Biology subjects

Pertsinidis, A.

Publications and source records attributed to Pertsinidis, A..

6 recordsLinked to original sources

bioRxiv2025

NIPBL-mediated 3D genome folding translates enhancer priming into gene activation and safeguards lineage fidelity during embryonic transitions

Precise gene control by complex regulatory landscapes is fundamental to embryo development, yet the instructive role of 3D genome architecture remains controversial. While acute cohesin depletion completely disrupts genome folding, it yields modest transcriptional impacts, but these findings are often confounded by cohesins essential roles in cell division and proliferation. Here, we resolve this discrepancy by decoupling architectural functions from cell-cycle roles using an acute NIPBL degron system. By integrating single-gene imaging with single-cell and bulk multi-omics during mouse pluripotency transitions and germ-layer specification, we show that NIPBL-mediated cohesin function is required for proper de novo activation of lineage-specifying genes. Mechanistically, NIPBL translates epigenetic priming into transcriptional outputs by physically bringing distal enhancers and target promoters into proximity. We further uncover a dual regulatory role: an acute requirement for establishing new enhancer-promoter interactions during cell state transitions and a long-term role in safeguarding transcriptional fidelity by preventing ectopic gene de-repression. Our findings demonstrate that NIPBL/cohesin-orchestrated genome folding facilitates the faithful execution of developmental gene expression programs. HighlightsO_LIAcute NIPBL depletion decouples the architectural functions of cohesin from its essential roles in chromosome segregation and cell cycle progression. C_LIO_LINIPBL-mediated loop extrusion is required to translate the epigenetic priming of distal enhancers into de novo gene activation during embryonic state transitions. C_LIO_LINIPBL is a "rate-limiting physical relay" required to bring distal enhancers and target promoters into proximity to initiate transcription. C_LIO_LI3D genome architecture serves a dual role: enabling acute enhancer-promoter communication and safeguarding long-term lineage fidelity by preventing ectopic gene de-repression. C_LI

genetics↗

bioRxiv2025

Post-mitotic transcriptional activation and 3D regulatory interactions show locus- and differentiation-specific sensitivity to cohesin depletion

Prior studies showed that structural loops collapse upon acute cohesin depletion, while regulatory enhancer-promoter (E-P) loops largely persist, consistent with minimal transcriptional changes. However, these studies, conducted in asynchronous cells, could not resolve whether cohesin is required for the establishment of regulatory interactions and transcriptional activation during cell division or cell state transitions. To address this gap, we degraded RAD21, a core cohesin subunit, in naive mouse embryonic stem cells (ESCs) transitioning from mitosis to G1 either in self-renewal condition or during differentiation toward formative pluripotency. Although most structural loops failed to be re-established without cohesin, about 35% of regulatory loops reformed at normal or higher frequencies. Cohesin-independent loops showed characteristics of strong active enhancers and promoters and a significant association with H3K27ac mitotic bookmarks. However, inhibition of CBP/p300 during mitotic exit did not impact these cohesin-independent interactions, suggesting the presence of complex compensatory mechanisms. At the transcriptional level, cohesin depletion induced only minor changes, supporting that post-mitotic transcriptional reactivation is largely independent of cohesin. The few genes with impaired reactivation were directly bound by RAD21 at their promoters, engaged in many structural loops, and located within strongly insulated TADs with low gene density. Importantly, degrading cohesin during the M-to-G1 transition in the presence of EpiLC differentiation signals revealed a larger group of susceptible genes, including key signature genes and transcription factors. Impaired activation of these genes was partly due to the failure to establish de novo EpiLC-specific interactions in the absence of cohesin. These experiments revealed locus-specific and context-specific dependencies between cohesin, E-P interactions, and transcription.

molecular biology↗

bioRxiv2023

Mechanisms of transcription control by distal enhancers from high-resolution single-gene imaging

How distal enhancers physically control promoters over large genomic distances, to enable cell-type specific gene expression, remains obscure. Using single-gene super-resolution imaging and acute targeted perturbations, we define physical parameters of enhancer-promoter communication and elucidate processes that underlie target gene activation. Productive enhancer-promoter encounters happen at 3D distances {delta}200 nm - a spatial scale corresponding to unexpected enhancer-associated clusters of general transcription factor (GTF) components of the Pol II machinery. Distal activation is achieved by increasing transcriptional bursting frequency, a process facilitated by embedding a promoter into such GTF clusters and by accelerating an underlying multi-step cascade comprising early phases in the Pol II transcription cycle. These findings help clarify molecular/biochemical signals involved in long-range activation and their means of transmission from enhancer to promoter.

biophysics↗

bioRxiv2022

STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity

Transcription is a dynamic process that stochastically switches between the ON and OFF states. To detect the dynamic relationship among protein clusters of RNA polymerase II (RNAPII) and coactivators, gene loci, and transcriptional activity, we inserted an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site (TSS). By optimizing the individual elements, we have developed the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNAPII and BRD4 were observed proximally to the TSS of Nanog when the gene was transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 Mediator subunits were constitutively located near the TSS. Thus, the STREAMING-tag system revealed the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding dynamic transcriptional regulation in living cells.

molecular biology↗

bioRxiv2020

3D Interferometric Lattice Light-Sheet Imaging

Understanding cellular structure and function requires live-cell imaging with high spatio-temporal resolution and high detection sensitivity. Direct visualization of molecular processes using single-molecule/super-resolution techniques has thus been transformative. However, extracting the highest-resolution 4D information possible from weak and dynamic fluorescence signals in live cells remains challenging. For example, some of the highest spatial resolution methods, e.g. interferometric (4Pi) approaches1-6 can be slow, require high peak excitation intensities that accelerate photobleaching or suffer from increased out-of-focus background. Selective-plane illumination (SPIM)7-12 reduces background, but most implementations typically feature modest spatial, especially axial, resolution. Here we develop 3D interferometric lattice light-sheet (3D-iLLS) imaging, a technique that overcomes many of these limitations. 3D-iLLS provides, by virtue of SPIM, low light levels and photobleaching, while providing increased background suppression and significantly improved volumetric imaging/sectioning capabilities through 4Pi interferometry. We demonstrate 3D-iLLS with axial resolution and single-particle localization precision down to <100nm (FWHM) and <10nm (1{sigma}) respectively. 3D-iLLS paves the way for a fuller elucidation of sub-cellular phenomena by enhanced 4D resolution and SNR live imaging.

cell biology↗

bioRxiv2020

CAS-LiveFISH: Simple and versatile imaging of genomic loci in live mammalian cells and early pre-implantation embryos

Visualizing the 4D genome in live cells is essential for understanding its regulation. Programmable DNA-binding probes, such as fluorescent clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector (TALE) proteins have recently emerged as powerful tools for imaging specific genomic loci in live cells. However, many such systems rely on genetically-encoded components, often requiring multiple constructs that each must be separately optimized, thus limiting their use. Here we develop efficient and versatile systems, based on in vitro transcribed single-guide-RNAs (sgRNAs) and fluorescently-tagged recombinant, catalytically-inactivated Cas9 (dCas9) proteins. Controlled cell delivery of pre-assembled dCas9-sgRNA ribonucleoprotein (RNP) complexes enables robust genomic imaging in live cells and in early mouse embryos. We further demonstrate multiplex tagging of up to 3 genes, tracking detailed movements of chromatin segments and imaging spatial relationships between a distal enhancer and a target gene, with nanometer resolution in live cells. This simple and effective approach should facilitate visualizing chromatin dynamics and nuclear architecture in various living systems.

cell biology↗