Most plant development occurs post-embryonically, and is tied closely to environmental signals.  We use techniques of genetics, molecular biology, microscopy, physiology, and biochemistry to study how environmental and endogenous signals regulate plant development.  For these studies we use the model plant Arabidopsis thaliana, which has numerous technical advantages that facilitate experimental progress.  We hope in the long run to reconstruct how endogenous developmental programs and exogenous signals cooperate to determine plant form and the partitioning of growth among different organs.

The hormone auxin regulates multiple developmental processes in plants, including embryo and meristem patterning, organ growth, and flower maturation.   Auxin induces gene expression through a family of transcription factors called ARFs (Auxin Response Factors), whose activity is regulated by Aux/IAA proteins.  Auxin switches ARF proteins between gene repressing and gene activating states by promoting Aux/IAA protein turnover.  We are currently studying how this regulatory system controls seedling growth, ovule development, and flower opening and fertilization.

Among genes regulated by auxin are some (including those encoding Aux/IAA proteins) that feed back negatively on auxin response; and others encoding proteins that affect intercellular auxin transport.  We are interested in how feedback controls affect the dynamics of auxin response, and in how regulated intercellular auxin movement coordinates growth and differentiation of different cells during development.

At a Glance

• Epigenetic control of genome structure and function
• Cell cycle-regulated gene expression
• Developmental genetics

Synopsis

Our research focuses on understanding the molecular mechanisms that regulate DNA replication and cell proliferation during animal development.  An orderly process of events called the cell cycle, which in its most familiar form consists of four phases (G1-S-G2-M) controls cell proliferation.  The genome is replicated during the “S” or synthesis phase and duplicated chromosomes are segregated to daughter cells during the “M” or mitotic phase when cell division occurs.  G1 and G2 are “gap” phases during cells regulate the entry into S phase and M phase, respectively.  We study gene expression events that control how cells make the decision to enter S phase and proliferate, or to exit the cell cycle and differentiate. Without such control there would be no coordination between cell proliferation and the development and function of the many different types of tissues that make up an organism.  In addition, the breakdown of cell cycle regulation is one of the events that contribute to the generation of cancer.

We study this problem using the fruit fly Drosophila melanogaster, in part because the genes controlling cell proliferation in fruit flies have been highly conserved during evolution and function the same way as in other animals, including humans.  This allows us to exploit certain advantages that Drosophila has as a research tool, including the ease with which genetics (making and analyzing mutants) and cell biology (using microscopy to

observe cell proliferation) can be applied to the study of gene function in the context of a whole animal.  Some of the cell cycle regulatory pathways that we study become defective in virtually every human cancer.  Thus, one hope is that understanding how these pathways function in normal Drosophila development will give us clues to how they might malfunction in the deregulated growth typical of cancer.

Human congenital heart disease, the most common form of heart disease in childhood, occurs in about 1% of live births and up to 10% of stillbirths. Presently, the most effective therapy for cardiac diseases is heart transplantation. However, due to the shortage of organs, cost and inaccessibility of treatment for most affected individuals this remains a limited therapeutic option. Alternative treatment is the administration of drugs that improve myocardial contractility, though this treatment is only effective as a short term therapy, with the 5-year survival rate using current agents being less than 60%. An alternative therapeutic option is to treat patients with cardiac progenitor cell populations that could infiltrate and repair damaged heart tissue. Thus, the ability to isolate and propagate cell populations that can differentiate into cardiomyocytes in vivo offers the opportunity to treat a wide range of cardiac diseases. To this end, our lab is interested in understanding the relationship between cardiac progenitor proliferation and the onset of cardiac differentiation focusing on the endogenous roles of the transcription factors TBX5 and CST and the protein phosphatase SHP-2.

Cellular division, wound healing, chemotaxis, and neuronal outgrowth all rely on dynamic shape change and adaptability afforded via an ever-changing cellular scaffold termed the cytoskeleton. We examine two core components of the cytoskeleton: microtubules and actin filaments in concert with the molecules that regulate them and facilitate communication between them. We employ a combined approach of high resolution time-dependent imaging in parallel with atomic resolution protein crystallography and cryo-electron microscopy to understand, at multiple scales, the molecular processes that control cytoskeletal dynamics. Of particular interest are the +TIP protein families that dynamically localize to growing microtubule plus ends where they regulate microtubule dynamics, communicate with the actin cytoskeleton, capture kinetochores, and engage the cell cortex under polarity-based cues. Investigations proceed through three key areas.

1. Structure: Tertiary and quaternary molecular architecture of cytoskeletal regulators attained using x-ray crystallography and cryo-electron microscopy.
2. Cellular and Organismal Imaging: Time-dependent systems analysis via genetic, opto-genetic, and small molecule manipulation.
3. In Vitro Reconstitution: Microscopy-based physico-chemical analysis of cytoskeletal dynamics and convergent biological events (capture, signaling etc.) through titration of core components and regulators.

Interleaving these efforts, we aim to test, correlate, and bridge information gained from the organismal, cellular, sub-cellular and atomic levels. Of particular interest are the aberrant cytoskeletal molecular mechanisms at play in neuronal disorders and cancer biology.

The Vision lab studies genome evolution and the architecture of complex traits, with a (non-exclusive) focus on the flowering plants. Among the questions we ask are:

• What is the genetic basis for ecologically important differences between species, and what evolutionary forces generate that variation?
• What mutational and evolutionary processes are responsible for the structural rearrangement of chromosomes among taxa?
• What effect do genome structural changes have on organismal phenotypes?

To address these questions, we use the tools of both molecular and computational biology, and are actively involved in the development of new computational methods.

My laboratory interests are in the broad area of molecular biology. Before the advent of molecular cloning we successfully isolated a pure gene from sea urchins (the ribosomal rRNA gene). We also purified to near homogeneity a gene for one of the sea urchin histones prior to cloning. A paper by Bob Simpson and myself was the first to demonstrate nucleosome phasing.

My major interest at present is the study of protein-protein interactions. At present, we are investigating interactions of the blood coagulation proteins in coagulation and in the control of the pathways of coagulation. Our approach is to use molecular modeling to suggest mutations and to do in vitro mutagenesis to express the proteins and characterize their interactions.

We recently purified the g-glutamyl carboxylase from bovine liver and have now cloned its cDNA. This opens a new avenue of research for us and others.

The Laederach Lab is interested in better understanding the relationship between RNA structure and folding dynamics. We use a combination of computational and experimental approaches to study the process of RNA folding and assembly. In particular, we develop novel computational approaches to analyze and interpret chemical mapping data. Most recently, we have begun investigating the relationship between disease-associated Single Nucleotide Polymorphisms (SNPs) occurring in Human UTRs and their effect on RNA structure. In particular, we have been investigating regulatory RNA elements called “RiboSNitches,” which like bacterial Riboswitches undergo a large conformational change and regulate translation post-transcriptionally. SNPs in the RiboSNitch cause the conformational change.

Research in my laboratory is centered on the initial interactions of the plant pathogenic bacterium, Agrobacterium tumefaciens, with potential host cells. A. tumefaciens is a soil bacterium. Infections of wound sites of plants with this bacterium result in the formation of crown gall tumors. We are particularly interested in the initial attachment of the bacteria to plant host cells. This initial loose binding is required for bacterial virulence and T-DNA transfer to plant cells. Bacterial mutants which are unable to carry out this attachment are avirulent. We are currently engaged in cloning and sequencing the genes required for this attachment. Their expression is regulated by signals from the wounded plant tissue. We are characterizing the compounds which are involved in this signaling and the genes which are regulated. Our results suggest that there is an initial exchange of signals between the bacterium and the plant before the bacteria bind irreversibly to the plant and the transfer of T-DNA can begin. We are also interested in the plant receptor to which the bacteria bind. We have identified mutants of the plant Arabidopsis which appear to be unable to bind the bacteria and are in the process of identifying the plant genes and proteins involved in bacterial binding.

The double-stranded structure of DNA provides for an elegant mechanism allowing the accurate duplication and repair of DNA. However, to make use of the properties of complementary base pairing the hydrogen bonds holding the double helix together must be disrupted, and the helix unwound, to expose single-stranded DNA templates for use by DNA polymerases, primases and other DNA modifying enzymes. This unwinding reaction is accomplished by a class of enzymes called DNA helicases. These enzymes disrupt the double helical structure of DNA in an energy-requiring reaction that is dependent on the hydrolysis of nucleoside 5′-triphosphates. Examples of DNA helicase enzymes are now numerous in phage, bacteria, eukaryotic viruses, and in eukaryotic cells suggesting that these enzymes are ubiquitous. In addition, we now know that helicases are involved in all aspects of DNA metabolism. Individual cells contain multiple DNA helicases; each helicase apparently has a unique biochemical role in the cell.

For the last several years we have focused our studies on the enzymatic mechanisms and biological roles of DNA helicases. The bacterium E. coli and the budding yeast S. cerevisiae provide attractive systems in which to pursue these studies due to the ease of genetic manipulation and the ability to isolate enzymes for biochemical studies. A large body of literature now exists for the enzymes found in E. coli. Yeast, however, remains largely unexplored. We are directing our efforts toward characterizing DNA helicases from both E. coli and yeast. The long-range goal of the research program is to understand, in enzymatic and molecular terms, the mechanism of action of several helicase enzymes, and to define their individual roles in DNA metabolism.

The lab also has an interest in the process of DNA transfer by bacterial conjugation, defined as the unidirectional and horizontal transmission of genetic information between bacterial cells. Conjugation was discovered more than 50 years ago yet we still have only a rudimentary knowledge of the molecular details surrounding this important mechanism for DNA transfer. Previous studies have shown that DNA transfer begins at a site- and strand-specific nick in the conjugative plasmid. The double-stranded DNA molecule is then unwound from this nick, presumably by a DNA helicase, and a single-strand of DNA is transferred into the recipient cell. This laboratory has demonstrated, using the F plasmid as a paradigm, the participation of two F plasmid-encoded proteins, TraIp and TraYp, and one host-encoded protein, integration host factor (IHF), in the nicking reaction; subsequent unwinding has not yet been reconstituted in any system. The goal of this project is to arrive at a more detailed understanding of this key process.

Adaptations are central to the study of evolution. Thus it is surprising that we know so little about the molecular basis of adaptive evolution. The goal of my research is to identify, clone, and characterize the evolution of genes underlying natural adaptations in order to determine the types of genes involved, how many and what types of genetic changes occurred, and the evolutionary history of these changes. These data will address key questions. For example, do adaptations involve many genetic changes or only a few? How important are regulatory versu! s amino acid changes in adaptation? How often are “new” gene s involved in adaptations? Are most adaptive alleles new mutations or pre-existing alleles segregating at low to moderate frequency within a species? Clearly, a deeper understanding of how genes change during adaptation will give insight into the potential and limits of adaptive evolution.

Spatial patterns of genomic features

I am also using genomic data to address important evolutionary questions. Recently, I used D. melanogaster genome sequence data to estimate genome-wide levels of gene clustering and to contrast the amount of clustering among genes with similar motifs to the levels of clustering in general. All chromosomes, except the fourth, showed substantial levels of gene clusteri! ng. Although not more clustered than the average pair of adjacent genes, genes with the same primary motif occur adjacent to one another more often than expected by chance. These results may mean that these small local groups of genes share regulatory elements and evolutionary histories.

Detecting natural selection in DNA sequence data

Molecular evolutionists have long sought to determine which changes within the protein coding and regulatory regions of a gene were shap! ed by natural selection. If an adaptive substitution has occurred in the recent past, there should be a paucity of DNA polymorphism surrounding the site under selection. Taking advantage of this fact, Andrew Kern and I have developed a permutation approach for detecting selected sites using polarized DNA polymorphism and divergence data. This method is especially useful for detecting the effects of weak selected forces across several loci. We used this approach to analyze a large DNA polymorphism and divergence data set of D. simulans genes. Surprisingly, although replacement fixations do not on average appear to be driven by selection, preferred codons – those codons that use the most abundant tRNA – have on average been fixed by selection. We plan to apply this method to additional data sets and to look at spatial patterns of nucleotide fixation within and around genes. For instance, one could see if there is a bias in the types of polymorphism (sy! nonymous vs. non-synonymous) nearest to a type of fixation. This would give insight into the dynamics of the fixation process and its impacts on adjoining variation.