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Asymmetric cell divisions are essential for the development of all multicellular organisms. Many mechanisms are known to help secure differences in cell fate between daughter cells following the division of a parental stem cell. Current studies in the Lansdorp lab focus on three questions that can only (!) be answered using our single cell sequencing technique called Strand-seq:
- Investigate the molecular mechanisms that regulate stem cell self-renewal and differentiation.
- Elucidate molecular mechanisms involved in DNA repair and epigenetic asymmetry between sister chromatids
- Map polymorphic inversions in human chromosomes and combine this information with haplotype information to generate completely phased "personalized genomes" for "precision medicine".
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - May 2019)
Studies of genome heterogeneity and plasticity aim to resolve how genomic features underlie phenotypes and disease susceptibilities. Identifying genomic features that differ between individuals and cells can help uncover the functional variants that drive specific biological outcomes. For this, single cell studies are paramount, as characterizing the contribution of rare but functional cellular subpopulations is important for disease prognosis, management and progression. Until now, these studies have been challenged by our inability to map structural variants accurately and comprehensively. To overcome this, I employed the template strand sequencing method, Strand-seq, to preserve the organization and structure of individual homologues and visualize structural rearrangements in single cells. Using Strand-seq, I monitored homologue states in human genomes to quantify the degree of somatic rearrangements, and distinguished these from recurrent structural variants, such as inherited inversions. In so doing, I created an innovative tool to rapidly discover, map, and genotype structural polymorphisms with unprecedented resolution. Next, to facilitate systematic analyses of Strand-seq data, I developed novel bioinformatic software that locates putative genomic rearrangements in singles cells and identifies recurrent rearrangements across multiple cells. This provides an essential instrument for unbiased and non-targeted structural variant discovery in a high-throughput approach, helping to scale Strand-seq for population-based studies. Applying these tools, I explored the distribution and frequency of structural variation in a heterogeneous cell population to discover and genotype over 100 inversions in the human genome. I found significant structural heterogeneity resides in definable polymorphic domains and within complex and repetitive regions of our genome. Finally, I extended my strategy to comprehensively map the complete set of inversions in an individual’s genome and define their unique invertome. Comparing two invertomes, I found sets of inversions can be combined to make predictions about ancestry and health of an individual, and I characterized the architectural features of inversion breakpoints with base-pair resolution. Taken together, I describe a powerful new framework to study structural rearrangements and genomic heterogeneity in single cell samples, whether from individuals for population studies, or tissues for biomarker discovery.
G-quadruplex nucleic acids are a group of nucleic acids formed from the non-Watson-Crick base pairing of guanine nucleic acids. They can readily form at physiological pH and physiological temperatures within sufficiently long stretches of guanine-rich oligonucleotides. Although, the existence of the G-quartet (the fundamental unit of a G-quadruplex) in a Petri dish has been recognized since the early 60’s, the existence of G-quadruplex nucleic acids in mammalian cells remains unclear. Yet while unequivocal evidence of the existence of G-quadruplex nucleic acids in live cells remains unclear, interest in these potentially important biological structures continues to intensify. G-quadruplex nucleic acids have been suggested to play key roles in essential human molecular pathways including telomere biology, transcriptional regulation and disease development. One of the major obstacles in G-quadruplex nucleic acid research is a lack of tools for the in vivo detection of these structures.In our work, we have harnessed hybridoma technology to produce the first monoclonal antibodies to these unique nucleic acid structures. To our knowledge, these are the first hybridomas secreting monoclonal antibodies obtained through the immunization of mice with purified and validated G-quadruplex structures. Monoclonal antibodies have been approved for use in diagnostic tests and for therapeutic treatments in both cancer and autoimmune diseases, and continue to be very effective laboratory research tools. Using monoclonal antibodies to different G-quadruplex nucleic acids we have explored the existence of G-quadruplex nucleic acids in mammalian cells. One of our antibodies, termed 1H6, forms discrete nuclear foci in human and murine cells and strong nuclear staining in most cells of human tissues. Based on the specificity of the antibodies for defined G-quadruplex structures in vitro, these foci could represent the detection of G-quadruplex nucleic acid structures in mammalian cells. If so, the work presented here provides the first direct evidence for the existence of G-quadruplex nucleic acid structures in human cells.
Proper segregation of replicated chromosomes is essential for cell division in all organisms. Linear eukaryotic chromosomes contain specialized protective structures at the chromosome ends, called telomeres, which are essential for maintaining genome stability. Telomere associations have been observed during key cellular processes including mitosis, meiosis and carcinogenesis. These telomere associations need to be resolved prior to cell division to avoid loss of telomere function. TRF1, a core component of the telomere protein complex shelterin, has been implicated as a mediator of telomere associations. To determine the effect of TRF1 protein levels on telomere associations, we used live-cell fluorescence microscopy to visualize telomeres and chromosome dynamics in cells expressing defined levels of TRF1. Elevated levels of TRF1 induced anaphase bridges containing thin “thread-like” stretches of TRF1 foci connecting segregating chromosomes. We also observed telomere aggregates, mitotic bypass, and TRF1 bridges persisting into the following cell cycle. To examine the role of TRF1 in these telomere associations, we generated a TRF1 protein which can be inducibly cleaved by TEV protease. Telomere aggregates appeared to resolve upon cleavage of TRF1 proteins, suggesting that telomere associations result primarily from protein interactions mediated by TRF1. The essential helicase RTEL1 was observed at the extremities of persistent TRF1 bridges, possibly indicating a function for RTEL1 in the resolution of TRF1-induced telomere associations. Taken together, our results demonstrate that precise regulation of TRF1 levels is essential for telomere resolution and mitotic segregation.
Traditional cytogenetic approaches allow analysis of the chromosomal composition (karyotype) of mitotic cells fixed on slides cells by microscopy. The combination of karyotyping and Fluorescence In Situ Hybridization (FISH) enables the detection of specific target sequences on individual chromosomes. Disadvantages are that traditional cytogenetic approaches are very labor and time consuming and that chromosome specific information from only a few dozen cells has poor statistical power. An alternative is flow karyotyping, a method to analyze chromosomes in suspension by flow cytometry. For flow karyotyping, the DNA composition of specific chromosomes in suspension is measured based on the DNA-specific dyes Hoechst 33258 (HO) and Chromomycin A3 (CA3). My thesis work has focused on the development of a new method to analyze and sort chromosomes using FISH with labeled peptide nucleic acid (PNA) probes on chromosomes in suspension. I found that, following FISH, flow karyotyping can be used to detect and quantify repetitive DNA sequences within individual chromosomes. Using chromosome flow FISH (CFF), chromosomes isolated from cells of various species were hybridized to PNA probes and analyzed by flow cytometry. CFF was used to detect a variety of repeats; interstitial telomeric sequences in Chinese Hamster chromosomes, major satellite in mouse chromosomes and D18Z1 alpha satellite repeats in human chromosomes. Quantitative measurements of repeat length by CFF were validated by comparison with measurements obtained using Q-FISH. We found that parental homologs of human chromosome 18 with different D18Z1 satellite repeat array size could be purified using CFF and Fluorescence Activated Cell Sorting (FACS). Illumina short read sequencing of libraries built from these purified chromosomes enabled us to determine, with a high resolution, the allelic phasing of each homolog over the entire chromosome 18. Finally, CFF was modified to study sister chromatids separately. Using a cell model with inducible separation of sister chromatids, flow karyograms were generated. Using chromosome orientation FISH (CO-FISH) in suspension, we could identify sister chromatids according to the presence of DNA template strands. We anticipate that this approach will allow the purification of sister chromatids to study epigenetic differences between sister chromatids defined on the basis of DNA template strands.
Master's Student Supervision (2010 - 2018)
Template strand sequencing (Strand-seq) is a single cell sequencing approach which maintains 5’ -> 3’ directionality of sequence reads. I hypothesized that the directional information preserved can be used to map complex translocation events. Translocations often disrupt gene expression by reshuffling regulatory elements or by formation of novel fusion transcripts. Yet, detection is often difficult, confounded by complexities of the Structural Variations (SVs). I chose a cell line derived from a patient with pediatric Acute Lymphoblastic Leukemia (iALL) with a known complex karyotype. My aim was to explore Strand-seq’s ability in identifying breakpoints, linking translocation partners and resolving the configuration of SV, comparing low coverage Strand-seq data against high coverage Whole Genome Sequence (WGS) data as reference.The iALL cells selected for my study harbor complex translocations involving 4 chromosomes with 5 breakpoint positions that were previously validated by Fluorescent In Situ Hybridization (FISH). BreakpointR, a novel pipeline for Strand-seq analysis, was able to identify 18 breakpoints, 5 which were isolated for further analysis. These 5 breakpoints were identified with a resolution of 5-60kb, overlapping with the genomic positions of breakpoints identified by WGS analysis, validating the accuracy of BreakpointR. Despite the lower sequencing coverage of Strand-seq, 18 breakpoints were detected against WGS’s 119 breakpoints.Next, I developed a workflow to link translocation partners involved in the breakpoints, successfully linking 4 of 5 translocation partners; the final fragment remained unresolved due to lack of reads within the genomic interval, a limitation of low sequence coverage. By comparison, WGS successfully linked 4 of 5 translocation partners, its limitations of mapping across repetitive regions resulting in a different unresolved fragment.Post-translocation-partner matching and single cell resolution from Strand-seq allowed us to further interrogate expected breakpoints for each single cell. Strand-seq analysis identified an inversion of the 100kb fragment in chromosome 11, validated with Sanger sequencing, representing an additional layer of complexity not identified by the other approaches. I conclude that the application of Strand-seq should be further explored in the areas of SV mapping as it has been proven useful for complementing the inherent difficulties of complex SV mapping across repetitive regions.
Hutchinson-Gilford Progeria Syndrome (HGPS) is a premature aging disorder caused by mutations in the gene LMNA, which encodes the nuclear matrix protein, Lamin A. Lamin A is found predominantly at the nuclear periphery but also throughout the nucleus in a ‘nucleoplasmic veil’. The majority of HGPS patients have a single nucleotide mutation (1824 C→T) which results in the activation of a cryptic donor splice site causing a 150 nucleotide deletion in the mRNA and consequently a 50 amino acid in-frame deletion in the protein. The mutation results in aberrant processing and nuclear localization of the Lamin A protein. HGPS cells are characterized by misshapen nuclei, chromatin disorganization, accumulation of mutant Lamin A, short telomeres, DNA damage recruitment defect and early senescence.To measure the telomere length of individual chromosomes, Quantitative Fluorescence in-situ Hybridization was used. The average telomere length in HGPS fibroblasts was greatly decreased compared to controls as well as highly variable. In contrast, the telomere length in hematopoietic cells which do not express LMNA was within the normal range for three out of four HGPS patient samples. These results suggest that mutant Lamin A decreases telomere length via a direct effect and that expression of mutant LMNA is necessary for telomere loss in HGPS.Three different aspects of telomere biology were investigated: localization, mobility and attachment to the matrix. Telomeres were more localized to the nuclear periphery in HGPS fibroblasts than in wild type fibroblasts as well as having abnormal localization in regards to euchromatin/heterochromatin. To examine mobility, fluorescently tagged proteins were constructed to examine interactions between wild type and mutant Lamin A and telomeres during live cell imaging. Long telomeres in cells with the mutant protein did not move the same distance as those in wild type cells. Mutant Lamin A did not bind DNA with the same affinity as the wild type Lamin A did.These investigations show that telomeres and telomere dynamics are altered in HGPS cells. This is likely contributing to aspects of the pathology of the disease and would need to be taken into consideration in any therapeutic approach.