Biology subjects

Bunina, D.

Publications and source records attributed to Bunina, D..

7 recordsLinked to original sources

bioRxiv2025

High-throughput single-cell CRISPRi screens stratify neurodevelopmental functions of schizophrenia-associated genes

Schizophrenia is a complex neuropsychiatric disorder with strong genetic underpinnings, yet the molecular mechanisms linking genetic risk to disrupted brain development remain poorly understood. Transcription factors (TFs) and chromatin regulators (CRs) are increasingly implicated in neuropsychiatric disorders, where their dysregulation may disrupt neurodevelopmental programs. Despite this, systematic functional interrogation in human models has been limited. Here, we combine pooled CRISPR interference (CRISPRi) screens with high- throughput single-cell multiomic profiling in hiPSC-derived neural progenitors and neurons to functionally assess 65 schizophrenia-associated genes. Based on public datasets and literature review, we selected 55 TFs and CRs, along with ten additional risk genes whose loss-of-function has been linked to schizophrenia. Our single-cell CRISPRi readouts revealed that perturbations in TFs and CRs converge on disrupting neurodevelopmental timing. CRISPRi of several factors delayed neural differentiation, whereas others, such as the knockdown of MCRS1, drove precocious neural commitment. Validation screens combined with cell cycle and metabolic indicators confirmed the differentiation-restricting or -promoting roles of these TFs and CRs. Multimodal trajectory analysis uncovered discrete transcriptional and epigenomic states representing delayed and accelerated neurodevelopment, enriched for schizophrenia GWAS loci and disease-relevant pathways. Gene regulatory network (GRN) inference identified TCF4 and ZEB1 as critical mediators opposing the neural differentiation trajectory. Functional overexpression of these TFs followed by chromatin profiling demonstrated that TCF4 restrains, while ZEB1 promotes, neural differentiation in a stage-specific and competitive manner. Furthermore, we show that MCRS1 represses ZEB1 expression, positioning MCRS1 as a key brake on premature neurodevelopment. Together, our study establishes a scalable framework that integrates genetic perturbation, single- cell multiomics, and GRN modeling to functionally annotate disease-linked genes. We reveal convergent regulatory axes that underlie altered neurodevelopmental timing in schizophrenia, offering mechanistic insights into how chromatin misregulation contributes to disease pathogenesis.

neuroscience↗

bioRxiv2024

Distinct roles for MNK1 and MNK2 in social and cognitive behavior through kinase-specific regulation of the synaptic proteome and phosphoproteome

Local mRNA translation is required for adaptive changes in the synaptic proteome. The mitogen-activated protein kinase (MAPK) interacting protein kinases 1 and 2 (MNK1 and MNK2) have emerged as key regulators of neuronal translation, primarily through phosphorylation of the eukaryotic initiation factor 4E (eIF4E). The therapeutic benefits of targeting the MNKs are being investigated for nervous system conditions and disorders that affect translation, including autism, pain, and cancer. However, it is still unclear if MNK1 and MNK2 regulate distinct aspects of neuronal translation and how the activity of each kinase is associated with the synaptic and behavioral features linked to MNK signaling. To examine the individual neurobiological functions of each kinase, we used knockout mice lacking either MNK1 or MNK2. We found that the knockout of MNK1 and MNK2 leads to different social and cognitive behavioral profiles and distinct alterations of the cortical synaptic proteome, transcriptome, and phosphoproteome. Loss of MNK1 was associated with an increase in ribosomal protein expression, whereas deletion of MNK2 decreased the expression and phosphorylation of synaptic proteins. Together, our findings provide evidence for a high degree of functional specialization of the MNKs in synaptic compartments and suggest that pharmacological inhibition of individual MNK may provide more specific targets for neurological disorders.

neuroscience↗

bioRxiv2024

Glycolysis-Wnt signaling axis tunes developmental timing of embryo segmentation

The question of how metabolism impacts development is seeing a renaissance [1, 2]. How metabolism exerts instructive signaling functions is one of the central issues that need to be resolved. We tackled this question in the context of mouse embryonic axis segmentation. Previous studies have shown that changes in central carbon metabolism impact Wnt signaling [3-6] and the period of the segmentation clock [7], which controls the timing of axis segmentation. Here, we reveal that glycolysis tunes the segmentation clock period in an anti-correlated manner: higher glycolytic flux slows down the clock, and vice versa. Transcriptome and gene regulatory network analyses identified Wnt signaling and specifically the transcription factor Tcf7l2, previously associated with increased risk for diabetes [8, 9], as potential mechanisms underlying flux-dependent control of the clock period. Critically, we show that deletion of the Wnt antagonist Dkk1 rescued the slow segmentation clock phenotype caused by increased glycolysis, demonstrating that glycolysis instructs Wnt signaling to control the clock period. In addition, we demonstrate metabolic entrainment of the segmentation clock: periodic changes in the levels of glucose or glycolytic sentinel metabolite fructose 1,6-bisphosphate (FBP) synchronize signaling oscillations. Notably, periodic FBP pulses first entrained Wnt signaling oscillations and subsequently Notch signaling oscillations. We hence conclude that metabolic entrainment has an immediate, specific effect on Wnt signaling. Combined, our work identifies a glycolysis-FBP-Wnt signaling axis that tunes developmental timing, highlighting the instructive signaling role of metabolism in embryonic development.

developmental biology↗

bioRxiv2023

Histone H3.3 lysine 9 and 27 control repressive chromatin states at cryptic cis-regulatory elements and bivalent promoters in mouse embryonic stem cells

Histone modifications are associated with distinct transcriptional states, but it is unclear whether they instruct gene expression. To investigate this, we mutated histone H3.3 K9 and K27 residues in mouse embryonic stem cells (mESCs). Here, we find that H3.3K9 is essential for controlling specific distal intergenic regions and for proper H3K27me3 deposition at promoters. The H3.3K9A mutation resulted in decreased H3K9me3 at regions encompassing endogenous retroviruses and induced a gain of H3K27ac and nascent transcription. These changes in the chromatin environment unleashed cryptic enhancers, resulting in the activation of distinctive transcriptional programs and culminating in protein expression normally restricted to specialized immune cell types. The H3.3K27A mutant disrupted deposition and spreading of the repressive H3K27me3 mark, particularly impacting bivalent genes with higher basal level of H3.3 at promoters. Therefore, H3.3K9 and K27 crucially orchestrate repressive chromatin states at cis-regulatory elements and bivalent promoters, respectively, and instruct proper transcription in mESCs.

genomics↗

bioRxiv2021

GRaNIE and GRaNPA: Inference and evaluation of enhancer-mediated gene regulatory networks applied to study macrophages

Among the biggest challenges in the post-GWAS (genome-wide association studies) era is the interpretation of disease-associated genetic variants in non-coding genomic regions. Enhancers have emerged as key players in mediating the effect of genetic variants on complex traits and diseases. Their activity is regulated by a combination of transcription factors (TFs), epigenetic changes and genetic variants. Several approaches exist to link enhancers to their target genes, and others that infer TF-gene connections. However, we currently lack a framework that systematically integrates enhancers into TF-gene regulatory networks. Furthermore, we lack an unbiased way of assessing whether inferred regulatory interactions are biologically meaningful. Here we present two methods, implemented as user-friendly R packages: GRaNIE (Gene Regulatory Network Inference including Enhancers) for building enhancer-based gene regulatory networks (eGRNs) and GRaNPA (Gene Regulatory Network Performance Analysis) for evaluating GRNs. GRaNIE jointly infers TF-enhancer, enhancer-gene and TF-gene interactions by integrating open chromatin data such as ATAC-Seq or H3K27ac with RNA-seq across a set of samples (e.g. individuals), and optionally also Hi-C data. GRaNPA is a general framework for evaluating the biological relevance of TF-gene GRNs by assessing their performance for predicting cell-type specific differential expression. We demonstrate the power of our tool-suite by investigating gene regulatory mechanisms in macrophages that underlie their response to infection and cancer, their involvement in common genetic diseases including autoimmune diseases, and identify the TF PURA as putative regulator of pro-inflammatory macrophage polarisation. Availability- GRaNIE: https://bioconductor.org/packages/release/bioc/html/GRaNIE.html - GRaNPA: https://git.embl.de/grp-zaugg/GRaNPA Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

genomics↗

bioRxiv2021

Enhancer-priming in ageing human bone marrow mesenchymal stromal cells contributes to immune traits

Bone marrow mesenchymal stromal cells (BMSCs) can differentiate into adipocytes and osteoblasts, and are important regulators of the haematopoietic system. Ageing associates with an increased ratio of bone marrow adipocytes to osteoblasts and immune dysregulation. Here, we carried out an integrative multiomics analysis of ATAC-Seq, RNA-Seq and proteomics data from primary human BMSCs in a healthy cohort age between 20 - 60. We identified age-sensitive elements uniquely affecting each molecular level where transcription is mostly spared, and characterised the underlying biological pathways, revealing the interplay of age-related gene expression mechanism changes spanning multiple gene regulatory layers. Through data integration with enhancer-mediated gene regulatory network analysis, we discovered that enhancers and transcription factors influence cell differentiation potential in the ageing BMSCs. By combining our results with genome-wide association study data, we found that age-specific changes could contribute to common traits related to BMSC-derived tissues such as bone and adipose tissue, and to immune-related traits on a systemic level such as asthma. We demonstrate here that a multiomics approach is crucial for unravelling complex information, providing new insights on how ageing contributes to bone marrow- and immune-related disorders.

systems biology↗

bioRxiv2021

Pathological LSD1 mutations cause HDAC-mediated aberrant gene repression during early cell differentiation

Lysine-specific demethylase 1 (LSD1/KDM1A) demethylates both histone and non-histone substrates, recruits repressive chromatin complexes, and is increased in cancers. De novo LSD1 mutations impairing protein function lead to a rare developmental disorder, but the molecular details of the pathology remains unclear. Using patient-derived fibroblasts, reprogrammed pluripotent stem cells, and differentiated cells, we found over 4000 differentially expressed genes and 68 transcription factors (TFs) whose motif accessibilities changed upon LSD1 mutation. An enhancer-mediated gene regulatory network approach identified transcriptional repressors with impaired activity in fibroblast and stem cells, leading to erroneous activation of their target genes. We also revealed overall decreases in TF target gene expression during early lineage differentiation of LSD1 mutant stem cells, likely caused by increased activity of repressive histone deacetylases (HDACs), co-factors of LSD1. Indeed, an HDACs inhibitor restored changes in gene expression including downregulation phenotype. Our findings characterize the molecular pathogenesis of LSD1 mutations and suggest potential therapeutic strategies for the developmental disorder and cancers caused by LSD1 dysregulations.

genomics↗