Konrad Huppi

Molecular cloning of the human brain and gastric cholecystokinin receptor: Structure, functional expression and chromosomal localization☆

The receptors for the brain and gastrointestinal peptide, cholecystokinin, can be classified into CCKA and CCKB subtypes. Having recently cloned the rat CCKB receptor, we used its cDNA to isolate the human CCKB receptor homologue from brain and stomach which encodes a 447 amino acid protein with 90% identity to both rat CCKB and canine gastrin receptors. Northern hybridization identifies transcripts from stomach, pancreas, brain and gallbladder. The CCKB receptor gene maps to chromosome 11. Expression of the receptor cDNA in COS-7 cells was characteristic of a CCKB receptor subtype pharmacology. These data confirm that we have cloned a novel gene for the human brain and stomach CCKB receptor.

The identification of microRNAs in a genomically unstable region of human chromosome 8q24.

The PVT1 locus is identified as a cluster of T(2;8) and T(8;22) “variant” MYC-activating chromosomal translocation breakpoints extending 400 kb downstream of MYC in a subset (≈20%) of Burkitts lymphoma (vBL). Recent reports that microRNAs (miRNA) may be associated with fragile sites and cancer-associated genomic regions prompted us to investigate whether the PVT1 region on chromosome 8q24 may contain miRNAs. Computational analysis of the genomic sequence covering the PVT1 locus and experimental verification identified seven miRNAs. One miRNA, hsa-miR-1204, resides within a previously described PVT1 exon (1b) that is often fused to the immunoglobulin light chain constant region in vBLs and is present in high copy number in MYC/PVT1–amplified tumors. Like its human counterpart, mouse mmu-miR-1204 represents the closest miRNA to Myc (∼50 kb) and is found only 1 to 2 kb downstream of a cluster of retroviral integration sites. Another miRNA, mmu-miR-1206, is close to a cluster of variant translocation breakpoints associated with mouse plasmacytoma and exon 1 of mouse Pvt1. Virtually all the miRNA precursor transcripts are expressed at higher levels in late-stage B cells (including plasmacytoma and vBL cell lines) compared with immature B cells, suggesting possible roles in lymphoid development and/or lymphoma. In addition, lentiviral vector-mediated overexpression of the miR-1204 precursor (human and mouse) in a mouse pre–B-cell line increased expression of Myc. High levels of expression of the hsa-miR-1204 precursor is also seen in several epithelial cancer cell lines with MYC/PVT1 coamplification, suggesting a potentially broad role for these miRNAs in tumorigenesis. (Mol Cancer Res 2008;6(2):212–21)

p53-Dependent induction of PVT1 and miR-1204.

Background: p53 regulates myriad target genes, including non-coding RNAs that effect cellular outcomes consistent with tumor suppression. Results: p53 induces expression of the PVT1 locus, which encodes both long non-coding RNA and several microRNAs, one of which, miR-1204, is directly regulated by p53. Conclusion: miR-1204 may regulate key p53 outcomes, including cell death. Significance: p53-regulated miR-1204 expression may contribute to tumor suppression. p53 is a tumor suppressor protein that acts as a transcription factor to regulate (either positively or negatively) a plethora of downstream target genes. Although its ability to induce protein coding genes is well documented, recent studies have implicated p53 in the regulation of non-coding RNAs, including both microRNAs (e.g. miR-34a) and long non-coding RNAs (e.g. lincRNA-p21). We have identified the non-protein coding locus PVT1 as a p53-inducible target gene. PVT1, a very large (>300 kb) locus located downstream of c-myc on chromosome 8q24, produces a wide variety of spliced non-coding RNAs as well as a cluster of six annotated microRNAs: miR-1204, miR-1205, miR-1206, miR-1207-5p, miR-1207-3p, and miR-1208. Chromatin immunoprecipitation (ChIP), electrophoretic mobility shift assay (EMSA), and luciferase assays reveal that p53 binds and activates a canonical response element within the vicinity of miR-1204. Consistently, we demonstrate the p53-dependent induction of endogenous PVT1 transcripts and consequent up-regulation of mature miR-1204. Finally, we have shown that ectopic expression of miR-1204 leads to increased p53 levels and causes cell death in a partially p53-dependent manner.

Pvt1-encoded microRNAs in oncogenesis

BackgroundThe functional significance of the Pvt1 locus in the oncogenesis of Burkitts lymphoma and plasmacytomas has remained a puzzle. In these tumors, Pvt1 is the site of reciprocal translocations to immunoglobulin loci. Although the locus encodes a number of alternative transcripts, no protein or regulatory RNA products were found. The recent identification of non-coding microRNAs encoded within the PVT1 region has suggested a regulatory role for this locus.ResultsThe mouse Pvt1 locus encodes several microRNAs. In mouse T cell lymphomas induced by retroviral insertions into the locus, the Pvt1 transcripts, and at least one of their microRNA products, mmu-miR-1204 are overexpressed. Whereas up to seven co-mutations can be found in a single tumor, in over 2,000 tumors none had insertions into both the Myc and Pvt1 loci.ConclusionJudging from the large number of integrations into the Pvt1 locus – more than in the nearby Myc locus – Pvt1 and the microRNAs encoded by it are as important as Myc in T lymphomagenesis, and, presumably, in T cell activation. An analysis of the co-mutations in the lymphomas likely place Pvt1 and Myc into the same pathway.

Genomic instability in MycER-activated Rat1A-MycER cells

The deregulated expression of c-Myc protein is associated with the non-random locus-specific amplification of the dihydrofolate reductase (DHFR) gene. This study was performed to determine whether additional chromosomal aberrations occur when c-Myc protein levels are up-regulated for prolonged periods. To this end, we have used Rat1A-MycER cells, which allow the experimental regulation of Myc protein levels. We examined the genomic stability of Rat1A-MycER cells cultivated in either the absence or the presence of estrogen, which reportedly activates the chimeric MycER protein in these cells. Following prolonged periods of MycER activation, Rat1A-Mycer cells exhibited irreversible chromosomal aberrations. The aberrations included numerical changes, chromosome breakage, the formation of circular chromosomal structures, chromosome fusions, and extrachromosomal elements.

Mouse chromosome 4.

This year’s report incorporates 78 new genetic markers into the consensus linkage map. Of these markers, ten have a known, mapped human homolog. The murine gene, followed by the human homolog and the human chromosomal location in parenthesis are as follows: Cpt2 4 CPT2 (1p32); Cyp2j5 and Cyp2j6 4 CYP2J2 (1p31.3-p31.2); Guca1b 4 GUCA2B (1p34-p33); Htr6 4 HTR6 (1p36-1p35); Hub 4 ELAVL2 (9p21); Hud 4 ELAVL4 (1p34); Matn1 4 MATN1 (1p35); Mmp16 4 MMP16 (8q21.3-q22.1); Tgfbr1 4 TGFBR1 (9q33-q34). In the case of the Cyp genes, two genes have been identified in mouse while only a single gene has been identified in humans. Mouse Chromosome (Chr) 4 shares significant stretches of linkage homology with human Chr 1, 6, 8, 9 and 21 (34245; 23572). The entire distal half of mouse Chr 4 is homologous with human Chr 1p. There have been four changes in nomenclature of genetic loci on Chr 4. Gene names and/or symbols that have been changed by the Nomenclature Committee include: Cerr1 changed to Cer1; Dana changed to D4H1s1733E; Zie changed to Gklf; and Etl2 changed to Il11ra2.

Chromosomal localization of the gastric and brain receptors for cholecystokinin (CCKAR and CCKBR) in human and mouse

Receptors for cholcystokinin (CCK) can be pharmacologically classified into at least two distinct subtypes, CCKAR and CCKBR. In an effort to determine whether the CCKA and CCKB receptors may be associated with certain CNS or gastrointestinal diseases, we have localized and compared the human and mouse chromosomal loci encoded by the CCKAR and CCKBR genes. The gene encoding the CCKA receptor maps to a syntenic region of human chromosome 4 and mouse chromosome 5. The CCKB receptor gene, on the other hand, resides on a syntenic region of human chromosome 11 and distal mouse chromosome 7. Localization of the CCK receptors with two dopamine receptors, DRD5 (4p15.1-p15.3) and DRD4 (11p15), provides the interesting possibility of coinvolvement in neuropsychiatric or CNS illnesses.

Systems-wide RNAi analysis of CASP8AP2 / FLASH shows transcriptional deregulation of the replication-dependent histone genes and extensive effects on the transcriptome of colorectal cancer cells

BackgroundColorectal carcinomas (CRC) carry massive genetic and transcriptional alterations that influence multiple cellular pathways. The study of proteins whose loss-of-function (LOF) alters the growth of CRC cells can be used to further understand the cellular processes cancer cells depend upon for survival.ResultsA small-scale RNAi screen of ~400 genes conducted in SW480 CRC cells identified several candidate genes as required for the viability of CRC cells, most prominently CASP8AP2/FLASH. To understand the function of this gene in maintaining the viability of CRC cells in an unbiased manner, we generated gene specific expression profiles following RNAi. Silencing of CASP8AP2/FLASH resulted in altered expression of over 2500 genes enriched for genes associated with cellular growth and proliferation. Loss of CASP8AP2/FLASH function was significantly associated with altered transcription of the genes encoding the replication-dependent histone proteins as a result of the expression of the non-canonical polyA variants of these transcripts. Silencing of CASP8AP2/FLASH also mediated enrichment of changes in the expression of targets of the NFκB and MYC transcription factors. These findings were confirmed by whole transcriptome analysis of CASP8AP2/FLASH silenced cells at multiple time points. Finally, we identified and validated that CASP8AP2/FLASH LOF increases the expression of neurofilament heavy polypeptide (NEFH), a protein recently linked to regulation of the AKT1/ß-catenin pathway.ConclusionsWe have used unbiased RNAi based approaches to identify and characterize the function of CASP8AP2/FLASH, a protein not previously reported as required for cell survival. This study further defines the role CASP8AP2/FLASH plays in the regulating expression of the replication-dependent histones and shows that its LOF results in broad and reproducible effects on the transcriptome of colorectal cancer cells including the induction of expression of the recently described tumor suppressor gene NEFH.

Multiplexing siRNAs to compress RNAi-based screen size in human cells

Here we describe a novel strategy using multiplexes of synthetic small interfering RNAs (siRNAs) corresponding to multiple gene targets in order to compress RNA interference (RNAi) screen size. Before investigating the practical use of this strategy, we first characterized the gene-specific RNAi induced by a large subset (258 siRNAs, 129 genes) of the entire siRNA library used in this study (∼800 siRNAs, ∼400 genes). We next demonstrated that multiplexed siRNAs could silence at least six genes to the same degree as when the genes were targeted individually. The entire library was then used in a screen in which randomly multiplexed siRNAs were assayed for their affect on cell viability. Using this strategy, several gene targets that influenced the viability of a breast cancer cell line were identified. This study suggests that the screening of randomly multiplexed siRNAs may provide an important avenue towards the identification of candidate gene targets for downstream functional analyses and may also be useful for the rapid identification of positive controls for use in novel assay systems. This approach is likely to be especially applicable where assay costs or platform limitations are prohibitive.

Mouse Chromosome 15

This report is a supplement of the first Chr 15 report (Mock et al. 1991). It summarizes new and previously omitted data and contains (1) a locus list, (2) a list of microsatellite primer sequences, (3) a summary of new physical map data near Ly-6, (4) an updated cytogenetic map, (5) a summary of new linkage data and an updated composite map, (6) a list of new SDPs for RI strains and a map of Chr 15 based on RI data, and (7) new information concerning mutations in the mouse that produce visible phenotypes.