Keywords: a. chromatin structure; e. neuronal morphogenesis
The nervous system undergoes dramatic post-mitotic reorganization to become fully mature, but how chromatin 3D structure of neurons is regulated to assure proper transcriptomic dynamics during this essential process remains undefined in any organism. The tissue-specific chromatin insulator antagonist Alan Shepard (Shep) promotes Drosophila post-mitotic maturation by repressing expression of master regulator genes specifically in maturing neurons. To understand the mechanism of Shep repression of a key target gene, brain tumor (brat), we performed 4C-seq on CNS-derived BG3 cells and found that Shep depletion leads to increased brat promoter looping to proximal regions. Subsequent luciferase reporter assays verified enhancer activity of one of these candidate regions. Notably, depletion of Shep does not affect either enhancer or promoter activities in this artificial juxtaposed context, suggesting that Shep acts as a dedicated anti-looping factor to inhibit brat transcription. Interestingly, in vivo 3C also detected Shep inhibition of looping between the brat promoter and a proximal genomic region in pupal but not larval brains. Subsequent in vivo GFP reporter assays detected enhancer activity of this region specifically in pupal brains. This enhancer activity was not affected by Shep depletion, consistent with an anti-looping mechanism underlying Shep repression of brat transcription in pupal maturing neurons. Moreover, ATAC-seq and CUT&Tag for H3K4me1 on FACS-sorted neurons indicated that Shep is required to restrain chromatin accessibility of the brat enhancer as well as 1,126 other enhancers in pupal but not larval neurons, suggesting extensive Shep-mediated enhancer closure during neuronal maturation genome-wide. These enhancers are highly enriched for Shep-bound loci that are also transcriptionally inhibited by Shep, suggesting temporal Shep inhibition of enhancer accessibility to repress gene expression. Taken together, our results provide the first evidence for a chromatin anti-looping factor that regulates temporal enhancer accessibility and gene expression during organismal development.