The molecular basis for providing the identity and diversity of single neurons is a key for realizing the brain system. Diverse protocadherin isoforms encoded by the Pcdh-␣ and Pcdh-␥ gene clusters are expressed in all of the vertebrates studied. For the Pcdh-␣ isoforms, differential expression patterns have been found in single Purkinje cells by unusual monoallelic and combinatorial types of gene regulation. Here we investigated total allelic gene regulation in the Pcdh-␣ and -␥ clusters, including the C-type variable exons (C1 to C5) and the Pcdh-␥A and -␥B variable exons in single Purkinje cells. Using split single-cell reverse transcription-PCR analysis, almost all of the Purkinje cells at postnatal day 21 biallelically expressed all the C-type isoforms, whereas the Pcdh-␣ isoforms showed both monoallelic and combinatorial expression. The Pcdh-␥A and -␥B isoforms also showed differential regulation in each cell with both monoallelic and combinatorial gene regulation. These data indicated that different types of allelic gene regulation (monoallelic versus biallelic) occurred in the Pcdh-␣ and -␥ clusters, although they were spliced into the same constant exons. It has been reported that each C-type Pcdh-␣ or -␥ transcript has a different expression pattern during brain development, suggesting that the different C-type variable exons may code temporal diversity, although the Pcdh-␣, -␥A, and -␥B isoforms were differential and combinatorial gene regulation within a single cell. Thus, the multiple gene regulations in the Pcdh-␣ and -␥ clusters had a potential mechanism for increasing the diversity of individual neurons in the brain.

for trapoxin B, by X-ray analysis, mass spectrometric, NMR and chemical studies.

The clustered protocadherins (Pcdhs), Pcdh-␣, -␤, and -␥, are transmembrane proteins constituting a subgroup of the cadherin superfamily. Each Pcdh cluster is arranged in tandem on the same chromosome. Each of the three Pcdh clusters shows stochastic and combinatorial expression in individual neurons, thus generating a hugely diverse set of possible cell surface molecules. Therefore, the clustered Pcdhs are candidates for determining neuronal molecular diversity. Here, we showed that the targeted deletion of DNase I hypersensitive (HS) site HS5-1, previously identified as a Pcdh-␣ regulatory element in vitro, affects especially the expression of specific Pcdh-␣ isoforms in vivo. We also identified a Pcdh-␤ cluster control region (CCR) containing six HS sites (HS16, 17, 17, 18, 19, and 20) downstream of the Pcdh-␥ cluster. This CCR comprehensively activates the expression of the Pcdh-␤ gene cluster in cis, and its deletion dramatically decreases their expression levels. Deleting the CCR nonuniformly down-regulates some Pcdh-␥ isoforms and does not affect Pcdh-␣ expression. Thus, the CCR effect extends beyond the 320-kb region containing the Pcdh-␥ cluster to activate the upstream Pcdh-␤ genes. Thus, we concluded that the CCR is a highly specific regulatory unit for Pcdh-␤ expression on the clustered Pcdh genomic locus. These findings suggest that each Pcdh cluster is controlled by distinct regulatory elements that activate their expression and that the stochastic gene regulation of the clustered Pcdhs is controlled by the complex chromatin architecture of the clustered Pcdh locus.

The naked mole-rat (NMR, Heterocephalus glaber), which is the longest-lived rodent species, exhibits extraordinary resistance to cancer. Here we report that NMR somatic cells exhibit a unique tumour-suppressor response to reprogramming induction. In this study, we generate NMR-induced pluripotent stem cells (NMR-iPSCs) and find that NMR-iPSCs do not exhibit teratoma-forming tumorigenicity due to the species-specific activation of tumour-suppressor alternative reading frame (ARF) and a disruption mutation of the oncogene ES cell-expressed Ras (ERAS). The forced expression of Arf in mouse iPSCs markedly reduces tumorigenicity. Furthermore, we identify an NMR-specific tumour-suppression phenotype—ARF suppression-induced senescence (ASIS)—that may protect iPSCs and somatic cells from ARF suppression and, as a consequence, tumorigenicity. Thus, NMR-specific ARF regulation and the disruption of ERAS regulate tumour resistance in NMR-iPSCs. Our findings obtained from studies of NMR-iPSCs provide new insight into the mechanisms of tumorigenicity in iPSCs and cancer resistance in the NMR.

The protocadherin-␣ (Pcdh-␣) gene encodes diverse transmembrane proteins that are differentially expressed in individual neurons in the vertebrate central nervous system. The Pcdh-␣ genomic structure contains variable first exons, each regulated by its own promoter. Here, we investigated the effect of DNA methylation on gene regulation in the Pcdh-␣ gene cluster. We studied two mouse cell lines, C1300 and M3, that expressed different combinations of Pcdh-␣ isoforms and found that 1) the transcription of specific Pcdh-␣ isoforms correlated significantly with the methylation state of the promoter and the 5 (but not the 3) region of the first exon and 2) mosaic or mixed methylation states of the promoters were associated with both active and inactive transcription. Demethylation of C1300 cells up-regulated all of the Pcdh-␣ isoforms, and, in a promoter assay, hypermethylation of the promoters repressed their transcriptional activity. Cell lines subcloned from the demethylated C1300 cells transcribed different combinations of Pcdh-␣ isoforms than the parental, nondemethylated cells, and the promoters showed differential mosaic or mixed methylation patterns. In vivo, the promoter and 5-regions of the Pcdh-␣C1 and ␣C2 exons, which are transcribed in all neurons, were extensively hypomethylated. In contrast, the promoters of the Pcdh-␣1 to -␣12 isoforms, which are transcribed differentially by individual Purkinje cells, exhibited mosaic methylation patterns. Overall, our results demonstrated that mosaic or mixed DNA methylation states in the promoter and 5-region of the first exon may help regulate differential Pcdh-␣ transcription and that hypermethylation is sufficient to repress transcription.

Serotonergic axons extend diffuse projections throughout various brain areas, and serotonergic system disruption causes neuropsychiatric diseases. Loss of the cytoplasmic region of protocadherin-α (Pcdh-α) family proteins, products of the diverse clustered Pcdh genes, causes unbalanced distributions (densification and sparsification) of serotonergic axons in various target regions. However, which Pcdh-α member(s) are responsible for the phenotype is unknown. Here we demonstrated that Pcdh-αC2 (αC2), a Pcdh-α isoform, was highly expressed in serotonergic neurons, and was required for normal diffusion in single-axon-level analyses of serotonergic axons. The loss of αC2 from serotonergic neurons, but not from their target brain regions, led to unbalanced distributions of serotonergic axons. Our results suggest that αC2 expressed in serotonergic neurons is required for serotonergic axon diffusion in various brain areas. The αC2 extracellular domain displays homophilic binding activity, suggesting that its homophilic interaction between serotonergic axons regulates axonal density via αC2′s cytoplasmic domain.

The clustered protocadherin-␣ (Pcdha) genes, which are expressed in the vertebrate brain, encode diverse membrane proteins whose functions are involved in axonal projection and in learning and memory. The Pcdha cluster consists of 14 tandemly arranged genes (Pcdha1-Pcdha12, Pcdhac1, and Pcdhac2, from 5 to 3). Each first exon (the variable exons) is transcribed from its own promoter, and spliced to the constant exons, which are common to all the Pcdha genes. Cerebellar Purkinje cells show dual expression patterns for Pcdha. In individual Purkinje cells, different sets of the 5 genes in the cluster, Pcdha1-12, are randomly expressed, whereas both 3 genes, Pcdhac1 and Pcdhac2, are expressed constitutively. To elucidate the relationship between the genomic structure of the Pcdha cluster and their expression in Purkinje cells, we deleted or duplicated multiple variable exons and analyzed the expression of Pcdha genes in the mouse brain. In all mutant mice, transcript levels of the constant exons and the dual expression patterns were maintained. In the deletion mutants, the missing genes were flexibly compensated by the remaining variable exons. On the other hand, in duplication mutants, the levels of the duplicated genes were trimmed. These results indicate that the Pcdha genes are comprehensively regulated as a cluster unit, and that the regulators that randomly and constitutively drive Pcdha gene expression are intact in the deleted or duplicated mutant alleles. These dual regulatory mechanisms may play important roles in the diversity and fundamental functions of neurons.

PA48009,a lanthionine-containing peptide antibiotic was isolated from the culture broth of Streptoverticillium griseoverticillatum PA-48009, and identified as duramycin. Determination of the structure using both Edmandegradations and 2D NMR spectroscopy showed the need to revise the structure of duramycin given in literature. Duramycin (PA48009) was different from lanthiopeptin (Ro 09-0198, cinnamycin) only by a Lys/Arg exchange at position 2.During screening of new biologically active compounds, PA48009 (1), a lanthionine-containing peptide antibiotic, was isolated from the fermentation broth of Streptoverticillium griseoverticillatum PA-48009. It exhibited weak antimicrobial activity against Gram-positive organisms. Several lanthionine-containing peptide antibiotics have been reported already1~n). Recent development of NMRmethodology and instrumentation12 '13) make it possible to analyze NMRspectra and to determine the conformation of small proteins and oligopeptides. The structure of Ro 09-0198 was reported by Kessler et al.lt8\ but the reported structure showed some discrepancies with that of lanthiopeptin (3) ( = Ro 09-0198) reported by Wakamiya et al.3). Weconsidered that this problem had arisen from the difficulty in discriminating the amino acid residue as Ala or Phe in the sequence analysis using NMR. Therefore, we employed both NMRarid Edmandegradation methods to analyze the structure of 1. The discrimination of the Phe residue from the Ala residue was possible with Edmandegradation, and the sulfide bridges in the lanthionine (Lan) and methyllanthionine (MeLan) moiety and the NHbridge in the lysinoalanine (LysAla) moiety were easily determined by analyzing the NOESYspectra. As PA48009 (1) was identified as duramycin1'2) by physical measurements, wereport here the revised structure of duramycin (1) as shownin Fig. 1.