Abstract
The disruptor of telomeric silencing 1-like (DOT1L) mediates methylation of histone H3 at position lysine 79 (H3K79). Conditional knockout of Dot1l in mouse cerebellar granule cells (Dot1l-cKOAtoh1) led to a smaller external granular layer with fewer precursors of granule neurons. Dot1l-cKOAtoh1 mice had impaired proliferation and differentiation of granular progenitors, which resulted in a smaller cerebellum. Mutant mice showed mild ataxia in motor behavior tests. In contrast, Purkinje cell-specific conditional knockout mice showed no obvious phenotype. Genome-wide transcription analysis of Dot1l-cKOAtoh1 cerebella using microarrays revealed changes in genes that function in cell cycle, cell migration, axon guidance, and metabolism. To identify direct DOT1L target genes, we used genome-wide profiling of H3K79me2 and transcriptional analysis. Analysis of differentially methylated regions (DR) and differentially expressed genes (DE) revealed in total 12 putative DOT1L target genes in Dot1l-cKOAtoh1 affecting signaling (Tnfaip8l3, B3galt5), transcription (Otx1), cell migration and axon guidance (Sema4a, Sema5a, Robo1), cholesterol and lipid metabolism (Lss, Cyp51), cell cycle (Cdkn1a), calcium-dependent cell-adhesion or exocytosis (Pcdh17, Cadps2), and unknown function (Fam174b). Dysregulated expression of these target genes might be implicated in the ataxia phenotype observed in Dot1l-cKOAtoh1.
Highlights
Nissl staining of Dot1l-cKOAtoh1 cerebella revealed a thinner external granular layer (EGL) and a slightly disorganized granular layer (GL) (Fig. 1a)
BrdU-pulse labelling for 2 h revealed a reduced number of S-phase cells in the EGL of Dot1l-cKOAtoh1 compared to wild-type controls (Fig. 1b, d)
We observed fewer KI67-positive dividing cells as well as PAX6expressing cells in Dot1l-cKOAtoh1 (Fig. 1b–d). These findings indicated that the EGL of Dot1l-cKOAtoh1 contained less dividing progenitors than controls
Summary
It has been reported that DNA methylation and hydroxymethylation [11] as well as chromatin remodeling [12] are important for granule cell function and development. DOT1L deficiency in granule cells, but not in PCs, led to an ataxia phenotype and a smaller sized cerebellum in mice. We studied cell proliferation and differentiation in in vitro cultured CGNP and CGN under pharmacological inhibition of DOT1L activity. Microarray analysis of transcriptional alterations and H3K79me ChIP-seq identified differentially expressed genes (DE) as well as differentially methylated regions (DR) in either CGNP or CGN after pharmacological interference with DOT1L activity. This study reports on DOT1L target genes in the cerebellum that might account for the ataxia phenotype in mice with DOT1L-deficient granular cells in the cerebellum
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