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The B-lymphoid transcription factors PAX5 and IKZF1 restrict the supply of glucose and energy to B cells to levels that are not enough to fuel a driver-oncogene, thereby acting as tumour suppressors and sensitizing acute lymphoblastic leukaemia B cells to glucocorticoid therapy. Metabolic gatekeeper restricts B-cell malignancies This report examines how in B-cell malignancies, lymphoid transcriptional programs in early differentiation act as metabolic gatekeepers by restricting glucose transport, an important tumour-suppressor function that is subverted during the transformation to cancer. The findings provide a potential explanation for the selective efficacy of glucocorticoid therapy in B-cell malignancies, and suggest potential therapeutic avenues aimed at exploiting their metabolic vulnerabilities. B-lymphoid transcription factors, such as PAX5 and IKZF1, are critical for early B-cell development 1 , 2 , yet lesions of the genes encoding these transcription factors occur in over 80% of cases of pre-B-cell acute lymphoblastic leukaemia (ALL) 3 , 4 . The importance of these lesions in ALL has, until now, remained unclear. Here, by combining studies using chromatin immunoprecipitation with sequencing and RNA sequencing, we identify a novel B-lymphoid program for transcriptional repression of glucose and energy supply. Our metabolic analyses revealed that PAX5 and IKZF1 enforce a state of chronic energy deprivation, resulting in constitutive activation of the energy-stress sensor AMPK 5 , 6 , 7 . Dominant-negative mutants of PAX5 and IKZF1 , however, relieved this glucose and energy restriction. In a transgenic pre-B ALL mouse model, the heterozygous deletion of Pax5 increased glucose uptake and ATP levels by more than 25-fold. Reconstitution of PAX5 and IKZF1 in samples from patients with pre-B ALL restored a non-permissive state and induced energy crisis and cell death. A CRISPR/Cas9-based screen of PAX5 and IKZF1 transcriptional targets identified the products of NR3C1 (encoding the glucocorticoid receptor) 8 , TXNIP (encoding a glucose-feedback sensor) 9 and CNR2 (encoding a cannabinoid receptor) 10 as central effectors of B-lymphoid restriction of glucose and energy supply. Notably, transport-independent lipophilic methyl-conjugates of pyruvate and tricarboxylic acid cycle metabolites bypassed the gatekeeper function of PAX5 and IKZF1 and readily enabled leukaemic transformation. Conversely, pharmacological TXNIP and CNR2 agonists and a small-molecule AMPK inhibitor strongly synergized with glucocorticoids, identifying TXNIP, CNR2 and AMPK as potential therapeutic targets. Furthermore, our results provide a mechanistic explanation for the empirical finding that glucocorticoids are effective in the treatment of B-lymphoid but not myeloid malignancies. Thus, B-lymphoid transcription factors function as metabolic gatekeepers by limiting the amount of cellular ATP to levels that are insufficient for malignant transformation.

Genes are not simply turned on or off, but instead their expression is fine-tuned to meet the needs of a cell. How genes are modulated so precisely is not well understood. The glucocorticoid receptor (GR) regulates target genes by associating with specific DNA binding sites, the sequences of which differ between genes. Traditionally, these binding sites have been viewed only as docking sites. Using structural, biochemical, and cell-based assays, we show that GR binding sequences, differing by as little as a single base pair, differentially affect GR conformation and regulatory activity. We therefore propose that DNA is a sequence-specific allosteric ligand of GR that tailors the activity of the receptor toward specific target genes.

Glucocorticoid receptor (GR) binds as a dimer to DNA, with the canonical binding sequence featuring two half-sites, each bound by one monomer, separated by a 3-bp spacer. The specific bases contacted by GR are known to direct receptor activity. Now NMR and functional analyses show that nonspecific interactions, including with the spacer region, can also affect GR conformation and activity. Glucocorticoid receptor (GR) binds to genomic response elements and regulates gene transcription with cell and gene specificity. Within a response element, the precise sequence to which the receptor binds has been implicated in directing its structure and activity. Here, we use NMR chemical-shift difference mapping to show that nonspecific interactions with bases at particular positions in the binding sequence, such as those of the 'spacer', affect the conformation of distinct regions of the rat GR DNA-binding domain. These regions include the DNA-binding surface, the 'lever arm' and the dimerization interface, suggesting an allosteric pathway that signals between the DNA-binding sequence and the associated dimer partner. Disrupting this pathway by mutating the dimer interface alters sequence-specific conformations, DNA-binding kinetics and transcriptional activity. Our study demonstrates that GR dimer partners collaborate to read DNA shape and to direct sequence-specific gene activity.

The classical dogma of glucocorticoid-induced insulin resistance is that it is caused by the transcriptional activation of hepatic gluconeogenic and insulin resistance genes by the glucocorticoid receptor (GR). Here, we find that glucocorticoids also stimulate the expression of insulin-sensitizing genes, such as Irs2 . The transcriptional coregulator EHMT2 can serve as a transcriptional coactivator or a corepressor. Using male mice that have a defective EHMT2 coactivation function specifically, we show that glucocorticoid-induced Irs2 transcription is dependent on liver EHMT2’s coactivation function and that IRS2 play a key role in mediating the limitation of glucocorticoid-induced insulin resistance by EHMT2’s coactivation. Overall, we propose a model in which glucocorticoid-regulated insulin sensitivity is determined by the balance between glucocorticoid-modulated insulin resistance and insulin sensitizing genes, in which EHMT2 coactivation is specifically involved in the latter process. Glucocorticoids are known to induce insulin resistance via transcriptional activation of genes related to liver gluconeogenesis and insulin resistance. Here the authors report that in male mice treated with glucocorticoids, the transcriptional co-regulator EHMT2 is involved in the induction of Irs2 (a gene promoting insulin action) to restrict the extent of insulin resistance in the liver.

The glucocorticoid (GC) receptor (GR) suppresses inflammation by activating anti-inflammatory and repressing pro-inflammatory genes. GR-interacting protein-1 (GRIP1) is a GR corepressor in macrophages, however, whether GRIP1 mediates GR-activated transcription, and what dictates its coactivator versus corepressor properties is unknown. Here we report that GRIP1 loss in macrophages attenuates glucocorticoid induction of several anti-inflammatory targets, and that GC treatment of quiescent macrophages globally directs GRIP1 toward GR binding sites dominated by palindromic GC response elements (GRE), suggesting a non-redundant GRIP1 function as a GR coactivator. Interestingly, GRIP1 is phosphorylated at an N-terminal serine cluster by cyclin-dependent kinase-9 (CDK9), which is recruited into GC-induced GR:GRIP1:CDK9 hetero-complexes, producing distinct GRE-specific GRIP1 phospho-isoforms. Phosphorylation potentiates GRIP1 coactivator but, remarkably, not its corepressor properties. Consistently, phospho-GRIP1 and CDK9 are not detected at GR transrepression sites near pro-inflammatory genes. Thus, GR restricts actions of its own coregulator via CDK9-mediated phosphorylation to a subset of anti-inflammatory genes. Glucocorticoid reduces inflammation by both inducing anti-inflammatory genes and suppressing pro-inflammatory genes, but how these two functions are dictated is unclear. Here the authors show that phosphorylated glucocorticoid receptor-interacting protein 1 (GRIP1) serves as a coactivator for this response in macrophage.

Most endometrial cancers express the hormone receptor estrogen receptor alpha (ER) and are driven by excess estrogen signaling. However, evaluation of the estrogen response in endometrial cancer cells has been limited by the availability of hormonally responsive in vitro models, with one cell line, Ishikawa, being used in most studies. Here, we describe a novel, adherent endometrioid endometrial cancer (EEC) cell line model, HCI-EC-23. We show that HCI-EC-23 retains ER expression and that ER functionally responds to estrogen induction over a range of passages. We also demonstrate that this cell line retains paradoxical activation of ER by tamoxifen, which is also observed in Ishikawa and is consistent with clinical data. The mutational landscape shows that HCI-EC-23 is mutated at many of the commonly altered genes in EEC, has relatively few copy-number alterations, and is microsatellite instable high (MSI-high). In vitro proliferation of HCI-EC-23 is strongly reduced upon combination estrogen and progesterone treatment. HCI-EC-23 exhibits strong estrogen dependence for tumor growth in vivo and tumor size is reduced by combination estrogen and progesterone treatment. Molecular characterization of estrogen induction in HCI-EC-23 revealed hundreds of estrogen-responsive genes that significantly overlapped with those regulated in Ishikawa. Analysis of ER genome binding identified similar patterns in HCI-EC-23 and Ishikawa, although ER exhibited more bound sites in Ishikawa. This study demonstrates that HCI-EC-23 is an estrogen- and progesterone-responsive cell line model that can be used to study the hormonal aspects of endometrial cancer.

In addition to guiding proteins to defined genomic loci, DNA can act as an allosteric ligand that influences protein structure and activity. Here we compared genome-wide binding, transcriptional regulation, and, using NMR, the conformation of two glucocorticoid receptor (GR) isoforms that differ by a single amino acid insertion in the lever arm, a domain that adopts DNA sequence-specific conformations. We show that these isoforms differentially regulate gene expression levels through two mechanisms: differential DNA binding and altered communication between GR domains. Our studies suggest a versatile role for DNA in both modulating GR activity and also in directing the use of GR isoforms. We propose that the lever arm is a ”fulcrum” for bidirectional allosteric signaling, conferring conformational changes in the DNA reading head that influence DNA sequence selectivity, as well as conferring changes in the dimerization domain that connect functionally with remote regulatory surfaces, thereby influencing which genes are regulated and the magnitude of their regulation.

Cell signaling that culminates in posttranslational modifications directs protein activity. Here we report how multiple$Ca^{2+}-dependent$phosphorylation sites within the transcription activator Ets-1 act additively to produce graded DNA binding affinity. Nuclear magnetic resonance spectroscopic analyses show that phosphorylation shifts Ets-1 from a dynamic conformation poised to bind DNA to a well-folded inhibited state. These phosphates lie in an unstructured flexible region that functions as the allosteric effector of autoinhibition. Variable phosphorylation thus serves as a \"rheostat\" for cell signaling to fine-tune transcription at the level of DNA binding.

Autoinhibitory domains are regions of proteins that negatively regulate the function of other domains via intramolecular interactions. Autoinhibition is a potent regulatory mechanism that provides tight \"on-site\" repression. The discovery of autoinhibition generates valuable clues to how a protein is regulated within a biological context. Mechanisms that counteract the autoinhibition, including proteolysis, post-translational modifications, as well as addition of proteins or small molecules in trans, often represent central regulatory pathways. In this review, we document the diversity of instances in which autoinhibition acts in cell regulation. Seven well-characterized examples (e.g., sigma(70), Ets-1, ERM, SNARE and WASP proteins, SREBP, Src) are covered in detail. Over thirty additional examples are listed. We present experimental approaches to characterize autoinhibitory domains and discuss the implications of this widespread phenomenon for biological regulation in both the normal and diseased states.

Barrier-to-autointegration factor (BAF) is an essential component of the nuclear lamina. Encoded by BANF1, this DNA binding protein contributes to the regulation of gene expression, cell cycle progression, and nuclear integrity. A rare recessive BAF variant, Ala12Thr, causes the premature aging syndrome, Néstor–Guillermo progeria syndrome (NGPS). Here, we report the first dominant pathogenic BAF variant, Gly16Arg, identified in a patient presenting with progressive neuromuscular weakness. Although disease variants carry nearby amino acid substitutions, cellular and biochemical properties are distinct. In contrast to NGPS, Gly16Arg patient fibroblasts show modest changes in nuclear lamina structure and increases in repressive marks associated with heterochromatin. Structural studies reveal that the Gly16Arg substitution introduces a salt bridge between BAF monomers, reducing the conformation ensemble available to BAF. We show that this structural change increases the double-stranded DNA binding affinity of BAF Gly16Arg. Together, our findings suggest that BAF Gly16Arg has an increased chromatin occupancy that leads to epigenetic changes and impacts nuclear functions. These observations provide a new example of how a missense mutation can change a protein conformational equilibrium to cause a dominant disease and extend our understanding of mechanisms by which BAF function impacts human health.