06-863 Sigma-AldrichAnti-CREB Antibody

The study of late-onset/age-related Alzheimer's disease (AD)(sporadic AD, 95% of AD cases) has been hampered by a paucity of animal models. Oxidative stress is considered a causative factor in late onset/age-related AD, and aldehyde dehydrogenase 2 (ALDH2) is important for the catabolism of toxic aldehydes associated with oxidative stress. One such toxic aldehyde, the lipid peroxidation product 4-hydroxynonenal (HNE), accumulates in AD brain and is associated with AD pathology. Given this linkage, we hypothesized that in mice lacking ALDH2, there would be increases in HNE and the appearance of AD-like pathological changes.Changes in relevant AD markers in Aldh2 (-/-) mice and their wildtype littermates were assessed over a 1 year period. Marked increases in HNE adducts arise in hippocampi from Aldh2 (-/-) mice, as well as age-related increases in amyloid-beta, p-tau, and activated caspases. Also observed were age-related decreases in pGSK3β, PSD95, synaptophysin, CREB and pCREB. Age-related memory deficits in the novel object recognition and Y maze tasks begin at 3.5-4 months and are maximal at 6.5-7 months. There was decreased performance in the Morris Water Maze task in 6 month old Aldh2 (-/-) mice. These mice exhibited endothelial dysfunction, increased amyloid-beta in cerebral microvessels, decreases in carbachol-induced pCREB and pERK formation in hippocampal slices, and brain atrophy. These AD-associated pathological changes are rarely observed as a constellation in current AD animal models.We believe that this new model of age-related cognitive impairment will provide new insight into the pathogenesis and molecular/cellular mechanisms driving neurodegenerative diseases of aging such as AD, and will prove useful for assessing the efficacy of therapeutic agents for improving memory and for slowing, preventing, or reversing AD progression.

Microphthalmia-associated transcription factor (MITF) is the master regulator of the melanocyte lineage. To understand how MITF regulates transcription, we used tandem affinity purification and mass spectrometry to define a comprehensive MITF interactome identifying novel cofactors involved in transcription, DNA replication and repair, and chromatin organisation. We show that MITF interacts with a PBAF chromatin remodelling complex comprising BRG1 and CHD7. BRG1 is essential for melanoma cell proliferation in vitro and for normal melanocyte development in vivo. MITF and SOX10 actively recruit BRG1 to a set of MITF-associated regulatory elements (MAREs) at active enhancers. Combinations of MITF, SOX10, TFAP2A, and YY1 bind between two BRG1-occupied nucleosomes thus defining both a signature of transcription factors essential for the melanocyte lineage and a specific chromatin organisation of the regulatory elements they occupy. BRG1 also regulates the dynamics of MITF genomic occupancy. MITF-BRG1 interplay thus plays an essential role in transcription regulation in melanoma.

The methyl-DNA binding protein MeCP2 is emerging as an important regulator of drug reinforcement processes. Psychostimulants induce phosphorylation of MeCP2 at Ser421; however, the functional significance of this posttranslational modification for addictive-like behaviors was unknown. Here we show that MeCP2 Ser421Ala knock-in mice display both a reduced threshold for the induction of locomotor sensitization by investigator-administered amphetamine and enhanced behavioral sensitivity to the reinforcing properties of self-administered cocaine. These behavioral differences were accompanied in the knock-in mice by changes in medium spiny neuron intrinsic excitability and nucleus accumbens gene expression typically observed in association with repeated exposure to these drugs. These data show that phosphorylation of MeCP2 at Ser421 functions to limit the circuit plasticities in the nucleus accumbens that underlie addictive-like behaviors.

Transcription factors CREB, C/EBPβ and Jun regulate genes involved in keratinocyte proliferation and differentiation. We questioned if specific combinations of CREB, C/EBPβ and c-Jun bound to promoters correlate with RNA polymerase II binding, mRNA transcript levels and methylation of promoters in proliferating and differentiating keratinocytes.Induction of mRNA and RNA polymerase II by differentiation is highest when promoters are bound by C/EBP β alone, C/EBPβ together with c-Jun, or by CREB, C/EBPβ and c-Jun, although in this case CREB binds with low affinity. In contrast, RNA polymerase II binding and mRNA levels change the least upon differentiation when promoters are bound by CREB either alone or in combination with C/EBPβ or c-Jun. Notably, promoters bound by CREB have relatively high levels of RNA polymerase II binding irrespective of differentiation. Inhibition of C/EBPβ or c-Jun preferentially represses mRNA when gene promoters are bound by corresponding transcription factors and not CREB. Methylated promoters have relatively low CREB binding and, accordingly, those which are bound by C/EBPβ are induced by differentiation irrespective of CREB. Composite "Half and Half" consensus motifs and co localizing consensus DNA binding motifs are overrepresented in promoters bound by the combination of corresponding transcription factors.Correlational and functional data describes combinatorial mechanisms regulating the activation of promoters. Colocalization of C/EBPβ and c-Jun on promoters without strong CREB binding determines high probability of activation upon keratinocyte differentiation.

The transcription factor, ΔFosB, is robustly and persistently induced in striatum by several chronic stimuli, such as drugs of abuse, antipsychotic drugs, natural rewards, and stress. However, very few studies have examined the degree of ΔFosB induction in the two striatal medium spiny neuron (MSN) subtypes. We make use of fluorescent reporter BAC transgenic mice to evaluate induction of ΔFosB in dopamine receptor 1 (D1) enriched and dopamine receptor 2 (D2) enriched MSNs in ventral striatum, nucleus accumbens (NAc) shell and core, and in dorsal striatum (dStr) after chronic exposure to several drugs of abuse including cocaine, ethanol, Δ(9)-tetrahydrocannabinol, and opiates; the antipsychotic drug, haloperidol; juvenile enrichment; sucrose drinking; calorie restriction; the serotonin selective reuptake inhibitor antidepressant, fluoxetine; and social defeat stress. Our findings demonstrate that chronic exposure to many stimuli induces ΔFosB in an MSN-subtype selective pattern across all three striatal regions. To explore the circuit-mediated induction of ΔFosB in striatum, we use optogenetics to enhance activity in limbic brain regions that send synaptic inputs to NAc; these regions include the ventral tegmental area and several glutamatergic afferent regions: medial prefrontal cortex, amygdala, and ventral hippocampus. These optogenetic conditions lead to highly distinct patterns of ΔFosB induction in MSN subtypes in NAc core and shell. Together, these findings establish selective patterns of ΔFosB induction in striatal MSN subtypes in response to chronic stimuli and provide novel insight into the circuit-level mechanisms of ΔFosB induction in striatum.

E2F2 is essential for the maintenance of T lymphocyte quiescence. To identify the full set of E2F2 target genes, and to gain further understanding of the role of E2F2 in transcriptional regulation, we have performed ChIP-chip analyses across the genome of lymph node-derived T lymphocytes. Here we show that during quiescence, E2F2 binds the promoters of a large number of genes involved in DNA metabolism and cell cycle regulation, concomitant with their transcriptional silencing. A comparison of ChIP-chip data with expression profiling data on resting E2f2(-)(/)(-) T lymphocytes identified a subset of 51 E2F2-specific target genes, most of which are upregulated on E2F2 loss. Luciferase reporter assays showed a retinoblastoma-independent role for E2F2 in the negative regulation of these target genes. Importantly, we show that the DNA binding activity of the transcription factor CREB contributes to E2F2-mediated repression of Mcm5 and Chk1 promoters. siRNA-mediated CREB knockdown, expression of a dominant negative KCREB mutant or disruption of CREB binding by mutating a CRE motif on Mcm5 promoter, relieved E2F2-mediated transcriptional repression. Taken together, our data uncover a new regulatory mechanism for E2F-mediated transcriptional control, whereby E2F2 and CREB cooperate in the transcriptional repression of a subset of E2F2 target genes.

Systemic administration of amphetamine (AMPH) induces phosphorylation of MeCP2 at Ser421 (pMeCP2) in select populations of neurons in the mesolimbocortical brain regions. Because AMPH simultaneously activates multiple monoamine neurotransmitter systems, here we examined the ability of dopamine (DA), serotonin (5-HT), and norepinephrine (NE) to induce pMeCP2. Selective blockade of the DA transporter (DAT) or the 5-HT transporter (SERT), but not the NE transporter (NET), was sufficient to induce pMeCP2 in the CNS. DAT blockade induced pMeCP2 in the prelimbic cortex (PLC) and nucleus accumbens (NAc), whereas SERT blockade induced pMeCP2 only in the NAc. Administration of selective DA and 5-HT receptor agonists was also sufficient to induce pMeCP2; however, the specific combination of DA and 5-HT receptors activated determined the regional- and cell-type specificity of pMeCP2 induction. The D(1)-class DA receptor agonist SKF81297 induced pMeCP2 widely; however, coadministration of the D(2)-class agonist quinpirole restricted the induction of pMeCP2 to GABAergic interneurons of the NAc. Intra-striatal injection of the adenylate cyclase activator forskolin was sufficient to induce pMeCP2 in medium-spiny neurons, suggesting that the combinatorial regulation of cAMP by different classes of DA and 5-HT receptors may contribute to the cell-type specificity of pMeCP2 induction. Consistent with the regulation of pMeCP2 by multiple monoamine neurotransmitters, genetic disruption of any single monoamine transporter in DAT-, SERT-, and NET-knockout mice failed to eliminate AMPH-induced pMeCP2 in the NAc. Together, these studies indicate that combinatorial signaling through DA and 5-HT receptors can regulate the brain region- and cell-type specific pMeCP2 in the CNS.

Epigenetic changes are implicated in the persisting vascular effects of hyperglycemia. The precise mechanism whereby chromatin structure and subsequent gene expression are regulated by glucose in vascular endothelial cells remain to be fully defined.We have studied the molecular and functional mechanism whereby the Set7 methyltransferase associates with chromatin formation and histone methylation in vascular cells in response to current and previous exposure to glucose.To characterize the molecular and functional identity of the Set7 protein, we used vascular cells overexpressing or lacking Set7. Chromatin fractionation for mono-methylation of lysine 4 on histone H3 identified methyltransferase activity. Immunofluorescence experiments strongly suggest that Set7 protein accumulates in the nucleus in response to hyperglycemia. Moreover, activation of proinflammatory genes by high glucose is dependent on Set7 but distinguished by H3K4m1 gene patterns. We show that transient hyperglycemia regulates the expression of proinflammatory genes in vascular endothelial cells in vitro and the persistent increase in glucose-induced gene expression in the aorta of nondiabetic mice.This study uncovers that the response to hyperglycemia in vascular endothelial cells involves the H3K4 methyltransferase, Set7. This enzyme appears to regulate glucose-induced chromatin changes and gene expression not only by H3K4m1-dependent but also H3K4m1-independent pathways. Furthermore, Set7 appears to be responsible for sustained vascular gene expression in response to prior hyperglycemia and is a potential molecular mechanism for the phenomenon of hyperglycemic memory.

The cAMP response element binding protein (CREB) belongs to the CREB/cAMP response element binding modulator/activating transcription factor 1 family of cAMP-dependent transcription factors mediating a regulation of gene transcription in response to cAMP. Chronic stimulation of β-adrenergic receptors and the cAMP-dependent signal transduction pathway by elevated plasma catecholamines play a central role in the pathogenesis of heart failure. Ion channel remodeling, particularly a decreased transient outward current (I(to)), and subsequent action potential (AP) prolongation are hallmarks of the failing heart. Here, we studied the role of CREB for ion channel regulation in mice with a cardiomyocyte-specific knockout of CREB (CREB KO). APs of CREB KO cardiomyocytes were prolonged with increased AP duration at 50 and 70% repolarization and accompanied by a by 51% reduction of I(to) peak amplitude as detected in voltage-clamp measurements. We observed a 29% reduction of Kcnd2/Kv4.2 mRNA in CREB KO cardiomyocytes mice while the other I(to)-related channel subunits Kv4.3 and KChIP2 were not different between groups. Accordingly, Kv4.2 protein was reduced by 37% in CREB KO. However, we were not able to detect a direct regulation of Kv4.2 by CREB. The I(to)-dependent AP prolongation went along with an increase of I(Na) and a decrease of I(Ca,L) associated with an upregulation of Scn8a/Nav1.6 and downregulation of Cacna1c/Cav1.2 mRNA in CREB KO cardiomyocytes. Our results from mice with cardiomyocyte-specific inactivation of CREB definitively indicate that CREB critically regulates the AP shape and duration in the mouse ventricle, which might have an impact on ion channel remodeling in situations of altered cAMP-dependent signaling like heart failure.

LKB1 is a tumor susceptibility gene for the Peutz-Jeghers cancer syndrome and is a target for mutational inactivation in sporadic human malignancies. LKB1 encodes a serine/threonine kinase that has critical roles in cell growth, polarity and metabolism. A novel and important function of LKB1 is its ability to regulate the phosphorylation of CREB-regulated transcription co-activators (CRTCs) whose aberrant activation is linked with oncogenic activities. However, the roles and mechanisms of LKB1 and CRTC in the pathogenesis of esophageal cancer have not been previously investigated. In this study, we observed altered LKB1-CRTC signaling in a subset of human esophageal cancer cell lines and patient samples. LKB1 negatively regulates esophageal cancer cell migration and invasion in vitro. Mechanistically, we determined that CRTC signaling becomes activated because of LKB1 loss, which results in the transcriptional activation of specific downstream targets including LYPD3, a critical mediator for LKB1 loss-of-function. Our data indicate that de-regulated LKB1-CRTC signaling might represent a crucial mechanism for esophageal cancer progression.