Hongzhi Xu

The eukaryotic nucleus contains a variety of dynamic structures, yet studies into the functional relationship of one type of subnuclear domain to another have been limited. For example, PML bodies and Cajal bodies associate, but the functional consequence of this association and the mediating factors are unknown. Here we report that an associated PML body/Cajal body can co-localize to an snRNA gene locus, with the Cajal body invariably situated between the PML body and the snRNA locus. Binding studies demonstrate that coilin (a Cajal body protein) directly interacts with PIASy (a PML body protein). Cell biological experiments using coilin and PIASy knockout cell lines demonstrate that interactions between coilin and PIASy account in part for the observed association of Cajal bodies with PML bodies. When the PIASy interaction region on coilin is deleted, the frequency of the association between Cajal bodies and PML bodies is reduced. These studies provide another example of coilin's role in the functional organization of the nucleus.

Quiescent nuclei from differentiated somatic cells can reacquire pluripotence, the capacity to replicate, and reinitiate a program of differentiation after transplantation into amphibian eggs. The replication of quiescent nuclei is recapitulated in extracts derived from activated Xenopus eggs; therefore, we have exploited this cell-free system to explore the mechanisms that regulate initiation of replication in nuclei from terminally differentiated Xenopus erythrocytes. We find that these nuclei lack many, if not all, pre-replication complex (pre-RC) proteins. Pre-RC proteins from the extract form a stable association with the chromatin of permeable nuclei, which replicate in this system, but not with the chromatin of intact nuclei, which do not replicate, even though these proteins cross an intact nuclear envelope. During extract incubation, the linker histones H1 and H1(0) are removed from erythrocyte chromatin by nucleoplasmin. We show that H1 removal facilitates the replication of permeable nuclei by increasing the frequency of initiation most likely by promoting the assembly of pre-RCs on chromatin. These data indicate that initiation in erythrocyte nuclei requires the acquisition of pre-RC proteins from egg extract and that pre-RC assembly requires the loss of nuclear envelope integrity and is facilitated by the removal of linker histone H1 from chromatin.

Successful cloning by somatic cell nuclear transfer (NT) involves an oocyte-driven transition in gene expression from an inherited somatic pattern, to an embryonic form, during early development. This reprogramming of gene expression is thought to require the remodeling of somatic chromatin and as such, faulty and/or incomplete chromatin remodeling may contribute to the aberrant gene expression and abnormal development observed in NT embryos. We used a novel approach to supplement the oocyte with chromatin remodeling factors and determined the impact of these molecules on gene expression and development of bovine NT embryos. Nucleoplasmin (NPL) or polyglutamic acid (PGA) was injected into bovine oocytes at different concentrations, either before (pre-NT) or after (post-NT) NT. Pre-implantation embryos were then transferred to bovine recipients to assess in vivo development. Microinjection of remodeling factors resulted in apparent differences in the rate of blastocyst development and in pregnancy initiation rates in both NPL- and PGA-injected embryos, and these differences were dependent on factor concentration and/or the time of injection. Post-NT NPL-injected embryos that produced the highest rate of pregnancy also demonstrated differentially expressed genes relative to pre-NT NPL embryos and control NT embryos, both of which had lower pregnancy rates. Over 200 genes were upregulated following post-NT NPL injection. Several of these genes were previously shown to be downregulated in NT embryos when compared to bovine IVF embryos. These data suggest that addition of chromatin remodeling factors to the oocyte may improve development of NT embryos by facilitating reprogramming of the somatic nucleus.

Friedreich's ataxia (FRDA) is an autosomal recessive trinucleotide repeat disease with no effective therapy. Expanded GAA repeats in the first intron of the FRDA gene are thought to form unusual non-B DNA conformations that decrease transcription and subsequently reduce levels of the encoded protein, frataxin. Frataxin plays a crucial role in iron metabolism and detoxification. To discover small molecules that increase transcription through the GAA repeat region in FRDA, we have made stable cell lines containing a portion of expanded intron 1 fused to a GFP reporter. Small molecules identified using the competition dialysis method were found to increase FRDA-intron 1-reporter gene expression. One of these compounds, pentamidine, increases frataxin levels in patient cells. Thus our approach can be used to detect small molecules of potential therapeutic value in FRDA.