Autism candidate gene DIP2A regulates spine morphogenesis via acetylation of cortactin.

Dendritic spine development is crucial for the establishment of excitatory synaptic connectivity and functional neural circuits. Alterations in spine morphology and density have been associated with multiple neurological disorders. Autism candidate gene disconnected-interacting protein homolog 2 A (DIP2A) is known to be involved in acetylated coenzyme A (Ac-CoA) synthesis and is primarily expressed in the brain regions with abundant pyramidal neurons. However, the role of DIP2A in the brain remains largely unknown. In this study, we found that deletion of Dip2a in mice induced defects in spine morphogenesis along with thin postsynaptic density (PSD), and reduced synaptic transmission of pyramidal neurons. We further identified that DIP2A interacted with cortactin, an activity-dependent spine remodeling protein. The binding activity of DIP2A-PXXP motifs (P, proline; X, any residue) with the cortactin-Src homology 3 (SH3) domain was critical for maintaining the level of acetylated cortactin. Furthermore, Dip2a knockout (KO) mice exhibited autism-like behaviors, including excessive repetitive behaviors and defects in social novelty. Importantly, acetylation mimetic cortactin restored the impaired synaptic transmission and ameliorated repetitive behaviors in these mice. Altogether, our findings establish an initial link between DIP2A gene variations in autism spectrum disorder (ASD) and highlight the contribution of synaptic protein acetylation to synaptic processing.

SOX7 is involved in aspirin-mediated growth inhibition of human colorectal cancer cells.

AIM: To confirm the role of sex-determining region Y-box 7 (Sox7) in aspirin-mediated growth inhibition of COX-independent human colorectal cancer cells. METHODS: The cell survival percentage was examined by MTT (Moto-nuclear cell direc cytotoxicity) assay. SOX7 expression was assessed by using reverse transcription-polymerase chain reaction and Western blotting. SB203580 was used to inhibit the p38MAPK signal pathway. SOX7 promoter activity was detected by Luciferase reporter assay. RESULTS: SOX7 was upregulated by aspirin and was involved in aspirin-mediated growth inhibition of SW480 human colorectal cancer cells. The p38MAPK pathway played a role in aspirin-induced SOX7 expression, during which the AP1 transcription factors c-Jun and c-Fos upregulated SOX7 promoter activities. RESULTS: SOX7 is upregulated by aspirin and is involved in aspirin-mediated growth inhibition of human colorectal cancer SW480 cells.

Effects of histone deacetylase inhibitors on transcriptional regulation of the hsp70 gene in Drosophila.

Histone acetyltransferases/deacetylases contribute to the activation or inactivation of transcription by modifying the structure of chromatin. Here we examined the effects of histone deacetylase inhibitors (HDIs), trichostatin A, and sodium butyrate on hsp70 gene transcriptional regulation in Drosophila. The chromatin immunoprecipitation assays revealed that HDI treatments induced the hyperacetylation of histone H3 at the promoter and the transcribing regions of hsp70 gene, increased the accessibility of heat-shock factor to target heat-shock element, and promoted the RNA polymerase II-mediated transcription. Moreover, the quantitative real-time PCR confirmed that the HDI-induced hyperacetylation of histone H3 enhanced both the basal and the inducible expression of hsp70 mRNA level. In addition, the acetylation level of histone H3 at the promoter exhibited a fluctuated change upon the time of heat shock. These experimental data implicated a causal link between histone acetylation and enhanced transcription initiation of hsp70 gene in Drosophila.

Transcription factors Ets2 and Sp1 act synergistically with histone acetyltransferase p300 in activating human interleukin-12 p40 promoter.

There has been considerable interest in researching the regulatory mechanisms that control the synthesis of interleukin (IL)-12, which plays a central role in the differentiation of T-helper-1 cells. In this study, we performed a series of transient transfection experiments designed to elucidate the functional relationship between the IL-12 promoter-specific transcription factors (Ets2 and Sp1) and histone acetylation modification in IL-12 regulation mediated by p300 and various histone deacetylases (HDACs). Results presented in this report demonstrated that the transcription factors Ets2 and Sp1 acted synergistically with p300 to activate the human IL-12 promoter. The histone acetyltransferase (HAT) activity of p300 was required for this synergic effect, because the adenovirus E1A protein inhibited the synergy. Conversely, HDACs repressed the synergic effect of transcription factors and histone acetylation on the activation of IL-12, while p300 was able to rectify it. These data indicated that Ets2 and Sp1 worked concertedly and synergistically with p300 in the regulation of human IL-12 expression.

[The elp3 subunit of human elongator complex ignificantly complemented the growth defects of yeast elp3delta strain].

The human elongator complex was found to be very similar to the yeast Elongator both in composition and its interaction with RNA polymerase II. But little is known about its functions in vivo. In this study, we analyzed the functions of the help3, the catalytic subunit of human Elongator, using a yeast complementation system. The results indicated that help3 was able to significantly complement the growth defects of the elp3delta yeast strain under high temperature and caffeine conditions. Gene expression analysis showed that help3 significantly resumed the slow activation of the pho5 gene under the low phosphate condition, and when heat shocked it increased the expression level of ssa3 gene. The yhelp3 containing the HAT domain of human elp3 and the remainder of yeast elp3 had a higher complementation ability than human elp3, while the yhelp3HA T- with the catalytic HAT domain deleted had no complementation ability. These results implicate that human Elp3 subunit had similar functions to those of the yeast, and the HAT activity of human Elp3 was essential for its function.

Function of c-Fos-like and c-Jun-like proteins on trichostatin A-induced G2/M arrest in Physarum polycephalum.

The homologs of transcription factors c-Fos and c-Jun have been detected in slime mold Physarum polycephalum during progression of the synchronous cell cycle. Here we demonstrated that c-Fos-like and c-Jun-like proteins participated in G2/M transition by the regulation of the level of Cyclin B1 protein in P. polycephalum. The study of antibody neutralization revealed that interruption of the functions of c-Fos-like and c-Jun-like proteins resulted in G2/M transition arrest, implicating their functional roles in cell cycle control. When G2/M transition was blocked by histone deacetylase inhibitor trichostatin A, changes in c-Fos- and c-Jun-like protein levels, and hyperacetylation of c-Jun-like protein, were observed. The data suggest that in P. polycephalum, c-Fos- and c-Jun-like proteins may be the key factors in the regulation of histone acetylation-related G2/M transition, involving the coordinated expression and hyperacetylation of these proteins.

[The Elp4 subunit of human Elongator complex partially complements the growth defects of yeast ELP4 deletion strain].

In this study, we performed in vivo experiments to determine the function of human Elongator subunit Elp4 by using a yeast complementary system. Our results indicated that though human ELP4 was not able to complement the growth defects of the ELP4 deletion mutant strain to high concentration salt, it partially reduced the sensitivity of mutant strain to caffeine, high temperature and 6-AU. Gene expression analysis indicated that human ELP4 partially resumed the slow activation of the PHO5 gene caused by the deletion of yELP4 under the low phosphate concentration. Meanwhile, under the condition of heat shock treatment, hELP4 increased the expression level of SSA3 gene. All these data demonstrated that human ELP4 can partially complement the growth defects and restore the slow activation of certain genes of the yELP4 deletion strain. These results indicate that human Elp4 subunit has similar functions to that of the yeast.

Trichostatin A extends the lifespan of Drosophila melanogaster by elevating hsp22 expression.

The level of acetylation of histones in nucleosomes is related to the longevity of yeast and animals. However, the mechanisms by which acetylation and deacetylation affect longevity remain unclear. In present study, we investigated the influence of histone acetylation modification on the expression of hsp22 gene and the lifespan in Drosophila melanogaster using histone deacetylase (HDAC) inhibitor Trichostatin A (TSA). The results showed that TSA could extend the lifespan of Drosophila melanogaster. Furthermore, TSA significantly promoted the hsp22 gene transcription, and affected the chromatin morphology at the locus of hsp22 gene along the polytene chromosome. Present data implicate that TSA may affect the lifespan of Drosophila through changing the level of histone acetylation and influencing the expression of hsp22 gene that is related to aging.

[The histone deacetylase activity of SIR2 and chromatin silencing].

Chromatin silencing is the inactivation of large domains of DNA by packaging them into a specialized inaccessible chromatin structure. This type of inactivation is involved in the regulation of gene expression and is also associated with the chromosome structures required for chromosome maintenance and inheritance. Silent information protein 2(SIR2) is one of the important proteins involved in chromatin silencing. It is clear that SIR2 has two coupled enzymatic activities, histone deacetylation and NAD breakdown activities, and produces a novel compound, O-acetyl-ADP-ribose in the enzymatic reactions. The histone deacetylation activity of SIR2 provides the direct link between SIR2 and the hypoacetylation of silent chromatin. Moreover, the relationship between the NAD cleavage and the deacetylase activity of SIR2 shows that the histone deacetylase activity is not its only crucial function. The breakdown of NAD C-N bond and the synthesis of O-acetyl-ADP-ribose may also be involved in chromatin silencing.

[Cloning and prokaryotic expression of yeast GCN5 and RPD3].

The yeast histone acetyltransferase (HAT) gene gcn5 and histone deacetylase (HDAC) gene rpd3 were cloned from yeast genomic DNA by PCR amplification. The two genes,both with additional 6xHis tag,were subcloned into pBV220 vector to construct expression plasmids pBVgcn5 and pBVrpd3,respectively. Both pBVgcn5 and pBVrpd3 were over-expressed in Escherichia coli upon temperature induction,as revealed by SDS-PAGE. The recombinant GCN5 and RPD3 were purified by using a 6xHis affinity column. The purified GCN5 was tested to possess the HAT activity by using a (14)C-labeling assay. This work has laid down the basis for further in vitro studies into roles of histone acetylation/deacetylation in modulating chromatin conformation and transcription activity.

Expression of Recombinant alpha-Acetolactate Decarboxylase and Its Characterization.

alpha-Acetolactate decarboxylase(alpha-ALDC)gene has been cloned from Baillus brevis using PCR amplification. The amplified 0.97 kb DNA fragment was confirmed to be known alpha- ALDC gene by DNA sequencing. The fragment was inserted into the vector pBV220 to construct an expression plasmid pBVYI. This recombinant plasmid over expressed alpha-ALDC in E. coli DH5alpha. The alpha-ALDC activity of recombinant bacterium was 10 000-fold higher than that of Bacillus brevis. After purification, the properties of the recombinant alpha-ALDC were studied. The activity of this enzyme could be stimulated by Mn(2+), Sn(2+) and inhibited by Zn(2+), Cd(2+), Fe(2+), Co(2+) and Cu(2+). Moreover, amino acid modifiers could inhibit differently its activity. The optimum pH of the enzyme reaction was 5.5.