Biomedical subjects

R S Hansen

Publications and source records attributed to R S Hansen.

At least 19 recordsLinked to original sources

PubMed2006

Maintenance of X- and Y-inactivation of the pseudoautosomal (PAR2) gene SPRY3 is independent from DNA methylation and associated to multiple layers of epigenetic modifications.

Maintenance of X-inactivation is achieved through a combination of different repressive mechanisms, thus perpetuating the silencing message through many cell generations. The second human X-Y pseudoautosomal region 2 (PAR2) is a useful model to explore the features and internal relationships of the epigenetic circuits involved in this phenomenon. Recently, we demonstrated that DNA methylation plays an essential role for the maintenance of X- and Y-inactivation of the PAR2 gene SYBL1; here we report that the silencing of the second repressed PAR2 gene, SPRY3, appears to be independent of DNA methylation. In contrast to SYBL1, the inactive X and Y alleles of SPRY3 are not reactivated in cells treated with a DNA methylation inhibitor and in cells from ICF (immunodeficiency, centromeric instability, facial anomalies) syndrome patients, which have mutations in the DNA methyltransferase gene DNMT3B. SPRY3 X- and Y-inactivation is associated with a differential enrichment of repressive histone modifications and the recruitment of Polycomb 2 group proteins compared to the active X allele. Another major factor in SPRY3 repression is late replication; the inactive X and Y alleles of SPRY3 have delayed replication relative to the active X allele, even in ICF syndrome cells where the closely linked SYBL1 gene is reactivated and advanced in replication. The relatively stable maintenance of SPRY3 silencing compared with SYBL1 suggests that genes without CpG islands may be less prone to reactivation than previously thought and that genes with CpG islands require promoter methylation as an additional layer of repression.

Alleles↗

PubMed2002

ICF syndrome cells as a model system for studying X chromosome inactivation.

Mutations in the DNMT3B DNA methyltransferase gene cause the ICF immunodeficiency syndrome. The targets of this DNA methyltransferase are CpG-rich heterochromatic regions, including pericentromeric satellites and the inactive X chromosome. The abnormal hypomethylation in ICF cells provides an important model system for determining the relationships between replication time, CpG island methylation, chromatin structure, and gene silencing in X chromosome inactivation.

Abnormalities, Multiple↗

PubMed2001

Satellite 2 methylation patterns in normal and ICF syndrome cells and association of hypomethylation with advanced replication.

Mutation in the DNMT3B DNA methyltransferase gene is a common cause of ICF (immunodeficiency, centromeric heterochromatin, facial anomalies) immunodeficiency syndrome and leads to hypomethylation of satellites 2 and 3 in pericentric heterochromatin. This hypomethylation is associated with centromeric decondensation and chromosomal rearrangements, suggesting that these satellite repeats have an important structural role. In addition, the satellite regions may have functional roles in modifying gene expression. The extent of satellite hypomethylation in ICF cells is unknown because methylation status has only been determined with restriction enzymes that cut infrequently at these loci. We have therefore developed a bisulfite conversion-based method to determine the detailed cytosine methylation patterns at satellite 2 sequences in a quantitative manner for normal and ICF samples. From our sequence analysis of unmodified DNA, the internal repeat region analyzed for methylation contains an average of 17 CpG sites. The average level of methylation in normal lymphoblasts and fibroblasts is 69% compared with 20% in such cells from ICF patients with DNMT3B mutations and 29% in normal sperm. Although the mean satellite 2 methylation values for these groups do not overlap, there is considerable overlap at the level of individual DNA strands. Our analysis has also revealed a pattern of methylation specificity, suggesting that some CpGs in the repeat are more prone to methylation than other sites. Variation in satellite 2 methylation among lymphoblasts from different ICF patients has prompted us to determine the frequency of cytogenetic abnormalities in these cells. Although our data suggest that some degree of hypomethylation is necessary for pericentromeric decondensation, factors other than DNA methylation appear to play a major role in this phenomenon. Another such factor may be altered replication timing because we have discovered that the hypomethylation of satellite 2 in ICF cultures is associated with advanced replication.

Animals↗

PubMed2000

Escape from gene silencing in ICF syndrome: evidence for advanced replication time as a major determinant.

Chromosomal abnormalities associated with hypomethylation of classical satellite regions are characteristic for the ICF immunodeficiency syndrome. We, as well as others, have found that these effects derive from mutations in the DNMT3B DNA methyltransferase gene. Here we examine further the molecular phenotype of ICF cells and report several examples of extensive hypomethylation that are associated with advanced replication time, nuclease hypersensitivity and a variable escape from silencing for genes on the inactive X and Y chromosomes. Our analysis suggests that all genes on the inactive X chromosome may be extremely hypomethylated at their 5' CpG islands. Our studies of G6PD in one ICF female and SYBL1 in another ICF female provide the first examples of abnormal escape from X chromosome inactivation in untransformed human fibroblasts. XIST RNA localization is normal in these cells, arguing against an independent silencing role for this RNA in somatic cells. SYBL1 silencing is also disrupted on the Y chromosome in ICF male cells. Increased chromatin sensitivity to nuclease was found at all hypomethylated promoters examined, including those of silenced genes. The persistence of inactivation in these latter cases appears to depend critically on delayed replication of DNA because escape from silencing was only seen when replication was advanced to an active X-like pattern.

Alleles↗

PubMed2000

Genetic variation in ICF syndrome: evidence for genetic heterogeneity.

ICF syndrome is a rare autosomal recessive immunoglobulin deficiency, sometimes combined with defective cellular immunity. Other features that are frequently observed in ICF syndrome patients include facial dysmorphism, developmental delay, and recurrent infections. The most diagnostic feature of ICF syndrome is the branching of chromosomes 1, 9, and 16 due to pericentromeric instability. Positional candidate cloning recently discovered the de novo DNA methyltransferase 3B (DNMT3B) as the responsible gene by identifying seven different mutations in nine ICF patients. DNMT3B specifically methylates repeat sequences adjacent to the centromeres of chromosome 1, 9, and 16. Our panel of 14 ICF patients was subjected to mutation analysis in the DNMT3B gene. Mutations in DNMT3B were discovered in only nine of our 14 ICF patients. Moreover, two ICF patients from consanguineous families who did not show autozygosity (i.e. homozygosity by descent) for the DNMT3B locus did not reveal DNMT3B mutations, suggesting genetic heterogeneity for this disease. Mutation analysis revealed 11 different mutations, including seven novel ones: eight different missense mutations, two different nonsense mutations, and a splice-site mutation leading to the insertion of three aa's. The missense mutations occurred in or near the catalytic domain of DNMT3B protein, indicating a possible interference with the normal functioning of the enzyme. However, none of the ICF patients was homozygous for a nonsense allele, suggesting that absence of this enzyme is not compatible with life. Compound heterozygosity for a missense and a nonsense mutation did not seem to correlate with a more severe phenotype.

Abnormalities, Multiple↗

PubMed2000

Analysis of replication timing at the FRA10B and FRA16B fragile site loci.

The molecular basis for the cytogenetic appearance of chromosomal fragile sites is not yet understood. Late replication and further delay of replication at fragile sites expressing alleles has been observed for FRAXA, FRAXE and FRA3B fragile site loci. We analysed the timing of replication at the FRA10B and FRA16B loci to determine whether late replication is a feature which is shared by all fragile sites and, therefore, is a necessary condition for chromosomal fragile site expression. The FRA10B locus was located in a transitional region between early and late zones of replication. Fragile and non-fragile alleles exhibit a similar replication pattern proximal to the repeat but fragile alleles are delayed relative to non-fragile ones on the distal side. Although fragility at FRA10B appears to be caused by expansion of an AT-rich repeat in the region, replication time near the repeat was similar in fragile and non-fragile alleles. The FRA16B locus was late replicating and appeared to replicate even later on fragile chromosomes. While these observations are compatible with the hypothesis that delayed replication may play a role in fragile site expression, they suggest that replication delay may not need to occur at the expanded repeat region itself in order to be permissive for fragility.

Alleles↗

PubMed1999

The DNMT3B DNA methyltransferase gene is mutated in the ICF immunodeficiency syndrome.

DNA methylation is an important regulator of genetic information in species ranging from bacteria to humans. DNA methylation appears to be critical for mammalian development because mice nullizygous for a targeted disruption of the DNMT1 DNA methyltransferase die at an early embryonic stage. No DNA methyltransferase mutations have been reported in humans until now. We describe here the first example of naturally occurring mutations in a mammalian DNA methyltransferase gene. These mutations occur in patients with a rare autosomal recessive disorder, which is termed the ICF syndrome, for immunodeficiency, centromeric instability, and facial anomalies. Centromeric instability of chromosomes 1, 9, and 16 is associated with abnormal hypomethylation of CpG sites in their pericentromeric satellite regions. We are able to complement this hypomethylation defect by somatic cell fusion to Chinese hamster ovary cells, suggesting that the ICF gene is conserved in the hamster and promotes de novo methylation. ICF has been localized to a 9-centimorgan region of chromosome 20 by homozygosity mapping. By searching for homologies to known DNA methyltransferases, we identified a genomic sequence in the ICF region that contains the homologue of the mouse Dnmt3b methyltransferase gene. Using the human sequence to screen ICF kindreds, we discovered mutations in four patients from three families. Mutations include two missense substitutions and a 3-aa insertion resulting from the creation of a novel 3' splice acceptor. None of the mutations were found in over 200 normal chromosomes. We conclude that mutations in the DNMT3B are responsible for the ICF syndrome.

Amino Acid Sequence↗

PubMed1999

Inhibition of SV40 large T antigen induced apoptosis by small T antigen.

It is well established that the expression of simian virus 40 (SV40) early gene products causes oncogenic transformation of rodent cells. An important aspect of this process is the inactivation of the p53 and retinoblastoma (pRb) tumour suppressor proteins through interaction with the SV40 large tumour antigen (LT). In addition, the SV40 small tumour antigen (ST) may enhance LT induced transformation. Here we show that LT induces apoptotic cell death in rat embryo fibroblast (REF) cells and that ST functions to inhibit this effect by a mechanism which is different from other known anti-apoptotic proteins. Mutational analysis of LT indicates that mutants defective in the pRb-binding domain are unable to induce apoptosis whereas LT mutants defective in the p53-binding domain are still competent to induce apoptosis. Thus, interaction between LT and one or more pRb family members must occur for induction of apoptosis and that binding of p53 by LT is insufficient to inhibit LT induced apoptosis in REFs. The data presented herein suggest that the anti-apoptotic function of ST may explain, at least in part, how ST contributes to SV40 early region induced transformation of REF cells.

Animals↗

PubMed1999

DNA methylation in transcriptional repression of two differentially expressed X-linked genes, GPC3 and SYBL1.

Methylation of CpG islands is an established transcriptional repressive mechanism and is a feature of silencing in X chromosome inactivation. Housekeeping genes that are subject to X inactivation exhibit differential methylation of their CpG islands such that the inactive alleles are hypermethylated. In this report, we examine two contrasting X-linked genes with CpG islands for regulation by DNA methylation: SYBL1, a housekeeping gene in the Xq pseudoautosomal region, and GPC3, a tissue-specific gene in Xq26 that is implicated in the etiology of the Simpson-Golabi-Behmel overgrowth syndrome. We observed that in vitro methylation of either the SYBL1 or the GPC3 promoter resulted in repression of reporter constructs. In normal contexts, we found that both the Y and inactive X alleles of SYBL1 are repressed and hypermethylated, whereas the active X allele is expressed and unmethylated. Furthermore, the Y and inactive X alleles of SYBL1 were derepressed by treatment with the demethylating agent azadeoxycytidine. GPC3 is also subject to X inactivation, and the active X allele is unmethylated in nonexpressing leukocytes as well as in an expressing cell line, suggesting that methylation is not involved in the tissue-specific repression of this allele. The inactive X allele, however, is hypermethylated in leukocytes, presumably reflecting early X inactivation events that become important for gene dosage in expressing lineages. These and other data suggest that all CpG islands on Xq, including the pseudoautosomal region, are subject to X inactivation-induced methylation. Additionally, methylation of SYBL1 on Yq may derive from a process related to X inactivation that targets large chromatin domains for transcriptional repression.

Aza Compounds↗

PubMed1999

The timing of XIST replication: dominance of the domain.

Contiguous replicons are coordinately replicated and may be organized in temporal-spatial domains with early replication domains containing expressed genes and late ones carrying silent genes. XIST is silent on the active, early replicating X chromosome and expressed from the inactive, late replicating homolog. These circumstances potentially deviate from the aforementioned generalization and make studies of replication timing for XIST of special interest. Although earlier investigations of XIST replication in fibroblasts based on analysis of extracted DNA from cells at different stages of the cell cycle suggested that the silent gene does replicate before the expressed allele, studies using FISH technology produced the opposite results. Because the FISH replication studies could not directly distinguish between the active and inactive X chromosomes in the same cell, we undertook a re-investigation of this question utilizing FISH analysis under conditions that allowed us to make that distinction using cells sorted into different cell cycle stages by flow cytometry. The findings reported here indicate that the silent XIST gene on the active X chromosome does replicate before the expressed allele on the inactive X, supporting the view that the time of a gene's replication is determined by the large, multi-replicon domain in which it is located and not necessarily its expression state.

Alleles↗

PubMed1998

Very late DNA replication in the human cell cycle.

G2 was defined originally as the temporal gap between the termination of DNA replication and the beginning of mitosis. In human cells, the G2 period was estimated to be 3-4 h. However, the absence of replicative DNA synthesis during this period designated G2 has never been shown conclusively. In this report, we show that, at some autosomal and X linked loci, programmed DNA replication continues within 90 min of mitosis. Furthermore, the major accumulation of cyclin B1, a cell-cycle marker that is usually ascribed to G2, overlaps extensively with very late DNA replication. We conclude that the G2 period is much shorter than previously thought and may, in some cells, be nonexistent.

Cell Cycle↗

PubMed1998

Reactivation of XIST in normal fibroblasts and a somatic cell hybrid: abnormal localization of XIST RNA in hybrid cells.

The XIST gene, expressed only from the inactive X chromosome, is a critical component of X inactivation. Although apparently unnecessary for maintenance of inactivation, XIST expression is thought to be sufficient for inactivation of genes in cis even when XIST is located abnormally on another chromosome. This repression appears to involve the association of XIST RNA with the chromosome from which it is expressed. Reactivated genes on the inactive X chromosome, however, maintain expression in several somatic cell hybrid lines with stable expression of XIST. We describe here another example of an XIST-expressing human-hamster hybrid that lacks X-linked gene repression in which the human XIST gene present on an active X chromosome was reactivated by treatment with 5-aza-2'-deoxycytidine. These data raise the possibility that human XIST RNA does not function properly in human-rodent somatic cell hybrids. As part of our approach to address this question, we reactivated the XIST gene in normal male fibroblasts and then compared their patterns of XIST RNA localization by subcellular fractionation and in situ hybridization with those of hybrid cells. Although XIST RNA is nuclear in all cell types, we found that the in situ signals are much more diffuse in hybrids than in human cells. These data suggest that hybrids lack components needed for XIST localization and, presumably, XIST-mediated gene repression.

12E7 Antigen↗

PubMed1997

5-Azadeoxycytidine-induced chromatin remodeling of the inactive X-linked HPRT gene promoter occurs prior to transcription factor binding and gene reactivation.

During the process of 5-aza-2'-deoxycytidine (5aCdr)-induced reactivation of the X-linked human hypoxanthine phosphoribosyltransferase (HPRT) gene on the inactive X chromosome, acquisition of a nuclease-sensitive chromatin conformation in the 5' region occurs before the appearance of HPRT mRNA. In vivo footprinting experiments reported here show that the 5aCdr-induced change in HPRT chromatin structure precedes the appearance of three footprints in the immediate 5' flanking region that are characteristic of the active HPRT allele. These and other data suggest the following sequence of events that lead to the reactivation of the HPRT gene after 5aCdr treatment: (a) hemi-demethylation of the promoter, (b) an "opening" of chromatin structure detectable as increased nuclease sensitivity, (c) transcription factor binding to the promoter, (d) assembly of the transcription complex, and (e) synthesis of HPRT RNA. This sequence of events supports the view that inactive X-linked genes are silenced by a repressive chromatin structure that prevents the binding of transcriptional activators to the promoter.

Animals↗

PubMed1997

A variable domain of delayed replication in FRAXA fragile X chromosomes: X inactivation-like spread of late replication.

The timing of DNA replication in the Xq27 portion of the human X chromosome was studied in cells derived from normal and fragile X males to further characterize the replication delay on fragile X chromosomes. By examining a number of sequence-tagged sites (STSs) that span several megabases of Xq27, we found this portion of the normal active X chromosome to be composed of two large zones with different replication times in fibroblasts, lymphocytes, and lymphoblastoid cells. The centromere-proximal zone replicates very late in S, whereas the distal zone normally replicates somewhat earlier and contains FMR1, the gene responsible for fragile X syndrome when mutated. Our analysis of the region of delayed replication in fragile X cells indicates that it extends at least 400 kb 5' of FMR1 and appears to merge with the normal zone of very late replication in proximal Xq27. The distal border of delayed replication varies among different fragile X males, thereby defining three replicon-sized domains that can be affected in fragile X syndrome. The distal boundary of the largest region of delayed replication is located between 350 and 600 kb 3' of FMR1. This example of variable spreading of late replication into multiple replicons in fragile X provides a model for the spread of inactivation associated with position-effect variegation or X chromosome inactivation.

Cell Cycle↗

PubMed1996

The growth-inhibitory function of p53 is separable from transactivation, apoptosis and suppression of transformation by E1a and Ras.

p53 is known to suppress oncogenic cell transformation, inhibit cell growth, induce apoptosis and activate and repress gene transcription. To investigate the relationships between these functions, we have examined various mutant forms of p53 for their abilities to perform each activity. This study has shown that growth inhibition is not a prerequisite for apoptotic cell death as these two functions are separate and alternative activities of p53. Additionally, we have demonstrated that the ability of p53 to suppress transformation (by adenovirus E1a and activated Ras) correlates with its ability to induce apoptosis and not with its ability to inhibit cell growth. Although p53 is thought to inhibit growth through the transactivation of p21WAFI, our study has demonstrated that transcriptional activation and repression are neither sufficient nor necessary for growth inhibition. This indicates that p53 has more than one mechanism for inhibiting cell growth and that another type of biochemical function must be involved. Furthermore, we have shown that transcriptional activation and repression may each be necessary, and the combination of these activities may even be sufficient, for p53-dependent apoptosis. In summary, our results have provided new information about the cellular and biochemical mechanisms through which p53 acts as a tumor suppressor.

Adenovirus E1A Proteins↗

PubMed1996

Role of late replication timing in the silencing of X-linked genes.

Cytosine methylation at promoter regions and late replication timing have both been implicated in the regulation of genes subject to X chromosome inactivation. Reported here are studies of X-linked gene replication in normal male and female cells as well as in cell hybrids that contain either a normal active X, a normal inactive X, or an inactive X chromosome that has been treated with the demethylating agent, 5-azacytidine (5aC). The relationship between replication timing and transcriptional activity was examined for XIST, XPCT, PGK1, HPRT, F9, FMR1, IDS, and G6PD, and earlier replication was generally found to be associated with increased transcriptional activity. The HPRT and G6PD genes in an untreated inactive X hybrid were among the few exceptions to this correlation in that they remain inactive, yet replicate earlier than their inactive X alleles present in normal human diploid cells. This condition of earlier replication timing may contribute to the high rates of 5aC-induced reactivation for HPRT and G6PD in this hybrid relative to other inactive X hybrids. Other anomalous cases include 5aC-induced advances in replication time for genes such as XIST and F9 whose transcription was unaltered by treatment. These and other data support a model for regulation of X-inactivated genes that involves at least two levels of control: (i) large chromosomal domains are placed into a transcriptionally nonpermissive state by late replication and (ii) transcription is blocked at the local level by promoter methylation. In addition, our observations of continued XIST expression in 5aC-treated hybrids with reactivated genes indicates that such expression is not sufficient for the maintenance of X inactivation.

Base Sequence↗

PubMed1995

Allele-specific replication timing in imprinted domains: absence of asynchrony at several loci.

Using a bromodeoxyuridine incorporation method to detect replicated DNA, we studied allele-specific replication of several sites within the human Prader-Willi/Angelman and IGF2/H19 imprinted regions. No obvious allele-specific differences in time of replication were detected at most loci previously reported to replicate asynchronously in the same cell types as determined by a FISH-based replication assay. Our finding of an absence of allelic replication asynchrony may be related to low levels of imprinted gene expression near these loci in the examined cells (lymphocytes, fibroblasts and lymphoblastoid cells). This view is supported by our studies of the imprinted SNRPN gene in that cells with paternal allele-specific expression (lymphocytes and lymphoblasts) replicate SNRPN alleles asynchronously, whereas cells with a low level of expression (HeLa) replicate SNRPN later and with less allelic asynchrony. In lymphoblasts, the early replicating allele of SNRPN was identified as the paternal one based on the properties of maternal allele-specific methylation and paternal allele-specific expression. Our studies suggest that FISH data implying replication asynchrony in nonexpressing cells reflect structural differences between the maternal and paternal alleles rather than differences in replication timing.

Alleles↗

PubMed1995

Reverse replication timing for the XIST gene in human fibroblasts.

The timing of DNA replication appears to be an important epigenetic regulator of gene expression during development. Replication of active genes in expressing tissues occurs earlier than does replication of their inactive counterparts in nonexpressing tissues. This pattern is also observed for active and inactive alleles present in the same cell, as exemplified by genes subject to X chromosome inactivation in females. We find that the replication timing of the X-linked XIST gene in normal human fibroblasts provides a striking exception to this well-established pattern. Within the same cell, the expressed allele of XIST replicates late in S phase and the silent allele replicates early. This 'reverse' replication timing may have functional significance with respect to XIST or could be a passive consequence of the replication timing requirements of neighboring genes that are subject to X chromosome inactivation. Our finding of early replication for XIST in male fibroblasts contrasts with a report of late replication in such cells as determined by an in situ hybridization method [Torchia et al., (1994) Am. J. Hum. Genet. 55, 96-104]. We propose that our data and those obtained by the in situ method can be accommodated by the existence of structural features that differ between the silent and expressed alleles of XIST. Similar features may be important determinants of the replication asynchrony found by the in situ method for other genes subject to monoallelic expression.

Alleles↗