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Normal Rabbit IgG antibody

RRID:AB_1031062

Antibody ID

AB_1031062

Target Antigen

Normal Rabbit IgG rabbit, lepus cuniculus

Proper Citation

(Cell Signaling Technology Cat# 2729, RRID:AB_1031062)

Clonality

polyclonal antibody

Comments

Applications: IP, ChIP. Consolidation on 10/2018: AB_10284964, AB_1031062, AB_2617119.

Host Organism

rabbit

Vendor

Cell Signaling Technology

MicroRNAs Overcome Cell Fate Barrier by Reducing EZH2-Controlled REST Stability during Neuronal Conversion of Human Adult Fibroblasts.

  • Lee SW
  • Dev. Cell
  • 2018 Jul 2

Literature context:


Abstract:

The ability to convert human somatic cells efficiently to neurons facilitates the utility of patient-derived neurons for studying neurological disorders. As such, ectopic expression of neuronal microRNAs (miRNAs), miR-9/9∗ and miR-124 (miR-9/9∗-124) in adult human fibroblasts has been found to evoke extensive reconfigurations of the chromatin and direct the fate conversion to neurons. However, how miR-9/9∗-124 break the cell fate barrier to activate the neuronal program remains to be defined. Here, we identified an anti-neurogenic function of EZH2 in fibroblasts that acts outside its role as a subunit of Polycomb Repressive Complex 2 to directly methylate and stabilize REST, a transcriptional repressor of neuronal genes. During neuronal conversion, miR-9/9∗-124 induced the repression of the EZH2-REST axis by downregulating USP14, accounting for the opening of chromatin regions harboring REST binding sites. Our findings underscore the interplay between miRNAs and protein stability cascade underlying the activation of neuronal program.

Funding information:
  • NHLBI NIH HHS - P50 HL077107(United States)

Chromatin Binding of c-REL and p65 Is Not Limiting for Macrophage IL12B Transcription During Immediate Suppression by Ovarian Carcinoma Ascites.

  • Unger A
  • Front Immunol
  • 2018 Jul 13

Literature context:


Abstract:

Tumors frequently exploit homeostatic mechanisms that suppress expression of IL-12, a central mediator of inflammatory and anti-tumor responses. The p40 subunit of the IL-12 heterodimer, encoded by IL12B, is limiting for these functions. Ovarian carcinoma patients frequently produce ascites which exerts immunosuppression by means of soluble factors. The NFκB pathway is necessary for transcription of IL12B, which is not expressed in macrophages freshly isolated from ascites. This raises the possibility that ascites prevents IL12B expression by perturbing NFκB binding to chromatin. Here, we show that ascites-mediated suppression of IL12B induction by LPS plus IFNγ in primary human macrophages is rapid, and that suppression can be reversible after ascites withdrawal. Nuclear translocation of the NFκB transcription factors c-REL and p65 was strongly reduced by ascites. Surprisingly, however, their binding to the IL12B locus and to CXCL10, a second NFκB target gene, was unaltered, and the induction of CXCL10 transcription was not suppressed by ascites. These findings indicate that, despite its reduced nuclear translocation, NFκB function is not generally impaired by ascites, suggesting that ascites-borne signals target additional pathways to suppress IL12B induction. Consistent with these data, IL-10, a clinically relevant constituent of ascites and negative regulator of NFκB translocation, only partially recapitulated IL12B suppression by ascites. Finally, restoration of a defective IL-12 response by appropriate culture conditions was observed only in macrophages from a subset of donors, which may have important implications for the understanding of patient-specific immune responses.

Funding information:
  • NIGMS NIH HHS - GM71801(United States)

The SS18-SSX Fusion Oncoprotein Hijacks BAF Complex Targeting and Function to Drive Synovial Sarcoma.

  • McBride MJ
  • Cancer Cell
  • 2018 Jun 11

Literature context:


Abstract:

Synovial sarcoma (SS) is defined by the hallmark SS18-SSX fusion oncoprotein, which renders BAF complexes aberrant in two manners: gain of SSX to the SS18 subunit and concomitant loss of BAF47 subunit assembly. Here we demonstrate that SS18-SSX globally hijacks BAF complexes on chromatin to activate an SS transcriptional signature that we define using primary tumors and cell lines. Specifically, SS18-SSX retargets BAF complexes from enhancers to broad polycomb domains to oppose PRC2-mediated repression and activate bivalent genes. Upon suppression of SS18-SSX, reassembly of BAF47 restores enhancer activation, but is not required for proliferative arrest. These results establish a global hijacking mechanism for SS18-SSX on chromatin, and define the distinct contributions of two concurrent BAF complex perturbations.

Funding information:
  • NIAID NIH HHS - R01 AI076479(United States)

The Mitochondrial-Encoded Peptide MOTS-c Translocates to the Nucleus to Regulate Nuclear Gene Expression in Response to Metabolic Stress.

  • Kim KH
  • Cell Metab.
  • 2018 Jun 27

Literature context:


Abstract:

Cellular homeostasis is coordinated through communication between mitochondria and the nucleus, organelles that each possess their own genomes. Whereas the mitochondrial genome is regulated by factors encoded in the nucleus, the nuclear genome is currently not known to be actively controlled by factors encoded in the mitochondrial DNA. Here, we show that MOTS-c, a peptide encoded in the mitochondrial genome, translocates to the nucleus and regulates nuclear gene expression following metabolic stress in a 5'-adenosine monophosphate-activated protein kinase (AMPK)-dependent manner. In the nucleus, MOTS-c regulated a broad range of genes in response to glucose restriction, including those with antioxidant response elements (ARE), and interacted with ARE-regulating stress-responsive transcription factors, such as nuclear factor erythroid 2-related factor 2 (NFE2L2/NRF2). Our findings indicate that the mitochondrial and nuclear genomes co-evolved to independently encode for factors to cross-regulate each other, suggesting that mitonuclear communication is genetically integrated.

Funding information:
  • NIDDK NIH HHS - DK074310(United States)

Status epilepticus triggers long-lasting activation of complement C1q-C3 signaling in the hippocampus that correlates with seizure frequency in experimental epilepsy.

  • Schartz ND
  • Neurobiol. Dis.
  • 2018 Jun 25

Literature context:


Abstract:

Status epilepticus (SE) triggers a myriad of neurological alterations that include unprovoked seizures, temporal lobe epilepsy (TLE), and cognitive deficits. Although SE-induced loss of hippocampal dendritic structures and synaptic remodeling are often associated with this pathophysiology, the underlying mechanisms remain elusive. Recent evidence points to the classical complement pathway as a potential mechanism. Signaling through the complement protein C1q to C3, which is cleaved into smaller biologically active fragments including C3b and iC3b, contributes to the elimination of synaptic structures in the normal developing brain and in models of neurodegenerative disorders. We recently found increased protein levels of C1q and iC3b fragments in human drug-resistant epilepsy. Thus, to identify a potential role for C1q-C3 in SE-induced epilepsy, we performed a temporal analysis of C1q protein levels and C3 cleavage in the hippocampus along with their association to seizures and hippocampal-dependent cognitive functions in a rat model of SE and acquired TLE. We found significant increases in the levels of C1q, C3, and iC3b in the hippocampus at 2-, 3- and 5-weeks after SE relative to controls (p<0.05). In the SE group, greater iC3b levels were significantly correlated with higher seizure frequency (p<0.05). Together, these data support that hyperactivation of the classical complement pathway after SE parallels the progression of epilepsy. Future studies will determine whether C1q-C3 signaling contributes to epileptogenic synaptic remodeling in the hippocampus.

IRF8 Regulates Transcription of Naips for NLRC4 Inflammasome Activation.

  • Karki R
  • Cell
  • 2018 May 3

Literature context:


Abstract:

Inflammasome activation is critical for host defenses against various microbial infections. Activation of the NLRC4 inflammasome requires detection of flagellin or type III secretion system (T3SS) components by NLR family apoptosis inhibitory proteins (NAIPs); yet how this pathway is regulated is unknown. Here, we found that interferon regulatory factor 8 (IRF8) is required for optimal activation of the NLRC4 inflammasome in bone-marrow-derived macrophages infected with Salmonella Typhimurium, Burkholderia thailandensis, or Pseudomonas aeruginosa but is dispensable for activation of the canonical and non-canonical NLRP3, AIM2, and Pyrin inflammasomes. IRF8 governs the transcription of Naips to allow detection of flagellin or T3SS proteins to mediate NLRC4 inflammasome activation. Furthermore, we found that IRF8 confers protection against bacterial infection in vivo, owing to its role in inflammasome-dependent cytokine production and pyroptosis. Altogether, our findings suggest that IRF8 is a critical regulator of NAIPs and NLRC4 inflammasome activation for defense against bacterial infection.

Funding information:
  • NCI NIH HHS - R01 CA163507()
  • NIA NIH HHS - AG032375(United States)
  • NIAID NIH HHS - R01 AI124346()
  • NIAID NIH HHS - R37 AI101935()
  • NIAMS NIH HHS - R01 AR056296()

Aldose Reductase Mediates NLRP3 Inflammasome-Initiated Innate Immune Response in Hyperglycemia-Induced Thp1 Monocytes and Male Mice.

  • Pal PB
  • Endocrinology
  • 2017 Oct 1

Literature context:


Abstract:

Despite recent studies that show oxidative stress-generated reactive oxygen species (ROS) regulate NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome-mediated innate immune response in various diabetic complications, the mechanism by which ROS activate innate immune response is not well understood. We have shown previously that aldose reductase (AR), besides reducing glucose, reduces lipid aldehydes and their glutathione conjugates and participates in various oxidative stress-induced inflammatory pathways. To understand the role of AR in ROS-induced innate immune response, we have investigated the mechanism(s) by which AR activates hyperglycemia-induced NLRP3 inflammsome-initiated innate immune response in Thp1 monocytes and in streptozotocin (STZ)-induced diabetic mice. In Thp1 monocytes, inhibition or ablation of AR prevented high-glucose-induced activation of NLRP3 inflammasome and caspase-1 and release of the innate immune cytokines interleukin (IL)-1β and IL-18. AR inhibition in Thp1 cells also prevented the high-glucose-induced generation of ROS, influx of Ca2+, efflux of K+, and activation of Lyn, Syk, and PI3K. Furthermore, the AR inhibitor fidarestat prevented the expression of NLRP inflammasome components in STZ-induced diabetic mouse heart and aorta, and also prevented the release of various cytokines in the serum. Collectively, our data suggest that AR regulates hyperglycemia-induced NLRP3 inflammasome-mediated innate immune response by altering the ROS/Lyn/Syk/PI3K/Ca2+/K+ signals.

The Role of Sirt1 in Epileptogenesis.

  • Hall AM
  • eNeuro
  • 2017 Oct 30

Literature context:


Abstract:

The mechanisms by which brain insults lead to subsequent epilepsy remain unclear. Insults, including trauma, stroke, tumors, infections, and long seizures [status epilepticus (SE)], create a neuronal state of increased metabolic demand or decreased energy supply. Neurons express molecules that monitor their metabolic state, including sirtuins (Sirts). Sirtuins deacetylate cytoplasmic proteins and nuclear histones, and their epigenetic modulation of the chromatin governs the expression of many genes, influencing neuronal properties. Thus, sirtuins are poised to enduringly modulate neuronal properties following SE, potentially contributing to epileptogenesis, a hypothesis supported by the epilepsy-attenuating effects of blocking a downstream target of Sirt1, Neuron-Restrictive Silencer Factor (NRSF) also know as REST (RE1-Silencing Transcription factor). Here we used an adult male rat model of epileptogenesis provoked by kainic acid-induced SE (KA-SE). We assessed KA-SE-provoked Sirt1 activity, infused a Sirt1 inhibitor (EX-527) after KA-SE, and examined for epileptogenesis using continuous digital video-EEG. Sirt1 activity, measured using chromatin immunoprecipitation for Sirt1 binding at a target gene, increased rapidly after SE. Post hoc infusion of the Sirt1 inhibitor prevented Sirt1-mediated repression of a target gene. Blocking Sirt1 activity transiently after KA-SE did not significantly influence the time- course and all of the parameters of epilepsy development. Specifically, latency to first seizure and seizure number, duration, and severity (using the Racine scale and EEG measures) as well as the frequency and duration of interictal spike series, were all unchanged. KA-SE provoked a robust inflammatory response and modest cell loss, yet neither was altered by blocking Sirt1. In conclusion, blocking Sirt1 activity after KA-SE does not abrogate epilepsy development, suggesting that the mechanisms of such acquired epileptogenesis are independent of Sirt1 function.

Funding information:
  • NINDS NIH HHS - R01 NS035439()
  • NINDS NIH HHS - R01 NS078279()

Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function.

  • Sakamaki JI
  • Mol. Cell
  • 2017 May 18

Literature context:


Abstract:

Autophagy is a membrane-trafficking process that directs degradation of cytoplasmic material in lysosomes. The process promotes cellular fidelity, and while the core machinery of autophagy is known, the mechanisms that promote and sustain autophagy are less well defined. Here we report that the epigenetic reader BRD4 and the methyltransferase G9a repress a TFEB/TFE3/MITF-independent transcriptional program that promotes autophagy and lysosome biogenesis. We show that BRD4 knockdown induces autophagy in vitro and in vivo in response to some, but not all, situations. In the case of starvation, a signaling cascade involving AMPK and histone deacetylase SIRT1 displaces chromatin-bound BRD4, instigating autophagy gene activation and cell survival. Importantly, this program is directed independently and also reciprocally to the growth-promoting properties of BRD4 and is potently repressed by BRD4-NUT, a driver of NUT midline carcinoma. These findings therefore identify a distinct and selective mechanism of autophagy regulation.

Methyl CpG level at distal part of heat-shock protein promoter HSP70 exhibits epigenetic memory for heat stress by modulating recruitment of POU2F1-associated nucleosome-remodeling deacetylase (NuRD) complex.

  • Kisliouk T
  • J. Neurochem.
  • 2017 May 9

Literature context:


Abstract:

Depending on its stringency, exposure to heat in early life leads to either resilience or vulnerability to heat stress later in life. We hypothesized that epigenetic alterations in genes belonging to the cell proteostasis pathways are attributed to long-term responses to heat stress. Epigenetic regulation of the mRNA expression of the molecular chaperone heat-shock protein (HSP) 70 (HSPA2) was evaluated in the chick hypothalamus during the critical period of thermal-control establishment on day 3 post-hatch and during heat challenge on day 10. Both the level and duration of HSP70 expression during heat challenge a week after heat conditioning were more pronounced in chicks conditioned under harsh versus mild temperature. Analyzing different segments of the promoter in vitro indicated that methylation of a distal part altered its transcriptional activity. In parallel, DNA-methylation level of this segment in vivo was higher in harsh- compared to mild-heat-conditioned chicks. Hypermethylation of the HSP70 promoter in high-temperature-conditioned chicks was accompanied by a reduction in both POU Class 2 Homeobox 1 (POU2F1) binding and recruitment of the nucleosome remodeling deacetylase (NuRD) chromatin-remodeling complex. As a result, histone H3 acetylation levels at the HSP70 promoter were higher in harsh-temperature-conditioned chicks than in their mild-heat-conditioned counterparts. These results suggest that methylation level of a distal part of the HSP70 promoter and POU2F1 recruitment may reflect heat-stress-related epigenetic memory and may be useful in differentiating between individuals that are resilient or vulnerable to stress.

An Intrinsic Epigenetic Barrier for Functional Axon Regeneration.

  • Weng YL
  • Neuron
  • 2017 Apr 19

Literature context:


Abstract:

Mature neurons in the adult peripheral nervous system can effectively switch from a dormant state with little axonal growth to robust axon regeneration upon injury. The mechanisms by which injury unlocks mature neurons' intrinsic axonal growth competence are not well understood. Here, we show that peripheral sciatic nerve lesion in adult mice leads to elevated levels of Tet3 and 5-hydroxylmethylcytosine in dorsal root ganglion (DRG) neurons. Functionally, Tet3 is required for robust axon regeneration of DRG neurons and behavioral recovery. Mechanistically, peripheral nerve injury induces DNA demethylation and upregulation of multiple regeneration-associated genes in a Tet3- and thymine DNA glycosylase-dependent fashion in DRG neurons. In addition, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult CNS is attenuated upon Tet1 knockdown. Together, our study suggests an epigenetic barrier that can be removed by active DNA demethylation to permit axon regeneration in the adult mammalian nervous system.

Funding information:
  • NIGMS NIH HHS - T32 GM007814()

Selective Chemical Inhibition of PGC-1α Gluconeogenic Activity Ameliorates Type 2 Diabetes.

  • Sharabi K
  • Cell
  • 2017 Mar 23

Literature context:


Abstract:

Type 2 diabetes (T2D) is a worldwide epidemic with a medical need for additional targeted therapies. Suppression of hepatic glucose production (HGP) effectively ameliorates diabetes and can be exploited for its treatment. We hypothesized that targeting PGC-1α acetylation in the liver, a chemical modification known to inhibit hepatic gluconeogenesis, could be potentially used for treatment of T2D. Thus, we designed a high-throughput chemical screen platform to quantify PGC-1α acetylation in cells and identified small molecules that increase PGC-1α acetylation, suppress gluconeogenic gene expression, and reduce glucose production in hepatocytes. On the basis of potency and bioavailability, we selected a small molecule, SR-18292, that reduces blood glucose, strongly increases hepatic insulin sensitivity, and improves glucose homeostasis in dietary and genetic mouse models of T2D. These studies have important implications for understanding the regulatory mechanisms of glucose metabolism and treatment of T2D.

Funding information:
  • NHGRI NIH HHS - U54 HG005032()
  • NIDA NIH HHS - R03 DA032468()
  • NIDDK NIH HHS - F32 DK102293()
  • NIDDK NIH HHS - R01 DK040936()
  • NIDDK NIH HHS - R01 DK069966()
  • NIDDK NIH HHS - R24 DK080261()
  • NIDDK NIH HHS - U2C DK059635()