Abstract
During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. The chromatin insulator-associated protein Alan Shepard (Shep) promotes Drosophila post-mitotic neuronal remodeling by repressing transcription of master developmental regulators, such as brain tumor (brat), specifically in maturing neurons. Since insulator proteins can promote looping, we hypothesized that Shep antagonizes brat promoter interaction with an as yet unidentified enhancer. Using chromatin conformation capture and reporter assays, we identified two enhancer regions that increase in looping frequency with the brat promoter specifically in pupal brains after Shep depletion. The brat promoters and enhancers function independently of Shep, ruling out direct repression of these elements. Moreover, ATAC-seq in isolated neurons demonstrates that Shep restricts chromatin accessibility of a key brat enhancer as well as other enhancers genome-wide in remodeling pupal but not larval neurons. These enhancers are enriched for chromatin targets of Shep and are located at Shep-inhibited genes, suggesting direct Shep inhibition of enhancer accessibility and gene expression during neuronal remodeling. Our results provide evidence for temporal regulation of chromatin looping and enhancer accessibility during neuronal maturation.
Highlights
During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation
Forced E–P looping can result in ectopic activation of gene expression[3,4], suggesting that the E–P looping step itself may serve as a point of either positive or negative regulation during development
Shep temporally restricts the accessibility of a key brat enhancer and other enhancers genome-wide in pupal neurons that correspond to Shep-inhibited gene expression
Summary
During development, looping of an enhancer to a promoter is frequently observed in conjunction with temporal and tissue-specific transcriptional activation. Forced E–P looping can result in ectopic activation of gene expression[3,4], suggesting that the E–P looping step itself may serve as a point of either positive or negative regulation during development One such E–P looppromoting factor is the Lim domain-containing protein Ldb[1], which is expressed during the development of specific tissues and forms a complex with particular transcription factors[5]. The regulation of chromatin 3D structure during post-mitotic neuronal remodeling has not previously been studied Architectural proteins, such as insulator proteins, have been demonstrated to participate in the formation of topologically associating domains and cell type-specific E–P loops. We conclude that Shep inhibits chromatin looping and enhancer accessibility to regulate gene expression in a stage-specific manner to facilitate neuronal remodeling
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