Biology subjects

Selinger, M.

Publications and source records attributed to Selinger, M..

4 recordsLinked to original sources

bioRxiv2025

A conserved transcriptional backbone and rewiring of gene-regulatory networks in activated human CD4⁺ T cells

CD4+ T cells are components of the adaptive immune system with a plethora of subtype-specific functions. In order to further dissect the activation and differentiation regulatory program(s) of individual CD4+ T cell subsets, we performed an in vitro activation and differentiation of human primary naive CD4+ T cells towards Th1, Th2, Th17 and Treg subtypes followed by the single-cell RNA-seq and ATAC-seq (multiome) analysis. Resulting multiome data were used for constructing the subtype-specific gene regulatory networks, which were next assessed for their differences/similarities among the subtypes. Surprisingly, a conserved set of 8 "backbone" transcription factors (TFs) was identified as highly central in all subtypes, however, with unique differentiation-driven rewiring tendency. Subtype-specific "driver" TFs were identified in the case of Th1-Th1_17-Th17 lineage (EOMES, HLF), naive Tregs (ESR1, DACH1), and memory Tregs (SOX13). Finally, we applied community detection algorithms to identify potential non-obvious groups of genes that regulate diverse molecular functions within the differentiated subtypes, linked to the backbone TFs. Our atlas aims at providing a high resolution understanding of the gene regulatory networks and their rewiring in human primary CD4+ T cells, upon activation and differentiation.

immunology↗

bioRxiv2024

A scalable CRISPR-Cas9 gene editing system facilitates CRISPR screens in the malaria parasite Plasmodium berghei

Many Plasmodium genes remain uncharacterised due to low genetic tractability. Previous large scale knockout screens have only been able to target about half of the genome in the more genetically tractable rodent malaria parasite Plasmodium berghei. To overcome this limitation, we have developed a scalable CRISPR system called PbHiT, which uses a single cloning step to generate targeting vectors with 100 bp homology arms physically linked to a guide RNA (gRNA) that effectively integrate into the target locus. We show that PbHiT coupled with gRNA sequencing robustly recapitulates known knockout mutant phenotypes in pooled transfections. Furthermore, we provide vector designs and sequences to target the entire P. berghei genome and scale-up vector production using a pooled ligation approach. This work presents for the first time a tool for high-throughput CRISPR screens in Plasmodium for studying the parasites biology at scale.

molecular biology↗

bioRxiv2024

CRISPR-MIP replaces PCR and reveals GC and oversampling bias in pooled CRISPR screens

Pooled CRISPR screening is a powerful tool for finding the most important genes related to a biological process of interest. The quality of the generated gene list is however influenced by a range of technical parameters, such as CRISPR (single guide) sgRNA target efficiency, and further innovations are still called for. One open problem is the precise estimation of sgRNA abundances, as required for the statistical analysis. We do so using molecular inversion probes (MIPs) combined with the use of unique molecular identifiers (UMIs), thus enabling deduplication and absolute counting of cells. We show that this is a viable approach that eliminates sequencing depth bias. Furthermore, we find that GC% bias affects PCR, calling for a reanalysis of published CRISPR screen data and sgRNA efficiency estimates. We propose our method as a new gold standard for sgRNA quantification, especially for genes that are not top ranked but still of broad interest.

molecular biology↗

bioRxiv2024

The CD4 T cell epigenetic JUNB+ state is associated with proliferation and exhaustion

Adoptive cell therapy (ACT) requires the in vitro expansion of T cells, a process where currently several variables are poorly controlled. As the state and quality of the cells affects the treatment outcome, the lack of insight is problematic. To get a better understanding of the production process and its degrees of freedom, we have generated a multiome CD4 T cell single-cell atlas. We find in particular a JUNB+ epigenetic state, orthogonal to traditional CD4 T cell subtype categorization. This new state is present but overlooked in previous transcriptomic CD4 T cell atlases. We characterize it to be highly proliferative, having condensed and actively remodeled chromatin, and correlating with exhaustion. JUNB+ subsets are also linked to memory formation, as well as circadian rhythm, connecting several important processes into one state. To dissect JUNB regulation, we also derived a gene regulatory network (GRN) and developed a new explainable machine learning package, Nando. We propose potential upstream drivers of JUNB, verified by other atlases and orthogonal data. We expect our results to be relevant for optimizing in vitro ACT conditions as well as modulation of gene expression through novel gene editing.

immunology↗