Research in my lab mainly focuses on four areas of interest: 1. Elucidation of the function of the Myb complex (also known as the Myb-MuvB or dREAM complex), 2. Finding genes associated with human birth defects and disease, 3. Using a fruit fly model of myoclonic epilepsy to understand the genetic basis of the disease in addition to developing novel drug therapies, 4. Empirical annotation of a new model organism (Oikopleura dioica) using tiling microarrays and next gen sequencing methodologies. All of these projects take advantage of current genomics techniques to help answer fundamental questions in the biomedical sciences and are summarized below.
PROJECT 1: The importance of chromatin (and chromatin structure) in controlling nuclear processes such as gene transcriptional regulation and chromosome behavior, as well as its role in epigenetics, has come to light over the last several years. The Myb complex, which we have studied in fruit flies and is intimately involved in these processes, contains the fly homologue of the human c-Myb proto-oncogene (Dm-Myb). In addition, the complex contains the following components: 1) E2F2 and DP proteins which control the ability of a cell to progress through the cell cycle, 2) tumor suppressor proteins RBF1 and RBF2 whose human homologues are required to keep cells from proliferating uncontrollably, 3) proteins that modify or move around histones required to package or compact DNA. We generated the first null mutations of Dm-Myb in flies and showed that in the absence of Dm-Myb, abnormal mitoses occur such that incorrect numbers of chromosomes are passed to cells after division, a hallmark of cancer (Manak et al, 2002). We have subsequently shown that Dm-Myb is involved in a variety of chromatin-related processes including transcriptional regulation of target genes (Georlette et al, 2007), control of DNA replication of a specialized set of genes during egg cell development (Beall, Manak, et al, 2002), maintenance of chromatin integrity (Manak and Lipsick, unpublished results), and condensation of euchromatin during M phase (Manak et al, 2007). We have also shown that the Myb-MuvB complex binds to transcriptional start sites of a large number of genes in the genome. However, no studies to date have attempted on a genomic level to assess how the Myb-MuvB complex functions in an intact animal. We are now characterizing both expression patterns and chromatin structure of a variety of tissues in both the Myb mutant and controls. We find that Myb is directly functioning with the NURF nucleosome remodeliing complex to both activate and repress target genes in mitotic and non-mitotic tissue. However, one primary class of genes regulated by Myb, the M phase cell cycle genes, does not depend on NURF for their activation, suggesting that Myb can regulate targets in multiple ways. We have recently discovered that Myb is playing a role in repressing LTR-retrotransposon expression, and when Myb is absent, these transposons are mobilized in all tissues examined. Interestingly, NURF is also required for repression of retrotransposon transposition, suggesting that Myb and NURF act in concert to establish a repressive chromatin environment at the retrotransposon loci. We speculate that the transposition we observe in Myb mutants could help promote the genomic instability that is seen for many oncogenes. Classical techniques such as developmental and genetic analyses coupled with immunocytochemistry are strong components in this project.
PROJECT 2: Over the last several years, it has been demonstrated that genomic rearrangements play a role in human disease, which has in turn created new opportunities for finding the specific genes involved in the disease process. Genomic rearrangements can arise when interspersed repeat elements facilitate submicroscopic deletion (or duplication) events. Any gene caught in a genomic rearrangement could result in an alteration of that gene’s dosage. Tiling microarrays have been used to identify such changes using a procedure termed array-based Comparative Genomic Hybridization (aCGH). aCGH relies on competitively hybridizing to the tiling microarray a fluorescently labeled reference genomic DNA sample with a fluorescently labeled experimental sample from an individual afflicted with a disease. By comparing hybridization intensities of the reference and the experimental samples, it can be determined whether amplifications or deletions have occurred in the genomic region of interest. These changes are referred to as DNA copy number changes, or sometimes Copy Number Variants (CNVs). Importantly, CNVs are now considered common causes of human disease. aCGH has been used successfully to identify CNVs associated with a number of human diseases (for example, Kallioniemi, 2008; Walsh T. et al, 2008, Sharp et al, 2008, Ballif et al, 2007, Lenz et al, 2008). In collaboration with Drs. Alex Bassuk, Jeff Murray, Tom Wassink, Richard Smith and Patrick Brophy, we are now carrying out large-scale aCGH studies to identify causative deletions and amplications of the genome associated with spina bifida, cleft lip and palate, schizophrenia, and Branchio-Oto-Renal syndrome. Causative CNVs that we are identifying are being followed up using a variety of functional studies in model organisms to determine which genes uncovered by the CNVs are specifically involved in the disease. Currently, we have identified novel loci or rearrangements associated with spina bifida, cleft lip and palate, and BOR (see Bassuk et al, 2013 for identification of Glypican 5 as a gene associated with spina bifida or Brophy et al, 2013 for identification of a recombination hotspot associated with BOR).
PROJECT 3: We recently published our discovery that mutations in the prickle gene cause myoclonic epilepsy in fruit flies, mice and humans, and we are now using the fly model to perform genetic screens to identify other components in the epilepsy pathway, as well as elucidate the mechanism responsible for the disease phenotype. Additionally, we are using the fruit fly model to screen anti-epileptic drugs; since roughly two thirds of epilepsy patients have adverse effects from the drugs that are currently available, and since some of the more popular drugs have been associated with birth defects, there is a great need to develop safer and more effective anti-epileptic medications. Our pipeline from fly to mouse to human will allow us to move from one system to the other as we begin to not only identify key genes in the epilepsy pathway but also molecules that promote anti-seizure activity.
Recently, we have shown that mutations affecting different isoforms of the fly prickle gene have opposite phenotypes; remarkably, mutation of the pksple isoform makes flies susceptible to seizures, whereas mutation of the pkpk isoform actually protects the flies from seizures. The prickle gene has historically been associated with planar cell polarity (PCP) defects where structures such as hairs and bristles are formed with incorrect polarity. We have found that we can genetically separate the PCP and seizure phenotypes; specifically, heterozygous prickle mutants show the seizure phenotypes but none of the PCP defects. We have analyzed the electrophysiology of the prickle mutants in collaboration with Chun-Fang Wu's lab and have found that the pksple flies have a reduced seizure threshold compared to control flies, in addotion to increased seizure-associated spiking activity assessed by the electroconvulsive seizure stimulation paradigm. Recently, we have been able to create an epileptic fly simply by tipping the balance of prickle isoforms exclusively in neurons and muscles. Remarkably, overexpression of the pkpk isoform causes seizure activity whereas overexpression of the pksple isoform does not. These data are consistent with what we see in the prickle mutants; namely, that a lower amount of the pksple isoform relative to pkpk is critical for the neuronal hyperexcitability phenotype.
PROJECT 4: My laboratory is also collaborating on a project to use tiled genomic microarrays and RNA-seq to empirically annotate and characterize the genome of Oikopleura dioica. Oikopleura is a metazoan at the transition of invertebrate to vertebrate and this project is being done with the Thompson and Chourrout labs at the Sars International Centre for Marine Molecular Biology in Norway. Informing these studies, we have found through our work in Drosophila that traditional techniques to annotate genomes such as deep sequencing of cDNA libraries or in silico predictions of genes fails to reveal the entire transcriptome of a eukaryote. However, tiled microarray and RNA-seq studies can identify transcripts missed by these methodologies. We are currently mapping the transcripts of Oiko through the course of development as well as through ecological stressor experiments. Through these studies, we hope to identify nearly all transcribed sequences emanating from this genome, as well as learn about how such a genome responds to ecological stresses not usually encountered during development.