Biology subjects

Bulut-Karslioglu, A.

Publications and source records attributed to Bulut-Karslioglu, A..

9 recordsLinked to original sources

bioRxiv2026

Nuclear mTOR-Polycomb synergism at developmental gene promoters prone to activation

Pluripotent cells hold the capacity to differentiate into cells with diverse functions, enabling development and regeneration. The balance between local epigenetic repression and maintaining the potential for prompt transcriptional activation supports pluripotency while allowing imminent differentiation. How the activation of developmental pathways is correctly timed in sync with the environment remains poorly understood. Here we find that the cellular growth regulator mTOR selectively binds developmental gene promoters in pluripotent mouse embryonic stem cells. mTOR binding at target genes correlates with and depends on histone H2AK119 monoubiquitination (H2AK119ub1) deposited by the Polycomb Repressor Complex 1 (PRC1). Acute depletion of the whole PRC1 complex or its catalytic activity leads to depletion of mTOR at target gene promoters, whereas forced PRC1 recruitment to an artificial site brings along mTOR. At target genes, mTOR colocalizes with components of the transcription machinery and we find that mTOR-bound genes are distinctly characterized by high levels of RNA Polymerase II pausing. Our findings reveal a role of mTOR in pluripotency regulation and highlight the tight crosstalk between gene regulatory and cell growth machineries in stem cells.

molecular biology↗

bioRxiv2025

An advanced head-to-tail mouse embryo model with hypoxia-mediated neural patterning

The developing mammalian embryo is guided by the continuously changing signals that it receives from maternal tissues and its microenvironment. The dynamic cell-cell and cell-environment interactions that together shape the embryo largely remained unexplorable until the advance of stem cell-based embryo models. These revealed the self-organizing properties of cells in response to endogenous and exogenous cues. Among the latter, restricted oxygen (hypoxia) emerged as a critical microenvironmental regulator that influences cell type diversification in multicellular systems. Here we built a modular ESC-based head-to-tail model of mouse embryogenesis by developing an antero-posterior (AP) assembly strategy under hypoxia. These structures called HAP-gastruloids feature stage-appropriate anterior neural tissues that recapitulate the morphological organization and transcriptional identity of fore- and midbrain including spatial organizer regions such as the midbrain-hindbrain boundary. These anterior tissues develop in synchrony with posterior tissues such as the spinal cord, somites, and gut endoderm derivatives, ultimately yielding a unified structure. We show via genetic, environmental, and pharmacological perturbations that timed hypoxia is essential to boost anterior neural cell identities and their patterning through HIF1a and in part by modulating TGF{beta} activity. These results underline the key beneficial role of hypoxia in early development and offer a uniquely modular system to investigate antero-posterior phenotypes for basic discovery and translation.

developmental biology↗

bioRxiv2023

Combinatorial microRNA activity is essential for the transition of pluripotent cells from proliferation into dormancy

Dormancy is a key feature of stem cell function in adult tissues as well as embryonic cells in the context of diapause. The establishment of dormancy is an active process that involves extensive transcriptional, epigenetic, and metabolic rewiring1,2. How these processes are coordinated to successfully transition cells to the resting dormant state remains unclear. Here we show that microRNA activity, which is otherwise dispensable for pre-implantation development, is essential for the adaptation of early mouse embryos to the dormant state of diapause. In particular, the pluripotent epiblast depends on miRNA activity, the absence of which results in loss of pluripotent cells. Through integration of high-sensitivity small RNA expression profiling of individual embryos and protein expression of miRNA targets with public data of protein-protein interactions, we constructed the miRNA-mediated regulatory network of mouse early embryos specific to diapause. We find that individual miRNAs contribute to the combinatorial regulation by the network and the perturbation of the network compromises embryo survival in diapause. Without miRNAs, nuclear and cytoplasmic bodies show aberrant expression, concurrent with splicing defects. We identified the nutrient-sensitive transcription factor TFE3 as an upstream regulator of diapause-specific miRNAs, linking cytoplasmic mTOR activity to nuclear miRNA biogenesis. Our results place miRNAs as a critical regulatory layer for the molecular rewiring of early embryos to establish dormancy.

molecular biology↗

bioRxiv2023

Integrated Molecular-Phenotypic Profiling Reveals Metabolic Control of Morphological Variation in Stembryos

Mammalian stem-cell-based models of embryo development (stembryos) hold great promise in basic and applied research. However, considerable phenotypic variation despite identical culture conditions limits their potential. The biological processes underlying this seemingly stochastic variation are poorly understood. Here, we investigate the roots of this phenotypic variation by intersecting transcriptomic states and morphological history of individual stembryos across stages modeling post-implantation and early organogenesis. Through machine learning and integration of time-resolved single-cell RNA-sequencing with imaging-based quantitative phenotypic profiling, we identify early features predictive of the phenotypic end-state. Leveraging this predictive power revealed that early imbalance of oxidative phosphorylation and glycolysis results in aberrant morphology and a neural lineage bias that can be corrected by metabolic interventions. Collectively, our work establishes divergent metabolic states as drivers of phenotypic variation, and offers a broadly applicable framework to chart and predict phenotypic variation in organoid systems. The strategy can be leveraged to identify and control underlying biological processes, ultimately increasing the reproducibility of in vitro systems. HighlightsO_LITime-resolved single-cell RNA-sequencing and imaging-based quantitative charting of hundreds of individual stembryos generates molecular and phenotypic fingerprints C_LIO_LIMachine learning and integration of molecular and phenotypic fingerprints identifies features and biological processes predictive of phenotypic end-state C_LIO_LIEarly imbalance of oxidative phosphorylation and glycolysis results in aberrant morphology and cellular composition C_LIO_LIMetabolic interventions tune stembryo end-state and can correct derailment of differentiation outcomes C_LI

developmental biology↗

bioRxiv2023

Delay of human early development via in vitro diapause

Many mammals can control the timing of gestation and birth by pausing embryonic development at the blastocyst stage. It is unknown whether the capacity to pause development is conserved, in general across mammals, and more specifically in humans. Activity of the growth regulating mTOR pathway governs developmental pausing in the mouse (1). Here we show a stage-specific capacity to delay the progression of human development via mTOR inhibition. In this context, human blastoids and pluripotent stem cells in naive and naive-like, but not primed, states can be induced to enter a dormant state, which is reversible at the functional and molecular level. Comparative analysis of mouse and human naive cells longitudinal response to mTORi revealed distinct temporal dynamics and metabolic requirements of dormancy in each species. Mouse and human blastocysts show similar tissue-specific patterns of mTOR pathway activity, suggesting that the mTOR pathway may be a conserved regulator of blastocyst development and timing in both species. Our results raise the possibility that the developmental timing of the human embryo may be controllable, with implications for reproductive therapies.

developmental biology↗

bioRxiv2022

Metabolic enhancement of mammalian developmental pausing

The quest to model and modulate embryonic development became a recent cornerstone of stem cell and developmental biology. Mammalian developmental timing is adjustable in vivo by preserving preimplantation embryos in a dormant state called diapause. Inhibition of the growth regulator mTOR (mTORi) pauses mouse development in vitro, yet constraints to pause duration are unrecognized. By comparing the response of embryonic and extraembryonic stem cells to mTORi-induced pausing, we identified lipid usage as a bottleneck to developmental pausing. Enhancing fatty acid oxidation (FAO) boosts embryo longevity, while blocking it reduces the pausing capacity. Genomic and metabolic analyses of single embryos point toward a deeper dormant state in FAO-enhanced pausing and reveal a link between lipid metabolism and embryo morphology. Our results lift a constraint on in vitro embryo survival and suggest that lipid metabolism may be a critical metabolic transition relevant for longevity and stem cell function across tissues. One-Sentence SummaryFacilitating fatty acid oxidation by carnitine supplementation enhances mTOR inhibition-mediated developmental pausing.

developmental biology↗

bioRxiv2021

Hypoxia induces a transcriptional early primitive streak signature in pluripotent cells enhancing spontaneous elongation and lineage representation in gastruloids

The cellular microenvironment together with intrinsic regulators shapes stem cell identity and differentiation capacity. Mammalian early embryos are exposed to hypoxia in vivo and appear to benefit from hypoxic culture in vitro. Yet, components of the hypoxia response and how their interplay impacts stem cell transcriptional networks and lineage choices remain poorly understood. Here we investigated the molecular effects of acute and prolonged hypoxia on distinct embryonic and extraembryonic stem cell types as well as the functional impact on differentiation potential. We find a temporal and cell type-specific transcriptional response including an early primitive streak signature in hypoxic embryonic stem (ES) cells. Using a 3D gastruloid differentiation model, we show that hypoxia-induced T expression enables symmetry breaking and axial elongation in the absence of exogenous WNT activation. Importantly, hypoxia also modulates T levels in conventional gastruloids and enhances representation of endodermal and neural markers. Mechanistically, we identify Hif1 as a central factor that mediates the transcriptional response to hypoxia in balance with epigenetic and metabolic rewiring. Our findings directly link the microenvironment to stem cell function and provide a rationale supportive of applying physiological conditions in models of embryo development

developmental biology↗

bioRxiv2019

Chd1 regulates repair of promoter-proximal DNA breaks to sustain hypertranscription in embryonic stem cells

Stem and progenitor cells undergo a global elevation of nascent transcription, or hypertranscription, during key developmental transitions involving rapid cell proliferation. The chromatin remodeler Chd1 binds to genes transcribed by RNA Polymerase (Pol) I and II and is required for hypertranscription in embryonic stem (ES) cells in vitro and the early post-implantation epiblast in vivo. Biochemically, Chd1 has been shown to facilitate transcription at least in part by removing nucleosomal barriers to elongation, but its mechanism of action in stem cells remains poorly understood. Here we report a novel role for Chd1 in the repair of promoter-proximal endogenous double-stranded DNA breaks (DSBs) in ES cells. An unbiased proteomics approach revealed that Chd1 interacts with several DNA repair factors including Atm, Parp1, Kap1 and Topoisomerase 2{beta}. We show that wild-type ES cells display high levels of phosphorylated H2A.X and Kap1 at chromatin, notably at rDNA in the nucleolus, in a Chd1-dependent manner. Loss of Chd1 leads to an extensive accumulation of DSBs at Chd1-bound Pol II-transcribed genes and rDNA. Genes prone to DNA breaks in Chd1 KO ES cells tend to be longer genes with GC-rich promoters, a more labile nucleosomal structure and roles in chromatin regulation, transcription and signaling. These results reveal a vulnerability of hypertranscribing stem cells to endogenous DNA breaks, with important implications for developmental and cancer biology.

molecular biology↗

bioRxiv2017

The transcriptionally permissive chromatin state of ES cells is acutely tuned totranslational output

A permissive chromatin environment coupled to hypertranscription is critical to drive the rapid proliferation of Embryonic Stem (ES) cells and peri-implantation embryos. We carried out a genome-wide screen to systematically dissect the regulation of the euchromatic state of ES cells. The results reveal that activity of cellular growth pathways, prominently protein synthesis, perpetuates the euchromatic state and hypertranscription of ES cells. Acute, mild inhibition of translation results in rapid depletion of euchromatic marks in ES cells and blastocysts, concurrent with delocalization of RNA polymerase II and reduction in nascent transcription. Remarkably, reduced translational output leads to rewiring of open chromatin within 3 hours, including decreased accessibility at a subset of active developmental enhancers and increased accessibility at histone genes and transposable elements. Using a proteome-scale analysis, we show that several euchromatin regulators are unstable proteins and thus continuously depend on a high translational output. We propose that this mechanistic interdependence of euchromatin, transcription and translation sets the pace of proliferation at peri-implantation and may be employed generally by stem/progenitor cells.

molecular biology↗