Comprehensive analysis of two Shank3 and the Cacna1c mouse models of autism spectrum disorder.

To expand, analyze and extend published behavioral phenotypes relevant to autism spectrum disorder (ASD), we present a study of three ASD genetic mouse models: Feng's Shank3tm2Gfng model, hereafter Shank3/F, Jiang's Shank3tm1Yhj model, hereafter Shank3/J and the Cacna1c deletion model. The Shank3 models mimick gene mutations associated with Phelan-McDermid Syndrome and the Cacna1c model recapitulates the deletion underlying Timothy syndrome. This study utilizes both standard and novel behavioral tests with the same methodology used in our previously published companion report on the Cntnap2 null and 16p11.2 deletion models. We found that some but not all behaviors replicated published findings and those that did replicate, such as social behavior and overgrooming in Shank3 models, tended to be milder than reported elsewhere. The Shank3/F model, and to a much lesser extent, the Shank3/J and Cacna1c models, showed hypoactivity and a general anxiety-like behavior triggered by external stimuli which pervaded social interactions. We did not detect deficits in a cognitive procedural learning test nor did we observe perseverative behavior in these models. We did, however, find differences in exploratory patterns of Cacna1c mutant mice suggestive of a behavioral effect in a social setting. In addition, only Shank3/F showed differences in sensory-gating. Both positive and negative results from this study will be useful in identifying the most robust and replicable behavioral signatures within and across mouse models of autism. Understanding these phenotypes may shed light of which features to study when screening compounds for potential therapeutic interventions.

Targeted and extended acetylation of histones H4 and H3 at active and inactive genes in chicken embryo erythrocytes.

Affinity-purified polyclonal antibodies recognizing the most highly acetylated forms of histones H3 and H4 were used in immunoprecipitation assays with chromatin fragments derived from 15-day chicken embryo erythrocytes by micrococcal nuclease digestion. The distribution of hyperacetylated H4 and H3 was mapped at the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and the tissue-specific gene, carbonic anhydrase (CA). H3 and H4 acetylation was found targeted to the CpG island region at the 5' end of both these genes, falling off in the downstream direction. In contrast, at the beta(A)-globin gene, both H3 and H4 are highly acetylated throughout the gene and at the downstream enhancer, with a maximum at the promoter. Low level acetylation was observed at the 5' end of the inactive ovalbumin gene. Run-on assays to measure ongoing transcription showed that the GAPDH and CA genes are transcribed at a much lower rate than the adult beta(A)-globin gene. The extensive high level acetylation at the beta(A)-globin gene correlates most simply with its high rate of transcription. The targeted acetylation of histones H3 and H4 at the GAPDH and CA genes is consistent with a role in transcriptional initiation and implies that transcriptional elongation does not necessarily require hyperacetylation.

Independent dynamic regulation of histone phosphorylation and acetylation during immediate-early gene induction.

Induction of c-fos and c-jun is associated with phosphoacetylation of histone H3 and acetylation of histone H4. Upon induction, a large population of nucleosomes becomes highly acetylated on histones H3 and H4, whereas a much smaller population of comparable nucleosomes at similar positions along the gene becomes phosphoacetylated. Inhibiting histone H3 phosphorylation with kinase inhibitors does not measurably alter the enhanced acetylation of these nucleosomes. Finally, whereas H3 phosphorylation is a MAP kinase-mediated inducible event, we found acetylation to be continuously turning over by the targeted action of HATs and HDACs in the absence of any stimulation or gene transcription. These studies suggest that phosphorylation and acetylation are independently and dynamically regulated at these genes and reveal the complexity of multiple histone modifications at immediate-early gene chromatin.

Phosphoacetylation of histone H3 on c-fos- and c-jun-associated nucleosomes upon gene activation.

The induction of immediate-early (IE) genes, including proto-oncogenes c-fos and c-jun, correlates well with a nucleosomal response, the phosphorylation of histone H3 and HMG-14 mediated via extracellular signal regulated kinase or p38 MAP kinase cascades. Phosphorylation is targeted to a minute fraction of histone H3, which is also especially susceptible to hyperacetylation. Here, we provide direct evidence that phosphorylation and acetylation of histone H3 occur on the same histone H3 tail on nucleosomes associated with active IE gene chromatin. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies that specifically recognize the doubly-modified phosphoacetylated form of histone H3. Analysis of the associated DNA shows that histone H3 on c-fos- and c-jun-associated nucleosomes becomes doubly-modified, the same H3 tails becoming both phosphorylated and acetylated, only upon gene activation. This study reveals potential complications of occlusion when using site-specific antibodies against modified histones, and shows also that phosphorylated H3 is more sensitive to trichostatin A (TSA)-induced hyperacetylation than non-phosphorylated H3. Because MAP kinase-mediated gene induction is implicated in controlling diverse biological processes, histone H3 phosphoacetylation is likely to be of widespread significance.

The nucleosomal response associated with immediate-early gene induction is mediated via alternative MAP kinase cascades: MSK1 as a potential histone H3/HMG-14 kinase.

The nucleosomal response refers to the rapid phosphorylation of histone H3 on serine 10 and HMG-14 on serine 6 that occurs concomitantly with immediate-early (IE) gene induction in response to a wide variety of stimuli. Using antibodies against the phosphorylated residues, we show that H3 and HMG-14 phosphorylation is mediated via different MAP kinase (MAPK) cascades, depending on the stimulus. The nucleosomal response elicited by TPA is ERK-dependent, whereas that elicited by anisomycin is p38 MAPK-dependent. In intact cells, the nucleosomal response can be selectively inhibited using the protein kinase inhibitor H89. MAPK activation and phosphorylation of transcription factors are largely unaffected by H89, whereas induction of IE genes is inhibited and its characteristics markedly altered. MSK1 is considered the most likely kinase to mediate this response because (i) it is activated by both ERK and p38 MAPKs; (ii) it is an extremely efficient kinase for HMG-14 and H3, utilizing the physiologically relevant sites; and (iii) its activity towards H3/HMG-14 is uniquely sensitive to H89 inhibition. Thus, the nucleosomal response is an invariable consequence of ERK and p38 but not JNK/SAPK activation, and MSK1 potentially provides a link to complete the circuit between cell surface and nucleosome.

MAP kinase-mediated signalling to nucleosomes and immediate-early gene induction.

Extracellular signals are transduced into the nucleus through a variety of signalling mechanisms to elicit changes in patterns of gene expression. This review is focused in the MAP kinase cascades and the part they play in the induction of the immediate-early (IE) genes. We discuss the MAP kinases and their downstream effectors that are known to phosphorylate substrates in the nucleus. In addition to phosphorylating specific transcription factors, MAP kinases and their downstream kinases are implicated in eliciting rapidly targeted alterations in the chromatin environment of specific genes by modulating the phosphorylation and/or acetylation of nucleosomal and chromatin proteins.

Chromosomal mapping of core histone acetylation by immunoselection.

Acetylation of specific lysine residues in the N-terminal domains of core histones is a biochemical marker of active genes. To determine the spatial and temporal distribution of this reversible posttranslational modification, affinity-purified polyclonal antibodies recognizing the epitope epsilon-acetyllysine have been used in immunoselection procedures with mononucleosomes and salt-soluble chromatin fragments generated by micrococcal nuclease. The DNA of the antibody-selected chromatin was slot-blotted and probed with a variety of gene sequences: an enhanced hybridization signal, with respect to that from the DNA of the input chromatin, demonstrated elevated acetylation levels on the histones associated with the probing sequences. Using chicken embryonic erythrocytes as chromatin source and probes from the beta globin locus, it was shown that both the embryonic epsilon and adult beta genes are acetylated at 5 and 15 days, and the acetylation uniformly covers the whole of the locus, precisely comapping with the 33 kb of open chromatin structure. Studies with proliferating human K562 cells show that the inactive but poised PDGF-beta gene is already hyperacetylated and that its acetylation status is not enhanced on induction. These results indicate that acetylation is not a consequence of transcription but a prerequisite and that it may be responsible for either generating or maintaining the open structure of poised and active genes.

Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain.

The distribution of core histone acetylation across the chicken beta-globin locus has been mapped in 15 day chicken embryo erythrocytes by immunoprecipitation of mononucleosomes with an antibody recognizing acetylated histones, followed by hybridization probing at several points in the locus. A continuum of acetylation was observed, covering both genes and intergenic regions. Using the same probes, the generalized sensitivity to DNase I was mapped by monitoring the disappearance of intact genomic restriction fragments from Southern transfers. Close correspondence between the 33 kb of sensitive chromatin and the extent of acetylation indicates that one role of the modification could be the generation and/or maintenance of the open conformation. The precision of acetylation mapping makes it a possible approach to the definition of chromosomal domain boundaries.

Histone acetylation and gene induction in human cells.

An antibody recognising acetylated core histones was used to immunoprecipitate chromatin fragments from proliferating human K562 cells and from cells induced to differentiate with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The DNA of the acetylated chromatin was probed with sequences of platelet derived growth factor B chain (PDGF-B), a gene which is induced to strong expression upon differentiation. A high level of acetylation was observed before gene induction and no change seen following induction. This implies that core histone acetylation is an essential precondition for transcription.

Nucleosomal structure at hyperacetylated loci probed in nuclei by DNA-histone crosslinking.

Chemically induced histone-DNA crosslinking in nuclei is used to monitor structural changes in chromosomal domains containing hyperacetylated histones. Core particles harbouring the crosslinks are immunofractionated with antibodies specific for acetylated histones. Crosslinking is revealed by gel separation of tryptic peptides from core histones that carry 32P-labelled residual nucleotide. The large number of DNA-histone crosslinks retained indicates that acetylated core histone tails are not totally displaced from the DNA. Changes in the patterns of crosslinked peptides imply a restructuring of hyperacetylated histone-DNA interactions at several points within the nucleosome. This demonstrates that a distinct conformational state is adopted in acetylated nucleosomes, known to be concentrated at transcriptionally active loci.

Histone acetylation and globin gene switching.

An affinity-purified antibody that recognises the epitope epsilon-acetyl lysine has been used to fractionate chicken erythrocyte mononucleosomes obtained from 5 and 15 day embryos. The antibody bound chromatin was enriched in multiply acetylated forms of the core histones H3, H4 and H2B, but not in ubiquitinated H2A. The DNA of these modified nucleosomes was probed with genomic sequences from the embryonic beta rho gene (active at 5 days) and from the adult beta A gene (active at 15 days). Both genes were found to be highly enriched in the acetylated nucleosomes fractionated from both 5 day and from 15 day erythrocytes. We conclude that globin switching is not linked to a change in acetylation status of the genes and that a 'poised' gene carries histones acetylated to a similar level as a transcriptionally active gene. Core histone acetylation is not therefore a direct consequence of the transcriptional process and might operate at the level of the globin locus as a general enabling step for transcription.

Simplified test for detecting the resistance of herpes simplex virus to acyclovir.

The detection of herpes simplex virus (HSV) antigen by means of an enzyme amplified ELISA was investigated for rapid screening of acyclovir (ACV) resistance. Vero cell monolayers were inoculated in the presence of different concentrations of ACV. When cytopathic effect was present, the culture supernatants were tested by ELISA. The absorbance values were found to correlate with the results of virus yield and plaque reduction assays. The comparison between absorbance values obtained in the presence of 10 microM ACV and in the absence of drug provided the basis for a simplified sensitivity test. The use of a single ACV concentration allowed discrimination between ACV-resistant and ACV-sensitive reference strains, the detection of ACV-resistant virus mixed in the proportion of 10% with ACV-sensitive virus, and a study of the emergence of an ACV-resistant virus population in serial samples taken from experimental rabbit keratitis. The simplified susceptibility assay is a sensitive and convenient method for rapid screening of HSV resistance to ACV.

The selection and performance of monoclonal and polyclonal anti-respiratory syncytial virus (RS) antibodies in capture ELISAs for antigen detection.

Six monoclonal antibodies directed against fusion protein (F) or nucleoprotein (NP) of respiratory syncytial virus (RS) have been investigated in an antigen capture ELISA for virus detection. The potency, spectrum and pattern of reactivity were investigated with the intention of selecting antibodies reacting with RS-common antigen determinants and with complementary rather than competitive activity. Two anti-F protein antibodies satisfied these criteria and were used with enzyme amplified detection in a two site monoclonal assay (MCA/MCA) or as detectors with a polyclonal antibody as capture (PCA/MCA). Comparative studies were performed with immunofluorescence (FA) as the reference test and nasopharyngeal aspirates processed in different ways. The PCA/MCA assay was superior to that using monoclonal antibodies alone and gave results comparable to the reference method. However, the apparent sensitivity related to FA varied with the type of sample processing used. These results emphasise the need for a critical analysis of the factors which can influence the sensitivity of a particular assay system before judgements on relative sensitivity are made.

Antibodies to the five histones and poly(adenosine diphosphate-ribose) in drug induced lupus: implications for pathogenesis.

Certain drugs are a frequent source of antinuclear antibody (ANA) induction, and ANA is invariably present in the few patients who progress to the drug induced lupus syndrome. This report concerns the fine specificity of the ANA response to hydralazine, penicillamine, and sulphasalazine therapy. Using highly purified individual histones in fluorimetric assays, antihistone antibodies are always detectable, often in large amounts, but the pattern of response to individual histones is variable and not drug specific. In addition to the response to the three histones H1, H2B, and H3 reminiscent of idiopathic systemic lupus erythematosus, antibody to histone H2A predominates in some drug induced cases. Contrary to previous thought, histones are not the sole target of the antinuclear response: we also demonstrate a significant correlation between ANA titre and antibody to poly(adenosine diphosphate-ribose). Like the histones, this is a macromolecule that can bind to deoxyribonucleic acid (DNA). It is proposed that drug induced damage to chromatin leads to ANA production, while drug induced impairment of complement activity may then enable these autoantibodies to mediate the lupus syndrome.

Herpes simplex virus detection by ELISA: effect of enzyme amplification, nature of lesion sampled and specimen treatment.

The relative sensitivity of two enzyme detection procedures was investigated in a simultaneous "monoclonal" ELISA for herpes simplex virus (HSV). A cyclical enzyme amplified detection system with alkaline phosphatase, rather than horse-radish peroxidase and a conventional chromogenic substrate, gave an increase in absolute sensitivity and a 20 to 30% increase in the detection of HSV in routine isolation-positive genital specimens collected in transport medium. The HSV detection rate, with both procedures, was shown to vary with the site and clinical stage of lesion sampled; it was highest with penile vesicular lesions. Direct extraction of the swab specimen in a small volume of diluent further increased the sensitivity of antigen detection giving positive and negative predictive values of 100 and 96% respectively. The overall sensitivity of HSV detection was equivalent to that obtained by isolation in cell culture. The amplified ELISA offers an alternative, rapid, simple, non-culture technique for routine HSV diagnosis that does not rely upon retention of virus viability.

Factors influencing the sensitivity of herpes simplex virus detection in clinical specimens in a simultaneous enzyme-linked immunosorbent assay using monoclonal antibodies.

A rapid simultaneous enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies was investigated for herpes simplex virus (HSV) detection. All HSV isolated (n = 127) were detected, whereas no response was obtained with HSV negative preparations. Equivalent results were obtained from 275 of 277 clinical specimens in the monoclonal ELISA and in an ELISA using polyclonal antibodies, confirming that appropriately selected monoclonal antibodies may be as efficacious as polyclonal antibodies in antibody-based assays. In clinical specimens, the rate of HSV detection (sensitivity) relative to tissue culture isolation was low for both assays, and the major factor responsible for this was the low concentration of virus present in some specimens. The sensitivity of ELISA obtained in routine use varied with different panels of unselected specimens and was related to the speed of development of the cytopathic effect. These results emphasise the need for caution in assigning a definitive sensitivity level to ELISA tests evaluated on different panels of specimens.

Measurement of antibody to poly (adenosine diphosphate-ribose): its diagnostic value in systemic lupus erythematosus.

Poly (ADP-ribose) and dsDNA binding activity have been measured in sera from 61 patients with systemic lupus erythematosus (SLE) and 188 control sera from 20 normal individuals, 144 patients with clinically similar diseases and 24 patients with drug-induced anti-nuclear antibodies (ANA). Elevated poly (ADP-ribose) binding was not observed with normal sera. Five of 144 samples from diseases entering the differential diagnosis of SLE gave raised poly (ADP-ribose) binding compared with 12 in the 125I-dsDNA binding. Only two of these false positive samples gave elevated binding in the 14C-dsDNA assay. The apparent high specificity of the poly(ADP-ribose) assay was not observed with samples containing drug-induced ANA where 62% had elevated binding values. The frequency with which the poly(ADP-ribose) assay was positive with SLE sera (sensitivity) was lower than either of the dsDNA assays. This low sensitivity and the high rate of false positives in patients with drug-induced ANA limit the value of the poly(ADP-ribose) assay as a diagnostic test for SLE. However the restriction of poly(ADP-ribose) antibody to SLE and patients with drug-induced ANA together with the known role of poly(ADP-ribose) in DNA excision repair suggest that the antibody may be of fundamental significance.