Split single-cell RT-PCR analysis of Purkinje cells

Esumi, Shigeyuki; Kaneko, Ryosuke; Kawamura, Yoshimi; Yagi, Takeshi

doi:10.1038/nprot.2006.343

Cited by 22 publications

(16 citation statements)

References 19 publications

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“…Notably, this start site corresponded to the boundary of the TARE 3 element (Figure 4; Additional files 12 and 13). The intrinsic tendency of PCR amplification of multi-gene families to result in strongly biased representation of certain family members [43] likely explains the homogeneity of our cloned 5' RACE products.…”

Section: Resultsmentioning

confidence: 99%

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

et al. 2011

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BackgroundMounting evidence suggests a major role for epigenetic feedback in Plasmodium falciparum transcriptional regulation. Long non-coding RNAs (lncRNAs) have recently emerged as a new paradigm in epigenetic remodeling. We therefore set out to investigate putative roles for lncRNAs in P. falciparum transcriptional regulation.ResultsWe used a high-resolution DNA tiling microarray to survey transcriptional activity across 22.6% of the P. falciparum strain 3D7 genome. We identified 872 protein-coding genes and 60 putative P. falciparum lncRNAs under developmental regulation during the parasite's pathogenic human blood stage. Further characterization of lncRNA candidates led to the discovery of an intriguing family of lncRNA telomere-associated repetitive element transcripts, termed lncRNA-TARE. We have quantified lncRNA-TARE expression at 15 distinct chromosome ends and mapped putative transcriptional start and termination sites of lncRNA-TARE loci. Remarkably, we observed coordinated and stage-specific expression of lncRNA-TARE on all chromosome ends tested, and two dominant transcripts of approximately 1.5 kb and 3.1 kb transcribed towards the telomere.ConclusionsWe have characterized a family of 22 telomere-associated lncRNAs in P. falciparum. Homologous lncRNA-TARE loci are coordinately expressed after parasite DNA replication, and are poised to play an important role in P. falciparum telomere maintenance, virulence gene regulation, and potentially other processes of parasite chromosome end biology. Further study of lncRNA-TARE and other promising lncRNA candidates may provide mechanistic insight into P. falciparum transcriptional regulation.

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Section: Resultsmentioning

confidence: 99%

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

et al. 2011

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“…Split Single Cell RT-PCR and SNP Analysis-The single cell RT-PCR was performed essentially as described previously, with small modifications (42). The details are provided under supplemental "Experimental Procedures".…”

Section: R/g16neomentioning

confidence: 99%

Total Expression and Dual Gene-regulatory Mechanisms Maintained in Deletions and Duplications of the Pcdha Cluster

Noguchi

Hirabayashi

Katori

et al. 2009

Journal of Biological Chemistry

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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.

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“…Populations-Purkinje cells were isolated as described by Esumi et al (16). The cerebellum of C57BL/6 mice at postnatal day 21 was dissociated by a brief treatment with papain.…”

Section: Dna Methylation Analysis Of Pcdh-␣ In Purkinje Cellmentioning

confidence: 99%

Relationship between DNA Methylation States and Transcription of Individual Isoforms Encoded by the Protocadherin-α Gene Cluster

Kawaguchi

Toyama

Kaneko

et al. 2008

Journal of Biological Chemistry

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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.

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Split single-cell RT-PCR analysis of Purkinje cells

Cited by 22 publications

References 19 publications

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

A global transcriptional analysis of Plasmodium falciparum malaria reveals a novel family of telomere-associated lncRNAs

Total Expression and Dual Gene-regulatory Mechanisms Maintained in Deletions and Duplications of the Pcdha Cluster

Relationship between DNA Methylation States and Transcription of Individual Isoforms Encoded by the Protocadherin-α Gene Cluster

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