RESUMEN
Immunodeficiency, Centromeric Instability, Facial Anomalies (ICF) syndrome is a rare autosomal recessive disorder that is characterized by a marked immunodeficiency, severe hypomethylation of the classical satellites 2 and 3 associated with disruption of constitutive heterochromatin, and facial anomalies. Sixty percent of ICF patients have mutations in the DNMT3B (DNA methyltransferase 3B) gene, encoding a de novo DNA methyltransferase. In the present study, we have shown that, in ICF lymphoblasts and peripheral blood, juxtacentromeric heterochromatic genes undergo dramatic changes in DNA methylation, indicating that they are bona fide targets of the DNMT3B protein. DNA methylation in heterochromatic genes dropped from about 80% in normal cells to approximately 30% in ICF cells. Hypomethylation was observed in five ICF patients and was associated with activation of these silent genes. Although DNA hypomethylation occurred in all the analyzed heterochromatic genes and in all the ICF patients, gene expression was restricted to some genes, every patient having his own group of activated genes. Histone modifications were preserved in ICF patients. Heterochromatic genes were associated with histone modifications that are typical of inactive chromatin: they had low acetylation on H3 and H4 histones and were slightly enriched in H3K9Me(3), both in ICF and controls. This was also the case for those heterochromatic genes that escaped silencing. This finding suggests that gene activation was not generalized to all the cells, but rather was restricted to a clonal cell population that may contribute to the phenotypic variability observed in ICF syndrome. A slight increase in H3K27 monomethylation was observed both in heterochromatin and active euchromatin in ICF patients; however, no correlation between this modification and activation of heterochromatic genes was found.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Epigénesis Genética , Cara/anomalías , Heterocromatina/genética , Adolescente , Adulto , Centrómero/ultraestructura , Niño , Preescolar , ADN (Citosina-5-)-Metiltransferasas/fisiología , Eucromatina/metabolismo , Femenino , Variación Genética , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Mutación , Fenotipo , Síndrome , ADN Metiltransferasa 3BRESUMEN
We showed earlier that BAGE (B melanoma antigen) loci are hypermethylated in normal tissues and hypomethylated in 98% of human cancers. More recently, we provided evidence that hypomethylation of BAGE loci represents an informative marker for colon cancer detection. In this study, we show that hypomethylation of BAGE loci was an early event that occurred in 43% of colorectal adenomas. Interestingly, hypomethylation of BAGE loci was frequent (50%) in tubulo-villous and villous adenomas, these adenomas having a high probability of being transformed into colorectal cancers.
RESUMEN
Early detection of colorectal cancer is a decisive step in the successful and complete cure of the disease. Epigenetic markers, in particular, those based on aberrant DNA methylation, can be used to diagnose cancer. B melanoma antigens (BAGE) are a family of genes and truncated genes located in the heterochromatic regions of several human chromosomes. Our previous work showed that BAGE loci (i.e., genes and truncated genes) were hypermethylated in normal tissues and hypomethylated in 98% of human cancers. In the present study, we analyzed DNA methylation of the BAGE loci in 54 colon cancers and in neighboring histopathologic normal tissue samples. Using a combined bisulfite restriction assay, we showed that BAGE loci were hypomethylated in 81% of carcinoma samples. Colon cancer could be diagnosed with 94% specificity, 83% sensitivity, and 89% accuracy. No correlation was found between DNA methylation of BAGE loci and age, gender of patients, nor with the tumor stage or site. Based on the hypothesis that during neoplastic transformation, hypomethylation occurs in juxtacentromeric CpG islands, we suggest that other genes located in the heterochromatic compartment should be tested. These new markers enrich the list of currently studied epigenetic alterations in colon cancer and could be associated with hypermethylation markers to develop reliable diagnostic tests.
Asunto(s)
Antígenos de Neoplasias/genética , Neoplasias Colorrectales/genética , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Islas de CpG , Metilación de ADN , Femenino , Humanos , Masculino , Persona de Mediana Edad , Sensibilidad y Especificidad , Estadísticas no ParamétricasRESUMEN
Euchromatin and heterochromatin are functional compartments of the genome. However, little is known about the structure and the precise location of the heterochromatin-euchromatin boundaries in higher eukaryotes. Constitutive heterochromatin in centromeric regions is associated with (1) specific histone methylation patterns, (2) high levels of DNA methylation, (3) low recombination frequency, and (4) the repression of transcription. All of this contrasts with the permissive structure of euchromatin found along chromosome arms. On the sequence level, the transition between these two domains consists most often of patchworks of segmental duplications. We present here a comprehensive analysis of gene expression, DNA methylation in CpG islands, distribution of histone isoforms, and recombination activity for the juxtacentromeric (or pericentromeric) region of the long arm of human chromosome 21. We demonstrate that most HapMap data are reliable within this region. We show that high linkage disequilibrium between pairs of SNPs extends 719-737 kb from the centromeric alpha-satellite. In the same region we find a peak of histone isoforms H3K9Me3 and H3K27Me (715-822 kb distal to the alpha-satellite). In normal somatic cells, CpG islands proximal to this peak are highly methylated, whereas distal CpG islands are not or very little methylated. This methylation profile undergoes dramatic changes in cancer cells and during spermatogenesis. As a consequence, transcription from heterochromatic genes is activated in the testis, and aberrant gene activation can occur during neoplastic transformation. Our data indicate that the frontier between the juxtacentromeric heterochromatic domain and euchromatic domain of the long arm of chromosome 21 is marked by a heterochromatic peak located approximately 750 kb distal to the alpha-satellite.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Humanos Par 21/genética , Metilación de ADN , Eucromatina/genética , Heterocromatina/genética , Inmunoprecipitación de Cromatina , Islas de CpG/genética , Humanos , Desequilibrio de Ligamiento , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
We characterized the mouse ortholog of the human MLL3 gene and a 10.6 kb-Mll3 transcript. The mouse Mll3 gene comprises 60 exons that encompass 226 kb in chromosome 5. The predicted protein of 3464 amino acids contains two PHD domains, an ATPase alpha_beta signature, an HMG, and a SET domain. We analyzed the expression of the Mll3 gene during the embryonic development of the mouse by whole-mount in situ hybridization. Low levels of expression throughout the embryo were first detected at 8.0 dpc. At this stage, the signal was already stronger in the forebrain neuroepithelium and absent in the heart. Next, expression outlined the ventral neural tube, the somites, the limbs, and the eye lens remaining at low levels throughout the embryo. By 13.0 dpc, expression became stronger in the spinal cord, in hand/foot plates, and in gonads. RT-PCR confirmed that Mll3 is expressed early during gametogenesis. We suggest that Mll3 is expressed early in pre-spermatogonia and then in spermatogonia.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , N-Metiltransferasa de Histona-Lisina/biosíntesis , Proteína de la Leucemia Mieloide-Linfoide/biosíntesis , Espermatogénesis/fisiología , Espermatogonias/metabolismo , Médula Espinal/embriología , Testículo/embriología , Animales , Extremidades/embriología , N-Metiltransferasa de Histona-Lisina/genética , Hibridación in Situ/métodos , Masculino , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Especificidad de Órganos/fisiología , Espermatogonias/citología , Médula Espinal/citología , Testículo/citologíaRESUMEN
We have analysed the genomic structure and transcriptional activity of a 2.3-Mb genomic sequence in the juxtacentromeric region of human chromosome 21. Our work shows that this region comprises two different chromosome domains. The 1.5-Mb proximal domain: (i) is a patchwork of chromosome duplications; (ii) shares sequence similarity with several chromosomes; (iii) contains several gene fragments (truncated genes having an intron/exon structure) intermingled with retrotransposed pseudogenes; and (iv) harbours two genes (TPTE and BAGE2) that belong to gene families and have a cancer and/or testis expression profile. The TPTE gene family was generated before the branching of Old World monkeys from the great ape lineage, by intra- and interchromosome duplications of the ancestral TPTE gene mapping to phylogenetic chromosome XIII. By contrast, the 0.8-Mb distal domain: (i) is devoid of chromosome duplications; (ii) has a chromosome 21-specific sequence; (iii) contains no gene fragments and only one retrotransposed pseudogene; and (iv) harbours six genes including housekeeping genes. G-rich sequences commonly associated with duplication termini cluster at the boundary between the two chromosome domains. These structural and transcriptional features lead us to suggest that the proximal domain has heterochromatic properties, whereas the distal domain has euchromatic properties.
Asunto(s)
Centrómero/genética , Cromosomas Humanos Par 21/genética , Monoéster Fosfórico Hidrolasas , Transportadoras de Casetes de Unión a ATP/genética , Proteínas Adaptadoras del Transporte Vesicular/genética , Empalme Alternativo , Animales , Antígenos de Neoplasias/genética , Composición de Base , Northern Blotting , Línea Celular , Mapeo Cromosómico , ADN Complementario/química , ADN Complementario/genética , Bases de Datos de Ácidos Nucleicos , Eucromatina/genética , Femenino , Duplicación de Gen , Expresión Génica , Heterocromatina/genética , Humanos , Hibridación Fluorescente in Situ , Masculino , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Fosfohidrolasa PTEN , Proteínas Tirosina Fosfatasas/genética , Seudogenes/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Secuencias Repetitivas de Ácidos Nucleicos , Retroelementos/genética , Análisis de Secuencia de ADNRESUMEN
In this paper, we show that the BAGE (B melanoma antigen) gene family was generated by chromosome rearrangements that occurred during the evolution of hominoids. An 84-kb DNA fragment derived from the phylogenetic 7q36 region was duplicated in the juxtacentromeric region of either chromosome 13 or chromosome 21. The duplicated region contained a fragment of the MLL3 gene, which, after juxtacentromeric reshuffling, generated the ancestral BAGE gene. Then, this ancestral gene gave rise to several independent genes through successive rounds of inter- and intrachromosome duplications. Comparison of synonymous and nonsynonymous mutations in putative coding regions shows that BAGE genes, but not the BAGE gene fragments, are under selective pressure. Our data strongly suggest that BAGE proteins have a function and that juxtacentromeric regions, whose plasticity is now largely proved, are not a simple junkyard of gene fragments, but may be the birth site of novel genes.
Asunto(s)
Antígenos de Neoplasias/genética , Centrómero/genética , Duplicación de Gen , Reordenamiento Génico/genética , Hominidae/genética , Selección Genética , Animales , Mapeo Cromosómico , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 7/genética , Análisis por Conglomerados , Cartilla de ADN , Electroforesis en Gel de Campo Pulsado , Humanos , Hibridación Fluorescente in Situ , FilogeniaRESUMEN
A first BAGE (B melanoma antigen) gene, BAGE1, was identified because it encodes a human tumour antigen recognised by a cytolytic T lymphocyte. Here, we characterised five new BAGE genes mapping to the juxtacentromeric regions of human chromosomes 13 and 21 and nine BAGE gene fragments mapping to the juxtacentromeric regions of chromosomes 9, 13, 18, and 21. Genes and gene fragments share extensive regions of 90-99% nucleotide identity. We analysed the expression of BAGE genes on 215 tumours of various histological types and on nine normal tissues. Similar to BAGE1, the new BAGE genes are expressed in melanomas, bladder and lung carcinomas and in a few tumours of other histological types. All the normal tissues were negative, with the exception of testis. Our results show that human juxtacentromeric regions harbour genes, which are transcribed and translated, in addition to gene fragments that are generally not expressed. We suggest that the pattern of expression restricted to cancer/testis is a feature of the few genes mapping to juxtacentromeric regions.
Asunto(s)
Antígenos de Neoplasias/genética , Centrómero/genética , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 21/genética , Perfilación de la Expresión Génica , Neoplasias/genética , Testículo/metabolismo , Secuencia de Aminoácidos , Antígenos de Neoplasias/química , Secuencia de Bases , Humanos , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Especificidad de Órganos , Mapeo Físico de Cromosoma , ARN Mensajero/genética , ARN Mensajero/metabolismo , Células Tumorales CultivadasRESUMEN
We characterized MLL3, a new human member of the TRX/MLL gene family. MLL3 is expressed in peripheral blood, placenta, pancreas, testes, and foetal thymus and is weakly expressed in heart, brain, lung, liver, and kidney. It encodes a predicted protein of 4911 amino acids containing two plant homeo domains (PHD), an ATPase alpha_beta signature, a high mobility group, a SET (Suppressor of variegation, Enhancer of zeste, Trithorax) and two FY (phenylalanine tyrosine)-rich domains. The amino acid sequence of the SET domain was used to obtain a phylogenetic tree of human MLL genes and their homologues in different species. MLL3 is closely related to human MLL2, Fugu mll2, a Caenorhabditis elegans predicted protein, and Drosophila trithorax-related protein. Interestingly, PHD and SET domains are frequently found in proteins encoded by genes that are rearranged in different haematological malignancies and MLL3 maps to 7q36, a chromosome region that is frequently deleted in myeloid disorders. Partial duplications of the MLL3 gene are found in the juxtacentromeric region of chromosomes 1, 2, 13, and 21.