Biology subjects

Tachtsidi, A.

Publications and source records attributed to Tachtsidi, A..

4 recordsLinked to original sources

bioRxiv2026

CRISPR-Mediated Linearization of IDLV Donor Enables Precise Targeted Integration in Human Hematopoietic Stem Cells

Ex vivo genome editing of human hematopoietic stem and progenitor cells (HSPCs) requires targeted integration strategies that support large therapeutic DNA payloads while preserving stem cell fitness. Although CRISPR/Cas9-mediated homology-directed repair using AAV donors is effective, it is constrained by limited cargo capacity and adverse effects on long-term HSPCs function. Integrase-defective lentiviral vectors (IDLVs) offer an alternative donor platform, yet their precise and controlled genomic integration remains inefficient. Here, we describe TILV (Targeted Integration of Lentiviral Vector), a CRISPR-assisted knock-in strategy that exploits Cas9-mediated linearization of episomal IDLV DNA to expose a single homology arm and engage homology-mediated end-joining repair pathways. TILV enables precise, directional and seamless integration of transgenes in multiple loci, enabling constitutive or physiological expression. Using single-cell clonal analyses and targeted long-read sequencing, we define the molecular features of TILV-mediated integration and demonstrate preferential use of CRISPR-linearized episomal substrates. TILV supports accurate insertion of large therapeutic transgenes, without compromising HSPC viability or multilineage potential. We further show that transient modulation of DNA repair pathway, in combination with extended homology arms, enhances integration efficiency and junctional precision. Importantly, optimized TILV enables targeted integration in phenotypically defined long-term HSPCs, highlighting its potential for scalable and durable gene therapy.

molecular biology↗

bioRxiv2021

OCT4 activates a Suv39h1-repressive antisense lncRNA to couple histone H3 Lysine 9 methylation to pluripotency

Histone H3 Lysine 9 (H3K9) methylation, a characteristic mark of heterochromatin, is progressively implemented during development to contribute to cell fate restriction as differentiation proceeds. For instance, in pluripotent mouse Embryonic Stem (ES) cells the global levels of H3K9 methylation are rather low and increase only upon differentiation. Conversely, H3K9 methylation represents an epigenetic barrier for reprogramming somatic cells back to pluripotency. How global H3K9 methylation levels are coupled with the acquisition and loss of pluripotency remains largely unknown. Here, we identify SUV39H1, a major H3K9 di- and tri-methylase, as an indirect target of the pluripotency network of Transcription Factors (TFs). We find that pluripotency TFs, principally OCT4, activate the expression of an uncharacterized antisense long non-coding RNA to Suv39h1, which we name Suv39h1as. In turn, Suv39h1as downregulates Suv39h1 transcription in cis via a mechanism involving the modulation of the chromatin status of the locus. The targeted deletion of the Suv39h1as promoter region triggers increased SUV39H1 expression and H3K9me2 and H3K9me3 levels, leading to accelerated and more efficient commitment into differentiation. We report, therefore, a simple genetic circuitry coupling the global levels of H3K9 methylation to pluripotency in mouse ES cells.

molecular biology↗

bioRxiv2019

CTCF confers local nucleosome resiliency after DNA replication and during mitosis

The access of Transcription Factors (TFs) to their cognate DNA binding motifs requires a precise control over nucleosome posi-tioning. This is especially important following DNA replication and during mitosis, both resulting in profound changes in nu-cleosome organization over TF binding regions. Using mouse Embryonic Stem (ES) cells, we show that the TF CTCF displaces nucleosomes from its binding site and locally organizes large and phased nucleosomal arrays, not only in interphase steady-state but also immediately after replication and during mitosis. While regions bound by other TFs, such as Oct4 and Sox2, display major rearrangement, the post-replication and mitotic nucleosome organization activity of CTCF is not likely to be unique: Esrrb binding regions are also characterized by persistent nucleosome positioning. Therefore, we propose that selected TFs, such as CTCF and Esrrb, govern the inheritance of nucleosome positioning at gene regulatory regions through-out the ES cell-cycle.

genomics↗

bioRxiv2018

Transcription factor activity and nucleosome organisation in mitosis

Mitotic bookmarking transcription factors (BFs) maintain the capacity to bind to their targets during mitosis, despite major rearrangements of the chromatin. While they were thought to propagate gene regulatory information through mitosis by statically occupying their DNA targets, it has recently become clear that BFs are highly dynamic in mitotic cells. This represents both a technical and a conceptual challenge to study and understand the function of BFs: first, formaldehyde has been suggested to be unable to efficiently capture these transient interactions, leading to profound contradictions in the literature; second, if BFs are not permanently bound to their targets during mitosis, it becomes unclear how they convey regulatory information to daughter cells. Here, comparing formaldehyde to alternative fixatives we clarify the nature of the chromosomal association of previously proposed BFs in embryonic stem cells: while Esrrb can be considered as a canonical BF that binds at selected regulatory regions in mitosis, Sox2 and Oct4 establish DNA sequence independent interactions with the mitotic chromosomes, either throughout the chromosomal arms (Sox2) or at pericentromeric regions (Oct4). Moreover, we show that ordered nucleosomal arrays are retained during mitosis at Esrrb book-marked sites, whereas regions losing transcription factor binding display a profound loss of order. By maintaining nucleosome positioning during mitosis, Esrrb might ensure the rapid post-mitotic re-establishment of functional regulatory complexes at selected enhancers and promoters. Our results provide a mechanistic framework that reconciles dynamic mitotic binding with the transmission of gene regulatory information across cell division.

genomics↗