479A Poster - 06. Regulation of gene expression
Thursday April 07, 2:00 PM - 4:00 PM

Authors:
Arjun Rajan ¹; Lucas Anhezini ¹; Megan Neville ²; Elizabeth Larson ³; Stephen Goodwin ²; Melissa Harrison ³; Cheng-Yu Lee ^1,4,5

Affiliations:
1) Life Sciences Institute, University of Michigan, Ann Arbor, MI; 2) Centre for Neural Circuits and Behaviour, University of Oxford, Oxford, UK; 3) Department of Biomolecular Chemistry, University of Wisconsin, Madison, WI; 4) Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, 48109 ; 5) Division of Molecular Medicine and Genetics, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI

Keywords:
d. repressors/corepressors; d. repressors/corepressors

A defined threshold of gene transcription prevents the accumulation of excess transcripts and proteins, allowing cells undergoing developmental transitions to rapidly rewire their gene regulatory circuits and assume new cell identities. The delay negative feedback loop that maintains the oscillatory pattern of Notch target gene expression in vertebrates exemplifies how post-transcriptional regulatory mechanisms function as a timer to indirectly regulate gene transcription levels. We investigated if repressive inputs could function in synchrony with transcriptional activators to modulate gene transcription thresholds in fly larval brain type II neuroblasts, which divide asymmetrically to generate a Notch^ON neuroblast and a Notch^OFF differentiating progeny. We hypothesize that factors eliciting repressive inputs should (1) be specifically expressed in type II neuroblasts, (2) bind Notch target gene loci and (3) repress Notch target gene expression. We performed single cell RNA-sequencing to establish a transcriptomic atlas of a wild-type type II neuroblast lineage that includes neuroblast progeny undergoing commitment to differentiation. We discovered that fruitless (fru) gene transcripts are uniquely detected in type II neuroblasts, and the C isoform of Fru (Fru^c) is specifically expressed in type II neuroblasts. Loss of fru^c function enhanced the supernumerary neuroblast phenotype in numb- or brat-hypomorphic brains in which reduced function of either gene leads to aberrant upregulation of Notch signaling in neuroblast progeny driving their reversion. We performed CUT&RUN to profile Fru^c-bound loci in type II neuroblast enriched brains and found that Fru^c binds most Notch target gene loci in the fly genome. These data suggest that Fru^c functions to antagonize Notch target gene expression in type II neuroblasts. Consistent with this model, reducing fru^c function increases Notch target gene expression levels in mitotic type II neuroblasts. Furthermore, overexpressing wild-type Fru^c or a constitutive repressor form of Fru^c is sufficient to downregulate Notch signaling and partially restore differentiation in brat-null brains. We propose that the balance between Fru^c-mediated chromatin modifications and Notch-induced histone acetylation specifies Notch target gene transcription thresholds in type II neuroblasts.