Active Development Research Articles

X-chromosome inactivation in development and cancer.

X-chromosome inactivation represents an epigenetics paradigm and a powerful model system of facultative heterochromatin formation triggered by a non-coding RNA, Xist, during development. Once established, the inactive state of the Xi is highly stable in somatic cells, thanks to a combination of chromatin associated proteins, DNA methylation and nuclear organization. However, sporadic reactivation of X-linked genes has been reported during ageing and in transformed cells and disappearance of the Barr body is frequently observed in cancer cells. In this review we summarise current knowledge on the epigenetic changes that accompany X inactivation and discuss the extent to which the inactive X chromosome may be epigenetically or genetically perturbed in breast cancer.

Reduced Fhit protein expression in human malignant mesothelioma.

Human malignant mesothelioma (MM) is an aggressive neoplasm related to occupational exposure to asbestos and characterised by a long latency time. Multiple chromosomal deletions and DNA losses have been revealed in MM by studies performed with karyotypic, comparative genomic hybridisation and loss of heterozygosity (LOH) analyses. Among frequently deleted chromosomal sites, LOH at chromosome 3p has been detected in MM, suggesting the presence of one or several tumour suppressor genes that have an important role in development of the disease. The FHIT (fragile histidine triad) tumour suppressor gene, located at 3p14.2, has been proposed to be a target to major human lung carcinogens, such as tobacco smoke and asbestos. Although many studies have indicated decreased Fhit protein expression in a variety of malignancies, there is no report of FHIT gene aberrations or Fhit protein abnormalities in MM. We examined expression of the Fhit protein and LOH at the FHIT gene in malignant mesothelioma. Altogether, 13 paraffin embedded MM tumours were analysed for Fhit protein expression, and 21 fresh tumours and 10 cell cultures for LOH at the FHIT gene with two intragenic microsatellite markers. All tumours showed less intense immunostaining than normal bronchial epithelium or mesothelium. Fhit expression was absent or reduced in 54% (7 of 13) of the tumours, with the weakest staining observed in poorly differentiated areas. Allele loss was seen in 3 of 10 (30%) of the MM cell lines, but only in 1 of the 21 fresh tumours studied, suggesting concealment of LOH by normal cells present in MM tumours. In conclusion, our present data indicate a frequent decrease of Fhit protein expression, thus supporting the significance of FHIT inactivation in development of MM.

Reduced Fhit protein expression and loss of heterozygosity at FHIT gene in tumours from smoking and asbestos-exposed lung cancer patients

The FHIT gene, at 3p14.2, has been suggested to form a molecular target to damage induced by human lung carcinogens. We examined aberrant expression of the Fhit protein and allele loss at the FHIT gene in a series of lung cancer cases, mainly of non-small cell carcinoma (NSCLC) histology. We had detailed data on tobacco smoke exposure and occupational asbestos exposure available for the cases. The principal aim of the present study was to investigate whether absent or reduced Fhit expression or FHIT allele loss was associated with exposure to these pulmonary carcinogens. We detected reduced Fhit expression in 62% (33/53) of the cases analysed. Prevalence of allele loss at the FHIT locus was 22% (20/89). Reduced protein expression was common both in the asbestos-exposed (67%) and non-exposed cases (59%); [odds ratio (OR) 1.4, 95% confidence interval (CI) 0.4-4.9]. LOH frequencies differed somewhat between the two groups and were 25% vs. 16%, respectively (OR 1.8; 95% CI 0.5-5.9). Absent or reduced expression was common in smokers, with no significant difference found between current smokers and non-smokers (mainly former smokers) (OR 1.4, 95% CI 0.5-4.5). NSCLCs with squamous cell histology exhibited both aberrant expression (OR 3.1, 95% CI 0.9-10.3) and allele loss (OR 3.3, 95% CI 0.9-12.7) more frequently than adenocarcinoma. Finally, we found that FHIT allele loss was increased in stage II or more advanced disease (OR 2.5, 95% CI 0.9-7.4), and in poorly differentiated tumours (grade 3, OR 2.6, 95% CI 0.8-8.1). In conclusion, our present data support significance of FHIT inactivation in development of lung cancer.

Regulation of X-chromosome inactivation in development in mice and humans.

Dosage compensation for X-linked genes in mammals is accomplished by inactivating one of the two X chromosomes in females. X-chromosome inactivation (XCI) occurs during development, coupled with cell differentiation. In somatic cells, XCI is random, whereas in extraembryonic tissues, XCI is imprinted in that the paternally inherited X chromosome is preferentially inactivated. Inactivation is initiated from an X-linked locus, the X-inactivation center (Xic), and inactivity spreads along the chromosome toward both ends. XCI is established by complex mechanisms, including DNA methylation, heterochromatinization, and late replication. Once established, inactivity is stably maintained in subsequent cell generations. The function of an X-linked regulatory gene, Xist, is critically involved in XCI. The Xist gene maps to the Xic, it is transcribed only from the inactive X chromosome, and the Xist RNA associates with the inactive X chromosome in the nucleus. Investigations with Xist-containing transgenes and with deletions of the Xist gene have shown that the Xist gene is required in cis for XCI. Regulation of XCI is therefore accomplished through regulation of Xist. Transcription of the Xist gene is itself regulated by DNA methylation. Hence, the differential methylation of the Xist gene observed in sperm and eggs and its recognition by protein binding constitute the most likely mechanism regulating imprinted preferential expression of the paternal allele in preimplantation embryos and imprinted paternal XCI in extraembryonic tissues. This article reviews the mechanisms underlying XCI and recent advances elucidating the functions of the Xist gene in mice and humans.

The X chromosome in development in mouse and man.

In mammals, dosage compensation for X-linked genes between males and females is achieved by the inactivation of one of the X chromosomes in females. The inactivation event occurs early in development in all cells of the female mouse embryo and is stable and heritable in somatic cells. However, in the primordial germ cells, reactivation occurs around the time of meiosis. Owing to random inactivation in somatic cells, all female mice and humans are mosaic for X-linked gene function. Variable mosaicism can result in expression of disease in human females heterozygous for an X-linked gene defect. In the extra-embryonic lineages of female mouse embryos, and in the somatic cells of female marsupials, the paternally inherited X chromosome is preferentially inactivated. The X chromosomes in the egg and sperm must be differentially marked or imprinted, so that they are distinguished by the inactivation mechanism in these tissues. Initiation of inactivation of an entire X chromosome appears to spread from a single X-inactivation centre and may involve the recently discovered gene, XIST, which is expressed only from the inactive X chromosome. The maintenance of inactivation of certain household genes on the inactive X chromosome involves methylation of CpG islands in their 5' regions. Critical CpG sites are methylated at, or very close to, the time of inactivation in development. The mouse and the human X chromosomes carry the same genes but their arrangement is different and there are some genes in the pairing segment and elsewhere on the human X chromosome which can escape inactivation. Regions of homology between the mouse and human X chromosomes allow prediction of the map positions of homologous genes and provide mouse models of genetic disease in the human.

Major changes in the 5′ and 3′ chromatin structure of sea urchin histone genes accompany their activation and inactivation in development

The major histone gene repeat (h22) of the sea urchin Psammechinus miliaris is transiently expressed for several hours during early embryonic development. Several major alterations in chromatin structure coincide with changes in the pattern of early histone gene expression and are reversed later. During the early (128-cell) blastula stages when h22 DNA is maximally expressed, promoter regions of all five histone genes are sensitive to both micrococcal nuclease and DNAase I. Hypersen-sitivity to micrococcal nuclease remains the same throughout the early hours of development, but disappears abruptly at hatching blastula stage when transcription has completely ceased. Some sequences near the 3′ end of active histone genes are very resistant to micrococcal nuclease cutting and map just downstream of the inverted DNA repeats essential for generating faithful 3′ ends of histone mRNAs. These same histone DNA sequences are readily cut in free DNA or when histone genes are transcriptionally inactive.