Biology subjects

Repele, A.

Publications and source records attributed to Repele, A..

5 recordsLinked to original sources

bioRxiv2025

The differentiation of myeloid progenitors is effected by cascading waves of coordinated gene expression that remodel cellular physiology in a characteristic sequence

The differentiation of hematopoietic progenitors into specialized types requires the transmittal of information from a few external or internal regulators to the thousands of genes that produce a cell types characteristic phenotypes. While the main signaling pathways, transcription factors, and the genes eliciting the terminal phenotypes are known, how information flows from a few regulators to thousands of genes to change the state of the cell remains to be fleshed out. To profile this information transfer process, we sampled the differentiation of the PUER myeloid cell line into macrophages and neutrophils at 29 time points over seven days. There is extensive transient regulation; the number of transcripts modulated in time is twice the number differentially expressed between endpoints. Differentiation is marked by two sharp transitions, at [~] 8h and [~] 80h, when transcriptomic state changes suddenly. We utilized non-negative matrix factorization to identify behaviors, characteristic temporal patterns of gene expression, and to classify transcripts by behavior. Only 10 distinct behaviors are sufficient to recapitulate the expression of [~]36,000 transcripts with high fidelity. Gene expression in most of the behaviors occurs in pulses of varying initiation times and durations. This implies that information transfer during differentiation occurs in cascading waves of gene expression culminating in the permanent turning on of certain genes after [~] 80h. Each behavior is enriched in specific biological processes, so that physiological remodeling proceeds in a characteristic order--signal transduction, translation and mRNA processing, metabolism, and, ultimately, myeloid phenotypic processes. The sharp transition at 8h corresponds to the completion of transcriptional and translational remodeling and the initiation of metabolic remodeling; the one at 80h corresponds to the elicitation of myeloid phenotypes. Our analysis shows that differentiation relies upon a series of transient, rapid, and complex gene regulatory events and highlights the importance of profiling it at a high temporal resolution. Author summaryThe maturation of hematopoietic progenitor cells into differentiated cell types occurs over a period of about a week. This process requires the progenitors to respond to external signals by changing the expression of thousands of genes to elicit the required phenotypes. We profiled how information is transferred from a few upstream regulators to thousands of genes by measuring genome-wide gene expression at high temporal resolution during white-blood cell differentiation. We show that the information transfer occurs in cascading waves, some as short as 8 hours and others lasting for 3 days, in which thousands of genes change expression coordinately. The physiological processes remodeled in each successive wave follow a characteristic order, starting with signal transduction pathways, followed by translation and mRNA processing, then metabolism, and culminating in the production of innate immunity phenotypes. Maturation is also punctuated with two sharp transitions, when genome-wide expression changes suddenly, associated with the initiation of metabolic remodeling and the production of terminal phenotypes. Our analysis shows that a complete description of differentiation requires the characterization of transient changes and not just those observable at the endpoints.

developmental biology↗

bioRxiv2024

Monoclonal antibodies derived from B cells in subjects with cystic fibrosis reduce Pseudomonas aeruginosa burden in mice

Pseudomonas aeruginosa (PA) is an opportunistic, frequently multidrug-resistant pathogen that can cause severe infections in hospitalized patients. Antibodies against the PA virulence factor, PcrV, protect from death and disease in a variety of animal models. However, clinical trials of PcrV-binding antibody-based products have thus far failed to demonstrate benefit. Prior candidates were derivations of antibodies identified using protein-immunized animal systems and required extensive engineering to optimize binding and/or reduce immunogenicity. Of note, PA infections are common in people with cystic fibrosis (pwCF), who are generally believed to mount normal adaptive immune responses. Here we utilized a tetramer reagent to detect and isolate PcrV-specific B cells in pwCF and, via single-cell sorting and paired-chain sequencing, identified the B cell receptor (BCR) variable region sequences that confer PcrV-specificity. We derived multiple high affinity anti-PcrV monoclonal antibodies (mAbs) from PcrV-specific B cells across 3 donors, including mAbs that exhibit potent anti-PA activity in a murine pneumonia model. This robust strategy for mAb discovery expands what is known about PA-specific B cells in pwCF and yields novel mAbs with potential for future clinical use.

immunology↗

bioRxiv2023

Binding-site resolution structure and dynamics of Cebpa regulatory regions during macrophage-neutrophil differentiation

The upregulation of gene expression by enhancers depends upon the interplay between the binding of sequence-specific transcription factors (TFs) and DNA accessibility. DNA accessibility is thought to limit the ability of TFs to bind to their sites, while TFs can increase accessibility to recruit additional factors that upregulate gene expression. Given this interplay, the causative regulatory events underlying the modulation of gene expression during cellular differentiation remain unknown for the vast majority of genes. We investigated the binding-site resolution dynamics of DNA accessibility and the expression dynamics of the enhancers of an important neutrophil gene, Cebpa, during macrophage-neutrophil differentiation. Reporter genes were integrated in a site-specific manner in PUER cells, which are progenitors that can be differentiated into neutrophils or macrophages in vitro by activating the pan-leukocyte TF PU.1. Time series data show that two enhancers upregulate reporter expression during the first 48 hours of neutrophil differentiation. Surprisingly, there is little or no increase in the total accessibility, measured by ATAC-Seq, of the enhancers during the same time period. Conversely, total accessibility peaks 96 hrs after PU.1 activation--consistent with its role as a pioneer--but the enhancers do not upregulate gene expression. Combining deeply sequenced ATAC-Seq data with a new bias-correction method allowed the profiling of acces-sibility at single-nucleotide resolution and revealed protected regions in the enhancers that match all previously characterized TF binding sites and ChIP-Seq data. Although the accessibility of most positions does not change during early differentiation, that of positions neighboring TF binding sites, an indicator of TF occupancy, did in-crease significantly. The localized accessibility changes are limited to nucleotides neighboring C/EBP-family TF binding sites, showing that the upregulation of enhancer activity during early differentiation is driven by C/EBP-family TF binding. These results show that increasing the total accessibility of enhancers is not sufficient for upregulating their activity and other events such as TF binding are necessary for upregulation. Also, TF binding can cause upregulation without a perceptible increase in total accessibility. Finally, this study demonstrates the feasibility of comprehensively mapping individual TF binding sites as footprints using high coverage ATAC-Seq and inferring the sequence of events in gene regulation by combining with time-series gene expression data.

genomics↗

bioRxiv2019

Robust Normalization of Luciferase Reporter Data

Transient Luciferase reporter assays are widely used in the study of gene regulation and intracellular cell signaling. In order to control for sample-to-sample variation in luminescence arising from variability in transfection efficiency and other sources, an internal control reporter is co-transfected with the experimental reporter. The luminescence of the experimental reporter is normalized against the control by taking the ratio of the two. Here we show that this method of normalization, \"ratiometric\", performs poorly when the transfection efficiency is low and leads to biased estimates of relative activity. We propose an alternative methodology based on linear regression that is much better suited for the normalization of reporter data, especially when transfection efficiency is low. We compare the ratiometric method against three regression methods on both simulated and empirical data. Our results suggest that robust errors-in-variables (REIV) regression performs the best in normalizing Luciferase reporter data. We have made the R code for Luciferase data normalization using REIV available on GitHub.

molecular biology↗

bioRxiv2018

The regulatory control of Cebpa enhancers and silencers in the myeloid and red-blood cell lineages

During development, cell identity is determined by lineage-specific transcriptional programs en-coded in the cis-regulatory DNA sequence of developmental genes. The sequence-level regulatory logic--identities of bound transcription factors (TFs), TF binding sites, and TF regulatory roles--of most developmental cis-regulatory modules (CRMs) is yet to be determined. We had previously developed an approach for inferring regulatory logic de novo by training sequence-based thermo-dynamic models on comprehensive reporter activity and gene expression datasets and applied it to Cebpa, an important hematopoietic gene. Here, we experimentally test thermodynamic models to decode the cis-regulatory logic of 4 enhancers and 3 silencers neighboring Cebpa at the resolution of individual binding sites. Cebpa is expressed at high and intermediate levels in neutrophils and macrophages respectively and downregulated in non-myeloid lineages. We tested the binding sites and functional roles of inferred TFs by designing and constructing mutated CRMs and comparing theoretical predictions of their activity against empirical measurements. Reporter activity was measured in PUER cells, which can be induced to differentiate into macrophages or neutrophils. All four enhancers were found to be simultaneously active in undifferentiated PUER cells and early-stage macrophages and neutrophils, and activated by combinations of PU.1, C/EBP family TFs, Egr1, and Gfi1. We show that silencers repress the activity of the proximal promoter in a dominant manner in G1ME cells, which are derived from the red-blood cell lineage. Dominant repression in G1ME cells can be traced to binding sites for GATA and Myb, a motif shared by all of the silencers. Finally, we demonstrate that GATA and Myb act redundantly to silence the proximal promoter. The result that silencers quench the promoter selectively in non-myeloid cells indicates that dominant repression is a novel mechanism for resolving hematopoietic lineages. Furthermore, Cebpa has a fail-safe cis-regulatory architecture, featuring several functionally similar CRMs, each of which contains redundant binding sites for multiple TFs. Lastly, by experimentally demonstrating the predictive ability of our sequence-based thermodynamic models, this work highlights the utility of this computational approach for decoding the logic of mammalian gene regulation.

developmental biology↗