Thanh H. Vu

Regulation of telomerase by alternate splicing of human telomerase reverse transcriptase (hTERT) in normal and neoplastic ovary, endometrium and myometrium

Telomerase extends telomeric repeats at the ends of linear chromosomes, thereby prolonging the replicative capacity of cells. To investigate possible regulatory mechanisms of telomerase, we measured telomerase enzyme activity, human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) mRNA in normal and neoplastic ovary, endometrium and myometrium. Telomerase activity was detected in most malignancies and in normal endometrium but not in myometrial leiomyoma, normal myometrium or normal ovary. hTR was expressed in all tissue samples. hTERT mRNA was expressed in many tissue samples, and no tissue sample exhibited telomerase activity without expressing hTERT mRNA. However, the presence of hTR and hTERT mRNA was not sufficient for telomerase activity. Alternate splicing of hTERT produced mRNAs lacking critical reverse transcriptase (RT) motifs in both normal and neoplastic tissues. Only tissues expressing hTERT containing complete A and B RT motifs demonstrated telomerase activity. Finally, several normal ovarian tissues and myometrial leiomyomas lacked telomerase activity despite expressing hTR and hTERT containing complete A and B RT motifs. This was not seen in ovarian and myometrial malignancies, where the expression of hTR and hTERT containing complete A and B RT motifs was sufficient for telomerase activity. We conclude that in ovarian and uterine tissues, the presence of a functional telomerase complex is regulated at multiple levels, including hTERT transcription and alternative splicing of hTERT transcripts. The lack of telomerase activity in several normal but not malignant tissues expressing hTR and hTERT containing complete A and B RT motifs suggests that there are further mechanisms for suppressing telomerase activity downstream of hTERT transcription and mRNA splicing, and these mechanisms have been lost during neoplastic transformation. Int. J. Cancer 85:330–335, 2000.©2000 Wiley‐Liss, Inc.

CTCF Regulates Allelic Expression of Igf2 by Orchestrating a Promoter-Polycomb Repressive Complex 2 Intrachromosomal Loop

ABSTRACT CTCF is a zinc finger DNA-binding protein that regulates the epigenetic states of numerous target genes. Using allelic regulation of mouse insulin-like growth factor II (Igf2) as a model, we demonstrate that CTCF binds to the unmethylated maternal allele of the imprinting control region (ICR) in the Igf2/H19 imprinting domain and forms a long-range intrachromosomal loop to interact with the three clustered Igf2 promoters. Polycomb repressive complex 2 is recruited through the interaction of CTCF with Suz12, leading to allele-specific methylation at lysine 27 of histone H3 (H3-K27) and to suppression of the maternal Igf2 promoters. Targeted mutation or deletion of the maternal ICR abolishes this chromatin loop, decreases allelic H3-K27 methylation, and causes loss of Igf2 imprinting. RNA interference knockdown of Suz12 also leads to reactivation of the maternal Igf2 allele and biallelic Igf2 expression. CTCF and Suz12 are coprecipitated from nuclear extracts with antibodies specific for either protein, and they interact with each other in a two-hybrid system. These findings offer insight into general epigenetic mechanisms by which CTCF governs gene expression by orchestrating chromatin loop structures and by serving as a DNA-binding protein scaffold to recruit and bind polycomb repressive complexes.

Tissue-specific alternate splicing of human telomerase reverse transcriptase (hTERT) influences telomere lengths during human development

Direct genetic manipulations have shown that telomerase‐mediated telomere elongation plays a key role in determining cellular replicative capacity and senescence. The mechanisms regulating the production of an active telomerase enzyme are still predominantly unknown, although roles for transcriptional control of hTERT, alternative‐splicing of hTERT transcripts, and post‐translational phosphorylation of hTERT protein have been advocated. Here we show that hTERT is alternatively spliced in specific patterns by different tissue types during human development. Alternative splicing often prohibits the expression of hTERT protein containing functional reverse transcriptase domains. In these instances, telomerase activity is absent, leading to shortening of telomeres. The specific pattern of hTERT mRNA variants in human development provides evidence that alternative splicing is non‐random and participates in the regulation of telomerase activity.

Promoter-specific Modulation of Insulin-like Growth Factor II Genomic Imprinting by Inhibitors of DNA Methylation

The insulin-like growth factor II (IGF-II) gene is maternally imprinted in most normal tissues with only the paternal allele being transcribed. In several human tumors, however, IGF-II is expressed from both parental alleles. To explore the underlying mechanism of IGF-II imprinting, we have examined the effect of DNA demethylation in cultured human and mouse astrocyte cells. An increased expression of IGF-II was observed when these cells were treated with the DNA demethylating agents, 5-azacytidine or 2-deoxy-5-azacytidine. Allelic analysis indicated that, following DNA demethylation, the increment in IGF-II mRNA was primarily derived from the normally suppressed maternal allele. Examination of promoter usage revealed that only the most proximal promoter (mP3 in mouse and hP4 in human) responded to DNA demethylating agents, whereas the expression of IGF-II from the other promoters remained unchanged. The enhanced expression of IGF-II from these promoters suggests the presence of a methylation-response element in or near mP3 and hP4. This study indicates that DNA demethylating agents increase IGF-II expression primarily by stimulating the normally imprinted allele through the activation of the most proximal IGF-II promoter.

Allele-specific histone acetylation accompanies genomic imprinting of the insulin-like growth factor II receptor gene.

The mouse insulin-like growth factor II receptor (Igf2r) gene encodes two reciprocally imprinted RNA transcripts: paternally imprinted Igf2r sense and maternally imprinted Igf2r antisense. Although DNA methylation has been implicated in the initiation and maintenance of genomic imprinting, acetylation of core histones has recently been appreciated as another important factor that regulates gene expression. To determine whether histone acetylation participates in the regulation of Igf2r imprinting, we examined the relative abundance of acetylated histones in interspecific mice (M. spretus 3 C57BL/6). Oligonucleosomes derived from liver were immunoprecipitated with acetyl-histone antiserum and were analyzed for the allelic distribution of DNA from the region of the sense and antisense Igf2r promoters. In nucleosomes associated with the Igf2r sense promoter, histone acetylation was demonstrated on the maternal allele, which is transcriptionally active. There was much less histone acetylation on the suppressed paternal allele. In nucleosomes associated with the Igf2r antisense promoter, the active paternal allele was heavily acetylated, whereas the suppressed maternal allele was underacetylated. Treatment of cultured fibroblasts with the histone deacetylase inhibitor Trichostatin A induces partial relaxation of genomic imprinting as well as decreased DNA methylation of both Igf2r sense and antisense promoters. These results demonstrate that increases in histone acetylation can lead to decreased DNA methylation, thereby modulating the regulation of the imprinted expression of Igf2r sense and antisense transcripts. (Endocrinology 141: 4428–4435, 2000) A DNA methylation is currently the favored mechanism to explain the initiation or maintenance of genomic imprinting. The expression of genes whose promoter regions are heavily methylated is usually decreased. DNA methyltransferase 1 (Dnmt1) can methylate CpG dinucleotides as well as provide maintenance methylation of the symmetrical cytosine in a hemi-methylated doublet (1). In mice that were made deficient in Dnmt 1 by gene targeting, there was no expression from the normally expressed maternal allele of Igf2r sense or from the normally expressed paternal allele of Igf2, whereas the normally imprinted H19 paternal allele was expressed, demonstrating the importance of DNA methylation in the transcription of imprinted genes (2). The degree of core histone acetylation has also been shown to modulate the expression of numerous genes. In general, histone deacetylation leads to transcriptional repression, whereas histone acetylation increases gene transcription. Histone acetylation is maintained during mitosis, so the acetylation pattern represents a heritable epigenetic imprint that can influence gene transcription (3). Thus, the degree of histone acetylation may represent another potential mechanism that could initiate or maintain genomic imprinting. DNA that is rich in methylated CpG is associated with hypoacetylated histone cores and increased histone H1, whereas DNA containing unmethylated CpG islands is associated with chromatin enriched in hyperacetylated histone cores and less histone H1 (4). Although the methylation of DNA may repress transcription by preventing the binding of transcription factors or by enhancing the binding of specific inhibitory proteins (5), it has also been shown that DNA methylation generates an inactive DNase-resistant local chromatin structure with hypoacetylated core histones. Moreover, the inactive chromatin structure generated by the methylated DNA can spread from the methylated area to adjacent nonmethylated DNA, thereby inhibiting gene transcription over a larger segment of the chromosome (6), and thus providing a potential mechanism for creating a region containing clusters of imprinted genes. Recent studies have demonstrated that the methyl-CpG-binding protein MeCP2 is in fact found in a complex with histone deacetylase and other proteins that might regulate transcription. Moreover, TSA, an inhibitor of histone deacetylase, overcomes DNA methylation-induced transcriptional repression, indicating a linkage between these two known mechanisms of transcriptional repression (7, 8). We previously demonstrated that inhibition of histone deacetylation by trichostatin A (TSA) induces the expression of the normally imprinted maternal IGF2 allele, leading to biallelic expression in human and murine cells (9). TSA-treated mouse conceptuses also demonstrated loss of H19 imprinting (10). In conjunction with DNA methylation, histone acetylation may represent a crucial molecular mechanism for initiating, maintaining and/or transmitting the genomic imprint. The reciprocally imprinted Igf2r sense and antisense tranReceived July 13, 2000. Address all correspondence and requests for reprints to: Andrew R. Hoffman, M.D., Medical Service, Building 101, Room B2–125, VA Palo Alto Health Care System, 3801 Miranda Avenue, Palo Alto, California 94304. E-mail: arhoffman@leland.stanford.edu. * Supported by NIH Grant DK-36054 and by the Research Service of the Department of Veterans Affairs. 0013-7227/00/

Dissociation of IGF2 and H19 imprinting in human brain

03.00/0 Vol. 141, No. 12 Endocrinology Printed in U.S.A. Copyright

Loss of IGF2 imprinting is associated with abrogation of long-range intrachromosomal interactions in human cancer cells

The human IGF2 and H19 genes are imprinted in most normal tissues. Alterations of genomic imprinting or loss of imprinting (LOI) have been observed in a number of malignant tumors. Although LOI has been linked to tumorigenesis, loss of IGF2 imprinting has also been observed in choroid plexus and leptomeninges in normal mouse brain. We have therefore analyzed the allelic expression of both IGF2 and H19 in human fetal brain and in different regions of human adult brain. In the brains of fetuses of 6-12 weeks gestation, both IGF2 and H19 were transcribed from both parental alleles. In contrast, strictly monoallelic expression of both IGF2 and H19 was observed in all other fetal tissues, suggesting a tissue-specific LOI in the central nervous system. In adult brain, LOI of IGF2 was region-specific. IGF2 was expressed from both parental alleles in the pons, but not in globus palludus, Raphe nucleus and hypothalamus. H19 expression was drastically reduced in adult brain compared to fetal brain, and was detectable only in the pons and globus palludus. In contrast to IGF2, the expression of H19 in adult pons was monoallelic. Examination of IGF2 promoter usage indicated predominant utilization of promoter P3 in all fetal and adult brain tissues. The LOI of IGF2 therefore reflects biallelic expression from the predominant promoter. IGF2 transcripts derived from the less abundant promoter P1, however, showed monoallelic expression in the adult pons. Our results suggest that IGF2 and H19 undergo ontogenetic changes in allelic expression and that there is dissociation of IGF2 and H19 imprinting in both fetal and adult human brain.

Epigenetic regulation of Igf2/H19 imprinting at CTCF insulator binding sites.

Nuclear architecture and chromatin geography are important factors in the regulation of gene expression, as these components may play a vital epigenetic role both in normal physiology as well as in the initiation and progression of malignancies. Using a modification of the chromosome conformation capture (3C) technique, we examined long-range chromatin interactions of the imprinted human IGF2 gene. We demonstrate that numerous intrachromosomal interactions occur along both parental alleles in normal tissues, where the IGF2 is paternally expressed, as well as in normal liver where gene expression is biallelic. Long-range and allele-specific interactions occur between the IGF2/H19 imprinting control region-1 (ICR1) and ICR2, a region which regulates an imprinted gene cluster nearly a megabase distant from IGF2. Loss of genomic imprinting is a common epigenetic event in cancer, and long-range interactions have not been examined in malignant cells. In cancer cell lines in which IGF2 imprinting is maintained (MOI), essentially all of the 3C interactions seen in normal cells were preserved. However, in cells in which IGF2 imprinting was lost (LOI), nearly all of the long-range chromatin interactions involving IGF2 were abrogated. A three-dimensional computer model depicts the physical interactions between the IGF2 promoter and ICR1 in MOI cells, while the model of LOI lung cancer cells is flattened with few long-range interactions. This dramatic change in the three-dimension configuration of the chromatin at the IGF2 locus in LOI cancer cells suggests that the loss of imprinting may lead to a variety of changes in gene expression in addition to changes in IGF2 transcription.

Effect of NADPH oxidase inhibition on endothelial cell ELAM-1 mRNA expression

The mouse insulin‐like growth factor II (Igf2) and H19 genes are located adjacent to each other on chromosome 7q11‐13 and are reciprocally imprinted. It is believed that the allelic expression of these two genes is regulated by the binding of CTCF insulators to four parent‐specific DNA methylation sites in an imprinting control center (ICR) located between these two genes. Although monoallelically expressed in peripheral tissues, Igf2 is biallelically transcribed in the CNS. In this study, we examined the allelic DNA methylation and CTCF binding in the Igf2/H19 imprinting center in CNS, hypothesizing that the aberrant CTCF binding as one of the mechanisms leads to biallelic expression of Igf2 in CNS. Using hybrid F1 mice (M. spretus males × C57BL/6 females), we showed that in CNS, CTCF binding sites in the ICR were methylated exclusively on the paternal allele, and CTCF bound only to the unmethylated maternal allele, showing no differences from the imprinted peripheral tissues. Among three other epigenetic modifications examined, histone H3 lysine 9 methylation correlated well with Igf2 allelic expression in CNS. These results suggest that CTCF binding to the ICR alone is not sufficient to insulate the Igf2 maternal promoter and to regulate the allelic expression of the gene in the CNS, thus challenging the aberrant CTCF binding as a common mechanism for lack of Igf2 imprinting in CNS. Further studies should be focused on the identification of factors that are involved in histone methylation and CTCF‐associated factors that may be needed to coordinate Igf2 imprinting.

A Complex Deoxyribonucleic Acid Looping Configuration Associated with the Silencing of the Maternal Igf2 Allele

Neutrophil adherence to endothelium is partially mediated by the expression of endothelial leukocyte adhesion molecule-1 (ELAM-1) on endothelial cells activated by agents such as lipopolysaccharide (LPS) and phorbol myristate acetate (PMA). To elucidate molecular mechanisms involved in the induction of ELAM-1 on endothelial cells, we investigated the effect of the NADPH oxidase inhibitor, apocynin (4-hydroxy-3-methoxyacetophenone), on ELAM-1 mRNA expression in human umbilical vein endothelial cells (HUVEC) by Northern blot analysis. Apocynin downregulated both LPS- and PMA-induced ELAM-1 mRNA expression in a dose-dependent manner. Our results suggest NADPH oxidase might play a key role in ELAM-1 mRNA expression in HUVEC.