Trimethylation of H3K27 during human cerebellar development in relation to medulloblastoma

Abstract

Medulloblastoma (MB), the most common malignant childhood brain tumor, encompasses a collection of four clinically and molecularly distinct tumor subgroups, i.e. WNT, SHH, Group 3 and Group 4. These tumors are believed to originate from precursor cells during cerebellar development. Although the exact etiology of these brain tumors is not yet known, histone modifications are increasingly recognized as key events during cerebellum development and MB tumorigenesis. Recent studies show that key components involved in post-translational modifications of histone H3 lysine 27 (H3K27) are commonly deregulated in MB. In this descriptive study, we have investigated the trimethylation status of H3K27, as well as the expression of the H3K27 methylase EZH2 and demethylases KDM6A and KDM6B, during human cerebellum development in relation to MB. H3K27 Trimethylation status differed between the MB subgroups. Moreover, trimethylation of H3K27 and expression of its modifiers EZH2, KDM6A and KDM6B were detected in a spatio-temporal manner during development of the human cerebellum, with consistent high occurrence in the four proliferative zones, which are believed to harbor the precursor cells of the different MB subgroups. Our results suggest that H3K27 trimethylation in MB is deregulated by EZH2, KDM6A and KDM6B. Moreover, we provide evidence that during development of the human cerebellum H3K27me3 and its regulators are expressed in a spatio-temporal manner.

Keywords: brain development; cerebellum; histone 3 trimethylation; immunohistochemistry; medulloblastoma.

Figure 1. Expression of H3K27 and its modifiers in medulloblastoma

Representative selection of immunohistochemical staining for KDM6A, KDM6B, EZH2 and H3K27me3 in MB samples of different subgroups.

Figure 2

Dot plot showing transcript levels of EZH2 (A), KDM6A (B) and KDM6B (C) during human development as reported in the Brainspan database. Depicted are stage 2A (corresponding with 9 weeks GSA; early prenatal – blue), stage 5 (corresponding to 28 and 33 weeks GSA; late prenatal – gray), and stage 6 (corresponding to 40 weeks GSA; birth-early infancy – yellow).

Figure 3. EZH2 expression during human cerebellum development

(a) Expression of EZH2 at 9 weeks GSA. (b-f) Higher magnifications (400x) of boxed areas showing high EZH2 expression in the proliferative zones of the developing cerebellum; upper RL (c), EGL (d), VZ (e), and lower RL (f). Expression of EZH2 is only detectable in the EGL layer between 28 weeks gestation (g) and 33 weeks gestation (h). Higher magnification of boxed area in (h) shows the EZH2 positive cells in the EGL layer. After 33 weeks no EZH2 expression can be detected in the cerebellum (i). Scale bars 1000 μm (b), 25 μm (c-i), 5 μm (inset in h).

Figure 4. KDM6A expression during human cerebellum development

(a) Expression of KDM6A expression at 9 weeks gestation. (b-f) Higher magnifications (400x) of boxed areas showing high KDM6A expression in the proliferative zones of the developing cerebellum; upper RL (c), EGL (d), VZ (e), and lower RL (f). At 28 weeks gestation, strong nuclear KDM6A expression was observed in Purkinje cells, and to a lesser extent in the granule neuron precursor cells In the EGL and in the granule cells in the IGL (g). After 28 weeks GSA KDM6A could not be detected in any layer of the human cerebellum (h-i). Scale bars 1000 μm (b), 100 μm (g), 50 μm (h), 25 μm (c-f and i).

Figure 5. KDM6B expression during human cerebellum development

(a) Expression of KDM6B at 9 weeks gestation. (b-f) Higher magnifications (400x) of boxed areas showing high KDM6B expression in the proliferative zones of the developing cerebellum; upper RL (c), EGL (d), VZ (e), and lower RL (f). During further development KDM6B is selectively expressed in the Purkinje cells. Staining intensity increases in time, and distribution changes from nuclear to more cytoplasmic (f-i). Scale bars 1000 μm (b), 100 μm (g), 50 μm (h), 25 μm (c-f and i).

Figure 6. H3K27me3 trimethylation during human cerebellum development

(a) Expression of H3K27me3 at 9 weeks gestation. (b-f) Higher magnifications (400x) of boxed areas showing high H3K27me3 expression in the proliferative zones of the developing cerebellum; upper RL (c), EGL (d), VZ (e), and lower RL (f). At 28 weeks gestation strong staining is present in Purkinje cells and some cells in the EGL and IGL layers (g). From 33 weeks gestation and further H3K27me3 expression is only present in Purkinje cells (h-i). Scale bars 1000 μm (B), 25 μm (c-i).