PAXIP1 and STAG2 converge to maintain 3D genome architecture and facilitate promoter/enhancer contacts to enable stress hormone-dependent transcription.

How steroid hormone receptors (SHRs) regulate transcriptional activity remains partly understood. Upon activation, SHRs bind the genome together with a co-regulator repertoire, crucial to induce gene expression. However, it remains unknown which components of the SHR-recruited co-regulator complex are essential to drive transcription following hormonal stimuli. Through a FACS-based genome-wide CRISPR screen, we functionally dissected the Glucocorticoid Receptor (GR) complex. We describe a functional cross-talk between PAXIP1 and the cohesin subunit STAG2, critical for regulation of gene expression by GR. Without altering the GR cistrome, PAXIP1 and STAG2 depletion alter the GR transcriptome, by impairing the recruitment of 3D-genome organization proteins to the GR complex. Importantly, we demonstrate that PAXIP1 is required for stability of cohesin on chromatin, its localization to GR-occupied sites, and maintenance of enhancer-promoter interactions. In lung cancer, where GR acts as tumor suppressor, PAXIP1/STAG2 loss enhances GR-mediated tumor suppressor activity by modifying local chromatin interactions. All together, we introduce PAXIP1 and STAG2 as novel co-regulators of GR, required to maintain 3D-genome architecture and drive the GR transcriptional programme following hormonal stimuli.

Multiparametric Analyses of Hepatocellular Carcinoma Somatic Mouse Models and Their Associated Tumor Microenvironment.

The rising incidence and increasing mortality of hepatocellular carcinoma (HCC), combined with its high tumor heterogeneity, lack of druggable targets, and tendency to develop resistance to chemotherapeutics, make the development of better models for this cancer an urgent challenge. To better mimic the high diversity within the HCC genetic landscape, versatile somatic murine models have recently been developed using the hydrodynamic tail vein injection (HDTVi) system. These represent novel in vivo tools to interrogate HCC phenotype and response to therapy, and importantly, allow further analyses of the associated tumor microenvironment (TME) shaped by distinct genetic backgrounds. Here, we describe several optimized protocols to generate, collect, and experimentally utilize various samples obtained from HCC somatic mouse models generated by HDTVi. More specifically, we focus on techniques relevant to ex vivo analyses of the complex liver TME using multiparameter flow cytometric analyses of over 21 markers, immunohistochemistry, immunofluorescence, and histochemistry. We describe the transcriptional assessment of whole tissue, or of isolated immune subsets by flow-cytometry-based cell sorting, and other protein-oriented analyses. Together, these streamlined protocols allow the optimal use of each HCC murine model of interest and will assist researchers in deciphering the relations between cancer cell genetics and systemic and local changes in immune cell landscapes in the context of HCC progression. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of HCC mouse models by hydrodynamic tail vein injection Basic Protocol 2: Assessment of HCC tumor progression by magnetic resonance imaging Basic Protocol 3: Mouse sacrifice and sample collection in HCC mouse models Support Protocol 1: Preparation of serum or plasma from blood Basic Protocol 4: Single-cell preparation and HCC immune landscape phenotyping by flow cytometry Alternate Protocol 1: Flow cytometric analysis of circulating immune cells Support Protocol 2: Generation, maintenance, and characterization of HCC cell lines Support Protocol 3: Fluorescence-activated cell sorting of liver single-cell preparation Basic Protocol 5: Preparation and immunohistochemical analysis of tumor tissues from HCC-bearing liver Alternate Protocol 2: Preparation and analyses for immunofluorescence staining of HCC-bearing liver Support Protocol 4: Liver-specific phenotypic analyses of liver sections Support Protocol 5: Immunohistochemical quantification in liver sections Basic Protocol 6: Preparation of snap-frozen tumor tissue from extracted liver and transcriptional analyses of bulk tumor or sorted cells Alternate Protocol 3: Protein analyses from HCC samples and serum or plasma.

Flagellin/TLR5 Stimulate Myeloid Progenitors to Enter Lung Tissue and to Locally Differentiate Into Macrophages.

Toll-like receptor 5 (TLR5) is the receptor of bacterial Flagellin. Reportedly, TLR5 engagement helps to combat infections, especially at mucosal sites, by evoking responses from epithelial cells and immune cells. Here we report that TLR5 is expressed on a previously defined bipotent progenitor of macrophages (MΦs) and osteoclasts (OCs) that resides in the mouse bone marrow (BM) and circulates at low frequency in the blood. In vitro, Flagellin promoted the generation of MΦs, but not OCs from this progenitor. In vivo, MΦ/OC progenitors were recruited from the blood into the lung upon intranasal inoculation of Flagellin, where they rapidly differentiated into MΦs. Recruitment of the MΦ/OC progenitors into the lung was likely promoted by the CCL2/CCR2 axis, since the progenitors expressed CCR2 and type 2 alveolar epithelial cells (AECs) produced CCL2 upon stimulation by Flagellin. Moreover, CCR2 blockade reduced migration of the MΦ/OC progenitors toward lung lavage fluid (LLF) from Flagellin-inoculated mice. Our study points to a novel role of the Flagellin/TLR5 axis in recruiting circulating MΦ/OC progenitors into infected tissue and stimulating these progenitors to locally differentiate into MΦs. The progenitor pathway to produce MΦs may act, next to monocyte recruitment, to fortify host protection against bacterial infection at mucosal sites.

The Ig heavy chain protein but not its message controls early B cell development.

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.

Plasticity of Lgr5-Negative Cancer Cells Drives Metastasis in Colorectal Cancer.

Colorectal cancer stem cells (CSCs) express Lgr5 and display extensive stem cell-like multipotency and self-renewal and are thought to seed metastatic disease. Here, we used a mouse model of colorectal cancer (CRC) and human tumor xenografts to investigate the cell of origin of metastases. We found that most disseminated CRC cells in circulation were Lgr5- and formed distant metastases in which Lgr5+ CSCs appeared. This plasticity occurred independently of stemness-inducing microenvironmental factors and was indispensable for outgrowth, but not establishment, of metastases. Together, these findings show that most colorectal cancer metastases are seeded by Lgr5- cells, which display intrinsic capacity to become CSCs in a niche-independent manner and can restore epithelial hierarchies in metastatic tumors.

Flow cytometry-based isolation of tumor-associated regulatory T cells and assessment of their suppressive potential.

Regulatory T cells (Tregs) play a major role in establishing an immunosuppressive tumor microenvironment. In order to fully uncover their role and molecular regulation in tumor-bearing hosts, it is critical to combine phenotypical characterization with functional analyses. A standard method to determine the suppressive potential of Tregs is with an in vitro suppression assay, in which the impact of freshly isolated Tregs on T cell proliferation is assessed. The assay requires the isolation of substantial numbers of Tregs from tissues and tumors, which can be challenging due to low yield or cell damage during sample preparation. In this chapter, we discuss a flexible suppression assay which can be used to assess the suppressive potential of low numbers of murine Tregs, directly isolated from tumors. We describe methods for tissue preparation, flow cytometry-based sorting of Tregs and optimal conditions to perform a suppression assay, to obtain reliable and reproducible results.