Faculty

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genes during egg cell development (Beall, Manak, et al, 2002), maintenance of chromatin integrity (Manak and Lipsick, unpublished results), and condensation of euchromatin during M phase (Manak et al, 2007).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 temporally determine how the Myb-MuvB complex functions.  We are now in the process of using cell culture techniques to synchronize Drosophila cells along with RNAi treatment targeting Myb-MuvB complex members to address how Myb modulates chromatin structure.  By using the ChIP on chip microarray methodology, we wish to determine over the course of the cell cycle whether (and where) the Myb complex is functioning to, 1) modulate deposition or positioning of both standard and variant histones, and, 2) modulate covalent modification of histones (such as acetylation, methylation, phosphorylation, etc).  We wish to demonstrate that a complex containing both proto-oncogene and tumor suppressor proteins is functioning at the most basic level; i.e., to create the proper chromatin structure that ensures the appropriate behavior of chromosomes.  We believe that alteration of these basic processes (which has global genomic consequences) can lead to catastrophic events, such as cancer and disease.  Classical techniques such as developmental and genetic analyses coupled with immunocytochemistry are strong components in this project. 

PROJECT 2A:  Additionally, my lab (in collaboration with human geneticists at the University of Iowa Carver College of Medicine and the Feinstein Institute for Medical Research) is using microarray technology to help find elusive mutations that cause human disease.  Their identification is critical to both understand the causative nature of a disease as well as develop strategies to identify afflicted individuals and potentially treat or cure them.  Through genetic studies, regions of the genome associated with disease are identified and characterized in hopes of finding those causative mutations.  However, many disease-causing mutations remain elusive even after sequencing all annotations (genes) in the region.  Using a Drosophila (fruit fly) model system, I developed a novel technique employing tiled genomic microarrays to efficiently map mutations to the genes they affect (Manak et al, 2006).  Approximately two thousand transcript isoforms were identified in flies, including a large number of novel exons, missed by traditional annotation strategies.  Using these data, we were able to map mutations that cause severe developmental defects, including death, to the genes that were affected by intersecting the known genomic locations of the mutations with the locations of the newly identified exons.  A survey of a small region of the human genome demonstrated that, remarkably, over 70% of the genes in that region contained putative novel exons, suggesting that the microarray-based strategy used in flies can be employed for mapping elusive human disease-causing mutations by first identifying novel exons of genes within a disease-causing genomic interval and then sequencing such regions to look for the causative mutations.

With the assistance of human geneticists (Drs. Alex Bassuk, Polly Ferguson and Jeff Murray at the University of Iowa Carver College of Medicine and Dr. Peter Gregersen at the Feinstein Institute for Medical Research), this technique is now being used to search for and map elusive disease-causing mutations responsible for autoinflammatory and autoimmune disorders, neural tube defects, epilepsy, and cranio-facial disorders.  Since originally utilized in flies and now refocused on identifying human mutations, this technique represents the development of a novel translational methodology.  The short term goal of this project will be to facilitate screening individuals for the identified disorders, using methods similar to those available for Down Syndrome and other known genetic diseases.  The long-term goal of the project will be to elucidate the genetic and molecular mechanisms underlying these conditions in order to develop novel treatment modalities for afflicted individuals.

PROJECT 2B:  In the last few years, recognition of genomic rearrangements as having a role in human diseases and disorders has created new opportunities for finding the causative genes.  Genomic rearrangements can arise when interspersed repeat elements, lying in tandem, facilitate submicroscopic deletion (or duplication) events. Any gene caught in a genomic rearrangement could be identified by data demonstrating a change in 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 a case individual.  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 refered to as DNA copy number changes, or sometimes Copy Number Variants (CNVs).  Importantly, aCGH has been used successfully to identify CNVs associated with a number of human diseases (Kallioniemi, 2008; Walsh T. et al, 2008, Sharp et al, 2008, Ballif et al, 2007, Lenz et al, 2008).  In collaboration with Drs. Bassuk, Gregersen and Murray, 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, and rheumatoid arthritis.  Once the causative CNVs are identified, we will follow up with a variety of functional studies to identify the specific genes involved in the disease.   

PROJECT 3:  My laboratory is also collaborating on several projects to use tiled genomic microarrays to empirically annotate and/or assemble a variety of genomes, including Oikopleura dioica, Tribolium castaneum, and Daphnia pulex.  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.  Tribolium (the flour beetle) is a model organism whose gene sequences are often more similar to their human counterparts than are their fly homologs (Tribolium White Paper, http://www.hgsc.bcm.tmc.edu/projects/tribolium/); in addition, beetles are the group of insects with the largest number of known species, and this is the first beetle genome to be sequenced and annotated.  This project is being done with the Brown lab at Kansas State University.  Daphnia is a model organism which has been used for many years to monitor environmental health due to its sensitivity to environmental toxins.  It is also the first crustacean to be sequenced.  This project is being done with the Colbourne and Cherbas labs at Indiana University.  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 expression studies using microarrays can identify transcripts missed by these methodologies.  We are currently mapping the transcripts of each of these organisms through the course of development and are finding novel transcripts for each one, including large numbers of new genes.  Through these studies, we hope to identify nearly all transcribed sequences emanating from these genomes.

We work on RNA interference based therapeutics which target hepatitis B virus RNAs and zinc-finger nucleases which are designer restriction endonucleases that recognize and cleave hepatitis B virus DNA genomes. Another part of the laboratory focuses on expression profiling of microRNAs. MicroRNAs are a newly discovered class of small RNAs that may be major regulators of gene expression in mammals.

A general problem in biology is the molecular basis for accuracy of cellular information transfer. Our special interests are the mechanisms and control of protein synthesis by cells and the impact of protein synthesis accuracy on cellular aging. We have studied the metabolic role of peptidyl-tRNA hydrolase, an enzyme that catalyzes the hydrolysis of peptides from peptidyl-tRNAs (ptRNAs) that have dissociated from the ribosome during protein synthesis. The "ribosome editing hypothesis" proposes that dissociation of ptRNA occurs preferentially after an error in protein synthesis. Perturbing E. coli cells in a way that affects the accuracy of their protein synthesis also alters the rate of dissociation of ptRNAs from their ribosomes. Starving the cells for an amino acid and treating them with certain antibiotics (aminoglycosides like streptomycin) enhances dissociation of ptRNA. Other antibiotics, notably erythromycin, other macrolides, and lincosamides, inhibit protein synthesis on ribosomes by stimulating the dissociation of ptRNA: peptide bonds continue to be made but the nascent proteins dissociate as ptRNA before becoming completed. These two lines of research have merged in the observation that macrolides and lincosamides enhance the accuracy of protein synthesis by stimulating the ribosome editing process. The effects of treatments that increase errors can be counteracted if the cells are also exposed to low doses of erythromycin or lincomycin. We have assessed the influence of erythromycin on the life span of Saccharomyces cerevisiae, brewers yeast. We hypothesize that this drug improves the accuracy of protein synthesis by yeast mitochondria and thus slows the aging process. Our results thus far show that appropriate doses of erythromycin do extend the lifespan of yeast. We are currently trying to extend our understanding of the erythromycin by assessing its effects on the accuracy of protein synthesis by yeast mitochondria. We are also developing a novel assay for measuring by time-lapse video microscopy the lifespan of yeast that are tethered to glass coverslips by covalent bonds. It seems likely that mRNA sequences and the ribosomes that translate them have co-evolved. The frequency of certain short sequences ("big losers") in the open reading frames of E. coli is much smaller than would be expected from the frequency of their individual codons. Careful analysis suggests that the disfavored sequences have been selected against during translation. The big loser sequences have much larger observed/expected ratios in the open reading frames of other microbial genomes. There are structural differences in the ribosomes of the various bacteria that could be related to differences in the response to big loser sequences during translation. We continue to analyze unusual usage of short mRNA sequences in microbial genomes.