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2.  PARP-1 ensures regulation of replication fork progression by homologous recombination on damaged DNA 
The Journal of Cell Biology  2008;183(7):1203-1212.
Poly-ADP ribose polymerase 1 (PARP-1) is activated by DNA damage and has been implicated in the repair of single-strand breaks (SSBs). Involvement of PARP-1 in other DNA damage responses remains controversial. In this study, we show that PARP-1 is required for replication fork slowing on damaged DNA. Fork progression in PARP-1−/− DT40 cells is not slowed down even in the presence of DNA damage induced by the topoisomerase I inhibitor camptothecin (CPT). Mammalian cells treated with a PARP inhibitor or PARP-1–specific small interfering RNAs show similar results. The expression of human PARP-1 restores fork slowing in PARP-1−/− DT40 cells. PARP-1 affects SSB repair, homologous recombination (HR), and nonhomologous end joining; therefore, we analyzed the effect of CPT on DT40 clones deficient in these pathways. We find that fork slowing is correlated with the proficiency of HR-mediated repair. Our data support the presence of a novel checkpoint pathway in which the initiation of HR but not DNA damage delays the fork progression.
PMCID: PMC2606964  PMID: 19103807
3.  Two-Step Regulation of Ad4BP/SF-1 Gene Transcription during Fetal Adrenal Development: Initiation by a Hox-Pbx1-Prep1 Complex and Maintenance via Autoregulation by Ad4BP/SF-1 
Molecular and Cellular Biology  2006;26(11):4111-4121.
The orphan nuclear receptor Ad4BP/SF-1 (adrenal 4 binding protein/steroidogenic factor 1) is essential for the proper development and function of reproductive and steroidogenic tissues. Although the expression of Ad4BP/SF-1 is specific for those tissues, the mechanisms underlying this tissue-specific expression remain unknown. In this study, we used transgenic mouse assays to examine the regulation of the tissue-specific expression of Ad4BP/SF-1. An investigation of the entire Ad4BP/SF-1 gene locus revealed a fetal adrenal enhancer (FAdE) in intron 4 containing highly conserved binding sites for Pbx-Prep, Pbx-Hox, and Ad4BP/SF-1. Transgenic assays revealed that the Ad4 sites, together with Ad4BP/SF-1, develop an autoregulatory loop and thereby maintain transcription, while the Pbx/Prep and Pbx/Hox sites initiate transcription prior to the establishment of the autoregulatory loop. Indeed, a limited number of Hox family members were found to be expressed in the adrenal primordia. Whether a true fetal-type adrenal cortex is present in mice remained controversial, and this argument was complicated by the postnatal development of the so-called X zone. Using transgenic mice with lacZ driven by the FAdE, we clearly identified a fetal adrenal cortex in mice, and the X zone is the fetal adrenal cells accumulated at the juxtamedullary region after birth.
PMCID: PMC1489093  PMID: 16705164
4.  Structure and evolution of the mouse pregnancy-specific glycoprotein (Psg) gene locus 
BMC Genomics  2005;6:4.
The pregnancy-specific glycoprotein (Psg) genes encode proteins of unknown function, and are members of the carcinoembryonic antigen (Cea) gene family, which is a member of the immunoglobulin gene (Ig) superfamily. In rodents and primates, but not in artiodactyls (even-toed ungulates / hoofed mammals), there have been independent expansions of the Psg gene family, with all members expressed exclusively in placental trophoblast cells. For the mouse Psg genes, we sought to determine the genomic organisation of the locus, the expression profiles of the various family members, and the evolution of exon structure, to attempt to reconstruct the evolutionary history of this locus, and to determine whether expansion of the gene family has been driven by selection for increased gene dosage, or diversification of function.
We collated the mouse Psg gene sequences currently in the public genome and expressed-sequence tag (EST) databases and used systematic BLAST searches to generate complete sequences for all known mouse Psg genes. We identified a novel family member, Psg31, which is similar to Psg30 but, uniquely amongst mouse Psg genes, has a duplicated N1 domain. We also identified a novel splice variant of Psg16 (bCEA). We show that Psg24 and Psg30 / Psg31 have independently undergone expansion of N-domain number. By mapping BAC, YAC and cosmid clones we described two clusters of Psg genes, which we linked and oriented using fluorescent in situ hybridisation (FISH). Comparison of our Psg locus map with the public mouse genome database indicates good agreement in overall structure and further elucidates gene order. Expression levels of Psg genes in placentas of different developmental stages revealed dramatic differences in the developmental expression profile of individual family members.
We have combined existing information, and provide new information concerning the evolution of mouse Psg exon organization, the mouse Psg genomic locus structure, and the expression patterns of individual Psg genes. This information will facilitate functional studies of this complex gene family.
PMCID: PMC546212  PMID: 15647114
5.  Methylation-Mediated Transcriptional Silencing in Euchromatin by Methyl-CpG Binding Protein MBD1 Isoforms 
Molecular and Cellular Biology  1999;19(9):6415-6426.
DNA methylation of promoter-associated CpG islands is involved in the transcriptional repression of vertebrate genes. To investigate the mechanisms underlying gene inactivation by DNA methylation, we characterized a human MBD1 protein, one of the components of MeCP1, which possesses a methyl-CpG binding domain (MBD) and cysteine-rich (CXXC) domains. Four novel MBD1 isoforms (MBD1v1, MBD1v2, MBD1v3, and MBD1v4) were identified by the reverse transcription-PCR method. We found that these transcripts were alternatively spliced in the region of CXXC domains and the C terminus. Green fluorescent protein-fused MBD1 was localized to multiple foci on the human genome, mostly in the euchromatin regions, and particularly concentrated in the pericentromeric region of chromosome 1. Both the MBD sequence and genome methylation were required for proper localization of the MBD1 protein. We further investigated whether MBD1 isoforms are responsible for transcriptional repression of human genes. A bacterially expressed MBD1 protein bound preferentially to methylated DNA fragments containing CpG islands from the tumor suppressor genes p16, VHL, and E-cadherin and from an imprinted SNRPN gene. All MBD1 isoforms inhibited promoter activities of these genes via methylation. Interestingly, MBD1 isoforms v1 and v2 containing three CXXC domains also suppressed unmethylated promoter activities in mammalian cells. These effects were further manifested in Drosophila melanogaster cells, which lack genome methylation. Sp1-activated transcription of methylated p16 and SNRPN promoters was inhibited by all of the MBD1 isoforms, whereas the isoforms v1 and v2 reduced Sp1-activated transcription from unmethylated promoters as well. These findings suggested that the MBD1 isoforms have different roles in methylation-mediated transcriptional silencing in euchromatin.
PMCID: PMC84611  PMID: 10454587

Results 1-5 (5)