Seq-Scope: repurposing Illumina sequencing flow cells for high-resolution spatial transcriptomics.

Spatial transcriptomics technologies aim to advance gene expression studies by profiling the entire transcriptome with intact spatial information from a single histological slide. However, the application of spatial transcriptomics is limited by low resolution, limited transcript coverage, complex procedures, poor scalability and high costs of initial setup and/or individual experiments. Seq-Scope repurposes the Illumina sequencing platform for high-resolution, high-content spatial transcriptome analysis, overcoming these limitations.

Seq-Scope Protocol: Repurposing Illumina Sequencing Flow Cells for High-Resolution Spatial Transcriptomics.

Spatial transcriptomics (ST) technologies represent a significant advance in gene expression studies, aiming to profile the entire transcriptome from a single histological slide. These techniques are designed to overcome the constraints faced by traditional methods such as immunostaining and RNA hybridization, which are capable of analyzing only a few target genes simultaneously. However, the application of ST in histopathological analysis is also limited by several factors, including low resolution, a limited range of genes, scalability issues, high cost, and the need for sophisticated equipment and complex methodologies.

Loss of synergistic transcriptional feedback loops drives diverse B-cell cancers.

Background: The most common B-cell cancers, chronic lymphocytic leukemia/lymphoma (CLL), follicular and diffuse large B-cell (FL, DLBCL) lymphomas, have distinct clinical courses, yet overlapping "cell-of-origin". Dynamic changes to the epigenome are essential regulators of B-cell differentiation. Therefore, we reasoned that these distinct cancers may be driven by shared mechanisms of disruption in transcriptional circuitry.

Activation of Mouse : Uncoupling RUNX1 Function from Its Cooperative Binding with ETS1.

T lineage commitment requires the coordination of key transcription factors (TFs) in multipotent progenitors that transition them away from other lineages and cement T cell identity. Two important TFs for the multipotent progenitors to T lineage transition are RUNX1 and ETS1, which bind cooperatively to composite sites throughout the genome, especially in regulatory elements for genes involved in T lymphopoiesis. Activation of the TCR β () locus in committed thymocytes is a critical process for continued development of these cells, and is mediated by an enhancer, Eβ, which harbors two RUNX-ETS composite sites.

cis-Regulatory Circuits Regulating NEK6 Kinase Overexpression in Transformed B Cells Are Super-Enhancer Independent.

Alterations in distal regulatory elements that control gene expression underlie many diseases, including cancer. Epigenomic analyses of normal and diseased cells have produced correlative predictions for connections between dysregulated enhancers and target genes involved in pathogenesis. However, with few exceptions, these predicted cis-regulatory circuits remain untested.

The Colonic Crypt Protects Stem Cells from Microbiota-Derived Metabolites.

In the mammalian intestine, crypts of Leiberkühn house intestinal epithelial stem/progenitor cells at their base. The mammalian intestine also harbors a diverse array of microbial metabolite compounds that potentially modulate stem/progenitor cell activity. Unbiased screening identified butyrate, a prominent bacterial metabolite, as a potent inhibitor of intestinal stem/progenitor proliferation at physiologic concentrations.

NKG2D-NKG2D Ligand Interaction Inhibits the Outgrowth of Naturally Arising Low-Grade B Cell Lymphoma In Vivo.

It is now clear that recognition of nascent tumors by the immune system is critical for survival of the host against cancer. During cancer immunoediting, the ability of the tumor to escape immune recognition is important for tumor development. The immune system recognizes tumors via the presence of classical Ags and also by conserved innate mechanisms.

Distinct Gene Regulatory Pathways for Human Innate versus Adaptive Lymphoid Cells.

Innate lymphoid cells (ILCs) serve as sentinels in mucosal tissues, sensing release of soluble inflammatory mediators, rapidly communicating danger via cytokine secretion, and functioning as guardians of tissue homeostasis. Although ILCs have been extensively studied in model organisms, little is known about these "first responders" in humans, especially their lineage and functional kinships to cytokine-secreting T helper (Th) cell counterparts. Here, we report gene regulatory circuitries for four human ILC-Th counterparts derived from mucosal environments, revealing that each ILC subset diverges as a distinct lineage from Th and circulating natural killer cells but shares circuitry devoted to functional polarization with their Th counterparts.

Short-Circuiting Gene Regulatory Networks: Origins of B Cell Lymphoma.

B cell lymphomas (BCLs) are characterized by widespread deregulation of gene expression compared with their normal B cell counterparts. Recent epigenomic studies defined cis-regulatory elements (REs) whose activities are altered in BCL to drive some of these pathogenic expression changes. During transformation, multiple mechanisms are employed to alter RE activities, including perturbations in the function of chromatin modifiers, which can lead to revision of the B cell epigenome.

Domain-Specific and Stage-Intrinsic Changes in Tcrb Conformation during Thymocyte Development.

Considerable cross-talk exists between mechanisms controlling genome architecture and gene expression. AgR loci are excellent models for these processes because they are regulated at both conformational and transcriptional levels to facilitate their assembly by V(D)J recombination. Upon commitment to the double-negative stage of T cell development, Tcrb adopts a compact conformation that promotes long-range recombination between Vβ gene segments (Trbvs) and their DβJβ targets.

Enhancer sequence variants and transcription-factor deregulation synergize to construct pathogenic regulatory circuits in B-cell lymphoma.

Most B-cell lymphomas arise in the germinal center (GC), where humoral immune responses evolve from potentially oncogenic cycles of mutation, proliferation, and clonal selection. Although lymphoma gene expression diverges significantly from GC B cells, underlying mechanisms that alter the activities of corresponding regulatory elements (REs) remain elusive. Here we define the complete pathogenic circuitry of human follicular lymphoma (FL), which activates or decommissions REs from normal GC B cells and commandeers enhancers from other lineages.

Lineage-specific compaction of Tcrb requires a chromatin barrier to protect the function of a long-range tethering element.

Gene regulation relies on dynamic changes in three-dimensional chromatin conformation, which are shaped by composite regulatory and architectural elements. However, mechanisms that govern such conformational switches within chromosomal domains remain unknown. We identify a novel mechanism by which cis-elements promote long-range interactions, inducing conformational changes critical for diversification of the TCRβ antigen receptor locus (Tcrb).

Unifying model for molecular determinants of the preselection Vβ repertoire.

The primary antigen receptor repertoire is sculpted by the process of V(D)J recombination, which must strike a balance between diversification and favoring gene segments with specialized functions. The precise determinants of how often gene segments are chosen to complete variable region coding exons remain elusive. We quantified Vβ use in the preselection Tcrb repertoire and report relative contributions of 13 distinct features that may shape their recombination efficiencies, including transcription, chromatin environment, spatial proximity to their DβJβ targets, and predicted quality of recombination signal sequences (RSSs).

Roles for common MLL/COMPASS subunits and the 19S proteasome in regulating CIITA pIV and MHC class II gene expression and promoter methylation.

Background: Studies indicate that the 19S proteasome contributes to chromatin reorganization, independent of the role the proteasome plays in protein degradation. We have previously shown that components of the 19S proteasome are crucial for regulating inducible histone activation events in mammalian cells. The 19S ATPase Sug1 binds to histone-remodeling enzymes, and in the absence of Sug1, a subset of activating epigenetic modifications including histone H3 acetylation, H3 lysine 4 trimethylation and H3 arginine 17 dimethylation are inhibited at cytokine-inducible major histocompatibilty complex (MHC)-II and class II transactivator (CIITA) promoters, implicating Sug1 in events required to initiate mammalian transcription.

The 19S ATPase S6a (S6'/TBP1) regulates the transcription initiation of class II transactivator.

Class II transactivator (CIITA) is the master regulator of the major histocompatibility class II transcription complex (MHC-II) and is critical for initiation of adaptive immune responses. We have previously demonstrated that the 19S proteasome ATPase Sug1 plays a significant role in regulating CIITA activity and MHC-II expression. We now show that an additional component of the 19S complex, the 19S ATPase S6a (S6'/Tat-binding protein 1), is crucial for regulating cytokine-inducible transcription of CIITA.

The 19S proteasome positively regulates histone methylation at cytokine inducible genes.

Studies indicate that the 19S proteasome functions in the epigenetic regulation of transcription. We have shown that as in yeast, components of the 19S proteasome are crucial for regulating inducible histone acetylation events in mammalian cells. The 19S ATPase Sug1 binds to histone acetyltransferases and to acetylated histone H3 and, in the absence of Sug1, histone H3 acetylation is dramatically decreased at mammalian promoters.

Regulation of acetylation at the major histocompatibility complex class II proximal promoter by the 19S proteasomal ATPase Sug1.

Recent studies have made evident the fact that the 19S regulatory component of the proteasome has functions that extend beyond degradation, particularly in the regulation of transcription. Although 19S ATPases facilitate chromatin remodeling and acetylation events in yeast (Saccharomyces cerevisiae), it is unclear if they play similar roles in mammalian cells. We have recently shown that the 19S ATPase Sug1 positively regulates the transcription of the critical inflammatory gene for major histocompatibility complex class II (MHC-II) by stabilizing enhanceosome assembly at the proximal promoter.