Summary 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. During homeostasis, differentiated colonocytes metabolized butyrate likely preventing it from reaching proliferating epithelial stem/progenitor cells within the crypt. Exposure of stem/progenitor cells in vivo to butyrate through either mucosal injury or application to a naturally crypt-less host organism led to inhibition of proliferation and delayed wound repair. The mechanism of butyrate action depended on the transcription factor Foxo3. Our findings indicate that mammalian crypt architecture protects stem/progenitor cell proliferation in part through a metabolic barrier formed by differentiated colonocytes that consume butyrate, and stimulate future studies on the interplay of host anatomy and microbiome metabolism.

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SUMMARY 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 studied extensively 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. Super-enhancers demarcate cohorts of cell identity genes in each lineage, uncovering new modes of regulation for signature cytokines, new molecules that likely impart important functions to ILCs, and potential mechanisms for autoimmune disease SNP associations within ILC–Th subsets.

Summary 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. Moreover, independent sets of transcription factors, whose expression was deregulated in FL, targeted commandeered versus decommissioned REs. Our approach revealed two distinct subtypes of low-grade FL, whose pathogenic circuitries resembled GC-B or activated B cells. FL-altered enhancers also were enriched for sequence variants, including somatic mutations, which disrupt transcription factor binding and expression of circuit-linked genes. Thus, the pathogenic regulatory circuitry of FL reveals distinct genetic and epigenetic etiologies for GC-B transformation.

Majumder et al. explore the large-scale looping architecture of the Tcrb locus early in murine thymocyte development during the generation of TCRβ diversity. They dissect novel DNA regulatory elements controlling V to D-J recombination and identify within an insulator region a distally located CTCF-containing element functioning as a tether, which facilitates looping of distal Vβ to Dβ-Jβ regions and promotes locus contraction. A second CTCF-containing element, proximal to the Dβ-Jβ region, acts as a boundary, preventing the spread of active chromatin associated with Dβ-Jβ regions. Removal of the proximal boundary element impairs the locus contraction capabilities of the tethering element.

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). We show that, in contrast to functional Vβ gene segments, all pseudo-Vβ segments are sequestered in transcriptionally silent chromatin, which effectively suppresses wasteful recombination. Importantly, computational analyses provide a unifying model, revealing a minimum set of five parameters that are predictive of Vβ use, dominated by chromatin modifications associated with transcription, but largely independent of precise spatial proximity to DβJβ clusters. This learned model-building strategy may be useful in predicting the relative contributions of epigenetic, spatial, and RSS features in shaping preselection V repertoires at other antigen receptor loci. Ultimately, such models may also predict how designed or naturally occurring alterations of these loci perturb the preselection use of variable gene segments.lymphocytes | T-cell receptor | gene regulation G ene activity is regulated at multiple levels to coordinate expression during development. At a most basic level, the collection of cis-acting elements for a genetic locus recruits transcription factors that alter its chromatin environment to either induce or repress gene activity. Emerging studies indicate that the 3D conformation of a locus also plays an important role in the regulation of its composite genes (1). At most genes, many levels of control are integrated to achieve the requisite gene expression state. For example, transcriptional promoters interact with their cognate enhancers over considerable distances in the linear genome to generate "hubs" where the two cis elements are in spatial proximity (1, 2).All of these regulatory strategies are used to generate functional Ig (Ig) and T-cell receptor (Tcr) genes during lymphocyte development (3). Each antigen receptor (AgR) locus is composed of multiple variable (V), joining (J), and sometimes diversity (D) gene segments that are assembled by the process of V (D)J recombination, creating a potential variable region exon (4). Recombination is mediated by the RAG-1/2 enzymatic complex, which is expressed in all developing lymphocytes and recognizes semiconserved recombination signal sequences (RSSs) flanking all AgR gene segments (5). On selection of two compatible gene segments by RAG-1/2, recombination proceeds via a DNA break/repair mechanism, ultimately fusing the two selected segments (4, 5).The assembly of AgR genes is strictly regulated despite a common collection of ge...

Quality control (QC) is a critical step in RNA sequencing (RNA-seq). Yet, it is often ignored or conducted on a limited basis. Here, we present a multi-perspective strategy for QC of RNA-seq experiments. The QC of RNA-seq can be divided into four related stages: (1) RNA quality, (2) raw read data (FASTQ), (3) alignment and (4) gene expression. We illustrate the importance of conducting QC at each stage of an RNA-seq experiment and demonstrate our recommended RNA-seq QC strategy. Furthermore, we discuss the major and often neglected quality issues associated with the three major types of RNA-seq: mRNA, total RNA and small RNA. This RNA-seq QC overview provides comprehensive guidance for researchers who conduct RNA-seq experiments.

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. We now show that Sug1 is crucial for regulating histone H3 acetylation at the MHC-II proximal promoter. Sug1 binds to acetylated histone H3 and, in the absence of Sug1, histone H3 acetylation is dramatically decreased at the proximal promoter, with a preferential loss of acetylation at H3 lysine 18. Sug1 also binds to the MHC-II histone acetyltransferase CREB-binding protein (CBP) and is critical for the recruitment of CBP to the MHC-II proximal promoter. Our current study strongly implicates the 19S ATPase Sug1 in modifying histones to initiate MHC-II transcription and provides novel insights into the role of the proteasome in the regulation of mammalian transcription.Major histocompatibility complex class II (MHC-II) molecules are cell surface glycoproteins which bind and present processed antigenic peptides to CD4ϩ T lymphocytes to initiate immune system protection against invading pathogens and tumors (57). Tight regulation of MHC-II expression is crucial to maintain a functional immune system and to limit the opportunity for the development of autoimmune diseases (32, 57). MHC-II is expressed constitutively on antigen-presenting cells and can be inducibly expressed on most nucleated cells by gamma interferon (IFN-␥) (7,20). Constitutive and IFN-␥-inducible MHC-II expression is regulated at the level of transcription by a series of elements in the MHC-II promoter. Nuclear factor Y, regulatory factor X, and cyclic AMP response element binding protein (CREB) bind, respectively, to the Y and X elements of the MHC-II proximal promoter, forming a multiprotein enhanceosome complex, which is necessary but not sufficient for transcription initiation. Once the enhanceosome is assembled on the MHC-II promoter, the class II transactivator (CIITA) can be recruited. CIITA binding stabilizes the enhanceosome complex and recruits basal transcriptional components, including the CDK7 subunit of TFIIH and the CDK9 subunit of P-TEFb, which phosphorylate polymerase II and initiate the switch to an elongation complex (12,41,48,50,68). CIITA is also known to interact with a variety of transcriptional cofactors, including multiple histone acetyltransferases (HATs) and histone deacetylases (HDACs) (8,29,76,81). Although much is known about the requirement of these basal and inducible transcription factors for MHC-II expression, less is known regarding the importance of epigenetic modifications required to open the chromatin structure and all...