A host of observations demonstrating the relationship between nuclear architecture and processes such as gene expression have led to a number of new technologies for interrogating chromosome positioning. Whereas some of these technologies reconstruct intermolecular interactions, others have enhanced our ability to visualize chromosomes in situ. Here, we describe an oligonucleotide-and PCR-based strategy for fluorescence in situ hybridization (FISH) and a bioinformatic platform that enables this technology to be extended to any organism whose genome has been sequenced. The oligonucleotide probes are renewable, highly efficient, and able to robustly label chromosomes in cell culture, fixed tissues, and metaphase spreads. Our method gives researchers precise control over the sequences they target and allows for single and multicolor imaging of regions ranging from tens of kilobases to megabases with the same basic protocol. We anticipate this technology will lead to an enhanced ability to visualize interphase and metaphase chromosomes.T he role of chromosome positioning in gene regulation and chromosome stability is fueling a growing interest in technologies that reveal the in situ organization of the genome. Among these technologies are chromosome conformation capture (3C) (1) and its several iterations, such as Hi-C (2), which are applied to populations of nuclei to identify chromosomal regions that are in close proximity to each other (3, 4). Another technology is fluorescence in situ hybridization (FISH), wherein nucleic acids are targeted by fluorescently labeled probes and then visualized via microscopy; this technology is an extension of methods that once used radioactive probes and autoradiography but have since been adapted to use nonradioactive labels (5-11). FISH is a single-cell assay, making it especially powerful for the detection of rare events that might otherwise be lost in mixed or asynchronous populations of cells. In addition, because FISH is applied to fixed cells, it can reveal the positioning of chromosomes relative to nuclear, cytoplasmic, and even tissue structures. FISH can also be used to visualize RNA, permitting the simultaneous assessment of gene expression, chromosome position, and protein localization.FISH probes are typically derived from cloned genomic regions or flow-sorted chromosomes, which are labeled directly via nick translation or PCR in the presence of fluorophore-conjugated nucleotides or labeled indirectly with nucleotide-conjugated haptens, such as biotin and digoxigenin, and then visualized with secondary detection reagents. Probe DNA is often fragmented into ∼150-to 250-bp pieces to facilitate its penetration into fixed cells (12) and, as many genomic clones contain repetitive sequences that occur abundantly in the genome, hybridization is typically performed in the presence of unlabeled repetitive DNA (13). Another limitation to clone-based probes is that the genomic regions that can be visualized with them are restricted by the availability of clones and the size of ...

Genome organization involves cis and trans chromosomal interactions, both implicated in gene regulation, development, and disease. Here, we focus on trans interactions in Drosophila, where homologous chromosomes are paired in somatic cells from embryogenesis through adulthood. We first address long-standing questions regarding the structure of embryonic homolog pairing and, to this end, develop a haplotype-resolved Hi-C approach to minimize homolog misassignment and thus robustly distinguish trans-homolog from cis contacts. This computational approach, which we call Ohm, reveals pairing to be surprisingly structured genome-wide, with trans-homolog domains, compartments, and interaction peaks, many coinciding with analogous cis features. We also find a significant genome-wide correlation between pairing, transcription during zygotic genome activation, and binding of the pioneer factor Zelda. Our findings reveal a complex, highly structured organization underlying homolog pairing, first discovered a century ago in Drosophila. Finally, we demonstrate the versatility of our haplotype-resolved approach by applying it to mammalian embryos.

Uterine leiomyomas (also known as uterine fibroids) are the most common benign tumors of female reproductive tract and are the single most common indication for hysterectomies. Despite their high prevalence, the exact pathogenesis of these benign tumors is still unknown. One possible mechanism for leiomyoma formation is dysregulation of mesenchymal stem cell activity. Mesenchymal stem cells have been identified in both human and murine uteri and cancer stem cells have been identified in female reproductive malignancies. We compared stem/progenitor cell characteristics in both normal myometrium and the corresponding leiomyoma of patient's undergoing hysterectomies. We found that leiomyoma cells form fewer mesenchymal stem cell colonies and exhibit less Hoechst dye-excluding side population activity, which is a function associated with progenitor cells in other tissues, than cells isolated from normal myometrium. Whereas in normal myometrium we observed heterogeneous expression of CD90, a cell surface marker associated the with differentiation potential of uterine fibroblasts, in leiomyomas, we observed homogenous expression of CD90, suggesting leiomyoma cells are more terminally differentiated. Furthermore, we found that while leiomyoma cells could only produce CD90 expressing cells, both CD90+ and CD90− myometrial cells could reestablish their original heterogeneous CD90 profile when expanded in vitro. These results suggest that normal myometrium contains cells with stem/progenitor cell activities that are absent in leiomyomas.