Biology subjects

Moene, C. J. I.

Publications and source records attributed to Moene, C. J. I..

4 recordsLinked to original sources

bioRxiv2026

Effects of CTCF on the regulatory landscape of the mouse Sox2 locus

Regulatory elements that control gene transcription are scattered along the genome, yet it is still poorly understood how their precise positioning affects the regulatory landscape. We previously reported a method to relocate a regulatory element to thousands of positions in a genomic locus and probe the position-dependent impact on gene regulation. Here, we apply this approach to systematically query the role and position-dependence of CTCF binding sites (CBSs), which are thought to modulate communication between distal regulatory elements. We focused on the mouse Sox2 locus in embryonic stem cells, where the Sox2 gene is activated by a potent distal enhancer. First, we found that a CBS that is naturally located immediately upstream of the Sox2 promoter endows this promoter with strong orientation-dependent activation, anywhere within the gene-enhancer interval. Second, throughout this interval, insertion of CBSs alone consistently reduces Sox2 expression in an orientation-dependent manner, suggesting a polarity in the loop extrusion process. Third, two native CBSs located within and downstream of the enhancer subtly help to confine the activation realm of the enhancer. Together, these results illuminate the interplay between CBSs and the detailed regulatory landscape of a genomic locus.

molecular biology↗

bioRxiv2025

Single-Molecule DNA Footprinting and Transcription Imaging Reveal the Molecular Mechanisms of Promoter Dynamics

Live cell RNA imaging revealed that transcription levels are encoded by the intrinsic dynamics of promoters. However, capturing both kinetic and molecular aspects of promoter fluctuations has been challenging. Here, we resolve this key issue by combining Single Molecule DNA footprinting (SMF) with live transcription imaging. Using HIV-1 as a model, SMF reveals that the promoter functions in two modes depending on the viral transactivator Tat. Without Tat, a nucleosome occupies the core promoter and prevents assembly of the pre-initiation complex. With Tat, this nucleosome is absent while TBP and initiating polymerases are frequently detected. Combining live imaging with SMF provides a mechanistic model of promoter dynamics, which estimates the rates of deposition and removal of promoter nucleosomes (0.7 h-1), TBP binding (0.04 min-1) and polymerase loading (seconds). The data further reveal a kinetic proofreading mechanism of initiating polymerases, which enables Tat to indirectly control promoter nucleosomes by promoting elongation.

molecular biology↗

bioRxiv2024

Mammalian promoters are characterised by low occupancy and high turnover of RNA polymerase II

The general transcription machinery and its occupancy at promoters are highly conserved across metazoans. This contrasts with the kinetics of mRNA production that considerably differ between model species such as Drosophila and mouse. The molecular basis for these kinetic differences is currently unknown. Here, we used Single Molecule Footprinting to measure RNA Polymerase II (Pol II) occupancy, the fraction of DNA molecules bound, at promoters in mouse and Drosophila cell lines. Single molecule data reveals that Pol II occupancy is on average 3-5 times more frequent at transcriptionally active Drosophila promoters than active mouse promoters. Kinetic modelling of the occupancy states suggests that these differences in Pol II occupancy are determined by the ratio between the transcription initiation and Pol II turnover rates. We used chemical perturbation of transcription initiation to determine Pol II turnover rate in both species. Integration of these data into the model shows that infrequent Pol II occupancy in mammals is explained by the combination of high Pol II turnover and low transcription initiation rates.

genomics↗

bioRxiv2024

Functional maps of a genomic locus reveal confinement of an enhancer by its target gene.

Genes are often activated by enhancers located at large genomic distances. The importance of this positioning is poorly understood. By relocating promoter-reporter constructs into >1,000 alternative positions within a single locus, we dissected the positional relationship between the mouse Sox2 gene and its distal enhancer. This revealed an intricate, sharply confined activation landscape, in which the native Sox2 gene occupies an optimal position for its activation. Deletion of the gene relaxes this confinement and broadly increases reporter activity. Surprisingly, the confining effect of the Sox2 gene is partially conferred by its [~]1 kb coding region. Our local relocation approach provides high-resolution functional maps of a genomic locus and reveals that a gene can strongly constrain the realm of influence of its enhancer.

molecular biology↗