Hiroyuki Kugoh

Functional expression and germline transmission of a human chromosome fragment in chimaeric mice.

Human chromosomes or chromosome fragments derived from normal fibroblasts were introduced into mouse embryonic stem (ES) cells via microcell-mediated chromosome transfer (MMCT) and viable chimaeric mice were produced from them. Transferred chromosomes were stably retained, and human genes, including immunoglobulin (Ig) κ, heavy, λ genes, were expressed in proper tissue-specific manner in adult chimaeric tissues. In the case of a human chromosome (hChr.) 2-derived fragment, it was found to be transmitted to the offspring through the germline. Our study demonstrates that MMCT allows for introduction of very large amounts of foreign genetic material into mice. This novel procedure will facilitate the functional analyses of human genomes in vivo.

Microdeletion of LIT1 in Familial Beckwith-Wiedemann Syndrome

Beckwith-Wiedemann syndrome (BWS), which causes prenatal overgrowth, midline abdominal wall defects, macroglossia, and embryonal tumors, is a model for understanding the relationship between genomic imprinting, human development, and cancer. The causes are heterogeneous, involving multiple genes on 11p15 and including infrequent mutation of p57(KIP2) or loss of imprinting of either of two imprinted gene domains on 11p15: LIT1, which is near p57(KIP2), or H19/IGF2. Unlike Prader-Willi and Angelman syndromes, no chromosomal deletions have yet been identified. Here we report a microdeletion including the entire LIT1 gene, providing genetic confirmation of the importance of this gene region in BWS. When inherited maternally, the deletion causes BWS with silencing of p57(KIP2), indicating deletion of an element important for the regulation of p57(KIP2) expression. When inherited paternally, there is no phenotype, suggesting that the LIT1 RNA itself is not necessary for normal development in humans.

Evidence for a putative telomerase repressor gene in the 3p14.2–p21.1 region

Telomeres, which are the repeated sequences located on both ends of chromosomes in eukaryotes, are known to shorten with each cell division, and their eventual loss is thought to result in cellular senescence. Unlike normal somatic cells, most tumor cells show activation of telomerase, a ribonucleoprotein enzyme that stably maintains telomere length by addition of the sequences of TTAGGG repeats to telomeres. The KC12 cell line derived from a renal cell carcinoma in a patient with von Hippel‐Lindau disease showed telomerase activity and loss of heterozygosity on the short arm of chromosome 3. Introduction of a normal human chromosome 3 into KC12 cells by microcell fusion induced cellular senescence, accompanied by suppression of telomerase activity and shortening of telomere length. Microcell hybrids that escaped from cellular senescence maintained telomere length and telomerase activity similar to those of the parental KC12 cells. We previously showed a similar suppression of telomerase activity by introduction of chromosome 3 into another renal cell carcinoma cell line, RCC23. The putative telomerase repressor gene was mapped to chromosome region 3p14.2–p21.1 by deletion mapping of KC12 + chromosome 3 revertants that escaped from cellular senescence and by transfer of subchromosomal fragments of chromosome 3 into RCC23 cells. Genes Chromosomes Cancer 23:123–133, 1998.

Expression profile of LIT1/KCNQ1OT1 and epigenetic status at the KvDMR1 in colorectal cancers

The human chromosome region 11p15.5 contains a number of maternally and paternally imprinted genes, and the LIT1/KCNQ1OT1 locus acts as an imprinting center in the proximal domain of 11p15.5. Loss of imprinting (LOI) of LIT1 and its correlation with methylation status at a differentially methylated region, the KvDMR1, were investigated in 69 colorectal cancer tissue specimens. LIT1 expression profiles were also examined by RNA‐fluorescence in situ hybridization in 13 colorectal cancer cell lines. In 69 colorectal cancer tissue specimens, LOI of LIT1 was observed in nine of the 17 (53%) informative cases. Moreover, LOI of LIT1 was only observed in tumor samples. In the cell lines, methylation status at the KvDMR1 correlated well with LIT1 expression profiles. Loss of expression of LIT1 also correlated with enrichment of H3 lysine 9 (H3‐K9) dimethylation and reduction of H3 lysine 4 (H3‐K4) dimethylation. Thus, LIT1 expression appears to be controlled by epigenetic modifications at the KvDMR1, although CDKN1C expression, which is considered to be controlled by LIT1, was not associated with epigenetic status at the KvDMR1 in some colorectal cancer cell lines. Therefore, these findings suggest that LOI of LIT1 via epigenetic disruption plays an important role in colorectal carcinogenesis, but it is not necessarily associated with CDKN1C expression. (Cancer Sci 2006; 97: 1147–1154)

Functional evidence for a telomerase repressor gene on human chromosome 10p15.1

Based on the sites of frequent allelic loss in hepatocellular carcinoma, five normal human chromosomes (2, 4, 5, 10 and 16) were transferred individually into a telomerase-positive human hepatocellular carcinoma cell line, Li7HM, by microcell-mediated chromosome transfer (MMCT). Chromosome 10, but not the others, repressed telomerase activity immediately and stopped cell growth after 50 population doublings (PDs). Loss of the transferred 10p loci resulted in the emergence of revertant cells that continued to proliferate and expressed telomerase activity, suggesting the presence of a telomerase repressor gene on this chromosomal arm. Transfer of a series of defined fragments from chromosome 10p successfully narrowed down the responsible region: a 28.9-cM region on 10p15 (between WI-4752 and D10S249), but not a 26.2-cM region (between D10S1728 and D10S249), caused repression of telomerase activity and progressive telomere shortening. A strong correlation between the expression level of telomerase catalytic subunit gene (hTERT) and telomerase activity was observed. These findings suggest that a novel telomerase repressor gene which controls the expression of hTERT is located on the 2.7-cM region (between WI-4752 and D10S1728) on chromosome 10p15.1.

Mapping of metastasis suppressor gene(s) for rat prostate cancer on the short arm of human chromosome 8 by irradiated microcell-mediated chromosome transfer

Our previous studies demonstrated that human chromosome 8 contains metastasis suppressor gene(s) for rat prostate cancer. However, it is still unknown which portion of human chromosome 8 is associated with suppression of metastatic ability, because all of the clones in which metastatic ability is suppressed contain at least one copy of intact human chromosome 8. In the present study, we used the irradiated microcell‐mediated chromosome transfer technique to enrich for specific chromosomal arm deletions of selected chromosomes. The resultant series of human chromosomes 8 with a variety of chromosomal deletions was introduced into highly metastatic Dunning rat prostate cancer cells. All of the resultant microcell hybrids showed reduced metastatic ability. To obtain a smaller size of human chromosome 8 and to locate further the region of metastasis suppressor gene(s), the most reduced size of human chromosome 8 that was generated with the initial irradiated chromosome transfer was retransferred into the Dunning cancer cells without irradiation. The resultant microcell hybrids were analyzed to determine which portion of human chromosome 8 suppressed the metastatic ability of the recipient cells. This analysis demonstrates that the portion of human chromosome 8 containing metastasis suppressor gene(s) for rat prostate cancer cells lies on human chromosome segment 8p21‐p12, where frequent allelic losses have been detected in allelotype analyses of human prostate cancer. This suggests that one of the metastasis suppressor genes for rat prostate cancer on human chromosome 8 may also play an important role in the progression of human prostate cancer. Genes Chromosom Cancer 17:260–268 (1996).

Identification of PITX1 as a TERT Suppressor Gene Located on Human Chromosome 5

ABSTRACT Telomerase, a ribonucleoprotein enzyme that maintains telomere length, is crucial for cellular immortalization and cancer progression. Telomerase activity is attributed primarily to the expression of telomerase reverse transcriptase (TERT). Using microcell-mediated chromosome transfer (MMCT) into the mouse melanoma cell line B16F10, we previously found that human chromosome 5 carries a gene, or genes, that can negatively regulate TERT expression (H. Kugoh, K. Shigenami, K. Funaki, J. Barrett, and M. Oshimura, Genes Chromosome Cancer 36:37–47, 2003). To identify the gene responsible for the regulation of TERT transcription, we performed cDNA microarray analysis using parental B16F10 cells, telomerase-negative B16F10 microcell hybrids with a human chromosome 5 (B16F10MH5), and its revertant clones (MH5R) with reactivated telomerase. Here, we report the identification of PITX1, whose expression leads to the downregulation of mouse tert (mtert) transcription, as a TERT suppressor gene. Additionally, both human TERT (hTERT) and mouse TERT (mtert) promoter activity can be suppressed by PITX1. We show that three and one binding site within the hTERT and mtert promoters, respectively, that express a unique conserved region are responsible for the transcriptional activation of TERT. Furthermore, we showed that PITX1 binds to the TERT promoter both in vitro and in vivo. Thus, PITX1 suppresses TERT transcription through direct binding to the TERT promoter, which ultimately regulates telomerase activity.

Phage ϕC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines

X‐linked severe combined immunodeficiency (SCID‐X1, X‐SCID) is a life‐threatening disease caused by a mutated common cytokine receptor γ chain (γc) gene. Although ex vivo gene therapy, i.e., transduction of the γc gene into autologous CD34+ cells, has been successful for treating SCID‐X1, the retrovirus vector‐mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the ϕC31 integrase‐based integration system using human T‐cell lines, including the γc‐deficient ED40515(‐).

CTCFL/BORIS is a methylation-independent DNA-binding protein that preferentially binds to the paternal H19 differentially methylated region

The CTCF paralog BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein thought to play a role in chromatin organization and gene expression. Under normal physiologic conditions, BORIS is predominantly expressed during embryonic male germ cell development; however, it is also expressed in tumors and tumor cell lines and, as such, has been classified as a cancer-germline or cancer-testis gene. It has been suggested that BORIS may be a pro-proliferative factor, whereas CTCF favors antiproliferation. BORIS and CTCF share similar zinc finger DNA-binding domains and seem to bind to identical target sequences. Thus, one critical question is the mechanism governing the DNA-binding specificity of these two proteins when both are present in tumor cells. Chromatin immunoprecipitation (ChIP) in HCT116 cells and their hypermethylated variant showed that BORIS binds to methylated DNA sequences, whereas CTCF binds to unmethylated DNA. Electromobility shift assays, using both whole-cell extracts and in vitro translated CTCF and BORIS protein, and methylation-specific ChIP PCR showed that BORIS is a methylation-independent DNA-binding protein. Finally, experiments in murine hybrid cells containing either the maternal or paternal human chromosome 11 showed that BORIS preferentially binds to the methylated paternal H19 differentially methylated region, suggesting a mechanism in which the affinity of CTCF for the unmethylated maternal allele directs the DNA binding of BORIS toward the paternal allele.

Suggestive evidence for chromosomal localization of non-coding RNA from imprinted LIT1.

AbstractThe non-coding RNA LIT1/KCNQ1OT1, itself the product of an imprinted gene, is involved in cis-limited silencing within an imprinted cluster on human chromosome 11p15.5. Although the locus serves as an imprinting center, the mechanism of transcriptional regulation is not clear. To help understand the function of the LIT1 non-coding RNA, we used fluorescence in situ hybridization (FISH) to examine the sub-cellular localization of LIT1 RNA molecules. LIT1 RNA signals were observed in most of the interphase human lymphoblast and fibroblast cells. The RNA also appeared to accumulate on neighboring regions of chromatin containing the SLC22A18/IMPT1 and CDKN1C/p57KIP2 genes, as shown by high-resolution fiber RNA FISH and modified RNA TRAP (tagging and recovery of associated proteins) methods. These results suggest that LIT1 RNA stably localizes to a specific chromatin region and plays an important role in the transcriptional silencing of the imprinting domain.