Regulation Of Immediate Early Genes Research Articles

Infusion of etoposide in the CA1 disrupts hippocampal immediate early gene expression and hippocampus-dependent learning

Tight regulation of immediate early gene (IEG) expression is important for synaptic plasticity, learning, and memory. Recent work has suggested that DNA double strand breaks (DSBs) may have an adaptive role in post-mitotic cells to induce IEG expression. Physiological activity in cultured neurons as well as behavioral training leads to increased DSBs and subsequent IEG expression. Additionally, infusion of etoposide—a common cancer treatment that induces DSBs—impairs trace fear memory. Here, we assessed the effects of hippocampal infusion of 60 ng of etoposide on IEG expression, learning, and memory in 3–4 month-old C57Bl/6J mice. Etoposide altered expression of the immediate early genes cFos and Arc in the hippocampus and impaired hippocampus-dependent contextual fear memory. These data add to the growing evidence that DSBs play an important role in IEG expression, learning, and memory, opening avenues for developing novel treatment strategies for memory-related disorders.

Bivalent-histone-marked immediate-early gene regulation is vital for VEGF-responsive angiogenesis.

Endothelial cells (ECs) are phenotypically heterogeneous, mainly due to their dynamic response to the tissue microenvironment. Vascular endothelial cell growth factor (VEGF), the best-known angiogenic factor, activates calcium-nuclear factor of activated Tcells (NFAT) signaling following acute angiogenic gene transcription. Here, we evaluate the global mapping of VEGF-mediated dynamic transcriptional events, focusing on major histone-code profiles using chromatin immunoprecipitation sequencing (ChIP-seq). Remarkably, the gene loci of immediate-early angiogenic transcription factors (TFs) exclusively acquire bivalent H3K4me3-H3K27me3 double-positive histone marks after the VEGF stimulus. Moreover, NFAT-associated Pax transactivation domain-interacting protein (PTIP) directs bivalently marked TF genes transcription through a limited polymerase II running. The non-canonical polycomb1 variant PRC1.3 specifically binds to and allows the transactivation of PRC2-enriched bivalent angiogenic TFs until conventional PRC1-mediated gene silencing is achieved. Knockdown of these genes abrogates post-natal aberrant neovessel formation via the selective inhibition of indispensable bivalent angiogenic TF gene transcription. Collectively, the reported dynamic histone mark landscape may uncover the importance of immediate-early genes and the development of advanced anti-angiogenic strategies.

Abstract 1088: Elucidating the regulation of delayed-early gene targets of sustained MAPK signaling

Abstract RAS and its downstream effector, BRAF, are commonly mutated proto-oncogenes in many types of human cancer. Mutationally activated RAS or BRAF signal through the MEK→ERK MAP kinase (MAPK) pathway to regulate key cancer cell hallmarks such as cell division cycle progression, reduced programmed cell death, and enhanced cell motility. Amongst the list of RAS/RAF-regulated genes are those encoding integrins, alpha-beta heterodimeric transmembrane proteins that regulate cell adhesion to the extracellular matrix. Altered integrin expression has been linked to the acquisition of more aggressive behavior by melanoma, lung, and breast cancer cells leading to diminished survival of cancer patients. We have previously documented the ability of the RAS-activated MAPK pathway to induce the expression of ITGB3 encoding integrin β3 in several different cell types. RAS/RAF-mediated induction of ITGB3 mRNA requires sustained, high-level activation of RAF→MEK→ERK signaling mediated by oncogene activation and is classified as “delayed-early”, in that it is sensitive to the protein synthesis inhibitor cycloheximide. However, to date, the regulatory mechanisms that allow for induced ITGB3 downstream of sustained, high-level activation of MAPK signaling remains obscure. We have identified over 300 DEGs, including those expressing additional cell surface proteins, that display similar regulatory characteristics as ITGB3. We use integrin β3 as a model to test our hypothesis that there is a different mechanism of regulation for delayed-early genes (DEG) compared to the canonical regulation of Immediate-Early genes. There are three regions in the chromatin upstream of the ITGB3 that become more accessible during RAF activation. We are relating the chromatin changes seen during RAF activation to active enhancer histone marks. To elucidate the essential genes of this regulation process, we are employing the use of a genome-wide CRISPR knockout screen. The work presented from this abstract will help elucidate the regulatory properties of oncogenic progression in BRAF mutated cancers that could lead to the identification of biomarkers. Citation Format: Kali J. Dale, Martin McMahon. Elucidating the regulation of delayed-early gene targets of sustained MAPK signaling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1088.

Evaluation of brain angiotensin and plasticity related genes following fear memory retrieval

BackgroundFear learning is an adaptive response to traumatic events and gradual decline of fear response on repeated exposure to stimuli can lead to fear extinction. However, a single presentation of a cue threat can reactive the fear memory through an alternative process called reconsolidation. Here we sought to investigate the role of the renin angiotensin systems (RAS) in reconsolidation to determine whether recent findings from RAS extinction studies in rodents and humans might also be explained by interference with reconsolidation.MethodsUsing Pavlovian auditory fear conditioning, a single retrieval tone was presented to initiate memory recall. It has been previously shown that fear memories, after retrieval, are in a labile state for a time‐limited window of six hours and require protein synthesis for consolidation. Without any pharmacological intervention, mRNA levels at early (40mins) and late (4hr) phases of reconsolidation were compared to examine gene expression pattern changes in common RAS and plasticity genes in different brain regions during memory reconsolidation.ResultsTo validate our behavior model of single retrieval presentation, we compared the expression of immediate early genes (IEGs) ‐ cfos, Egr‐1 and Arc in the basolateral amygdala (BLA) – critical for fear learning and memory. These IEG’s were induced rapidly at 40mins with 2–3‐fold (p<0.001; n=12) increases in mRNA expression and returned to the baseline within 4hrs comparable to animals which did not receive the retrieval tone. The RAS distribution in the brain follows a distinct pattern which we confirmed in our lab by measuring the expression level of angiotensinogen (Agt) in telencephalon (forebrain) and diencephalon (mid and hindbrain) relative to liver. There is a 3‐fold increase in Agt (p=0.025, n=6) mRNA levels at 40mins. Angiotensin converting enzyme (Ace) mRNA levels decreased by half (p = 0.0169, n=6) and consequently Ace2 levels increased 2‐fold at early reconsolidation phase in BLA. The angiotensin type1 receptor (Agtr1) showed increased expression (p=0.0213, n=10) at 4hrs whereas angiotensin type 2 receptor (Agtr2) had steady rise at both 40mins and 4hrs (p=0.0016, n=10). The BLA mRNA levels for tachykinin receptor 3 (Tac3r), previously shown to modulate fear memory and angiotensin II, was elevated by 3‐fold at both 40mins and 4hrs. We also examined the CA1 hippocampal region, but unlike the BLA, there was no significant change in the expression levels of these.ConclusionHere we identified two distinct pathways for brain RAS in the BLA during a time course of memory reconsolidation, including early phase activation of Ace2 and a later phase increase in angiotensin receptors (Agtr1 and Agtr2) mRNA levels. These data suggest that, similar to transcriptional regulation of IEGs in the BLA, angiotensin‐related genes undergo dynamic regulation during reconsolidation following fear memory retrieval.Support or Funding InformationNIH1R01HL137103‐01, AHA15CSA24340001 Evaluation of brain angiotensin and plasticity related genes following fear memory retrieval in BLA (− decrease; + increase) Gene Gene Symbol Changes Peak Time 1. Angiotensin Type 1 Receptor AT1R + 4 hour 2. Angiotensin Type 2 Receptor AT2R + 40 mins and 4hrs 3. Angiotensin Converting Enzyme Ace − 40 mins 4. Angiotensin Converting Enzyme 2 Ace 2 + 40 mins 5. Angiotensinogen Agt + 40 mins 6. Tachykinin Receptor 3 Tac3R ++ 40mins and 4 hrs 7. IEG Fos ++ 40mins 8. IEG Arc ++ 40mins 9. IEG Egr‐1 ++ 40mins

Immune modulation by chronic exposure to waterpipe smoke and immediate-early gene regulation in murine lungs

ObjectiveWe investigated the effects of chronic waterpipe (WP) smoke on pulmonary function and immune response in a murine model using a research-grade WP and the effects of acute exposure on...

Epigenetic regulation of immediate-early gene Nr4a2/Nurr1 in the medial habenula during reinstatement of cocaine-associated behavior.

Propensity to relapse following long periods of abstinence is a key feature of substance use disorder. Drugs of abuse, such as cocaine, cause long-term changes in the neural circuitry regulating reward, motivation, and memory processes through dysregulation of various molecular mechanisms, including epigenetic regulation of activity-dependent gene expression. Underlying drug-induced changes to neural circuit function are the molecular mechanisms regulating activity-dependent gene expression. Of note, histone acetyltransferases and histone deacetylases (HDACs), powerful epigenetic regulators of gene expression, are dysregulated following both acute and chronic cocaine exposure and are linked to cocaine-induced changes in neural circuit function. To better understand the effect of drug-induced changes on epigenetic function and behavior, we investigated HDAC3-mediated regulation of Nr4a2/Nurr1 in the medial habenula, an understudied pathway in cocaine-associated behaviors. Nr4a2, a transcription factor critical in cocaine-associated behaviors and necessary for MHb development, is enriched in the cholinergic cell-population of the MHb; yet, the role of NR4A2 within the MHb in the adult brain remains elusive. Here, we evaluated whether epigenetic regulation of Nr4a2 in the MHb has a role in reinstatement of cocaine-associated behaviors. We found that HDAC3 disengages from Nr4a2 in the MHb in response to cocaine-primed reinstatement. Whereas enhancing HDAC3 function in the MHb had no effect on reinstatement, we found, using a dominant-negative splice variant (NURR2C), that loss of NR4A2 function in the MHb blocked reinstatement behaviors. These results show for the first time that regulation of NR4A2 function in the MHb is critical in relapse-like behaviors.

The DNA Repair-Associated Protein Gadd45γ Regulates the Temporal Coding of Immediate Early Gene Expression within the Prelimbic Prefrontal Cortex and Is Required for the Consolidation of Associative Fear Memory.

We have identified a member of the growth arrest and DNA damage (Gadd45) protein family, Gadd45γ, which is known to be critically involved in DNA repair, as a key player in the regulation of immediate early gene (IEG) expression underlying the consolidation of associative fear memory in adult male C57BL/6 mice. Gadd45γ temporally influences learning-induced IEG expression in the prelimbic prefrontal cortex (PLPFC) through its interaction with DNA double-strand break (DSB)-mediated changes in DNA methylation. Our findings suggest a two-hit model of experience-dependent IEG activity and learning that comprises (1) a first wave of IEG expression governed by DSBs and followed by a rapid increase in DNA methylation, and (2) a second wave of IEG expression associated with the recruitment of Gadd45γ and active DNA demethylation at the same site, which is necessary for memory consolidation.SIGNIFICANCE STATEMENT How does the pattern of immediate early gene transcription in the brain relate to the storage and accession of information, and what controls these patterns? This paper explores how Gadd45γ, a gene that is known to be involved with DNA modification and repair, regulates the temporal coding of IEGs underlying associative learning and memory. We reveal that, during fear learning, Gadd45γ serves to act as a coordinator of IEG expression and subsequent memory consolidation by directing temporally specific changes in active DNA demethylation at the promoter of plasticity-related IEGs.

Smoking alters the normal transcriptome of healthy human gingiva

1. Immune modulation by chronic exposure to waterpipe smoke and immediate-early gene regulation in murine lungs Hermes Reyes-Caballero, Bongsoo Park, Jeffrey Loube, Ian Sanchez, Vinesh Vinayachandran, Youngshim Choi, Juhyung Woo, Justin Edwards, Marielle Brinkman, Thomas Sussan, Wayne Mitzner, Shyam Biswal Tobacco Control CrossRef

Gene regulation in the immediate-early response process.

Immediate-early genes (IEGs) can be activated and transcribed within minutes after stimulation, without the need for de novo protein synthesis, and they are stimulated in response to both cell-extrinsic and cell-intrinsic signals. Extracellular signals are transduced from the cell surface, through receptors activating a chain of proteins in the cell, in particular extracellular-signal-regulated kinases (ERKs), mitogen-activated protein kinases (MAPKs) and members of the RhoA-actin pathway. These communicate through a signaling cascade by adding phosphate groups to neighboring proteins, and this will eventually activate and translocate TFs to the nucleus and thereby induce gene expression. The gene activation also involves proximal and distal enhancers that interact with promoters to simulate gene expression. The immediate-early genes have essential biological roles, in particular in stress response, like the immune system, and in differentiation. Therefore they also have important roles in various diseases, including cancer development. In this paper we summarize some recent advances on key aspects of the activation and regulation of immediate-early genes.

Cyclic AMP pathways leading to immediate‐early gene activation in neuroendocrine cells: relevance to neurotransmitter signaling through the D1 dopamine and PAC1 neuropeptide receptors

Gs‐coupled GPCR signaling in the NS‐1 pheochromocytoma cell line leads to the activation of three distinct cyclic AMP sensors, Epac, PKA and NCS/Rapgef2 mediate distinct cellular outputs: p38‐dependent growth arrest; CREB‐dependent cell survival and neuron‐specific gene expression; and ERK‐dependent neuritogenesis, respectively (Emery et al., Sci. Sig. 6: ra51, 2013; Emery et al., JBC 289: 10126, 2014). Expression of D1 dopamine receptors from a stably‐integrated, CMV promoter‐driven expression cassette in NS‐1 cells causes cyclic AMP elevation and activation of all three of these pathways, leading to D1 receptor‐dependent activation of all three downstream pathways, as previously reported for activation of the endogenous PAC1hop receptor by the neuropeptide PACAP. To investigate which of the three pathways might mediate cAMP‐dependent immediate‐early gene (IEG) transcription relevant to neurotransmitter action in vivo, we examined genome‐wide transcriptional regulation by cyclic AMP in NS‐1 cells, using microarray analysis. Treatment of NS‐1 cells for one hour with the cell‐permeant cyclic AMP analog 8‐CPT‐cAMP (100 micromolar), an equipotent activator of Epac2, PKA and NCS/Rapgef2 (Emery et al., JBC 289: 10126, 2014) resulted in significant up‐regulation of 174 mRNAs. Of these, ten encoded transcription factors, and six of these encoded IEGs: Ier‐3/Egr3, Zif268/Egr1, Nr4a1, Nr4a3, c‐Fos, and JunB. Cyclic AMP‐dependent up‐regulation of the Ier‐3/Egr2 and Zig268/Egr1 genes, but not the Nr1a1, Nr4a3, or JunB genes, was ERK‐dependent (blocked by the MEK inhibitor U0126, at 10 micromolar), while up‐regulation of the c‐Fos gene appeared to be partially ERK‐dependent. These results suggest that regulation of IEG expression by D1 and PAC1 receptor stimulation may involve two cAMP‐dependent pathways, one mediated by NCS/Rapgef2, and the other by either Epac or PKA. These novel signaling pathways may be relevant to IEG regulation by dopamine in the striatum in vivo (Gerfen et al., J. Neurosci. 15: 8167, 1995) and by the stress‐regulatory neuropeptide PACAP in hypothalamus and extended amygdala (Stroth and Eiden, Neuroscience 165: 1025, 2010; Emery and Eiden, FASEB J. 26: 3199, 2012).Support or Funding InformationSupported by NIMH‐IRP project ZO1‐MH002386

Growth differentiation factor-15/macrophage inhibitory cytokine-1 induction after kidney and lung injury.

The immunoregulatory cytokine macrophage inhibitory cytokine-1 (MIC-1), a divergent TGF-beta family member, and its murine ortholog, growth/differentiation factor-15 (GDF-15) are induced in hepatocytes by surgical and chemical injury and heat shock. To better understand the in vivo role this factor plays in organ injury, we examined the regulation of GDF-15 in murine models of kidney and lung injury. We demonstrate herein induction of GDF-15/MIC-1 after surgical, toxic/genotoxic, ischemic, and hyperoxic kidney or lung injury. Gdf15 induction was independent of protein synthesis, a hallmark of immediate-early gene regulation. Although TNF induced GDF-15 expression, injury-elicited Gdf15 expression was not reduced in mice deficient for both TNF receptor subtype. Furthermore, although the stress sensor p53 is known to induce GDF-15/MIC-1 expression, injury-elicited Gdf15 expression was unchanged in p53-null mice. Our results demonstrate that GDF-15 induction after organ injury is a hallmark of many tissues. These data demonstrate that GDF-15/MIC-1 is an early mediator of the injury response in kidney and lung that might regulate inflammation, cell survival, proliferation, and apoptosis in a variety of injured tissues and disease processes.

Photic stimulation of the suprachiasmatic nucleus via the non-visual optic system. A gene expression study in the blind Crx-/- mouse.

The visual system of vertebrates consists of an image-forming and a non-image-forming optic system; the image-forming optic system involves the classic photoreceptors, the rods and cones, whereas the non-image-forming optic system involves the melanopsin-containing retinal ganglion cells. Both optic systems make direct neuroanatomical connections to the suprachiasmatic nucleus (SCN) in the hypothalamus in which the biological clock of vertebrates is located. The rhythmic output from SCN neurons is entrained by light via the retina and the retinohypothalamic tract. The response of exposure to light during the subjective night is an immediate expression of several early response genes in the SCN. We show, by quantitative real-time polymerase chain reaction, that the amount of melanopsin mRNA in the retinal ganglion cells is preserved in the blind Crx(-/-) mouse with degenerated classic photoreceptors. At zeitgeber time 16, the Crx(-/-) and wild-type mice were exposed to 1 h of light. This resulted in a strong up-regulation of the immediate early genes Nr4a1, Erg, and Rrad in the SCN of both genotypes. Light stimulation during the subjective night resulted in a strong up-regulation of c-fos in both genotypes with a significantly higher up-regulation in the blind Crx(-/-) mouse. Expression of Grp and Vip, the genes for two classic peptides located in the SCN, was not influenced by light stimulation. The data strongly indicate the involvement of the melanopsin-based non-visual optic system in the regulation of immediate early genes in the SCN.

Alterations in Brain-Derived Neurotrophic Factor in the Mouse Hippocampus Following Acute but Not Repeated Benzodiazepine Treatment

Benzodiazepines (BZs) are safe drugs for treating anxiety, sleep, and seizure disorders, but their use also results in unwanted effects including memory impairment, abuse, and dependence. The present study aimed to reveal the molecular mechanisms that may contribute to the effects of BZs in the hippocampus (HIP), an area involved in drug-related plasticity, by investigating the regulation of immediate early genes following BZ administration. Previous studies have demonstrated that both brain derived neurotrophic factor (BDNF) and c-Fos contribute to memory- and abuse-related processes that occur within the HIP, and their expression is altered in response to BZ exposure. In the current study, mice received acute or repeated administration of BZs and HIP tissue was analyzed for alterations in BDNF and c-Fos expression. Although no significant changes in BDNF or c-Fos were observed in response to twice-daily intraperitoneal (i.p.) injections of diazepam (10 mg/kg + 5 mg/kg) or zolpidem (ZP; 2.5 mg/kg + 2.5 mg/kg), acute i.p. administration of both triazolam (0.03 mg/kg) and ZP (1.0 mg/kg) decreased BDNF protein levels within the HIP relative to vehicle, without any effect on c-Fos. ZP specifically reduced exon IV-containing BDNF transcripts with a concomitant increase in the association of methyl-CpG binding protein 2 (MeCP2) with BDNF promoter IV, suggesting that MeCP2 activity at this promoter may represent a ZP-specific mechanism for reducing BDNF expression. ZP also increased the association of phosphorylated cAMP response element binding protein (pCREB) with BDNF promoter I. Future work should examine the interaction between ZP and DNA as the cause for altered gene expression in the HIP, given that BZs can enter the nucleus and intercalate into DNA directly.

Anesthesia-induced hypothermia mediates decreased ARC gene and protein expression through ERK/MAPK inactivation

Several anesthetics have been reported to suppress the transcription of a number of genes, including Arc, also known as Arg3.1, an immediate early gene that plays a significant role in memory consolidation. The purpose of this study was to explore the mechanism of anesthesia-mediated depression in Arc gene and protein expression. Here, we demonstrate that isoflurane or propofol anesthesia decreases hippocampal Arc protein expression in rats and mice. Surprisingly, this change was secondary to anesthesia-induced hypothermia. Furthermore, we confirm in vivo and in vitro that hypothermia per se is directly responsible for decreased Arc protein levels. This effect was the result of the decline of Arc mRNA basal levels following inhibition of ERK/MAPK by hypothermia. Overall, our results suggest that anesthesia-induced hypothermia leads to ERK inhibition, which in turns decreases Arc levels. These data give new mechanistic insights on the regulation of immediate early genes by anesthesia and hypothermia.

Inositol polyphosphate multikinase is a transcriptional coactivator required for immediate early gene induction

Profound induction of immediate early genes (IEGs) by neural activation is a critical determinant for plasticity in the brain, but intervening molecular signals are not well characterized. We demonstrate that inositol polyphosphate multikinase (IPMK) acts noncatalytically as a transcriptional coactivator to mediate induction of numerous IEGs. IEG induction by electroconvulsive stimulation is virtually abolished in the brains of IPMK-deleted mice, which also display deficits in spatial memory. Neural activity stimulates binding of IPMK to the histone acetyltransferase CBP and enhances its recruitment to IEG promoters. Interestingly, IPMK regulation of CBP recruitment and IEG induction does not require its catalytic activities. Dominant-negative constructs, which prevent IPMK-CBP binding, substantially decrease IEG induction. As IPMK is ubiquitously expressed, its epigenetic regulation of IEGs may influence diverse nonneural and neural biologic processes.

Serum regulation of Id1 expression by a BMP pathway and BMP responsive element

Immediate early genes (IEGs) are expressed upon re-entry of quiescent cells into the cell cycle following serum stimulation. These genes are involved in growth control and differentiation and hence their expression is tightly controlled. Many IEGs are regulated through Serum Response Elements (SREs) in their promoters, which bind Serum Response Factor (SRF). However, many other IEGs do not have SREs in their promoters and their serum regulation is poorly understood. We have identified SRF-independent IEGs in SRF-depleted fibroblasts. One of these, Id1, was examined more closely. We mapped a serum responsive element in the Id1 promoter and find that it is identical to a BMP responsive element (BRE). The Id1 BRE is necessary and sufficient for the serum regulation of Id1. Inhibition of the BMP pathway by siRNA depletion of Smad 4, treatment with the BMP antagonist noggin, or the BMP receptor inhibitor dorsomorphin blocked serum induction of Id1. Further, BMP2 is sufficient to induce Id1 expression. Given reports that SRC inhibitors can block Id1 expression, we tested the SRC inhibitor, AZD0530, and found that it inhibits the serum activation of Id1. Surprisingly, this inhibition is independent of SRC or its family members. Rather, we show that AZD0530 directly inhibits the BMP type I receptors. Serum induction of the Id1 related gene Id3 also required the BMP pathway. Given these and other findings we conclude that the Id family of IEGs is regulated by BMPs in serum through similar BREs. This represents a second pathway for serum regulation of IEGs.

209 TRANSCRIPTOME ANALYSIS IDENTIFIES A NOVEL MECHANISM OF CAMP-MEDIATED GROWTH ARREST IN VSMC: PKA AND EPAC SYNERGISE TO SUPPRESS EGR1 EXPRESSION

Cyclic AMP signalling promotes VSMC quiescence in healthy vessels and during vascular healing following injury. We recently demonstrated that cAMP-sensitive PKA and Epac signalling pathways synergise to inhibit VSMC proliferation but the underlying mechanisms are not understood. Using PKA and Epac-selective agonists and microarray analysis we tested the hypothesis that PKA and Epac block VSMC proliferation via coordinate regulation of immediate early genes required for cell-cycle progression. Epac agonist significantly enhanced PKA-dependent inhibition of EGR1 mRNA (4.6±0.38 fold for PKA vs 13.8±1.09 fold for PKA+EPAC, p cAMP-dependent inhibition (3.9±0.8 fold, p In summary, our results demonstrate that PKA and Epac synergise to inhibit VSMC proliferation by rapidly inhibiting EGR1 expression via a mechanism that probably involves inhibition of SRF activity. This also provides a mechanistic explanation for the CREB and MAPK-independent anti-mitogenic effects of cAMP in VSMC.

Histone H3 phosphorylation, immediate-early gene expression, and the nucleosomal response: a historical perspective1This article is part of Special Issue entitled Asilomar Chromatin and has undergone the Journal’s usual peer review process.

Histone H3 is modified at serines 10 and 28 in interphase cells following activation of the RAS-MAPK or p38-MAPK pathways by growth factors or stress. These modifications are involved in the regulation of immediate-early genes, including Jun and Fos, whose increased expression is a trademark of various cancers. This review outlines the series of discoveries that led to the characterization of these modifications, the kinase, MSK1/2, which is activated by both MAPK pathways and directs phosphorylation of H3, and the mechanistic function of these modifications in transcriptional activation. Research examining the effect of deregulated MSK1/2 in human disorders, namely cancer, is evaluated. Recently, a number of reports proposed novel, intervening pathways leading to enrichment of phosphorylated serine 10 and 28 and the activation of MSK1/2. These novel pathways predict an even more complicated signalling mechanism for cell growth, apoptosis, and the immune response, suggesting that MSK1/2 is intrinsically responsible for an even greater number of biological processes. This review proposes that MSK1/2 is an optimal target for cancer therapy, based on its fundamental role in transmitting external signals into varied responses involved in cancer development.

Mutation of MeCP2 alters transcriptional regulation of select immediate-early genes

Loss-of-function mutations in the methyl-DNA binding protein MeCP2 are associated with neurological dysfunction and impaired neural plasticity. However, the transcriptional changes that underlie these deficits remain poorly understood. Here, we show that mice bearing a C-terminal truncating mutation in Mecp2 (Mecp2308) are hypersensitive to the locomotor stimulating effects of cocaine. Furthermore, these mice have gene-specific alterations in striatal immediate-early gene (IEG) induction following cocaine administration. MeCP2 mutant mice show normal levels of baseline and cocaine-induced striatal Fos expression compared with their wild-type littermates. However, the mutant mice have enhanced cocaine-induced transcription of Junb and Arc. At the chromatin level, we find increased histone H3 acetylation at gene promoters in the Mecp2 mutant mice compared with their wild-type littermates, whereas two sites of repressive histone methylation are unchanged. Interestingly, we find that MeCP2 mutant mice show increased steady-state association of elongation-competent RNA Polymerase II (RNAP II) with the Junb and Arc promoters, whereas levels of RNAP II association at the Fos promoter are unchanged. These data reveal a gene-specific effect of MeCP2 on the recruitment of RNAP II to gene promoters that may modulate the inducibility of IEGs. In addition, our findings raise the possibility that aberrant regulation of IEGs including Junb and Arc may contribute to altered cocaine-induced neuronal and behavioral plasticity in Mecp2 mutant mice.

Application of multiple forms of mechanical loading to human osteoblasts reveals increased ATP release in response to fluid flow in 3D cultures and differential regulation of immediate early genes

ATP is actively released into the extracellular environment from a variety of cell types in response to mechanical stimuli. This is particularly true in bone where mechanically induced ATP release leads to immediate early gene activation to regulate bone remodelling; however there is no consensus as to which mechanical stimuli stimulate osteoblasts the most. To elucidate which specific type(s) of mechanical stimuli induce ATP release and gene activation in human osteoblasts, we performed an array of experiments using different mechanical stimuli applied to both monolayer and 3D cultures of the same osteoblast cell type, SaOS-2. ATP release from osteoblasts cultured in monolayer significantly increased in response to turbulent fluid flow, laminar fluid flow and substrate strain. No significant change in ATP release could be detected in 3D osteoblast cultures in response to cyclic or static compressive loading of osteoblast-seeded scaffolds, whilst turbulent fluid flow increased ATP release from 3D cultures of osteoblasts to a greater degree than observed in monolayer cultures. Cox-2 expression quantified using real time PCR was significantly lower in cells subjected to turbulent fluid flow whereas c-fos expression was significantly higher in cells subjected to strain. Load-induced signalling via c-fos was further investigated using a SaOS-2 c-fos luciferase reporter cell line and increased in response to substrate strain and turbulent fluid flow in both monolayer and 3D, with no significant change in response to laminar fluid flow or 3D compressive loading. The results of this study demonstrate for the first time strain-induced ATP release from osteoblasts and that turbulent fluid flow in 3D up regulates the signals required for bone remodelling.