Dynamic recruitment of Ets1 to both nucleosome-occupied and -depleted enhancer regions mediates a transcriptional program switch during early T-cell differentiation.

Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4(-)/CD8(-) double negative (DN) to CD4(+)/CD8(+) double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1(-/-) thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity.

High-Quality Brain and Bone Marrow Nuclei Preparation for Single Nuclei Multiome Assays.

Single-cell analysis has become the approach of choice for unraveling the complexity of biological processes that require assessing the variability of individual cellular responses to treatment or infection with single-cell resolution. Many techniques for single-cell molecular profiling have been developed over the past 10 years, and several dedicated technologies have been commercialized. The 10X Genomics droplet-based single-cell profiling is a widespread technology that offers ready-to-use reagents for transcriptomic and multi-omic single-cell profiling. The technology includes workflows for single-cell and single-nuclei RNA sequencing (scRNA-Seq and snRNA-Seq, respectively), scATAC-Seq, single-cell immune profiling (BCR/TCR sequencing), and multiome. The latter combines transcriptional (scRNA-Seq) and epigenetic information (scATAC-Seq) coming from the same cell. The quality (viability, integrity, purity) of single-cell or single-nuclei suspensions isolated from tissues and analyzed by any of these approaches is critical for generating high-quality data. Therefore, the sample preparation protocols should be adapted to the particularities of each biological tissue and ensure the generation of high-quality cell and nuclei suspensions. This article describes two protocols for preparing brain and bone marrow samples for the downstream multiome 10X Genomics pipeline. The protocols are performed stepwise and cover tissue dissociation, cell sorting, nuclei isolation, and quality control of prepared nuclei suspension that is used as starting material for cell partitioning and barcoding, library preparation, and sequencing. These standardized protocols produce high-quality nuclei libraries and robust and reliable data.

Altered Immune Phenotypes and HLA-DQB1 Gene Variation in Multiple Sclerosis Patients Failing Interferon ß Treatment.

Background: Interferon beta (IFNß) has been prescribed as a first-line disease-modifying therapy for relapsing-remitting multiple sclerosis (RRMS) for nearly three decades. However, there is still a lack of treatment response markers that correlate with the clinical outcome of patients. Aim: To determine a combination of cellular and molecular blood signatures associated with the efficacy of IFNß treatment using an integrated approach. Methods: The immune status of 40 RRMS patients, 15 of whom were untreated and 25 that received IFNß1a treatment (15 responders, 10 non-responders), was investigated by phenotyping regulatory CD4+ T cells and naïve/memory T cell subsets, by measurement of circulating IFNα/ß proteins with digital ELISA (Simoa) and analysis of ~600 immune related genes including 159 interferon-stimulated genes (ISGs) with the Nanostring technology. The potential impact of HLA class II gene variation in treatment responsiveness was investigated by genotyping HLA-DRB1, -DRB3,4,5, -DQA1, and -DQB1, using as a control population the Milieu Interieur cohort of 1,000 French healthy donors. Results: Clinical responders and non-responders displayed similar plasma levels of IFNß and similar ISG profiles. However, non-responders mainly differed from other subject groups with reduced circulating naïve regulatory T cells, enhanced terminally differentiated effector memory CD4+ TEMRA cells, and altered expression of at least six genes with immunoregulatory function. Moreover, non-responders were enriched for HLA-DQB1 genotypes encoding DQ8 and DQ2 serotypes. Interestingly, these two serotypes are associated with type 1 diabetes and celiac disease. Overall, the immune signatures of non-responders suggest an active disease that is resistant to therapeutic IFNß, and in which CD4+ T cells, likely restricted by DQ8 and/or DQ2, exert enhanced autoreactive and bystander inflammatory activities.