Biology subjects

Mekerishvili, L.

Publications and source records attributed to Mekerishvili, L..

5 recordsLinked to original sources

bioRxiv2026

A CRISPR-Based Humanized Model Reveals Cooperative Role of STAG2 Loss in Familial GATA2-Deficient MDS Progression

Myelodysplastic syndrome (MDS) is a heterogeneous myeloid malignancy driven by hematopoietic stem cell dysfunction, leading to ineffective hematopoiesis and cytopenias. Familial GATA2 deficiency is the most common cause of Myelodysplastic syndrome in adolescents, with progression often accelerated by co-occurring mutations, notably STAG2 loss-of-function. Using CRISPR/Cas9-mediated genome engineering in primary human fetal liver-derived hematopoietic stem cells and xenotransplantation in mice, we modeled GATA2-deficient Myelodysplastic syndrome with acquired STAG2 loss to investigate disease initiation and progression. While GATA2 deficiency alone had minimal short-term impact in our model, combined GATA2 and STAG2 loss increased hematopoietic stem cell maintenance and self-renewal, induced a myeloid-lineage bias, and expanded primitive progenitors. Single-cell transcriptional profiling revealed upregulation of stemness genes and inflammatory pathways. This humanized model faithfully recapitulates high-risk GATA2-deficient Myelodysplastic syndrome, providing mechanistic insight into how cooperative mutations drive stem cell expansion, inflammatory signaling, and myeloid skewing. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/702879v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1972beforg.highwire.dtl.DTLVardef@1c57886org.highwire.dtl.DTLVardef@16bd582org.highwire.dtl.DTLVardef@8e728a_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIHumanized model of familial GATA2-deficiency requires the loss of STAG2 for progression to an MDS disease phenotype C_LIO_LIGATA2-ko+STAG2-ko increase HSC self-renewal, induce a myeloid-lineage bias, and trigger an inflammatory transcriptional program C_LI

cancer biology↗

bioRxiv2025

Ontogeny Dictates Oncogenic Potential, Lineage Hierarchy, and Therapy Response in Pediatric Leukemia

Accumulating evidence links pediatric cancers to prenatal transformation events, yet the influence of the developmental stage on oncogenesis remains elusive. We investigated how hematopoietic stem cell developmental stages affect leukemic transformation, disease progression, and therapy response using a novel, humanized model of NUP98::NSD1-driven pediatric acute myeloid leukemia, that is particularly aggressive with WT1 co-mutations. Fetal-derived hematopoietic stem cells readily transform into leukemia, and WT1 mutations further enhance stemness and alter lineage hierarchy. In contrast, stem cells from later developmental stages become progressively resistant to transformation. Single-cell analyses revealed that fetal-origin leukemia stem cells exhibit greater quiescence and reliance on oxidative phosphorylation than their postnatal counterparts. These differences drive distinct therapeutic responses, despite identical oncogenic mutations. In patients, onco-fetal transcriptional programs correlate with worse outcomes. By targeting key vulnerabilities of fetal-origin leukemia cells, we identified combination therapies that significantly reduce aggressiveness, highlighting the critical role of ontogeny in pediatric cancer treatment.

cancer biology↗

bioRxiv2025

Single-cell mapping of regulatory DNA:Protein interactions

Gene expression is coordinated by a multitude of transcription factors (TFs), whose binding to the genome is directed through multiple interconnected epigenetic signals, including chromatin accessibility and histone modifications. These complex networks have been shown to be disrupted during aging, disease, and cancer. However, profiling these networks across diverse cell types and states has been limited due to the technical constraints of existing methods for mapping DNA:Protein interactions in single cells. As a result, a critical gap remains in understanding where TFs or other chromatin remodelers bind to DNA and how these interactions are perturbed in pathological contexts. To address this challenge, we developed a transformative single-cell immuno-tethering DNA:Protein mapping technology. By coupling a species-specific antibody-binding nanobody to a cytosine base editing enzyme, this approach enables profiling of even weak or transient factor binding to DNA, a task that was previously unachievable in single cells. Thus, our Docking & Deamination followed by sequencing (D&D-seq) technique induces cytosine-to-uracil edits in genomic regions bound by the target protein, offering a novel means to capture DNA:Protein interactions with unprecedented resolution. Importantly, this technique can be seamlessly incorporated into common single-cell multiomics workflows, enabling multimodal analysis of gene regulation in single cells. We tested the ability of D&D-seq to record TF binding both in bulk and at the single-cell level by profiling CTCF and GATA family members, obtaining high specificity and efficiency, with clear identification of TF footprint and signal retention in the targeted cell subpopulations. Furthermore, the deamination reaction showed minimal off-target activity, with high concordance to bulk ChIP-seq reference data. Applied to primary human peripheral blood mononuclear cells (PBMCs), D&D-seq successfully identified CTCF binding sites and enabled integration with advanced machine-learning algorithms for predicting 3D chromatin structure. Furthermore, we integrated D&D-seq with single-cell genotyping to assess the impact of IDH2 mutations on CTCF binding in a human clonal hematopoiesis sample, uncovering altered binding and chromatin co-accessibility patterns in mutant cells. Altogether, D&D-seq represents an important technological advance enabling the direct mapping of TF or chromatin remodeler binding to the DNA in primary human samples, opening new avenues for understanding chromatin and transcriptional regulation in health and disease.

genomics↗

bioRxiv2022

Integrated Single-Cell Genotyping and Chromatin Accessibility Charts JAK2V617F Human Hematopoietic Differentiation

In normal somatic tissue differentiation, changes in chromatin accessibility govern priming and commitment of precursors towards cellular fates. In turn, somatic mutations can disrupt differentiation topologies leading to abnormal clonal outgrowth. However, defining the impact of somatic mutations on the epigenome in human samples is challenging due to admixed mutated and wildtype cells. To chart how somatic mutations disrupt epigenetic landscapes in human clonal outgrowths, we developed Genotyping of Targeted loci with single-cell Chromatin Accessibility (GoT-ChA). This high-throughput, broadly accessible platform links genotypes to chromatin accessibility at single-cell resolution, across thousands of cells within a single assay. We applied GoT-ChA to CD34+ cells from myeloproliferative neoplasm (MPN) patients with JAK2V617F-mutated hematopoiesis, where the JAK2 mutation is known to perturb hematopoietic differentiation. Differential accessibility analysis between wildtype and JAK2V617F mutant progenitors revealed both cell-intrinsic and cell state-specific shifts within mutant hematopoietic precursors. An early subset of mutant hematopoietic stem and progenitor cells (HSPCs) exhibited a cell-intrinsic pro-inflammatory signature characterized by increased NF-{kappa}B and JUN/FOS transcription factor motif accessibility. In addition, mutant HSPCs showed increased myeloid/erythroid epigenetic priming, preceding increased erythroid and megakaryocytic cellular output. Erythroid progenitors displayed aberrant regulation of the {gamma}-globin locus, providing an intrinsic epigenetic basis for the dysregulated fetal hemoglobin expression observed in MPNs. In contrast, megakaryocytic progenitors exhibited a more specialized inflammatory chromatin landscape relative to early HSPCs, with increased accessibility of pro-fibrotic JUN/FOS transcription factors. Notably, analysis of myelofibrosis patients treated with JAK inhibitors revealed an overall loss of mutant-specific phenotypes without modifying clonal burden, consistent with clinical responses. Finally, expansion of the multi-modality capability of GoT-ChA to integrate mitochondrial genome profiling and cell surface protein expression measurement enabled genotyping imputation and discovery of aberrant cellular phenotypes. Collectively, we show that the JAK2V617F mutation leads to epigenetic rewiring in a cell-intrinsic and cell type-specific manner. We envision that GoT-ChA will thus serve as a foundation for broad future explorations to uncover the critical link between mutated somatic genotypes and epigenetic alterations across clonal populations in malignant and non-malignant contexts.

cancer biology↗

bioRxiv2022

Nanobody-tethered transposition allows for multifactorial chromatin profiling at single-cell resolution

Chromatin states are functionally defined by a complex combination of histone modifications, transcription factor binding, DNA accessibility, and other factors. However, most current single-cell-resolution methods are unable to measure more than one aspect of chromatin state in a single experiment, limiting our ability to accurately measure chromatin states. Here, we introduce nanobody-tethered transposition followed by sequencing (NTT-seq), a new assay capable of measuring the genome-wide presence of multiple histone modifications and protein-DNA binding sites at single-cell resolution. NTT-seq utilizes recombinant Tn5 transposase fused to a set of secondary nanobodies (nb). Each nb-Tn5 fusion protein specifically binds to different immunoglobulin-G antibodies, enabling a mixture of primary antibodies binding different epitopes to be used in a single experiment. We apply bulk- and single-cell NTT-seq to generate high-resolution multimodal maps of chromatin states in cell culture and in cells of the human immune system, demonstrating the high accuracy and sensitivity of the method. We further extend NTT-seq to enable simultaneous profiling of cell-surface protein expression alongside multimodal chromatin states to study cells of the immune system.

genomics↗