Nuclear Structure Studied by Fluorescence Hybridization: Visualization of Individual Gene Transcription and RNA Splicing: A Thesis

Abstract

The overall objective of this study has been to address some of the longstanding questions concerning functional organization of the interphase nucleus. This was achieved by using recently developed high-resolution fluorescence in situ hybridization techniques for a precise localization of specific DNA and RNA sequences in conjunction with immunocytochemistry and biochemical fractionation. This study is based on the philosophy that new insights may be gained by an approach that attempts to interrelate genomic organization, spatial arrangement of RNA metabolism, and nuclear substructure within the mammalian cell nucleus. The nuclear distribution of an exogenous, viral RNA (Epstein-Barr Virus, EBV) within nuclear matrix preparations was studied by developing an approach which couples in situhybridization with biochemical fractionation procedures. EBV RNA molecules accumulate in highly localized foci or elongated tracks within the nucleus of lymphoma cells. These RNA tracks were retained with spatial and quantitative fidelity in nuclear matrix preparations even after biochemical fractionation which removes 95% of cellular protein, DNA, and phospholipid. This provided direct evidence that the primary transcripts are localized via their binding to, or comprising part of, a non-chromatin nuclear substructure. Then the nuclear distribution of RNA from an endogenous gene, fibronectin, was investigated using fluorescence techniques modified for more sensitive detection of endogenous RNAs within nuclear morphology. A series of in situhybridization experiments were performed using different combinations of intron, cDNA, and genomic probes for RNA/RNA or RNA/DNA analysis in intact cells. Fibronectin RNAs were highly localized in the nucleus, forming foci or tracks. Both intron and exon sequences were highly concentrated at the same site within the nucleus, indicating the presence of primary unspliced transcripts. Double-color hybridization using a nontranscribed 5' flanking sequence probe and a genomic DNA probe showed that the gene and RNA track for fibronectin were spatially overlapped, with the gene consistently towards one end of the track. These results provided evidence that the accumulation of RNA molecules occurs directly at or near the site of transcription, and further indicated a structural polarity to the RNA track formation with the gene towards one end. It was further discovered that within a single cell, cDNA probes produced longer tracks than those formed with intron probes, i.e. intron signals were generally confined to a smaller part of the track than the exon signals, indicating that splicing occurs within the RNA track. Additional experiments using poly(A) RNA hybridization or anti-SC-35 antibody staining combined with fibronectin RNA hybridization have shown that the fibronectin tracks were associated with recently discovered transcript domains enriched in poly(A) RNA and splicing factors. To further determine whether other specific genes and RNAs are functionally organized within the nucleus, the nuclear distribution of several active or inactive genes was analyzed in terms of their spatial relationship to transcript domains. The results indicated that in addition to fibronectin, the genes or their primary transcripts from two other active genes, collagen and actin, were also closely associated with the domains. For both of these, over 90% of the gene/RNA sites were either overlapping or directly contacting the domains. In contrast. for two inactive genes, cardiac myosin heavy chain and neurotensin, it was found that both genes were separated from the domains in the majority of nuclei. Histone genes, which have several unique features, showed a relatively complex result with about half of the gene signals extremely close to the domains. Therefore, three actively expressed genes were demonstrated to be tightly associated with the domains and, moreover, their RNAs showed distinct and characteristic spatial relationships with the domains. In contrast, two inactive genes were not associated with the domains. One potential implication of these finding is that active genes may be preferentially localized in and around these transcript domains. The nuclear localization of another RNA, XIST, standing for X-inactivation specific transcript, was studied because of its potentially unique biological role. XIST is the only gene which is known to be expressed from the inactive human X chromosome but not from the active X chromosome, and was believed to be important in X inactivation. Using fluorescence in situhybridization, it was found that XIST RNA was highly localized within the nucleus and always completely overlapped the Barr body which is the condensed, inactive X chromosome. The different fine distribution pattern of XIST RNA within the nucleus as compared to other protein coding RNAs suggested a unique function for this RNA, possibly involving a structural role in inactivating the X chromosome. The final area of my thesis research was to study and acquire expertise in the applications of fluorescence in situ hybridization in gene mapping and cancer genetics. A retinoblastoma (RB)-related putative tumor suppressor gene, p107, was mapped to human chromosome 20 in band q11.2. Localization of p107 to 20q11.2 was of particular interest because of the correlation of breakpoints in this area with specific myeloid disorders such as acute nonlymphocytic leukemia and myelodysplastic syndrome. Other applications of in situ hybridization including the search for unknown genes at a known chromosomal breakpoint, detection of deletions, translocations or other chromosomal rearrangements associated with specific tumors were also explored and reviewed.