Stephanie M. Cohen

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. J. Cell. Biochem. 88: 923–931, 2003.

BRG1 co-localizes with DNA replication factors and is required for efficient replication fork progression

For DNA replication to occur, chromatin must be remodeled. Yet, we know very little about which proteins alter nucleosome occupancy at origins and replication forks and for what aspects of replication they are required. Here, we demonstrate that the BRG1 catalytic subunit of mammalian SWI/SNF-related complexes co-localizes with origin recognition complexes, GINS complexes, and proliferating cell nuclear antigen at sites of DNA replication on extended chromatin fibers. The specific pattern of BRG1 occupancy suggests it does not participate in origin selection but is involved in the firing of origins and the process of replication elongation. This latter function is confirmed by the fact that Brg1 mutant mouse embryos and RNAi knockdown cells exhibit a 50% reduction in replication fork progression rates, which is associated with decreased cell proliferation. This novel function of BRG1 is consistent with its requirement during embryogenesis and its role as a tumor suppressor to maintain genome stability and prevent cancer.

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). J. Cell. Biochem. 85: 346–356, 2002.

Performance of three-biomarker immunohistochemistry for intrinsic breast cancer subtyping in the AMBER consortium

Background: Classification of breast cancer into intrinsic subtypes has clinical and epidemiologic importance. To examine accuracy of IHC-based methods for identifying intrinsic subtypes, a three-biomarker IHC panel was compared with the clinical record and RNA-based intrinsic (PAM50) subtypes. Methods: Automated scoring of estrogen receptor (ER), progesterone receptor (PR), and HER2 was performed on IHC-stained tissue microarrays comprising 1,920 cases from the African American Breast Cancer Epidemiology and Risk (AMBER) consortium. Multiple cores (1–6/case) were collapsed to classify cases, and automated scoring was compared with the clinical record and to RNA-based subtyping. Results: Automated analysis of the three-biomarker IHC panel produced high agreement with the clinical record (93% for ER and HER2, and 88% for PR). Cases with low tumor cellularity and smaller core size had reduced agreement with the clinical record. IHC-based definitions had high agreement with the clinical record regardless of hormone receptor positivity threshold (1% vs. 10%), but a 10% threshold produced highest agreement with RNA-based intrinsic subtypes. Using a 10% threshold, IHC-based definitions identified the basal-like intrinsic subtype with high sensitivity (86%), although sensitivity was lower for luminal A, luminal B, and HER2-enriched subtypes (76%, 40%, and 37%, respectively). Conclusion: Three-biomarker IHC-based subtyping has reasonable accuracy for distinguishing basal-like from nonbasal-like, although additional biomarkers are required for accurate classification of luminal A, luminal B, and HER2-enriched cancers. Impact: Epidemiologic studies relying on three-biomarker IHC status for subtype classification should use caution when distinguishing luminal A from luminal B and when interpreting findings for HER2-enriched cancers. Cancer Epidemiol Biomarkers Prev; 25(3); 470–8. ©2015 AACR.

DNA replication in early S phase pauses near newly activated origins.

During the S phase of the cell cycle, the entire genome is replicated. There is a high level of orderliness to this process through the temporally and topologically coordinated activation of many replication origins situated along chromosomes. We investigated the program of replication from origins initiating in early S phase by labeling synchronized normal human fibroblasts (NHF1) with nucleotide analogs for various pulse times and measuring labeled tracks in combed DNA fibers. Our analysis showed that replication forks progress 9-35 kilobases from newly initiated origins, followed by a pause in synthesis before replication resumes. Pausing was not observed near origins that initiated in the middle of S phase. No evidence for pausing near origins was found at the beginning of the S phase in glioblastoma T98G cells. Treatment with the S phase checkpoint inhibitor caffeine abrogated pausing in NHF1 cells in early S phase. This suggests that pausing may comprise a novel aspect of the intra-S phase checkpoint pathway or a related new early S checkpoint. Further, it is possible that the loss of this regulatory process in cancer cells such as T98G could be a contributing factor in the genetic instability that typifies cancers.

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.

Early Replication and the Apoptotic Pathway

In higher eukaryotes there is a link between time of replication and transcription. It is generally accepted that genes that are actively transcribed are replicated in the first half of S phase while inactive genes replicate in the second half of S phase. We have recently reported that in normal human fibroblasts there are some functionally related genes that replicate at the same time in S phase. This had been previously reported for functionally related genes that are located in clusters, for example the α‐ and β‐globin complexes. We have shown, however, that this also occurs with some functionally related genes that are not organized in a cluster, but rather are distributed throughout the genome. For example, using GOstat analysis of data from our and other groups, we found an overrepresentation of genes involved in the apoptotic process among sequences that are replicated very early (approximately in the first hour of S phase) in both fibroblasts and lymphoblastoid cells. This finding leads us to question how and why the replication of genes in the apoptotic pathway is temporally organized in this manner. Here we discuss the possible explanations and implications of this observation. J. Cell. Physiol. 213: 434–439, 2007.

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 (Cohen et al. [1998]: Exp Cell Res. 245:321–329). 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 (Giacca et al. [1994]: Proc Natl Acad Sci USA. 91:7119), 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.

Construction of a cosmid library of DNA replicated early in the S phase of normal human fibroblasts.

We constructed a subgenomic cosmid library of DNA replicated early in the S phase of normal human diploid fibroblasts. Cells were synchronized by release from confluence arrest and incubation in the presence of aphidicolin. Bromodeoxyuridine (BrdUrd) was added to aphidicolin‐containing medium to label DNA replicated as cells entered S phase. Nuclear DNA was partially digested with Sau 3AI, and hybrid density DNA was separated in CsCl gradients. The purified early‐replicating DNA was cloned into sCos1 cosmid vector. Clones were transferred individually into the wells of 96 microtiter plates (9,216 potential clones). Vigorous bacterial growth was detected in 8,742 of those wells. High‐density colony hybridization filters (1,536 clones/filter) were prepared from a set of replicas of the original plates. Bacteria remaining in the wells of replica plates were combined, mixed with freezing medium, and stored at −80°C. These pooled stocks were analyzed by polymerase chain reaction to determine the presence of specific sequences in the library. Hybridization of high‐density filters was used to identify the clones of interest, which were retrieved from the frozen cultures in the 96‐well plates. In testing the library for the presence of 14 known early‐replicating genes, we found sequences at or near 5 of them: APRT, β‐actin, β‐tubulin, c‐myc, and HPRT. This library is a valuable resource for the isolation and analysis of certain DNA sequences replicated at the beginning of S phase, including potential origins of bidirectional replication. J. Cell. Biochem. 78:509–517, 2000.

Racial Differences in PAM50 Subtypes in the Carolina Breast Cancer Study

Background African American breast cancer patients have lower frequency of hormone receptor-positive (HR+)/human epidermal growth factor receptor 2 (HER2)-negative disease and higher subtype-specific mortality. Racial differences in molecular subtype within clinically defined subgroups are not well understood. Methods Using data and biospecimens from the population-based Carolina Breast Cancer Study (CBCS) Phase 3 (2008-2013), we classified 980 invasive breast cancers using RNA expression-based PAM50 subtype and recurrence (ROR) score that reflects proliferation and tumor size. Molecular subtypes (Luminal A, Luminal B, HER2-enriched, and Basal-like) and ROR scores (high vs low/medium) were compared by race (blacks vs whites) and age (≤50 years vs > 50 years) using chi-square tests and analysis of variance tests. Results Black women of all ages had a statistically significantly lower frequency of Luminal A breast cancer (25.4% and 33.6% in blacks vs 42.8% and 52.1% in whites; younger and older, respectively). All other subtype frequencies were higher in black women (case-only odds ratio [OR] = 3.11, 95% confidence interval [CI] = 2.22 to 4.37, for Basal-like; OR = 1.45, 95% CI = 1.02 to 2.06, for Luminal B; OR = 2.04, 95% CI = 1.33 to 3.13, for HER2-enriched). Among clinically HR+/HER2- cases, Luminal A subtype was less common and ROR scores were statistically significantly higher among black women. Conclusions Multigene assays highlight racial disparities in tumor subtype distribution that persist even in clinically defined subgroups. Differences in tumor biology (eg, HER2-enriched status) may be targetable to reduce disparities among clinically ER+/HER2- cases.