Keystone Molecular Basis for Chromatin Structure and Regulation
Keystone Conferences make our head hurt...more because of the amount of knowledge that is dropped over five days than the altitude. Thanks to loyal EpiGenie subscriber and guest correspondent, Christopher Ricupero from the Rutgers Stem Cell Research Center for covering Keystone’s Molecular Basis for Chromatin Structure for us and providing you this killer summary.
Getting to Taos, located at the base of the Rocky Mountains included a 2 ½ hour ride from Albuquerque to an elevation of 6,950 feet. The symposium was held at the Sagebrush Inn and housing accommodations were within the center and nearby hotels. The staff was both courteous and organized, making the entire stay a pleasure. The event had an underlying Native American vibe which as an east-coaster, was a welcome change that we don’t often get to experience. It was apparent that the organizers and the locals were proud of the rich history of the Taos Pueblo (Native American Village) surrounding the area. A variety of excursions were available including trips to Taos Ski Valley, art galleries downtown, or tours of the neighboring Pueblo Reservation and Rio Grande Gorge. Overall, when we weren’t digesting the science, the conference organizers kept us well fed and occupied with interesting activities. Now, onto the science…
Within the last decade, progress in Epigenetics has exploded on a variety of fronts due to pioneering technologies built upon earlier research. While insights into the atomic structure of chromatin modifiers and nucleosomes have been rapidly growing, the mechanisms underlying these observations have been lacking. The goal of this keystone symposium was to bring together researchers from diverse areas of chromatin biology to help bridge the gap between chromatin structure and the mechanisms responsible for function.
In an attempt to fulfill this ambitious goal, the organizers put together a lineup of discussions that fit together like “beads on a string”. In addition, over 150 posters were presented throughout the conference.
Beyond the Double Helix: Writing and Reading the Histone Code
C. David Allis, Rockefeller University
Dr. Allis was one of two keynote talks to kick off the symposia on day 1. He started off with a brief history of chromatin research and quote from Alan Colfe stating “The future will increasingly emphasize not the monotony of chromatin structure but the capacity of nucleosomes to adopt a fascinating variety for form and function”. Dr. Allis proceeded to discuss recent studies that have delineated various post translational modifications of histone tails that correlate with both activation and repressive gene activities. “Interest in histone variants is heating up!” Allis claimed, and described recent research focusing on histone H3 variants and how Histone H3.3 is the “deviant” variant. A favored hypothesis of the Allis lab is that Histone H3.3 is important during embryonic stem cell (ES) differentiation. Their approach to overcome distinguishing H3.3 from the other variants was to add tags to H3.3, therefore replacing the endogenous allele in mouse ES cells. ES cells were subsequently differentiated into neural precursor cells (NPCs) followed by ChIP-Seq. Results showed that pluripotency genes enriched with H3.3 in ES, lost it during differentiation while the bivalent genes became enriched post differentiation.
So, how does H3.3 get to TF Binding sites? Great question and the Allis lab is still investigating, so stay tuned...
Dr. Allis concluded his keynote with the message that variety is the spice of nucleosomes, and that it will turn out that every amino acid in histones DOES matter.
Coordinated Control of PRC2 Enzymatic Activity and Target Gene Occupancy in Pluripotent Cells
Joanna Wysocka, Stanford School of Medicine
Dr. Wysocka originally planned to discuss how the polycomb Repressive Complex 2 (PRC2) regulates key developmental genes in pluripotent cells during development. Instead, she decided to tell us a new story about turning back the epigenetic clock. Wysocka described the remarkable capability of how migratory neural crest cells (originally ectodermal origin); undergo transcriptional reprogramming allowing diverse lineage potential of both ectoderm and mesoderm. These neural crest descendants migrate and differentiate into cells of the peripheral nervous system, bone, cartilage, and cardiac structures.
A particular genetic defect hypothesized to be the result of neural crest malfunction is CHARGE syndrome. A spontaneous genetic disease affecting multiple tissues and organs of neural crest origin, CHARGE syndrome results in a host of tissue specific abnormalities, however the underlying mechanism are still unknown, and is caused by heterozygous mutations in the gene encoding CHD family protein CHD7, an ATP-dependent chromatin remodeller.
Dr. Wyscoka's group investigated the relationships between CHD7, multipotential neural crest lineages and CHARGE syndrome and established a system of human embryonic (hESC) derived neural rossettes that spontaneously attach and give rise to a migratory cell population behaving as early neural crest cells. They first knocked down CHD7 in vitro and later in Xenopus embryos to observe the effect of CHD7 down regulation on the formation of these human ES derived neural crest precursors. They witnessed an incredible effect on the formation of the migratory cells. Results show the emergence of CHARGE traits such as craniofacial malformations, and other CHARGE related defects.
In summary, the Wysocka group has demonstrated that CHD7 is essential for the activation of the multipotent migratory neural crest cells. Furthermore, they propose a tissue specific model for the CHD7/PBAF complex functioning to activate the transcriptional circuitry of essential neural crest genes.
Nucleosome Occupancy and Transcriptional Regulation
Robert Kingston, Mass General Hospital
Dr. Kingston began his talk by highlighting the importance of the spatial organization and protein composition of chromatin during development. The Polycomb and Trithorax group genes play key roles by both responding to and initiating chromatin changes during regulatory events. Some of these gene families can hold and move nucleosomes around and Dr. Kingston stressed the importance of understanding how and why nucleosomes shift. These differential occupancy regions are important for learning and predicting downstream regulatory events.
His group used microarray-based technology to perform large scale mapping of nucleosome occupancy to assess potential regulatory regions, specifically polycomb repressive elements (PREs). His regions of choice were specific HOX clusters predicted to contain dynamic nucleosome occupancies during human embryonic to mesenchymal stem cell differentiation. The group focused on locating a putative PRE region that contains big dips in occupancy within HOX clusters during differentiation. One region, HOX D11.12 was dubbed a potential PRE by satisfying multiple criteria: low nucleosome occupancy, repressive histone modifications (H3K27me3) flanking the region, and the binding of polycomb complex proteins. In summary, these putative PREs located by nucleosome occupancy screens may shed light on polycomb recruitment and function. Future discoveries on human PREs will clarify the similarities and/or differences from previous research in flies.
The Dynamics of Accessing DNA
Michelle D. Wang, Cornell University
Dr. Wang is applying cutting edge optical trapping techniques to probe the motions and dynamics of molecular motors that translocate along DNA. Dr. Wang described a new optical tweezer technique, a fantastic tool to observe nucleosomal positioning that is accurate within 2.5 base pairs. It is an unzipping technique that converts double stranded to single stranded DNA and is a powerful single-molecule method to explore protein-DNA interactions. When unzipping through a nucleosome, there is a large increase in the tension of DNA. The changes in force are monitored throughout the unzipping process resulting in the precise spatial location of the protein-DNA complex.
Dr. Wang focused on the mechanism of transcription through nucleosomes and specifically understanding how eukaryotic PolII carries out transcription. By using their single molecule techniques, the Wang group tackled this mechanism in three short parts.
- Where does RNA polymerase pause at a nucleosome? Strong histone-DNA interactions induce RNA polymerase pausing. PolII slows when it encounters nucleosomes, significantly reducing transcription rate.
- Why does Polymerase pause at a nucleosome? PolII pauses due to backtracking. Dr. Wang's lab looked at how backtracking was occurring by mapping nucleosomal positioning using their DNA unzipping assay again to locate PolII. They found a displacement, and backtracking of approximately 15 bps.
- How does PolII overcome the nucleosome barrier? Dr. Wang postulated that many factors may contribute to overcoming this transcriptional barrier, including making nucleosomes more accessible via histone modifications. However, she ended with the interesting notion that a trailing polymerase assists the 1st polymerase to overcome the nucleosomal barrier.
In summary, Dr. Wang's approach to transcriptional dynamics is a fresh and highly accurate technique, and will be interesting to track her lab's progress in the near future.
Next Generation Quantitative Proteomic Tools for Analyzing Histone Modifications
Benjamin A. Garcia, Princeton University
Dr. Ben Garcia rounded out the last day of talks by describing a bottom up approach to studying histone modifications. Dr. Garcia's lab is pioneering novel mass-spectrometry based proteomic methodologies for quantitatively measuring changes in protein expression and post-translational modification state of chromatin associated proteins. Their approach observes systems-wide analysis of key molecular events during epigenetic processes. His lab has developed a new approach that involves N Terminal labeling followed by rapid quantification of the labeled protein of choice.
He has since scaled his techniques to investigate combinatorial modifications. This method can quantify all histone H3 and H4 post translational modifications in a single two hour experiment. Add in an extra hour for bioinformatics and you have a massive amount of data providing you with a systems level view of chromatin signatures!
Recently, the Garcia lab has documented 100s of combinatorial signatures during a single experiment. In addition, these studies do not require an exorbitant amount of starting material, approximately one million cells. These novel approaches have the potential to identify vast novel combinatorial “Histone Codes” that may be responsible for a wide variety of developmental processes and diseases.
Highlights from some other great talks include:
Dr. John Lis described the kinetics of nucleosome depletion in activated heat shock genes and concluded that this structural change precedes movement of Pol II. In addition, his team concluded that Poly (ADP)-Ribose Polymerase (PARP1) is critical for this rapid chromatin alteration. Dr. Shelley Berger gave an interesting talk on how her team is using genetic approaches in yeast to link post translational histone modifications to genome function. Specifically, she discussed how these modifications have implications in gametogenesis and aging. Berger’s group is actively screening a library of over 400 histone mutants. Early results have identified mutants responsible for both shortening and extending life spans.
Dr. Cheryl Arrowsmith began by introducing the many crystal structures of histone methyltransferase bromodomains that her group is investigating. By using structural analysis, one of their goals is to understand histone methyltransferase substrate recognition. Arrowsmith concluded with the announcement of The Structural Genomics Consortium, a non-for-profit organization with the aim of determining the three dimensional structures of proteins with medical relevance. One area of interest is the development of chemical probes that can be used to exploit the variability of histone methyltransferase activities. Newly designed probes will be made publicly available without restriction.
Dr. Jennifer Ottesen’s group is taking a synthetic histone approach to probe histone modifications in the nucleosome core. Over 30 known nucleosome modifications have been identified in the core. To generate synthetic histones and insert modifications, the Ottesen lab employs both expressed protein and multi-step chemical ligation strategies. These methods involve splitting the histone into three pieces and then linking them all back together. This approach has yielded the 1st fully modified synthetic histone (H3K56ac).
Coverage provided by Christopher Ricupero-IGERT Graduate Fellow in Dr. Ron Hart’s lab at The Rutgers Stem Cell Research Center.