Chromatin accessibility profiling of developing cerebellar granule neurons reveals novel neuronal enhancers and regulatory scheme for ZIC transcription factors. C. L. Frank1,2, F. Liu3, R. Wijayatunge3, L. Song2, C. M. Vockley2,4, A. Safi2, G. E. Crawford2,5, A. E. West2,3 1) Department of Molecular Genetics and Microbiology, Duke University, Durham, NC; 2) Institute for Genome Sciences and Policy, Duke University, Durham, NC; 3) Department of Neurobiology, Duke University, Durham, NC; 4) Department of Cell Biology, Duke University, Durham, NC; 5) Department of Pediatrics, Division of Medical Genetics, Duke University, Durham, NC.

   Chromatin regulation mediates the cell-type specific expression of genes by establishing differential accessibility to transcription factor binding sites across the genome in cells of distinct fate lineages. However, the dynamics of chromatin regulation over the time-course of cellular differentiation within a single fate lineage remain poorly understood. We set out to characterize the temporal relationship between chromatin regulation and gene expression in the developing mouse cerebellar cortex, which is dominantly comprised of a single type of neuron, cerebellar granule neurons (CGNs). We used DNase-seq to globally map chromatin accessibility of cis-regulatory elements and RNA-seq to profile transcript abundance at three key time-points in postnatal development. We observed widespread chromatin accessibility changes at 24,886 regulatory elements (FDR<.05). Equivalent profiling for three time-points of purified CGNs differentiating in culture further improves temporal resolution of early chromatin events. The majority of these dynamic elements appear to be developmental stage-specific neuronal enhancers, which is supported by these regions being (i) primarily located outside of proximal promoters, (ii) overlapping H3K27ac and H3K4me1 ChIP-seq peaks from adult cerebellum, (iii) enriched nearby developmentally-regulated genes, (iv) enriched for a publicly available set of confirmed hindbrain enhancer regions that function in embryonic mice, and (v) sufficient to drive reporter gene expression in cultured mouse neurons. Motif discovery in the differentially accessible elements revealed several transcription factor families, including zinc finger proteins of the cerebellum (ZIC), that likely bind these elements. We confirmed ZIC involvement by globally mapping ZIC binding sites in early and adult postnatal cerebellum by ChIP-seq. Despite consistent expression of Zic genes, binding patterns are highly dynamic across development. Knockdown of Zic1 and Zic2 resulted in loss of up-regulation of key CGN maturity marker genes, suggesting that ZIC chromatin-gated access to the DNA template is required for driving mature neuronal gene expression patterns, a previously unappreciated role for these factors. Together this study has revealed chromatin dynamics at thousands of novel enhancers that facilitate expression patterns necessary for neuronal differentiation and function.

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