Differential gene expression mechanisms ensure cellular differentiation and plasticity to shape ontogenetic and phylogenetic diversity of cell types. A key regulator of differential gene expression programs are the enhancers, the gene-distal cis-regulatory sequences that govern spatiotemporal and quantitative expression dynamics of target genes. Enhancers are widely believed to physically contact the target promoters to effect transcriptional activation. However, our understanding of the full complement of regulatory proteins and the definitive mechanics of enhancer action is incomplete. Here, we review recent findings to present some emerging concepts on enhancer action and also outline a set of outstanding questions.

The PTPN22 genetic variant 1858T, encoding Lyp620W, is associated with multiple autoimmune disorders for which the production of autoantibodies is a common feature, suggesting a loss of B cell tolerance. Lyp620W results in blunted BCR signaling in memory B cells. Because BCR signal strength is tightly coupled to central and peripheral tolerance, we examined whether Lyp620W impacts peripheral B cell homeostasis in healthy individuals heterozygous for the PTPN221858T variant. We found that these subjects display alterations in the composition of the B cell pool that include specific expansion of the transitional and anergic IgD+IgM−CD27− B cell subsets. The PTPN22 1858T variant was further associated with significantly diminished BCR signaling and a resistance to apoptosis in both transitional and naive B cells. Strikingly, parallel changes in both BCR signaling and composition of B cell compartment were observed in type 1 diabetic subjects, irrespective of PTPN22 genotype, revealing a novel immune phenotype and likely shared mechanisms leading to a loss of B cell tolerance. Our combined findings suggest that Lyp620W-mediated effects, due in part to the altered BCR signaling threshold, contribute to breakdown of peripheral tolerance and the entry of autoreactive B cells into the naive B cell compartment.

Specific metabolic underpinnings of androgen receptor (AR)-driven growth in prostate adenocarcinoma (PCa) are largely undefined, hindering the development of strategies to leverage the metabolic dependencies of this disease when hormonal manipulations fail. Here we show that the mitochondrial pyruvate carrier (MPC), a critical metabolic conduit linking cytosolic and mitochondrial metabolism, is transcriptionally regulated by AR. Experimental MPC inhibition restricts proliferation and metabolic outputs of the citric acid cycle (TCA) including lipogenesis and oxidative phosphorylation in AR-driven PCa models. Mechanistically, metabolic disruption resulting from MPC inhibition activates the eIF2α/ATF4 integrated stress response (ISR). ISR signaling prevents cell cycle progression while coordinating salvage efforts, chiefly enhanced glutamine assimilation into the TCA, to regain metabolic homeostasis. We confirm that MPC function is operant in PCa tumors in-vivo using isotopomeric metabolic flux analysis. In turn, we apply a clinically viable small molecule targeting the MPC, MSDC0160, to pre-clinical PCa models and find that MPC inhibition suppresses tumor growth in hormone-responsive and castrate-resistant conditions. Collectively, our findings characterize the MPC as a tractable therapeutic target in AR-driven prostate tumors.

To better understand whether autoimmunity in Lyn-deficient mice arises from compromised central or peripheral B cell tolerance, we examined BCR signaling properties of wild-type and Lyn-deficient B cells at different stages of development. Wild-type mature follicular B cells were less sensitive to BCR stimulation than were immature transitional stage 1 B cells with regard to BCRinduced calcium elevation and ERK MAPK activation. In the absence of Lyn, mature B cell signaling was greatly enhanced, whereas immature B cell signaling was minimally affected. Correspondingly, Lyn deficiency substantially enhanced the sensitivity of mature B cells to activation via the BCR, but minimally affected events associated with tolerance induction at the immature stage. The effects of CD22 deficiency on BCR signaling were very similar in B cells at different stages of maturation. These results indicate that the Lyn-CD22-Src homology region 2 domain-containing phosphatase-1 inhibitory pathway largely becomes operational as B cell mature, and sets a threshold for activation that appears to be critical for the maintenance of tolerance in the B cell compartment. The Journal of Immunology, 2009, 182: 5382-5392. Bcell receptor hyperresponsiveness is associated with a breakdown in tolerance and the development of autoantibodies in several genetically modified mouse strains (1, 2). A good example of this is the Lyn-deficient mouse (3). Lyn is a Src family kinase (SFK) 3 that, like two other SFKs expressed by B cells, Blk and Fyn, phosphorylates ITAMs on BCR Ig␣/Ig␤ chains following Ag binding (4 -6). Lyn also functions to phosphorylate ITIMs on inhibitory receptors that negatively regulate BCR signaling, including CD22 (5, 7-9), Fc␥RIIb (7, 8), and perhaps others (10 -13). In this way, Lyn facilitates recruitment of the Src homology region 2 domain-containing phosphatase-1 (SHP-1) and SHIP phosphatases to the plasma membrane, which downmodulate BCR signaling (5,8,9). In the absence of Lyn, BCR signaling is supported by Blk and Fyn, but inhibitory receptors are ineffective at down-regulating BCR signaling, thereby leading to BCR hyperresponsiveness (3).Lyn-deficient mice exhibit increased plasma cell numbers and serum Ig levels. Surprisingly, these mice also produce autoantibodies to nuclear Ags (4,14,15). Why elevated BCR signaling would lead to a loss of tolerance in lyn Ϫ/Ϫ mice is not entirely self-evident, because whereas BCR signaling drives activation of mature B cells, it also promotes tolerance-inducing mechanisms in immature B cells. For example, binding of Ag by immature B cells in the bone marrow (BM) induces expression of RAG 1 and 2, which can generate L chain rearrangement and thereby change the specificity of the BCR (receptor editing) (16 -18). Continued recognition of Ag by the BCR may induce apoptosis of self-reactive B cells (clonal deletion) (16). Therefore, genetic alterations that enhance BCR signaling should increase the sensitivity of immature B cells to self Ags, causing more thorough removal of autoreactive B...

B cells undergo a random process of V(D)J recombination to generate the many distinct receptors needed to recognize a vast array of antigens. An inevitable consequence of this random process is the production of autoreactive B cells ( 1 ). An important mechanism for tolerizing autoreactive B cells is receptor editing ( 2 ). Receptor editing results in the alteration of B cell receptor specifi city and is achieved by ongoing Ig gene rearrangement, most commonly at the light chain loci ( 3 -5 ). Light chain rearrangement proceeds in an ordered fashion as B cells develop in the bone marrow, with genes recombining fi rst, followed by rearrangement of the recombining sequence (RS) and ( 6, 7 ). The RS (also known as the deleting element [KDE] in humans) is a noncoding gene segment located 25 kb downstream of C in the locus that is rearranged during continued Ig light chain gene rearrangement ( 8,9 ).Because of the unique structure of the locus, primary V -J rearrangements that are nonfunctional or autoreactive can be replaced via " leap-frogging " recombination of unrearranged upstream V and downstream J gene segments to form new light chains ( Fig. 1 a ). Additional rearrangement attempts can be made through recombination at the second allele or at . Recombination of RS to upstream V gene segments or a recombination signal sequence within the J -C intron results in the deletion or inversion of C and functional inactivation of the locus ( Fig. 1 a ). Because RS rearrangements do not encode any functional proteins ( 10 ), monitoring RS rearrangement provides a specifi city-independent means of measuring repeated rearrangement attempts at (receptor editing).The original studies characterizing RS recombination postulated that it served to promote rearrangement by either repressing rearrangement or activating the locus ( 7, 11 ). However, -expressing B cells can form without undergoing RS rearrangement, indicating that RS is not required for the production of ( 12 ). When RS rearrangement is prevented in RS knockout mice, receptor editing is ineffi cient and autoreactive B cells are found among peripheral cells ( 13 ) Continued antibody gene rearrangement, termed receptor editing, is an important mechanism of central B cell tolerance that may be defective in some autoimmune individuals. We describe a quantitative assay for recombining sequence (RS) rearrangement that we use to estimate levels of antibody light chain receptor editing in various B cell populations. RS rearrangement is a recombination of a noncoding gene segment in the antibody light chain locus. RS rearrangement levels are highest in the most highly edited B cells, and are inappropriately low in autoimmune mouse models of systemic lupus erythematosus (SLE) and type 1 diabetes (T1D), including those without overt disease. Low RS rearrangement levels are also observed in human subjects with SLE or T1D.