Biology subjects

Chlebowski, A.

Publications and source records attributed to Chlebowski, A..

5 recordsLinked to original sources

bioRxiv2023

Epiregulon: Inference of single-cell transcription factor activity to dissect mechanisms of lineage plasticity and drug response

Transcription factors (TFs) and transcriptional coregulators represent an emerging class of therapeutic targets in oncology. Gene regulatory networks (GRNs) can be used to evaluate pharmacological agents targeting these factors and to identify drivers of disease and drug resistance. However, GRN methods that rely solely on gene expression often fail to account for post-transcriptional modulation of TF function. We present Epiregulon, a method that constructs GRNs from single-cell ATAC-seq and RNA-seq data for accurate prediction of TF activity. This is achieved by considering the co-occurrence of TF expression and chromatin accessibility at TF binding sites in each cell. We leverage ChIP-seq data to extend inference to transcriptional coregulators lacking defined motifs or TF harboring neomorphic mutations. Epiregulon accurately predicted the effects of AR inhibition across various drug modalities including an AR antagonist and an AR degrader, delineated the mechanisms of a SMARCA4 degrader by identifying context-dependent interaction partners and prioritized known and novel drivers of lineage reprogramming and tumorigenesis. By mapping gene regulation across various cellular contexts, Epiregulon can accelerate the discovery of therapeutics targeting transcriptional regulators.

bioinformatics↗

bioRxiv2020

Cytoplasmic polyadenylation by TENT5A is required for proper bone formation

Osteoblasts orchestrate bone formation by secreting dense, highly cross-linked type I collagen and other proteins involved in osteogenesis. Mutations in Col11, Col12, or collagen biogenesis factors lead to the human genetic disease, osteogenesis imperfecta (OI). Herein, we show that the TENT5A gene, whose mutation is responsible for poorly characterized type XVIII OI, encodes an active cytoplasmic poly(A) polymerase regulating osteogenesis. TENT5A is induced during osteoblast differentiation and TENT5A KO osteoblasts are defective in mineralization. The TENT5A KO mouse recapitulates OI disease symptoms such as bone fragility and hypomineralization. Direct RNA sequencing revealed that TENT5A polyadenylates and increases expression of Col11 and Col12 RNAs, as well as those of other genes mutated in OI, resulting in lower production and improper folding of collagen chains. Thus, we have identified the specific pathomechanism of XVIII OI and report for the first time a biologically relevant post-transcriptional regulator of collagen production. We further postulate that TENT5A, possibly together with its paralogue TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization.

genetics↗

bioRxiv2020

High-throughput siRNA screening reveals functional interactions and redundancies of human processive exoribonucleases

Processive exoribonucleases, the executors of RNA decay, participate in multiple physical and functional interactions. Unlike physical ones, functional relationships have not been investigated in human cells. Here we have screened cells deficient in DIS3, XRN2, EXOSC10, DIS3L, and DIS3L2 with a custom siRNA library and determined their functional interactions with diverse pathways of RNA metabolism. We uncover a complex network of positive interactions that buffer alterations in RNA degradation. We reveal important reciprocal actions between RNA decay and transcription and explore alleviating interactions between RNA splicing and DIS3 mediated degradation. We also use a large scale library of genes associated with RNA metabolism to determine genetic interactions of nuclear DIS3 and cytoplasmic DIS3L, revealing their unique functions in RNA degradation and uncovering cooperation between the cytoplasmic degradation and nuclear processing of RNA. Finally, genome-wide siRNA screening of DIS3 reveals processes such as microtubule organization and regulation of telomerase activity that are also functionally associated with nuclear exosome-mediated RNA degradation.

systems biology↗

bioRxiv2018

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation

BackgroundPollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level.\n\nResultsHere, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis.\n\nConclusionsOur findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and embryo development.

plant biology↗

bioRxiv2017

Versatile approach for functional analysis of human proteins and efficient stable cell line generation using FLP-mediated recombination system

Deciphering a function of a given protein requires investigating various biological aspects. Usually, the protein of interest is expressed with a fusion tag that aids or allows subsequent analyses. Additionally, downregulation or inactivation of the studied gene enables functional studies. Development of the CRISPR/Cas9 methodology opened many possibilities but in many cases it is restricted to non-essential genes. It may also be time-consuming if a homozygote is needed. Recombinase-dependent gene integration methods, like the Flp-In system, are very good alternative. The system is widely used in different research areas, which calls for the existence of compatible vectors and efficient protocols that ensure straightforward DNA cloning and creation of stable cell lines. We have created and validated a robust series of 52 vectors for streamlined generation of stable mammalian cell lines using the FLP recombinase-based methodology. Using the sequence-independent DNA cloning method all constructs for a given coding-sequence can be made with just three universal PCR primers. The collection allows tetracycline-inducible expression of proteins with various tags suitable for protein localization, FRET, bimolecular fluorescence complementation (BiFC), protein dynamics studies (FRAP), co-immunoprecipitation, the RNA tethering assay and cell sorting. Some of the vectors contain a bidirectional promoter for concomitant expression of miRNA and mRNA, so that a gene can be silenced and its product replaced by a mutated miRNA-insensitive version. We demonstrate the efficacy of our vectors by creating stable cell lines with various tagged proteins (numatrin, fibrillarin, coilin, centrin, THOC5, PCNA). We have analysed transgene expression over time to provide a guideline for future experiments and compared the utility of commonly used inducers of tetracycline-responsive promoters. We determined the protein interaction network of the exoribonuclease XRN2 and examined the role of the protein in transcription termination by RNAseq analysis of cells devoid of its ribonucleolytic activity. In total we created more than 500 DNA constructs which proves high efficiency of our strategy.

molecular biology↗