Retinol binding protein 4 primes the NLRP3 inflammasome by signaling through Toll-like receptors 2 and 4.

Adipose tissue (AT) inflammation contributes to systemic insulin resistance. In obesity and type 2 diabetes (T2D), retinol binding protein 4 (RBP4), the major retinol carrier in serum, is elevated in AT and has proinflammatory effects which are mediated partially through Toll-like receptor 4 (TLR4). We now show that RBP4 primes the NLRP3 inflammasome for interleukin-1ß (IL1ß) release, in a glucose-dependent manner, through the TLR4/MD2 receptor complex and TLR2. This impairs insulin signaling in adipocytes. IL1ß is elevated in perigonadal white AT (PGWAT) of chow-fed RBP4-overexpressing mice and in serum and PGWAT of high-fat diet-fed RBP4-overexpressing mice vs. wild-type mice. Holo- or apo-RBP4 injection in wild-type mice causes insulin resistance and elevates PGWAT inflammatory markers, including IL1ß. TLR4 inhibition in RBP4-overexpressing mice reduces PGWAT inflammation, including IL1ß levels and improves insulin sensitivity. Thus, the proinflammatory effects of RBP4 require NLRP3-inflammasome priming. These studies may provide approaches to reduce AT inflammation and insulin resistance in obesity and diabetes.

TERRA RNA binding to TRF2 facilitates heterochromatin formation and ORC recruitment at telomeres.

Telomere-repeat-encoding RNA (referred to as TERRA) has been identified as a potential component of yeast and mammalian telomeres. We show here that TERRA RNA interacts with several telomere-associated proteins, including telomere repeat factors 1 (TRF1) and 2 (TRF2), subunits of the origin recognition complex (ORC), heterochromatin protein 1 (HP1), histone H3 trimethyl K9 (H3 K9me3), and members of the DNA-damage-sensing pathway. siRNA depletion of TERRA caused an increase in telomere dysfunction-induced foci, aberrations in metaphase telomeres, and a loss of histone H3 K9me3 and ORC at telomere repeat DNA. Previous studies found that TRF2 amino-terminal GAR domain recruited ORC to telomeres. We now show that TERRA RNA can interact directly with the TRF2 GAR and ORC1 to form a stable ternary complex. We conclude that TERRA facilitates TRF2 interaction with ORC and plays a central role in telomere structural maintenance and heterochromatin formation.

RNA-dependent recruitment of the origin recognition complex.

The origin recognition complex (ORC) has an important function in determining the initiation sites of DNA replication. In higher eukaryotes, ORC lacks sequence-specific DNA binding, and the mechanisms of ORC recruitment and origin determination are poorly understood. ORC is recruited with high efficiency to the Epstein-Barr virus origin of plasmid replication (OriP) through a complex mechanism involving interactions with the virus-encoded EBNA1 protein. We present evidence that ORC recruitment to OriP and DNA replication function depends on RGG-like motifs, referred to as LR1 and LR2, in the EBNA1 amino-terminal domain. Moreover, we show that LR1 and LR2 recruitment of ORC is RNA dependent. HMGA1a, which can functionally substitute for LR1 and LR2 domain, can also recruit ORC in an RNA-dependent manner. EBNA1 and HMGA1a RGG motifs bound to structured G-rich RNA, as did ORC1 peptides, which interact with EBNA1. RNase A treatment of cellular chromatin released a fraction of the total ORC, suggesting that ORC association with chromatin, and possibly cellular origins, is stabilized by RNA. We propose that structural RNA molecules mediate ORC recruitment at some cellular and viral origins, similar to OriP.

Regulation of Epstein-Barr virus origin of plasmid replication (OriP) by the S-phase checkpoint kinase Chk2.

The Epstein-Barr virus (EBV) origin of plasmid replication (OriP) is required for episome stability during latent infection. Telomere repeat factor 2 (TRF2) binds directly to OriP and facilitates DNA replication and plasmid maintenance. Recent studies have found that TRF2 interacts with the DNA damage checkpoint protein Chk2. We show here that Chk2 plays an important role in regulating OriP plasmid stability, chromatin modifications, and replication timing. The depletion of Chk2 by small interfering RNA (siRNA) leads to a reduction in DNA replication efficiency and a loss of OriP-dependent plasmid maintenance. This corresponds to a change in OriP replication timing and an increase in constitutive histone H3 acetylation. We show that Chk2 interacts with TRF2 in the early G(1)/S phase of the cell cycle. We also show that Chk2 can phosphorylate TRF2 in vitro at a consensus acceptor site in the amino-terminal basic domain of TRF2. TRF2 mutants with a serine-to-aspartic acid phosphomimetic substitution mutation were reduced in their ability to recruit the origin recognition complex (ORC) and stimulate OriP replication. We suggest that the Chk2 phosphorylation of TRF2 is important for coordinating ORC binding with chromatin remodeling during the early S phase and that a failure to execute these events leads to replication defects and plasmid instability.

Role for G-quadruplex RNA binding by Epstein-Barr virus nuclear antigen 1 in DNA replication and metaphase chromosome attachment.

Latent infection by Epstein-Barr virus (EBV) requires both replication and maintenance of the viral genome. EBV nuclear antigen 1 (EBNA1) is a virus-encoded protein that is critical for the replication and maintenance of the genome during latency in proliferating cells. We have previously demonstrated that EBNA1 recruits the cellular origin recognition complex (ORC) through an RNA-dependent interaction with EBNA1 linking region 1 (LR1) and LR2. We now show that LR1 and LR2 bind to G-rich RNA that is predicted to form G-quadruplex structures. Several chemically distinct G-quadruplex-interacting drugs disrupted the interaction between EBNA1 and ORC. The G-quadruplex-interacting compound BRACO-19 inhibited EBNA1-dependent stimulation of viral DNA replication and preferentially blocked proliferation of EBV-positive cells relative to EBV-negative cell lines. BRACO-19 treatment also disrupted the ability of EBNA1 to tether to metaphase chromosomes, suggesting that maintenance function is also mediated through G-quadruplex recognition. These findings suggest that the EBNA1 replication and maintenance function uses a common G-quadruplex binding capacity of LR1 and LR2, which may be targetable by small-molecule inhibitors.

Genome-wide analysis of host-chromosome binding sites for Epstein-Barr Virus Nuclear Antigen 1 (EBNA1).

The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.

Downregulation of STRA6 in adipocytes and adipose stromovascular fraction in obesity and effects of adipocyte-specific STRA6 knockdown in vivo.

To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6(-/-) mice. Adipose-Stra6(-/-) mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the expression of retinoid homeostatic genes (encoding retinoic acid receptor ß [RARß], CYP26A1, and lecithin retinol acyltransferase) were not altered in adipocytes from adipose-Stra6(-/-) mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6(-/-) mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated insulin resistance.

Retinol-binding protein 4 inhibits insulin signaling in adipocytes by inducing proinflammatory cytokines in macrophages through a c-Jun N-terminal kinase- and toll-like receptor 4-dependent and retinol-independent mechanism.

Retinol-binding protein 4 (RBP4), the sole retinol transporter in blood, is secreted from adipocytes and liver. Serum RBP4 levels correlate highly with insulin resistance, other metabolic syndrome factors, and cardiovascular disease. Elevated serum RBP4 causes insulin resistance, but the molecular mechanisms are unknown. Here we show that RBP4 induces expression of proinflammatory cytokines in mouse and human macrophages and thereby indirectly inhibits insulin signaling in cocultured adipocytes. This occurs through activation of c-Jun N-terminal protein kinase (JNK) and Toll-like receptor 4 (TLR4) pathways independent of the RBP4 receptor, STRA6. RBP4 effects are markedly attenuated in JNK1-/- JNK2-/- macrophages and TLR4-/- macrophages. Because RBP4 is a retinol-binding protein, we investigated whether these effects are retinol dependent. Unexpectedly, retinol-free RBP4 (apo-RBP4) is as potent as retinol-bound RBP4 (holo-RBP4) in inducing proinflammatory cytokines in macrophages. Apo-RBP4 is likely to be physiologically significant since RBP4/retinol ratios are increased in serum of lean and obese insulin-resistant humans compared to ratios in insulin-sensitive humans, indicating that higher apo-RBP4 is associated with insulin resistance independent of obesity. Thus, RBP4 may cause insulin resistance by contributing to the development of an inflammatory state in adipose tissue through activation of proinflammatory cytokines in macrophages. This process reveals a novel JNK- and TLR4-dependent and retinol- and STRA6-independent mechanism of action for RBP4.

ORC binding to TRF2 stimulates OriP replication.

In higher eukaryotes, the origin recognition complex (ORC) lacks sequence-specific DNA binding, and it remains unclear what other factors specify an origin of DNA replication. The Epstein-Barr virus origin of plasmid replication (OriP) recruits ORC, but the precise mechanism of ORC recruitment and origin activation is not clear. We now show that ORC is recruited selectively to the dyad symmetry (DS) region of OriP as a consequence of direct interactions with telomere repeat factor 2 (TRF2) and ORC1. TRF-binding sites within DS stimulate replication initiation and facilitate ORC recruitment in vitro and in vivo. TRF2, but not TRF1 or hRap1, recruits ORC from nuclear extracts. The amino-terminal domain of TRF2 associated with a specific region of ORC1 and was necessary for stimulation of DNA replication. These results support a model in which TRF2 stimulates OriP replication activity by direct binding with ORC subunits.

Regulated translation of listeriolysin O controls virulence of Listeria monocytogenes.

Listeria monocytogenes is an intracytosolic bacterial pathogen that escapes from the phagosome using a secreted cytolysin, listeriolysin O (LLO). In the host cytosol, LLO activity is minimized to prevent pore formation in the host plasma membrane; premature lysis of the infected host cell exposes the bacteria to extracellular immune defences of the host and is detrimental to infection. Here we identified nucleotide substitutions in the coding sequence of the LLO gene (hly) that did not alter the protein sequence, yet caused over-production of LLO, cytotoxicity and loss of virulence. These phenotypes were independent of the promoter and, under conditions in which the mutants produced more LLO protein than wild type, levels of hly mRNA were similar. Finally, negative regulation of LLO was maintained even when bacteria were engineered to produce elevated levels of the wild-type hly transcript. Together, our data demonstrate that translational regulation of LLO is critical for L. monocytogenes pathogenesis.