able ad libitum under a 12-h light/dark cycle. All animal nology), anti-H3 antibody (Millipore, 05-499, 1:2,000), experiments were performed in accordance with the pro- anti-H3K9me2 antibody (Millipore, 07-441, 1:1,000), tocols approved by the Animal Care and Use Committee anti-α-tubulin antibody (Santa Cruz, TU-02, 1:2,000), of Seoul National University. and anti-β-actin antibody (Santa Cruz, AC-15, 1:2,000). Behavioral test Tissue sample preparation Te whole procedure, including surgical and behav- To prepare the granule cell-enriched (GCE) and Purkinje- ioral processes, was performed according to a previ- cell-enriched (PCE) tissue samples, sagittal slices of the ously described method with minor modifcations [18, cerebellar vermis (250 μm thick) were obtained using a 20]. Briefy, a headpost was mounted on the head of the vibratome (VT1200, Leica). Vermal slices were manually mouse using nuts, screws, and dental cement. Mice were dissected into GCE and PCE samples under a stereomi- administered for at least 24 h after surgery. Optokinetic croscope, as previously described [18]. Kim et al. Mol Brain (2021) 14:106 Page 3 of 11 and vestibular stimuli were generated by a patterned comparisons, only signifcant results were collected screen and a custom-made turntable with a restrainer (adjusted p < 0.05). Tese results were used to identify the attached. Right eye movements were monitored using related genes and pass on to the network construction an infrared video-oculography system.
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