Identification and characterization of transcribed enhancers during cerebellar development through enhancer RNA analysis.

BACKGROUND: The development of the brain requires precise coordination of molecular processes across many cell-types. Underpinning these events are gene expression programs which require intricate regulation by non-coding regulatory sequences known as enhancers. In the context of the developing brain, transcribed enhancers (TEs) regulate temporally-specific expression of genes critical for cell identity and differentiation. Transcription of non-coding RNAs at active enhancer sequences, known as enhancer RNAs (eRNAs), is tightly associated with enhancer activity and has been correlated with target gene expression. TEs have been characterized in a multitude of developing tissues, however their regulatory role has yet to be described in the context of embryonic and early postnatal brain development. In this study, eRNA transcription was analyzed to identify TEs active during cerebellar development, as a proxy for the developing brain. Cap Analysis of Gene Expression followed by sequencing (CAGE-seq) was conducted at 12 stages throughout embryonic and early postnatal cerebellar development. RESULTS: Temporal analysis of eRNA transcription identified clusters of TEs that peak in activity during either embryonic or postnatal times, highlighting their importance for temporally specific developmental events. Functional analysis of putative target genes identified molecular mechanisms under TE regulation revealing that TEs regulate genes involved in biological processes specific to neurons. We validate enhancer activity using in situ hybridization of eRNA expression from TEs predicted to regulate Nfib, a gene critical for cerebellar granule cell differentiation. CONCLUSIONS: The results of this analysis provide a valuable dataset for the identification of cerebellar enhancers and provide insight into the molecular mechanisms critical for brain development under TE regulation. This dataset is shared with the community through an online resource ( https://goldowitzlab.shinyapps.io/trans-enh-app/ ).

Temporal analysis of enhancers during mouse cerebellar development reveals dynamic and novel regulatory functions.

We have identified active enhancers in the mouse cerebellum at embryonic and postnatal stages which provides a view of novel enhancers active during cerebellar development. The majority of cerebellar enhancers have dynamic activity between embryonic and postnatal development. Cerebellar enhancers were enriched for neural transcription factor binding sites with temporally specific expression. Putative gene targets displayed spatially restricted expression patterns, indicating cell-type specific expression regulation. Functional analysis of target genes indicated that enhancers regulate processes spanning several developmental epochs such as specification, differentiation and maturation. We use these analyses to discover one novel regulator and one novel marker of cerebellar development: Bhlhe22 and Pax3, respectively. We identified an enrichment of de novo mutations and variants associated with autism spectrum disorder in cerebellar enhancers. Furthermore, by comparing our data with relevant brain development ENCODE histone profiles and cerebellar single-cell datasets we have been able to generalize and expand on the presented analyses, respectively. We have made the results of our analyses available online in the Developing Mouse Cerebellum Enhancer Atlas, where our dataset can be efficiently queried, curated and exported by the scientific community to facilitate future research efforts. Our study provides a valuable resource for studying the dynamics of gene expression regulation by enhancers in the developing cerebellum and delivers a rich dataset of novel gene-enhancer associations providing a basis for future in-depth studies in the cerebellum.