Darrell D. Norton

Phosphorylation of p66Shc and forkhead proteins mediates Aβ toxicity

Excessive accumulation of amyloid β-peptide (Aβ) plays an early and critical role in synapse and neuronal loss in Alzheimers Disease (AD). Increased oxidative stress is one of the mechanisms whereby Aβ induces neuronal death. Given the lessened susceptibility to oxidative stress exhibited by mice lacking p66Shc, we investigated the role of p66Shc in Aβ toxicity. Treatment of cells and primary neuronal cultures with Aβ caused apoptotic death and induced p66Shc phosphorylation at Ser36. Ectopic expression of a dominant-negative SEK1 mutant or chemical JNK inhibition reduced Aβ-induced JNK activation and p66Shc phosphorylation (Ser36), suggesting that JNK phosphorylates p66Shc. Aβ induced the phosphorylation and hence inactivation of forkhead transcription factors in a p66Shc-dependent manner. Ectopic expression of p66ShcS36A or antioxidant treatment protected cells against Aβ-induced death and reduced forkhead phosphorylation, suggesting that p66Shc phosphorylation critically influences the redox regulation of forkhead proteins and underlies Aβ toxicity. These findings underscore the potential usefulness of JNK, p66Shc, and forkhead proteins as therapeutic targets for AD.

Cutting Edge: A cis-Acting DNA Element Targets AID-Mediated Sequence Diversification to the Chicken Ig Light Chain Gene Locus

Somatic hypermutation and gene conversion are two closely related processes that increase the diversity of the primary Ig repertoire. Both processes are initiated by the activation-induced cytidine deaminase that converts cytosine residues to uracils in a transcription-dependent manner; these lesions are subsequently fixed in the genome by direct replication and error-prone DNA repair. Two alternative mechanisms were proposed to explain why this mutagenic activity is targeted almost exclusively to Ig loci: 1) specific cis-acting DNA sequences; or 2) very high levels of Ig gene transcription. In this study we now identify a novel 3′ regulatory region in the chicken Ig light chain gene containing not only a classical transcriptional enhancer but also cis-acting DNA elements essential for targeting activation-induced cytidine deaminase-mediated sequence diversification to this locus.

Single-stranded DNA-binding Proteins and Neuron-restrictive Silencer Factor Participate in Cell-specific Transcriptional Control of the NMDAR1 Gene

Our previous studies revealed that a proximal region of the N-methyl-d-aspartate receptor 1 (NMDAR1) promoter is important for cell-type-specific expression. We have now explored the contributions of several regulatory elements to this specificity. Deletion of the neuron-restrictive silencer element partially relieved the suppression of promoter activity in C6 glioma and HeLa cells. An overlapping G(C/G)G/tandem Sp1-containing region crucial for both basal and nerve growth factor (NGF)-regulated promoter activity specifically bound nuclear proteins on its purine-rich sense strand. A faster migrating complex, single-stranded binding protein complex 1 (SBPC1), was highly enriched in HeLa cells, whereas a slower migrating complex, SBPC2, was enriched in PC12 cells. A high ratio of 2/1 complex correlated with a high level of promoter activity. NGF treatment of PC12 cells reduced SBPC1 but increased SBPC2. Competition experiments showed that the SBPC1 binding required a dG4sequence and the SBPC2 needed a core of TG3A plus a 5′-flanking sequence. Single-stranded DNA encompassing TG3A and/or dG4 specifically suppressed cotransfected NMDAR1 promoter activity. UV cross-linking studies indicated that a 31.5-kDa protein mainly formed SBPC1, whereas SBPC2 contained several larger proteins. Our results suggest that neuron-restrictive silencer factor and single-stranded DNA-binding proteins may both play a role in cell-type specificity of the NMDAR1 gene, and the latter may also be involved in basal and NGF-regulated activity.

Synthesis and Characterization of a SIRT6 Open Tubular Column: Predicting Deacetylation Activity using Frontal Chromatography

SIRT6 is a histone deacetylase that has been proposed as a potential therapeutic target for metabolic disorders and the prevention of age-associated diseases. Thus the identification of compounds that modulate SIRT6 activity could be of great therapeutic importance. We have previously reported on the identification of quercetin and vitexin as SIRT6 inhibitors, using SIRT6-coated magnetic beads. In this study, we have immobilized SIRT6 onto the surface of an open tubular capillary and characterized the quercetin binding site using frontal displacement chromatography. Structurally related flavonoids were tested for their activity on SIRT6, including apigenin, naringenin, luteolin, and kaempferol. In addition to obtaining their binding activity using frontal affinity chromatographic techniques, we also ranked the compounds based on their ability to displace quercetin. The data suggest that a single displacement curve is representative of the enzymatic activity of the tested ligand. In addition, using the inhibition data obtained in this study, we developed a preliminary pharmacophore model that confirmed the experimental data.

A splice variant of the N-methyl-D-aspartate (NMDAR1) receptor

A splice variant of the NMDA receptor (NMDAR1) was discovered containing a deletion of 37 amino acids near the carboxyl tail and has been designated NMDAR1b. The 111 nucleotides corresponding to the deleted amino acid sequence were found in a separate exon bounded by consensus intron/exon junction sequences in rat genomic DNA. A partial restriction map of genomic DNA bounding this region placed the deleted exon approximately 600 base pairs (bp) downstream of the upstream exon. RT/PCR analysis of RNA from different brain regions showed that the deletion variant is more abundantly expressed in the brain stem and cerebellum while the full-length form is expressed more abundantly in the olfactory bulb, striatum, hippocampus, and cortex. Northern analysis of poly(A)+ RNA from different brain regions with probes specific for the deleted exon (i.e., full-length form) and for the splice junction (deletion form) indicated approximately 4.4 kb transcripts. The probe for the deleted exon hybridized to transcripts in olfactory bulb, cortex, striatum, and hippocampus while the splice junction probe hybridized most strongly to transcripts in cerebellum. The results suggest an interesting rostral to caudal shift in the expression of splice variants of the NMDAR1 which may signify important functional differences in native forms of NMDA receptors.

Separation of Mutational and Transcriptional Enhancers in Ig Genes

Secondary Ig gene diversification relies on activation-induced cytidine deaminase (AID) to create U:G mismatches that are subsequently fixed by mutagenic repair pathways. AID activity is focused to Ig loci by cis-regulatory DNA sequences named targeting elements. In this study, we show that in contrast to prevailing thought in the field, the targeting elements in the chicken IGL locus are distinct from classical transcriptional enhancers. These mutational enhancer elements (MEEs) are required over and above transcription to recruit AID-mediated mutagenesis to Ig loci. We identified a small 222-bp fragment in the chicken IGL locus that enhances mutagenesis without boosting transcription, and this sequence represents a key component of an MEE. Lastly, MEEs are evolutionarily conserved among birds, both in sequence and function, and contain several highly conserved sequence modules that are likely involved in recruiting trans-acting targeting factors. We propose that MEEs represent a novel class of cis-regulatory elements for which the function is to control genomic integrity.

The PHD domain of the sea urchin RAG2 homolog, SpRAG2L, recognizes dimethylated lysine 4 in histone H3 tails

V(D)J recombination is a somatic gene rearrangement process that assembles antigen receptor genes from individual segments during lymphocyte development. The access of the RAG1/RAG2 recombinase to these gene segments is regulated at the level of chromatin modifications, in particular histone tail modifications. Trimethylation of lysine 4 in histone H3 (H3K4me3) correlates with actively recombining gene elements, and this mark is recognized and interpreted by the plant homeodomain (PHD) of RAG2. Here we report that the PHD domain of the only known invertebrate homolog of RAG2, the SpRAG2L protein of the purple sea urchin (Strongylocentrotus purpuratus) also binds to methylated histones, but with a unique preference for H3K4me2. While the cognate substrate for the sea urchin RAG1L/RAG2L complex remains elusive, the affinity for histone tails and the nuclear localization of ectopically expressed SpRAG2L strongly support the model that this enzyme complex exerts its activity on DNA in the context of chromatin.

Classical Mus musculus Igκ enhancers support transcription but not high level somatic hypermutation from a V-lambda promoter in chicken DT40 cells.

Somatic hypermutation (SHM) of immunoglobulin genes is initiated by activation-induced cytidine deaminase (AID) in activated B cells. This process is strictly dependent on transcription. Hence, cis-acting transcriptional control elements have been proposed to target SHM to immunoglobulin loci. The Mus musculus Igκ locus is regulated by the intronic enhancer (iE/MAR) and the 3′ enhancer (3′E), and multiple studies using transgenic and knock-out approaches in mice and cell lines have reported somewhat contradictory results about the function of these enhancers in AID-mediated sequence diversification. Here we show that the M. musculus iE/MAR and 3′E elements are active solely as transcriptional enhancer when placed in the context of the IGL locus in Gallus gallus DT40 cells, but they are very inefficient in targeting AID-mediated mutation events to this locus. This suggests that either key components of the cis-regulatory targeting elements reside outside the murine Igκ transcriptional enhancer sequences, or that the targeting of AID activity to Ig loci occurs by largely species-specific mechanisms.