Nicole A. Becker

Alterations in the common fragile site gene Parkin in ovarian and other cancers.

The cloning and characterization of the common fragile site (CFS) FRA6E (6q26) identified Parkin, the gene involved in the pathogenesis of many cases of juvenile, early-onset and, rarely, late-onset Parkinsons disease, as the third large gene to be localized within a large CFS. Initial analyses of Parkin indicated that in addition to playing a role in Parkinsons disease, it might also be involved in the development and/or progression of ovarian cancer. These analyses also indicated striking similarities among the large CFS-locus genes: fragile histidine triad gene (FHIT; 3p14.2), WW domain-containing oxidoreductase gene (WWOX; 16q23), and Parkin (6q26). Analyses of FHIT and WWOX in a variety of different cancer types have identified the presence of alternative transcripts with whole exon deletions. Interestingly, various whole exon duplications and deletions have been identified for Parkin in juvenile and early-onset Parkinsons patients. Therefore, we performed mutational/exon rearrangement analysis of Parkin in ovarian cancer cell lines and primary tumors. Four (66.7%) cell lines and four (18.2%) primary tumors were identified as being heterozygous for the duplication or deletion of a Parkin exon. Additionally, three of 23 (13.0%) nonovarian tumor-derived cell lines were also identified as having a duplication or deletion of one or more Parkin exons. Analysis of Parkin protein expression with antibodies revealed that most of the ovarian cancer cell lines and primary tumors had diminished or absent Parkin expression. While functional analyses have not yet been performed for Parkin, these data suggest that like FHIT and WWOX, Parkin may represent a tumor suppressor gene.

Characterization of FRA6E and its potential role in autosomal recessive juvenile parkinsonism and ovarian cancer

Characterization of FRA6E (6q26), the third most frequently observed common fragile site (CFS) in the human population, determined that aphidicolin‐induced instability at FRA6E extends over a very large region (3.6 Mb). Sequence analysis identified eight genes (IGF2R, SLC22A1, SLC22A2, SLC22A3, PLG, LPA, MAP3K4, and PARK2) as mapping within the large FRA6E region. PARK2, the gene associated with autosomal recessive juvenile parkinsonism (ARJP), accounts for more than half of the CFS. Homozygous deletions and large heterozygous deletions have been observed in PARK2 in ARJP patients. RT‐PCR analysis of the eight genes localizing to FRA6E indicated that 50% of the genes, including PARK2, were down‐regulated in one or more of the primary ovarian tumors analyzed. PARK2 expression was down‐regulated in 60.0% of the primary ovarian tumors analyzed. Additionally, we found tumor‐specific alternative transcripts of PARK2. Loss of heterozygosity analysis of primary ovarian tumors by use of polymorphic markers in the 6q26 region demonstrated 72% LOH in the center of the PARK2 gene, the highest of any of the markers tested. FRA6E shares many similarities with FRA3B (3p14.2) and FRA16D (16q23.2) in representing a large region of genomic instability and containing an extremely large gene that may play a role in the development of ovarian and many other cancers.

Evidence that instability within the FRA3B region extends four megabases

FRA3B is the most frequently expressed common fragile site localized within human chromosomal band 3p14.2, which is frequently deleted in many different cancers, including cervical cancer. Previous reports indicate aphidicolin-induced FRA3B instability occurs over ∼500 kb which is spanned by the 1.5 Mb fragile histidine triad (FHIT) gene. Recently an HPV16 cervical tumor integration, 2 Mb centromeric to the published FRA3B region, has been identified. FISH-based analysis with a BAC spanning the integration has demonstrated this integration occurs within the FRA3B region of instability. These data suggest that the unstable FRA3B region is much larger than previously reported. FISH-based analysis of aphidicolin-induced metaphase chromosomes allowed for a complete characterization of instability associated with FRA3B. This analysis indicates that fragility extends for 4 Mb. Within this region are a total of five genes, including FHIT. FRA3B gene expression analysis on a panel of cervical tumor-derived cell lines revealed that three of the five genes within FRA3B were aberrantly regulated. A similar analysis of genes outside of FRA3B indicated that the surrounding genes were not aberrantly expressed. These data provide additional support that regions of instability associated with CFSs and the genes contained within them, may play an important role in cancer development.

Novel insights from hybrid LacI/GalR proteins: family-wide functional attributes and biologically significant variation in transcription repression

LacI/GalR transcription regulators have extensive, non-conserved interfaces between their regulatory domains and the 18 amino acids that serve as ‘linkers’ to their DNA-binding domains. These non-conserved interfaces might contribute to functional differences between paralogs. Previously, two chimeras created by domain recombination displayed novel functional properties. Here, we present a synthetic protein family, which was created by joining the LacI DNA-binding domain/linker to seven additional regulatory domains. Despite ‘mismatched’ interfaces, chimeras maintained allosteric response to their cognate effectors. Therefore, allostery in many LacI/GalR proteins does not require interfaces with precisely matched interactions. Nevertheless, the chimeric interfaces were not silent to mutagenesis, and preliminary comparisons suggest that the chimeras provide an ideal context for systematically exploring functional contributions of non-conserved positions. DNA looping experiments revealed higher order (dimer–dimer) oligomerization in several chimeras, which might be possible for the natural paralogs. Finally, the biological significance of repression differences was determined by measuring bacterial growth rates on lactose minimal media. Unexpectedly, moderate and strong repressors showed an apparent induction phase, even though inducers were not provided; therefore, an unknown mechanism might contribute to regulation of the lac operon. Nevertheless, altered growth correlated with altered repression, which indicates that observed functional modifications are significant.

Mechanism of promoter repression by Lac repressor–DNA loops

The Escherichia coli lactose (lac) operon encodes the first genetic switch to be discovered, and lac remains a paradigm for studying negative and positive control of gene expression. Negative control is believed to involve competition of RNA polymerase and Lac repressor for overlapping binding sites. Contributions to the local Lac repressor concentration come from free repressor and repressor delivered to the operator from remote auxiliary operators by DNA looping. Long-standing questions persist concerning the actual role of DNA looping in the mechanism of promoter repression. Here, we use experiments in living bacteria to resolve four of these questions. We show that the distance dependence of repression enhancement is comparable for upstream and downstream auxiliary operators, confirming the hypothesis that repressor concentration increase is the principal mechanism of repression loops. We find that as few as four turns of DNA can be constrained in a stable loop by Lac repressor. We show that RNA polymerase is not trapped at repressed promoters. Finally, we show that constraining a promoter in a tight DNA loop is sufficient for repression even when promoter and operator do not overlap.

Quantitative Methods for Measuring DNA Flexibility In Vitro and In Vivo

The double-helical DNA biopolymer is particularly resistant to bending and twisting deformations. This property has important implications for DNA folding in vitro and for the packaging and function of DNA in living cells. Among the outstanding questions in the field of DNA biophysics are the underlying origin of DNA stiffness and the mechanisms by which DNA stiffness is overcome within cells. Exploring these questions requires experimental methods to quantitatively measure DNA bending and twisting stiffness both in vitro and in vivo. Here, we discuss two classical approaches: T4 DNA ligase-mediated DNA cyclization kinetics and lac repressor-mediated DNA looping in Escherichia coli. We review the theoretical basis for these techniques and how each can be applied to quantitate biophysical parameters that describe the DNA polymer. We then show how we have modified these methods and applied them to quantitate how apparent DNA physical properties are altered in vitro and in vivo by sequence-nonspecific architectural DNA-binding proteins such as the E. coli HU protein and eukaryotic HMGB proteins.

Potential for H-DNA in the Human MUC1 Mucin Gene Promoter

Similar imperfect purine/pyrimidine mirror repeat (PMR) elements have previously been identified upstream of the human MUC1 mucin and CFTR genes. These elements confer S1 nuclease sensitivity on isolated plasmid DNA at low pH. We now present a detailed characterization of the non-B DNA structure responsible for S1 nuclease sensitivity upstream of the MUC1 gene. A ∼90-base pair (bp) DNA fragment containing a 32-bp PMR element termed M-PMR3 was subcloned into a recombinant vector. This fragment conferred S1 nuclease sensitivity on the resulting supercoiled plasmid. High resolution mapping of sites reactive to S1 and P1 nucleases demonstrates that cleavage occurs within the M-PMR3 element. High resolution mapping with chemical agents selective for non-B DNA provides evidence that M-PMR3 adopts an H-DNA structure (intramolecular triple helix) in the less common H-y5 isomer at low pH. This result is observed in the presence or absence of Mg2+. Mutation of the native M-PMR3 element to create perfect homopurine/homopyrimidine mirror symmetry alters the preferred folding to the more common H-y3 triplex DNA isomer. These results demonstrate that imperfections in mirror symmetry can alter the relative stabilities of different H-DNA isomers.

Enhancement of DNA flexibility in vitro and in vivo by HMGB box A proteins carrying box B residues

HMGB proteins are abundant non-histone components of eukaryotic chromatin. The biological function of DNA sequence-nonspecific HMGB proteins is obscure. These proteins are composed of one or two conserved HMG box domains, each forming three alpha-helices that fold into a sequence-nonspecific DNA-binding module recognizing the DNA minor groove. Box A and box B homology domains have subtle sequence differences such that box B domains bend DNA strongly while DNA bending by isolated box A domains is weaker. Both box A and box B domains preferentially bind to distorted DNA structures. Here we show using DNA cyclization kinetics assays in vitro and Escherichia coli DNA looping assays in vivo that an isolated HMG box A domain derived from human HMGB2 folds poorly and does not enhance apparent DNA flexibility. Surprisingly, substitution of a small number of cationic residues from the N-terminal leader of a functional yeast box B protein, Nhp6Ap, confers the ability to enhance DNA flexibility. These results demonstrate important roles for cationic leader amino acids in HMGB folding, DNA interaction, and DNA bending.

Transcriptional profiling reveals that several common fragile-site genes are downregulated in ovarian cancer.

Previous transcriptional profiling analysis of 14 primary ovarian tumors identified approximately 12,000 genes as decreased in expression by at least twofold in one or more of the tumors sampled. Among those genes were several known to be mapped to common fragile sites (CFSs), some of which had previously been shown to exhibit a loss of expression in ovarian carcinoma. Therefore, we selected a subset of genes to determine whether they localized within CFSs. Of the 262 genes that were downregulated at least twofold in 13 of 14 tumors, 10 genes were selected based on the following criteria: localization to a CFS band; documented aberrations in at least one malignancy; and feasibility of scoring breakage at the specific CFS. Fluorescence in situ hybridization analysis was performed using bacterial artificial chromosome clones encompassing portions of the genes to determine the position of the genes relative to their corresponding CFSs. Nine genes were determined to localize within seven previously uncloned CFSs. Semiquantitative reverse‐transcription/polymerase chain reaction analysis of the cell lines and primary ovarian tumors validated the downregulation of seven of the 10 genes. We identified portions of seven uncloned CFSs and provide data to suggest that several of the genes mapping within CFSs may be inactivated in ovarian cancer.

Cyclin A2 is an RNA binding protein that controls Mre11 mRNA translation

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding–dependent role that ensures adequate repair of common replication errors.