Biomedical subjects

David G Kaufman

Publications and source records attributed to David G Kaufman.

11 recordsLinked to original sources

PubMed2006

Mapping of an origin of DNA replication in the promoter of fragile X gene FMR1.

An origin of bidirectional DNA replication was mapped to the promoter of the FMR1 gene in human chromosome Xq27.3, which has been linked to the fragile X syndrome. This origin is adjacent to a CpG island and overlaps the site of expansion of the triplet repeat (CGG) at the fragile X instability site, FRAXA. The promoter region of FMR2 in the FRAXE site (approximately 600 kb away, in chromosome band Xq28) also includes an origin of replication, as previously described [Chastain II, P.D., Cohen, S.M., Brylawski, B.P., Cordeiro-Stone, M., Kaufman, D.G., 2006. A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene. Cell Cycle 5, 869-872]. FMR1 transcripts were detected in foreskin and male fetal lung fibroblasts, while FMR2 transcripts were not. However, both FMR1 and FMR2 were found to replicate late in S phase (approximately 6 h into the S phase of normal human fibroblasts). The position of the origin of replication relative to the CGG repeat, and perhaps the late replication of these genes, might be important factors in the susceptibility to triplet repeat amplification at the FRAXA and FRAXE sites.

Cell Line↗

PubMed2006

Genome-wide sequence and functional analysis of early replicating DNA in normal human fibroblasts.

BACKGROUND: The replication of mammalian genomic DNA during the S phase is a highly coordinated process that occurs in a programmed manner. Recent studies have begun to elucidate the pattern of replication timing on a genomic scale. Using a combination of experimental and computational techniques, we identified a genome-wide set of the earliest replicating sequences. This was accomplished by first creating a cosmid library containing DNA enriched in sequences that replicate early in the S phase of normal human fibroblasts. Clone ends were then sequenced and aligned to the human genome. RESULTS: By clustering adjacent or overlapping early replicating clones, we identified 1759 "islands" averaging 100 kb in length, allowing us to perform the most detailed analysis to date of DNA characteristics and genes contained within early replicating DNA. Islands are enriched in open chromatin, transcription related elements, and Alu repetitive elements, with an underrepresentation of LINE elements. In addition, we found a paucity of LTR retroposons, DNA transposon sequences, and an enrichment in all classes of tandem repeats, except for dinucleotides. CONCLUSION: An analysis of genes associated with islands revealed that nearly half of all genes in the WNT family, and a number of genes in the base excision repair pathway, including four of ten DNA glycosylases, were associated with island sequences. Also, we found an overrepresentation of members of apoptosis-associated genes in very early replicating sequences from both fibroblast and lymphoblastoid cells. These data suggest that there is a temporal pattern of replication for some functionally related genes.

Cell Proliferation↗

PubMed2006

Checkpoint regulation of replication dynamics in UV-irradiated human cells.

At any moment during S phase, regions of genomic DNA are in various stages of replication (i.e., initiation, chain elongation, and termination). These stages may be differentially inhibited after treatment with various carcinogens that damage DNA such as UV. We used visualization of active replication units in combed DNA fibers, in combination with quantitative analyses of the size distributions of nascent DNA, to evaluate the role of S-checkpoint proteins in UV-induced inhibition of DNA replication. When HeLa cells were exposed to a low fluence (1 J/m(2)) of 254 nm UV light (UVC), new initiation events were severely inhibited (5-6-fold reduction). A larger fluence of UVC (10 J/m(2)) resulted in stronger inhibition of the overall rate of DNA synthesis without decreasing further the frequency of replicon initiation events. Incubation of HeLa cells with caffeine and knockdown of ATR or Chk1 kinases reversed the UVC-induced inhibition of initiation of new replicons. These findings illustrate the concordance of data derived from different experimental approaches, thus strengthening the evidence that the activation of the intra-S checkpoint by UVC is dependent on the ATR and Chk1 kinases.

Ataxia Telangiectasia Mutated Proteins↗

PubMed2006

A late origin of DNA replication in the trinucleotide repeat region of the human FMR2 gene.

We confirmed that the replication of the fragile-X E site (FRAXE) in human chromosomal band Xq28 occurs at six hours into the eight-hour S phase of normal human fibroblasts. In this late-replicating region, we mapped an origin of DNA replication within the promoter of FMR2. This origin is coincident with CpG islands, the trinucleotide repeat, and exon 1 of the FMR2 gene. Identification of this origin may aid in the investigation of the mechanism of trinucleotide repeat expansion and its effect on FMR2 expression. In addition, knowledge of the chromosomal locations and sequence characteristics of both early and late origins of DNA replication, such as the one described in this report, will facilitate studies of the molecular determinants of the time of activation of different origins of replication and allow us to refine our insights concerning origin inactivation in response to the DNA damage-induced intra-S checkpoint.

Cell Line↗

PubMed2005

Decidual differentiation of stromal cells promotes Proprotein Convertase 5/6 expression and lefty processing.

Lefty/Ebaf polypeptides, novel members of the TGF-beta superfamily, are involved in endometrial differentiation and embryo implantation. Recently, we showed that, during undisturbed estrous cycle, lefty is present in mouse uterine horn primarily in a precursor form. Here, we show that decidual differentiation of endometrial stroma leads to increased lefty (approximately 3.1- to 3.6-fold in vivo and 5- to 8-fold in vitro) and processing of its precursor primarily to its long form. This event occurs on d 5 of pregnancy, and is paralleled by proprotein convertase (PC)5/6 up-regulation (approximately 6-fold increase for PC5A and 3-fold increase for PC5B) in decidualized uterine horn, independent of embryo implantation. Among the known convertases, only PC5/6A processes lefty to its long form. Taken together, the findings show that decidualized differentiation of stroma, which is a prerequisite for embryo implantation, leads to processing of lefty by PC5/6A.

Animals↗

PubMed2005

Expression of exogenous human telomerase in cultures of endometrial stromal cells does not alter their hormone responsiveness.

In the human endometrium, stromal cells mediate the proliferative response of epithelial cells to the steroid hormones estrogen and progesterone. These stromal-epithelial interactions are readily studied in vitro by coculture of both cell types. A major impediment to such studies is the rapid senescence of normal stromal cells. To circumvent this problem, we tested whether human endometrial stromal cells immortalized by expressing a transduced human telomerase reverse transcriptase (TERT) subunit retained the ability to mediate hormonal control of epithelial proliferation in the coculture assay. We found that the telomerized stromal cells were very similar to the parental strain from which they were derived according to criteria of proliferation, karyotype, cellular localization of cytoskeletal markers and nuclear staining, and basal gene expression based on microarray analysis. We also showed that expression of estrogen and progesterone receptors, as assessed by immunodetection, was similar in both telomerized and parental stromal cells. Importantly, the telomerized stromal cells were shown in coculture assay to be as effective as normal stromal cells in regulating the proliferation of endometrial epithelial cells in response to estrogen or progesterone. The availability of these long-lived stromal cells may advance studies addressing the mechanistic, regulatory, and cell structural basis of stromal-epithelial interactions and hormonal responses in normal, preneoplastic, and neoplastic human endometrial tissue.

Blotting, Western↗

PubMed2004

Transitions in replication timing in a 340 kb region of human chromosomal R-Band 1p36.1.

DNA replication is initiated within a few chromosomal bands as normal human fibroblasts enter the S phase. In the present study, we determined the timing of replication of sequences along a 340 kb region in one of these bands, 1p36.13, an R band on chromosome 1. Within this region, we identified a segment of DNA (approximately 140 kb) that is replicated in the first hour of the S phase and is flanked by segments replicated 1-2 h later. Using a quantitative PCR-based assay to measure sequence abundance in size-fractionated (900-1,700 nt) nascent DNA, we mapped two functional origins of replication separated by 54 kb and firing 1 h apart. One origin was found to be functional during the first hour of S and was located within a CpG island associated with a predicted gene of unknown function (Genscan NT_004610.2). The second origin was activated in the second hour of S and was mapped to a CpG island near the promoter of the aldehyde dehydrogenase 4A1 (ALDH4A1) gene. At the opposite end of the early replicating segment, a more gradual change in replication timing was observed within the span of approximately 100 kb. These data suggest that DNA replication in adjacent segments of band 1p36.13 is organized differently, perhaps in terms of replicon number and length, or rate of fork progression. In the transition areas that mark the boundaries between different temporal domains, the replication forks initiated in the early replicated region are likely to pause or delay progression before replication of the 340 kb contig is completed.

Aldehyde Dehydrogenase↗

PubMed2004

Complementation of replication origin function in mouse embryonic stem cells by human DNA sequences.

A functional origin of replication was mapped to the transcriptional promoter and exon 1 of the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in the mouse and human genomes. This origin was lost in mouse embryonic stem (ES) cells with a spontaneous deletion (approximately 36 kb) at the 5' end of the HPRT locus. Restoration of HPRT activity by homologous recombination with human/mouse chimeric sequences reconstituted replication origin activity in two independent ES cell lines. Quantitative PCR analyses of abundance of genetic markers in size-fractionated nascent DNA indicated that initiation of DNA replication coincided with the site of insertion in the mouse genome of the 335 bp of human DNA containing the HPRT exon 1 and a truncated promoter. The genetic information contained in the human sequence and surrounding mouse DNA was analyzed for cis-acting elements that might contribute to selection and functional activation of a mammalian origin of DNA replication.

Animals↗

PubMed2003

Same origins of DNA replication function on the active and inactive human X chromosomes.

We previously characterized a functional origin of DNA replication at the transcriptional promoter of the human hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene (Cohen et al. [2002] J. Cell. Biochem. 85:346-356). This origin was mapped using a quantitative PCR assay to evaluate the relative abundance of HPRT markers in short nascent DNA strands isolated from asynchronous cultures of male fibroblasts. The HPRT gene on the X chromosome is transcriptionally active in male human fibroblasts. It is known that on the heterochromatic X chromosome in female cells the HPRT gene is transcriptionally silenced and its replication timing changes from early to late in S phase. This change in replication timing could indicate that replication of the HPRT gene is under the control of different origins of DNA replication in the active (euchromatic, early replicating) and the inactive (heterochromatic, late replicating) X chromosomes. In the present study, we identified the location of the origin of replication of a second X chromosome gene, glucose-6-phosphate dehydrogenase (G6PD), which we mapped to its transcriptional promoter, in normal male human fibroblasts. Then, we determined the activity of the previously identified HPRT and the G6PD human origins in hybrid hamster cells carrying either the active or the inactive human X chromosome. The results of these studies clearly demonstrated that the human HPRT and G6PD origins of replication were utilized to the same extent in the active and the inactive X chromosomes. Therefore, transcription activity at the HPRT and G6PD genes is not necessary for initiation of DNA replication at the origins mapped to these chromosomal loci.

Animals↗

PubMed2002

Effect of normal endometrial stroma on growth and differentiation in Ishikawa endometrial adenocarcinoma cells.

Endometrial cancer is characterized by alterations in the stromal cells and the supporting extracellular matrix in addition to the intrinsic alterations of the malignant epithelial cells. We have developed a cell culture model that demonstrates the role of stromal cells in the regulation of proliferation, hormone responsiveness, and differentiation of an endometrial adenocarcinoma cell line (Ishikawa). Conditioned medium (CM) was collected from normal primary human endometrial stromal cells grown on plastic or within the basement membrane extract, Matrigel. The CM produced by stromal cells cultured in contact with Matrigel markedly inhibited Ishikawa cell proliferation compared with CM from stromal cells cultured on plastic. Ishikawa cell proliferation varied with steroid hormone treatment in the presence of CM from stromal cells embedded in Matrigel. When the Ishikawa cells were placed in coculture in contact with stromal cells in Matrigel, production of a differentiated epithelial secretory product, glycodelin, was induced. Gene expression of stromal cell hormone receptors, growth factors, and integrins was analyzed by reverse transcription-PCR in the presence of Matrigel to determine the potential factors involved in stromal regulatory function. These combined studies imply that the phenotype of the Ishikawa cells can be induced to differentiate to more closely resemble normal endometrial epithelium by reintroduction of stromal factors and appropriate extracellular matrix. Additionally, the study shows that basement membrane proteins influence the regulatory function of stromal cells as they mediate epithelial cell growth.

Adenocarcinoma↗

PubMed2002

Mapping of an origin of DNA replication near the transcriptional promoter of the human HPRT gene.

A quantitative PCR method was used to map a functional origin of DNA replication in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in normal human fibroblasts. This PCR method measures the abundance of specific sequences in short fragments of newly replicated DNA from logarithmically growing cells. Quantitative measurements rely on synthetic molecules (competitors) that amplify with the same primer sets as the target molecules, but generate products of different sizes. This method was first utilized to determine the position of the replication origin near the lamin B2 gene (Giacca et al. [1994] Proc. Natl. Acad. Sci. U S A. 91:7119-7123). In the present study, primer sets were tested along a 16-kb region near exon 1 of the HPRT gene. The most abundant fragment was found to be located in the first intron of HPRT, just downstream of the promoter and exon 1 of the gene, and approximately 3.5 kb upstream of a previously reported autonomously replicating sequence (Sykes et al. [1988] Mol. Gen. Genet. 212:301-309).

Chromosome Mapping↗