Cellular Structural Biology Research Articles

Abstract A47: Investigating Zbtb protein recognition of epigenetically modified DNA

Abstract Methylation of CpG dinucleotides is essential for genomic stability, control of gene expression and the regulation of chromatin structure in normal eukaryotic cells. Aberrant alterations in these genomic methylation patterns have been linked with many diseases, including cancer. This correlation has prompted the need to better understand the regulatory mechanisms of DNA methylation in gene transcription. The Zbtb family, consisting of Zbtb33, Zbtb4 and Zbtb38, is a set of methyl-CpG binding proteins (MBPs), which recognize both methylated as well as sequence-specific non-methylated DNA sites through conserved Cys2His2 zinc fingers. DNA recognition subsequently recruits chromatin remodeling machinery to the target site resulting in chromatin compaction and gene silencing. While sufficient evidence suggests that the Zbtb proteins play a role in cancer, an extensive analysis of the gene targets and signaling pathways regulated by each protein has yet to be investigated. Here we utilize an interdisciplinary approach combining cellular biology, molecular biology and structural biology to determine the epigenetic targets regulated by the Zbtb MBPs and to identify the protein region(s) necessary for high-affinity DNA recognition. Utilizing western blot analysis, we have determined that the three Zbtb proteins exhibit differential endogenous expression levels in a variety of cancer cell lines. Further, ChIP qRT-PCR analysis demonstrated that the occupancy of Zbtb33 at various methylated and sequence-specific genes was highly variable between the different cancer cell lines. Combined, these results suggest that the Zbtb proteins likely mediate differential signaling pathways in different cancers, possibly by targeting specific regulatory gene elements. Mapping of the global gene occupation for Zbtb33 in various cancer cell lines is in progress. Additionally, we have designed multiple protein constructs around the conserved Cys2His2 zinc finger DNA binding region as well as additional zinc fingers located in the C-terminal regions of Zbtb4 and Zbtb38, as the functions of the latter have not yet been identified. The constructs have been analyzed by solution NMR and EMSA to determine whether they are amenable for further structural analysis and to characterize their DNA binding interactions. This multidisciplinary approach provides the means for advancing our understanding of the molecular basis by which these proteins interpret and translate epigenetic signals in the cancerous state. Citation Format: Tommy W. Terooatea, Maya J. Pandya, Alan C. Chugg, Bethany A. Buck-Koehntop. Investigating Zbtb protein recognition of epigenetically modified DNA. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A47.

It's the Technology, Stupid

Living organisms are incredibly complex. Almost every biological function arises from the combined action of multiple components whose interactions generate functions and properties that are not currently predictable from the attributes of the individual parts. Because they evolved, living systems solve design problems in idiosyncratic ways that can only be resolved by experiment. Unraveling this complexity, moving toward the prediction of phenotype from genotype, is the major challenge that life scientists, including cell biologists, will face over the next 50 years. As always in biological research, it will be new technologies that will determine what is achievable. But predicting new technologies is impossible, so here I describe currently foreseeable developments. Iain W. Mattaj Cellular parts lists, RNAs, proteins, protein modifications, lipids, will be increasingly complete, as will catalogs of the interactions that occur between these components. Single cells will continue to be amenable to a variety of genetic, biochemical, and imaging approaches that cannot be simply applied to complex organisms and intracellular processes coupled to cell–cell interactions provide the basis for the development, structure, and function of tissues, organs, and multicellular organisms. Therefore, the cell will undoubtedly remain at the center of biology in the next few decades, even though the nature of cell biology will change significantly. Cell biology will become more interdisciplinary. The boundaries with other biomedical disciplines have already blurred and physics, chemistry, mathematics, and computer science are also beginning to have a heavy influence. The recent and ongoing radical advances in imaging have turned molecular cell biology from a discipline dominated by reductionist experimental methods and descriptive morphology into a quantitative science geared at measuring, dissecting, and understanding molecular mechanisms in their in vivo context. This trend will continue. Imaging in four dimensions captures real-time data that reflects the physiological conditions and dynamics of the biological system under study. Fluorescence-based light microscopy techniques use reporter molecules to monitor the localization, and in an increasing number of cases the activity, of molecules inside cells. These methods will allow biochemical and biophysical parameters of cellular processes, such as signaling cascades or the assembly and disassembly of subcellular structures, to be quantitatively determined and thus provide the raw material for the type of modeling and simulation methods that, when applied together with experimental manipulation, will enable detailed understanding of the information flow that underlies cellular processes and functions. Thus, I foresee a broad, significant increase in our understanding of “how cells work.” The major current limitation is the small variety of available in vivo reporters and the small number that can be studied simultaneously. I confidently expect both of these limitations to yield to energetic attack over the next two decades. The recent advent of super-resolution microscopy and electron microscopic tomography are important steps in bridging the resolution gaps between cell and structural biology. These and other developments have led to a new approach, sometimes called cellular structural biology, that integrates across scales to move toward a detailed, structure-based understanding of cellular organization and function. In my view, the insight gained will change our way of thinking about cellular processes and how to manipulate them. In parallel, automated high-throughput and high-content microscopy have revolutionized the scale at which cell biology experiments can be done. Phenotypes of thousands of cells can now be screened in parallel, and image-processing software recognizes patterns and phenotypes more reliably than the human eye. Microfluidics will enhance single-cell study methods and the parallelization of biochemical assays. This will have a big impact in the years to come, for example, in systematically studying aspects of cellular phenotype based on interindividual or interspecies genetic variation. All of these developments suggest that cell biology will probably be substantially redefined in terms of its scope, scale, and toolbox and in the questions it addresses over the next 50 years. I encourage those entering the field to train as broadly as possible, studying chemistry, physics, or mathematics as well as biology. However, given that cells have already been under study for 400 years, we shouldn't be too surprised if there are still more questions than answers in 2060.

My Dream of a Fantastic Voyage to See the Inner Workings of a Cell

What would I give to be allowed to embark on a Fantastic Voyage at the nanometer scale inside the cell! In the 1960's film, humans were shrunk down to merely micrometer scale, but we now know that “nano” is the ticket. Imagine being able to observe cellular structural biology in real time and to see proteins and nucleic acids in action, in the concentrated environment of the nucleus, let alone in the cytosol. I imagine a complex molecular ballroom full of many intricately choreographed dancers, changing partners, switching locations, dying and being born again. Imagine the purpose and chaos governing the survival of the cellular microcosm, there for me (for you) to see and ultimately understand. Eva Nogales. Photo by Paul Fetters for HHMI. As a structural biologist, initially trained as a physicist, cells often seem too complex and far afield from my own research. My scientific presentations typically borrow images from cell biologists' websites, and I warn my audience after the first introductory slides that no more cells will be seen for the rest of the talk. However, as a molecular electron microscopist working on the cytoskeleton and large macromolecular complexes, I boast in front of crystallographers and nuclear magnetic resonance experts that we study fully functional complexes, in as close to physiological conditions as one can possibly expect outside of a cell. However, this is obviously not as physiological as if we were visualizing the native complexes in their cellular context! With fully annotated genomes providing nearly complete cellular parts lists, and with ever more powerful light microscopy, among other 21st century techniques, we have come a long way toward understanding the inner workings of the cell. Still, the dream of visualizing the intact cell's anatomy with molecular resolution seems like science fiction. Is there any hope for visualizing these molecular machines where they function and when it matters, even if it is not through our atomically shrunk, craving eyes? I really want to think so. It may not come from a single instrument, like the Star Trek scope where magnification can be cranked until the bar code on individual atoms becomes clear. However, combining currently available methodologies and then taking them to the next level may, step by step, get us there. The first combined electron and light microscopes are already producing data wherein the fluorescent colors of choice are superimposed on the gray levels of individual molecular density. Spectroscopy readings, with nanometer cube mapping capability, are being carved out of cellular slices in futuristic attempts at spatial metabolomics. Three-dimensional maps of hydrated cells will soon become routine, and as the resolution moves from 100 to 10 Å in my life time (if I take good care of myself), we will be able to identify macromolecules by shapes, in snapshots of cellular life. We will be able to follow the paths of single proteins as they meander their way along the chaotic cellular freeways of the cytoskeleton, or fluidly traffic between cellular compartments. We are almost there. My own personal dream of “visualizing” the complete kinetochore protein network, carrying out the dynamic engagement of chromosomes to the mitotic spindle and thus revealing the force coupling mechanism, may be just around the corner. It is not that images are worth a thousand gels, or movies a million pulldowns (although perhaps they are), it's just my own preferred means of getting to know and understand nature. My wish for the future of cell biology: I want to “see” the cell (I leave smelling it and listening to it to biochemists and theoretical physicists, respectively).

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The future is hybrid

On the occasion of the 50th anniversary of the Journal of Structural Biology, we review some of the major advances that have taken place in molecular and cellular structural biology over this timeframe and consider some current trends, as well as promising new directions. While the primary experimental techniques of X-ray diffraction, electron microscopy and NMR spectroscopy continue to improve and other powerful new techniques have come on-line, it appears that the most comprehensive analyses of large, dynamic, macromolecular machines will rely on integrated combinations of different methodologies, viz. "hybrid approaches". The same prospect applies to the challenge of integrating observations of isolated macromolecules with data pertaining to their distributions and interaction networks in living cells. Looking ahead, computation in its diverse aspects may be expected to assume an increasingly important role in structural biology, as the prediction of molecular structures, the computation of dynamic properties, and quantitative time-resolved models of intracellular molecular populations (structural systems biology) move towards functional maturity.