Biology subjects

Arnosti, D. N.

Publications and source records attributed to Arnosti, D. N..

3 recordsLinked to original sources

bioRxiv2023

A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor

The C-terminal Binding Protein (CtBP) is a transcriptional corepressor that plays critical roles in development, tumorigenesis, and cell fate. CtBP proteins are structurally similar to alpha hydroxyacid dehydrogenases and feature a prominent intrinsically disordered region in the C-terminus. In the mammalian system, CtBP proteins lacking the C-terminal Domain (CTD) are able to function as transcriptional regulators and oligomerize, putting into question the significance of this unstructured domain for gene regulation. Yet, the presence of an unstructured CTD of [~]100 residues, including some short motifs, is conserved across Bilateria, indicating the importance of maintaining this domain over evolutionary time. To uncover the significance of the CtBP CTD, we functionally tested naturally occurring Drosophila isoforms of CtBP that possess or lack the CTD, namely CtBP(L) and CtBP(S). We used the CRISPRi system to recruit dCas9-CtBP(L) and dCas9-CtBP(S) to endogenous promoters to directly compare their transcriptional impacts in vivo. Interestingly, CtBP(S) was able to significantly repress transcription of the Mpp6 promoter, while CtBP(L) was much weaker, suggesting that the long CTD may modulate CtBPs repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfected Mpp6 reporter gene. The context-specific differences in activity of these two developmentally-regulated isoforms suggests that the CTD may help provide a spectrum of repression activity suitable for developmental programs.

molecular biology↗

bioRxiv2023

Retinoblastoma protein activity revealed by CRISPRi study of divergent Rbf1 and Rbf2 paralogs

Retinoblastoma tumor suppressor proteins regulate the key transition from G1 to S phase of the cell cycle. The mammalian Rb family comprises Rb, p107, and p130, with overlapping and unique roles in gene regulation. Drosophila experienced an independent gene duplication event, leading to the Rbf1 and Rbf2 paralogs. To uncover the significance of paralogy in the Rb family, we used CRISPRi. We engineered dCas9 fusions to Rbf1 and Rbf2, and deployed them to gene promoters in developing Drosophila tissue to study their relative impacts on gene expression. On some genes, both Rbf1 and Rbf2 mediate potent repression, in a highly distance-dependent manner. In other cases, the two proteins have different effects on phenotype and gene expression, indicating different functional potential. In a direct comparison of Rb activity on endogenous genes and transiently transfected reporters, we found that only qualitative, but not key quantitative aspects of repression were conserved, indicating that the native chromatin environment generates context-specific effects of Rb activity. Our study uncovers the complexity of Rb-mediated transcriptional regulation in a living organism, which is clearly impacted by the different promoter landscapes and the evolution of the Rb proteins themselves.

molecular biology↗

bioRxiv2022

The cynosure of CtBP: evolution of a bilaterian transcriptional corepressor

Evolution of sequence-specific transcription factors clearly drives lineage-specific innovations, but less is known about how changes in the central transcriptional machinery may contribute to evolutionary transformations. In particular, transcriptional regulators are rich in intrinsically disordered regions that appear to be magnets for evolutionary innovation. The C-terminal Binding Protein (CtBP) is a transcriptional corepressor derived from an ancestral lineage of alpha hydroxyacid dehydrogenases; it is found in mammals and invertebrates, and features a core NAD-binding domain as well as an unstructured C-terminus (CTD) of unknown function. CtBP can act on promoters and enhancers to repress transcription through chromatin-linked mechanisms. Our comparative phylogenetic study shows that CtBP is a bilaterian innovation whose CTD of about 100 residues is present in almost all orthologs. CtBP CTDs contain conserved blocks of residues and retain a predicted disordered property, despite having variations in the primary sequence. Interestingly, the structure of the C-terminus has undergone radical transformation independently in certain lineages including flatworms and nematodes. Also contributing to CTD diversity is the production of myriad alternative RNA splicing products, including the production of "short" tailless forms of CtBP in Drosophila. Additional diversity stems from multiple gene duplications in vertebrates, where up to five CtBP orthologs have been observed. Vertebrate lineages show fewer major modifications in the unstructured CTD, possibly because gene regulatory constraints of the vertebrate body plan place specific constraints on this domain. Our study highlights the rich regulatory potential of this previously unstudied domain of a central transcriptional regulator.

evolutionary biology↗