A novel TREX1 inhibitor, VB-85680, upregulates cellular interferon responses.

Activation of the cGAS-STING pathway plays a key role in the innate immune response to cancer through Type-1 Interferon (IFN) production and T cell priming. Accumulation of cytosolic double-stranded DNA (dsDNA) within tumor cells and dying cells is recognized by the DNA sensor cyclic GMP-AMP synthase (cGAS) to create the secondary messenger cGAMP, which in turn activates STING (STimulator of INterferon Genes), resulting in the subsequent expression of IFN-related genes. This process is regulated by Three-prime Repair EXonuclease 1 (TREX1), a 3' → 5' exonuclease that degrades cytosolic dsDNA, thereby dampening activation of the cGAS-STING pathway, which in turn diminishes immunostimulatory IFN secretion. Here, we characterize the activity of VB-85680, a potent small-molecule inhibitor of TREX1. We first demonstrate that VB-85680 inhibits TREX1 exonuclease activity in vitro in lysates from both human and mouse cell lines. We then show that treatment of intact cells with VB-85680 results in activation of downstream STING signaling, and activation of IFN-stimulated genes (ISGs). THP1-Dual™ cells cultured under low-serum conditions exhibited an enhanced ISG response when treated with VB-85680 in combination with exogenous DNA. Collectively, these findings suggest the potential of a TREX1 exonuclease inhibitor to work in combination with agents that generate cytosolic DNA to enhance the acquisition of the anti-tumor immunity widely associated with STING pathway activation.

Myeloid-specific deletion of Zfp36 protects against insulin resistance and fatty liver in diet-induced obese mice.

Obesity is associated with adipose tissue inflammation that contributes to insulin resistance. Zinc finger protein 36 (Zfp36) is an mRNA-binding protein that reduces inflammation by binding to cytokine transcripts and promoting their degradation. We hypothesized that myeloid-specific deficiency of Zfp36 would lead to increased adipose tissue inflammation and reduced insulin sensitivity in diet-induced obese mice. As expected, wild-type (Control) mice became obese and diabetic on a high-fat diet, and obese mice with myeloid-specific loss of Zfp36 [knockout (KO)] demonstrated increased adipose tissue and liver cytokine mRNA expression compared with Control mice. Unexpectedly, in glucose tolerance testing and hyperinsulinemic-euglycemic clamp studies, myeloid Zfp36 KO mice demonstrated improved insulin sensitivity compared with Control mice. Obese KO and Control mice had similar macrophage infiltration of the adipose depots and similar peripheral cytokine levels, but lean and obese KO mice demonstrated increased Kupffer cell (KC; the hepatic macrophage)-expressed Mac2 compared with lean Control mice. Insulin resistance in obese Control mice was associated with enhanced Zfp36 expression in KCs. Compared with Control mice, KO mice demonstrated increased hepatic mRNA expression of a multitude of classical (M1) inflammatory cytokines/chemokines, and this M1-inflammatory hepatic milieu was associated with enhanced nuclear localization of IKKß and the p65 subunit of NF-κB. Our data confirm the important role of innate immune cells in regulating hepatic insulin sensitivity and lipid metabolism, challenge-prevailing models in which M1 inflammatory responses predict insulin resistance, and indicate that myeloid-expressed Zfp36 modulates the response to insulin in mice.

Glucocorticoid receptor-mediated cis-repression of osteogenic genes requires BRM-SWI/SNF.

Glucocorticoids are an effective therapy for a variety of severe inflammatory and autoimmune disorders; however, the therapeutic use of glucocorticoids is severely limited by their negative side effects, particularly on osteogenesis. Glucocorticoids regulate transcription by binding to the glucocorticoid receptor (GR), which then binds the promoters of target genes to induce either activation or repression. The gene activation effects of nuclear hormone receptors broadly require the cooperation of the chromatin remodeling complex known as SWI/SNF, which is powered by an ATPase core. The well-studied SWI/SNF ATPase, BRG1, is required for gene activation by a spectrum of nuclear hormone receptors including GR. However, glucocorticoid-induced side effects specifically related to impaired osteogenesis are mostly linked with GR-mediated repression. We have considered whether cis-repression of osteogenic genes by GR may be mediated by a distinct subclass of SWI/SNF powered by the alternative ATPase, BRM. BRM does not have an essential role in mammalian development, but plays a repressor role in osteoblast differentiation and favors adipogenic lineage selection over osteoblast commitment, effects that mirror the repressor effects of GR. The studies reported here examine three key GR cis-repression gene targets, and show that GR association with these promoters is sharply reduced in BRM deficient cells. Each of these GR-targeted genes act in a different way. Bglap encodes osteocalcin, which contributes to normal maturation of osteoblasts from committed pre-osteoblasts. The Per3 gene product acts in uncommitted mesenchymal stem cells to influence the osteoblast/adipocyte lineage selection point. Fas ligand, encoded by FasL, is a means by which osteoblasts can modulate bone degradation by osteoclasts. Repression of each of these genes by glucocorticoid favors bone loss. The essential role of BRM in cooperation with GR at each of these control points offers a novel mechanistic understanding of the role of GR in bone loss.

SWI/SNF-Mediated Lineage Determination in Mesenchymal Stem Cells Confers Resistance to Osteoporosis.

Redirecting the adipogenic potential of bone marrow-derived mesenchymal stem cells to other lineages, particularly osteoblasts, is a key goal in regenerative medicine. Controlling lineage selection through chromatin remodeling complexes such as SWI/SNF, which act coordinately to establish new patterns of gene expression, would be a desirable intervention point, but the requirement for the complex in essentially every lineage pathway has generally precluded selectivity. However, a novel approach now appears possible by targeting the subset of SWI/SNF powered by the alternative ATPase, mammalian brahma (BRM). BRM is not required for development, which has hindered understanding of its contributions, but knockdown genetics here, designed to explore the hypothesis that BRM-SWI/SNF has different regulatory roles in different mesenchymal stem cell lineages, shows that depleting BRM from mesenchymal stem cells has a dramatic effect on the balance of lineage selection between osteoblasts and adipocytes. BRM depletion enhances the proportion of cells expressing markers of osteoblast precursors at the expense of cells able to differentiate along the adipocyte lineage. This effect is evident in primary bone marrow stromal cells as well as in established cell culture models. The altered precursor balance has major physiological significance, which becomes apparent as protection against age-related osteoporosis and as reduced bone marrow adiposity in adult BRM-null mice.

p107-Dependent recruitment of SWI/SNF to the alkaline phosphatase promoter during osteoblast differentiation.

The retinoblastoma protein family is intimately involved in the regulation of tissue specific gene expression during mesenchymal stem cell differentiation. The role of the following proteins, pRB, p107 and p130, is particularly significant in differentiation to the osteoblast lineage, as human germ-line mutations of RB1 greatly increase susceptibility to osteosarcoma. During differentiation, pRB directly targets certain osteogenic genes for activation, including the alkaline phosphatase-encoding gene Alpl. Chromatin immunoprecipitation (ChIP) assays indicate that Alpl is targeted by p107 in differentiating osteoblasts selectively during activation with the same dynamics as pRB, which suggests that p107 helps promote Alpl activation. Mouse models indicate overlapping roles for pRB and p107 in bone and cartilage formation, but very little is known about direct tissue-specific gene targets of p107, or the consequences of targeting by p107. Here, the roles of p107 and pRB were compared using shRNA-mediated knockdown genetics in an osteoblast progenitor model, MC3T3-E1 cells. The results show that p107 has a distinct role along with pRB in induction of Alpl. Deficiency of p107 does not impede recruitment of transcription factors recognized as pRB co-activation partners at the promoter; however, p107 is required for the efficient recruitment of an activating SWI/SNF chromatin-remodeling complex, an essential event in Alpl induction.

Cooperative activation of tissue-specific genes by pRB and E2F1.

The retinoblastoma tumor suppressor protein pRB is conventionally regarded as an inhibitor of the E2F family of transcription factors. Conversely, pRB is also recognized as an activator of tissue-specific gene expression along various lineages including osteoblastogenesis. During osteoblast differentiation, pRB directly targets Alpl and Bglap, which encode the major markers of osteogenesis alkaline phosphatase and osteocalcin. Surprisingly, p130 and repressor E2Fs were recently found to cooccupy and repress Alpl and Bglap in proliferating osteoblast precursors before differentiation. This raises the further question of whether these genes convert to E2F activation targets when differentiation begins, which would constitute a remarkable situation wherein pRB and E2F would be cotargeting genes for activation. Chromatin immunoprecipitation analysis in an osteoblast differentiation model shows that Alpl and Bglap are indeed targeted by an activator E2F, i.e., is E2F1. Promoter occupation of Alpl and Bglap by E2F1 occurs specifically during activation, and depletion of E2F1 severely impairs their induction. Mechanistically, promoter occupation by E2F1 and pRB is mutually dependent, and without this cooperative effect, activation steps previously shown to be dependent on pRB, including recruitment of RNA polymerase II, are impaired. Myocyte- and adipocyte-specific genes are also cotargeted by E2F1 and pRB during differentiation along their respective lineages. The finding that pRB and E2F1 cooperate to activate expression of tissue-specific genes is a paradigm distinct from the classical concept of pRB as an inhibitor of E2F1, but is consistent with the observed roles of these proteins in physiological models.

Essential role of ARID2 protein-containing SWI/SNF complex in tissue-specific gene expression.

Unfolding of the gene expression program that converts precursor cells to their terminally differentiated counterparts is critically dependent on the nucleosome-remodeling activity of the mammalian SWI/SNF complex. The complex can be powered by either of two alternative ATPases, BRM or BRG1. BRG1 is critical for development and the activation of tissue specific genes and is found in two major stable configurations. The complex of BRG1-associated factors termed BAF is the originally characterized form of mammalian SWI/SNF. A more recently recognized configuration shares many of the same subunits but is termed PBAF in recognition of a unique subunit, the polybromo protein (PBRM1). Two other unique subunits, BRD7 and ARID2, are also diagnostic of PBAF. PBAF plays an essential role in development, apparent from the embryonic lethality of Pbmr1-null mice, but very little is known about the role of PBAF, or its signature subunits, in tissue-specific gene expression in individual differentiation programs. Osteoblast differentiation is an attractive model for tissue-specific gene expression because the process is highly regulated and remains tightly synchronized over a period of several weeks. This model was used here, with a stable shRNA-mediated depletion approach, to examine the role of the signature PBAF subunit, ARID2, during differentiation. This analysis identifies a critical role for ARID2-containing complexes in promoting osteoblast differentiation and supports a view that the PBAF subset of SWI/SNF contributes importantly to maintaining cellular identity and activating tissue-specific gene expression.

Tissue-specific gene targeting by the multiprotein mammalian DREAM complex.

The mammalian DP, RB-like, E2F, and MuvB-like proteins (DREAM) complex, whose key components include p130 and E2F4, plays a fundamental role in repression of cell cycle-specific genes during growth arrest. Mammalian DREAM is well conserved with Drosophila and Caenorhabditis elegans complexes that repress pivotal developmental genes, but the mammalian complex has been thought to exist only in quiescent cells and not to be linked with development. However, new findings here identify tissue-specific promoters repressed by DREAM in proliferating precursors, revealing a new connection between control of growth arrest and terminal differentiation. Mechanistically, tissue-specific promoter occupation by DREAM is dependent on the integrity of a repressor form of the SWI/SNF chromatin-remodeling complex.

FOXO1 modulates osteoblast differentiation.

Forkhead box O1 (FOXO1) is upregulated during bone formation and in response to stimulation by bone morphogenetic proteins. Studies presented here examined the functional role of FOXO1 in a well defined culture system in which pre-osteoblastic cells undergo terminal differentiation in vitro. Mineralizing cultures of MC3T3-E1 cells were examined with or without FOXO1 knockdown by RNAi. Normal cells show the upregulation of FOXO1 and RUNX2 DNA binding activity, alkaline phosphatase activity, and mRNA levels of FOXO1, RUNX2, type 1 collagen, osteocalcin and MMP13 during formation of mineralizing nodules. In FOXO1 depleted cells each of these measurements was significantly reduced compared to values in control cells transfected with scrambled siRNA (P<0.05). Depletion of FOXO1 also reduced the number of mineralized nodules formed. Moreover, chromatin immunoprecipitation assays revealed a direct interaction of FOXO1 with the RUNX2 promoter. Overexpression of FOXO1 reduced the MC3T3-E1 cell number and the number of PCNA positive cells with little effect on apoptosis. These findings indicate that FOXO1 plays an important role in promoting osteoblast differentiation and suppressing proliferation in differentiating cells.

Transcriptional activation by pRB and its coordination with SWI/SNF recruitment.

A central question in cancer biology is why most tumor susceptibility genes are linked with only limited types of cancer. Human germ-line mutation of the retinoblastoma susceptibility gene Rb1 is closely linked with just retinoblastoma and osteosarcoma, although the gene is universally expressed. Functional analysis of pRB and its close relatives, p107 and p130, has largely focused on their roles in repression of proliferation across all tissue types, but genetic evidence indicates an active requirement for pRB in osteoblast differentiation that correlates more directly with osteosarcoma susceptibility. Still, potential promoter targets of pRB and its role in normally differentiating osteoblasts remain insufficiently characterized. Here, an early marker of osteoblast differentiation, alkaline phosphatase, is identified as a direct promoter activation target of pRB. One role of pRB on this promoter is to displace the histone lysine demethylase KDM5A, thereby favoring trimethylation of H3K4, a promoter activation mark. A major new aspect of pRB-mediated transcriptional activation revealed in this promoter analysis is its role in recruitment of an activating SWI/SNF chromatin-remodeling complex. SWI/SNF is a critical coordinator of tissue-specific gene expression. In osteoblasts, SWI/SNF complexes containing the BRM ATPase repress osteoblast-specific genes to maintain the precursor state, whereas the alternative ATPase BRG1 distinguishes an activating SWI/SNF complex necessary for RNA polymerase-II recruitment. A switch from BRM to BRG1 on the alkaline phosphatase promoter marks the onset of differentiation and is accomplished in a precise two-step mechanism. Dissociation of BRM-containing SWI/SNF depends on p300, and association of BRG1-containing SWI/SNF depends on pRB.

Antagonistic roles for BRM and BRG1 SWI/SNF complexes in differentiation.

The mammalian SWI/SNF chromatin-remodeling complex is essential for the multiple changes in gene expression that occur during differentiation. However, the basis within the complex for specificity in effecting positive versus negative changes in gene expression has only begun to be elucidated. The catalytic core of the complex can be either of two closely related ATPases, BRM or BRG1, with the potential that the choice of alternative subunits is a key determinant of specificity. Short hairpin RNA-mediated depletion of the ATPases was used to explore their respective roles in the well characterized multistage process of osteoblast differentiation. The results reveal an unexpected role for BRM-specific complexes. Instead of impeding differentiation as was seen with BRG1 depletion, depletion of BRM caused accelerated progression to the differentiation phenotype. Multiple tissue-specific differentiation markers, including the tightly regulated late stage marker osteocalcin, become constitutively up-regulated in BRM-depleted cells. Chromatin immunoprecipitation analysis of the osteocalcin promoter as a model for the behavior of the complexes indicates that the promoter is a direct target of both BRM- and BRG1-containing complexes. BRG1 complexes, which are required for activation, are associated with the promoter well before induction, but the concurrent presence of BRM-specific complexes overrides their activation function. BRM-specific complexes are present only on the repressed promoter and are required for association of the co-repressor HDAC1. These findings reveal an unanticipated degree of specialization of function linked with the choice of ATPase and suggest a new paradigm for the roles of the alternative subunits during differentiation.

Expression of p270 (ARID1A), a component of human SWI/SNF complexes, in human tumors.

Human SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromosomes and alter gene expression patterns. The activity of the complexes generally promotes tissue-specific gene expression and restricts cell proliferation. The ATPase that drives the complexes, BRG1, is essential for tumor suppression in mice and deficient in a variety of established human tumor cell lines. The complex contains at least 7 other core components, one of which is a large subunit designated p270. p270 RNA is expressed in all normal human tissues examined, but protein expression is severely reduced in at least 2 human tumor lines, C33A and T47D. We show here that loss of p270 in the C33A and T47D cell lines is evident at the RNA level as well as the protein level. The implication that p270 can be informatively screened at the RNA level made a high-efficiency cancer profiling array approach to screening human tumors feasible. Expression was screened in an array containing RNA-derived cDNA from 241 tumor and corresponding matched normal tissues from individual patients. p270 deficiency was observed at a higher overall frequency than BRG1 deficiency, but all tissues were not equally affected. Deficiency of p270 was observed most frequently in carcinomas of the breast and kidney. The results were most striking in kidney, where p270 expression was deficient in 30% of carcinoma samples screened. Screening of a panel of established human renal carcinoma-derived cell lines supports the frequency observed in the primary tumor tissue samples.

Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli.

In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC-DNA and DnaC-DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6*DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein.

Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching.

DnaB helicase of E. coli unwinds duplex DNA in the replication fork using the energy of ATP hydrolysis. We have analyzed structural and conformational changes in the DnaB protein in various nucleotides and DNA bound intermediate states by fluorescence quenching analysis of intrinsic fluorescence of native tryptophan (Trp) residues in DnaB. Fluorescence quenching analysis indicated that Trp48 in domain alpha is in a hydrophobic environment and resistant to fluorescence quenchers such as potassium iodide (KI). In domain beta, Trp294 was found to be in a partially hydrophobic environment, whereas Trp456 in domain gamma appeared to be in the least hydrophobic environment. Binding of oligonucleotides to DnaB helicase resulted in a significant attenuation of the fluorescence quenching profile, indicating a change in conformation. ATPgammaS or ATP binding appeared to lead to a conformation in which Trp residues had a higher degree of solvent exposure and fluorescence quenching. However, the most dramatic increase of Trp fluorescence quenching was observed with ADP binding with a possible conformational relaxation. Site-specific Trp --> Cys mutants of DnaB helicase demonstrated that conformational change upon ADP binding could be attributed exclusively to a conformational transition in the alpha domain leading to an increase in the solvent exposure of Trp48. However, formation of DnaB.ATPgammaS.DNA ternary complex led to a conformation with a fluorescence quenching profile similar to that observed with DnaB alone. The DnaB.ADP.DNA ternary complex produced a quenching curve similar to that of DnaB.ADP complex pointing to a change in conformation due to ATP hydrolysis. There are at least four identifiable structural/conformational states of DnaB helicase that are likely important in the helicase activity. The noncatalytic alpha domain in the N-terminus appeared to undergo the most significant conformational changes during nucleotide binding and hydrolysis. This is the first reported elucidation of the putative role of domain alpha, which is essential for DNA helicase action. We have correlated these results with partial structural models of alpha, beta, and gamma domains