ICAM Workshop:

Chromatin Dynamics, Gene Regulation and Silencing

Aug 11-13, 2006

Snowmass, CO

Organizers: Jonathan Widom, Northwestern University

Scope:

Dynamic properties of the chromatin play an essential role in mechanisms of eukaryotic gene expression regulation. This workshop is intended for physical scientists who want to understand the biophysical underpinnings of such mechanisms, both in the context of local regulation of gene expression as well regulation of whole loci by mechanisms such as chromatin silencing. The topics covered would include, among other things, in vitro single molecule experiments on the fluctuations in chromatin structure, efforts to model its three-dimensional structure as well as mechanical properties and single cell measurements of epigenetic silencing effects.

The meeting is sponsored by ICAM. ICAM strongly encourages participation by junior members of the scientific community. We have set aside 16-20 places for junior participants-graduate students, postdocs, junior faculty members-from US institutions, and can offer substantial travel and living expense support to those willing to share a room. Moreover, junior participants coming from ICAM branches abroad may apply for an I2CAM travel grant - go to the web site i2cam.org to apply.

Speakers:

Jim Broach, Princeton Brad Cairns, U. of Utah

Alex Grosberg, U. of Minnesota Terrence Hwa, UCSD

Wilma Olson, Rutgers Rob Phillips, Caltech

Vincenzo Pirotta, Rutgers Oliver Rando, Harvard

Jasper Rine, Berkeley Anirvan Sengupta, Rutgers

Boris Shklovskii, U. of Minnesota Vasily Studitsky, UMDNJ

Jose Vilar, Sloan-Kettering Jonathan Widom, Northwestern

Titles and Abstracts:

As they come in, the titles and abstracts of the talks would be found here.

Venue and Schedule

The workshop will take place in the Silvertree Hotel. It would begin with a welcome dinner on Thursday evening.

Formal talks start Friday morning 9.30AM and conclude before Sunday lunch hours at 12.30PM.

According to the current schedule (see below) talks would be over by Saturday, late evening. This is due to a cancellation and to requests from some speakers.

Travel Info

Snowmass is very close to the Aspen airport. For cheaper travel arrangements, one might also fly to Denver and take a bus from there.

Hotel and Registration Information

Please make your reservations at the Silvertree Hotel (100 Elbert Lane, Snowmass Village), Snowmass at (1-800-525-9402) or by fax at (970-923-5494). The reservation block is under "ICAM Chromatin Workshop"; please refer to the above title when making your reservations. The rate is $99.00 single or $99.00 double occupancy excluding any taxes. The cut off date for making these reservations is July 10, 2006. The hotel check in time is 4:00 pm and the check out time is 10:00 am.

For further information about the Silvertree Hotel, you can go to their website. (Click on "Getting here" under the heading "About our hotel" for travel instructions.)

In order to register for the meeting, please download the registration form, fill it out and return it to Rose Romero at your earliest convenience at (rbromero@lanl.gov), or by fax at (505) 665-9030.

There will be a registration fee of $175 for all workshop attendees, payable by check on arrival, the funds from which will be used to cover the cost of meals and beverages and the meeting rooms.

Schedule:

Friday

9.30AM Widom

10.30AM Rando

11.30AM Cairns

12.30-2.00PM Lunch Break

2.00PM Vilar

3.00PM Hwa

Break 4.00-4.30PM

4.30PM Studitsky

5.30PM Grosberg

Saturday

9.30AM Rine

10.30AM Broach

11.30AM Sengupta

Lunch 12.30-2.00PM

2.00PM Olson

3.00PM Pirrotta

Break 4.00-4.30PM

4.30PM Shklovskii

5.30PM Phillips

Sunday

Nothing scheduled

Talks:

Analysis of Heterchromatin Formation and Inheritance by Single Cell Observations

Eugenia Y. Xu, Karl A. Zawadzki and James R. Broach

Princeton University

Analysis of transcriptional silencing in Saccharomyces has provided valuable insights into heterochromatin formation and function. However, most of these studies revealed only the average behaviors of populations of cells. We have examined transcriptional silencing by monitoring individual yeast cells carrying distinguishable fluorescent reporter genes inserted at two different silent loci. These studies showed that two silent loci in a single cell behave independently, demonstrating that heterochromatin formation is locus autonomous. Furthermore, some silencing mutants with an intermediate phenotype, such as sir1, consist of two distinct populations, one repressed and one derepressed, while other mutants, such as those inactivating the SAS-I histone H4 K16 acetylase, consist of cells all with an intermediate level of expression. Finally, both establishment and decay of silencing can be influenced by specific gene activators, with establishment occurring stochastically over several generations. Thus, quantifying silencing in individual cells reveals aspects of silencing not evident from population-wide measurements.

DNA translocation as a mechanism for nucleosome remodeling

Brad Cairns, HCI, Utah

Proteins searching for their targets on DNA

Alexander Grosberg, Department of Physics, University of

Minnesota, 116 Church Street SE, Minneapolis, MN 55403, USA

Abstract: Proteins locate their specific targets on DNA up to two orders of magnitude faster than the Smoluchowski 3D diffusion rate. A widely accepted explanation of this fact is that proteins are non-specifically adsorbed on DNA, and sliding along DNA provides for the faster 1D search. Surprisingly, the role of DNA conformation was never considered in this context. By explicitly addressing the relative role of 3D diffusion and 1D sliding along coiled or globular DNA and the possibility of correlated re-adsorption of desorbed proteins, we have identified a plethora of new different scaling regimes and made quantitative predictions for the macroscopic diffusion of proteins through a semi-dilute DNA system.

Quantitative characterization of the Lac promoter -- cooperativity and sensitivity mediated by DNA looping

Terrence Hwa, Dept. of Physics, UCSD

It is the goal of systems biology to understand the behavior of the whole in terms of the knowledge of the parts. This is hard to achieve in many cases so far due to the difficulty of characterizing the many constituents and their complex web of interactions involved. The lac operon of E. coli, being one of the most extensively studied systems of molecular biology, offers a possibility of confronting “system-level” properties of transcriptional regulation with the known biochemistry of the molecular constituents and their mutual interactions. Such confrontations can reveal previously unknown constituents and interactions, as well as offering new insight into how the components work together as a whole. Here we study the combinatorial control of the lac promoter by the regulators LacR and CRP. A previous in vivo study [Setty et al, PNAS 100: 7702-7 (2003)] found gross disagreement between the observed promoter activities and the expected behavior based on the known molecular mechanisms. We repeated the study by identifying and removing several extraneous factors which significantly modulated the expression of the lac promoter. Through quantitative, systematic characterization of promoter activity for a number of key mutants and guided by the thermodynamic model of transcriptional regulation, we are able to account for the combinatorial control of the lac promoter quantitatively, in term of a cooperative interaction between CRP and LacR-mediated DNA looping. Specifically, our analysis indicates that the sensitivity of the inducer response results from LacR-mediated DNA looping, which is significantly enhanced by CRP. Thus CRP plays a triple role, in elevating the overall transcription level through direct activation, and in increasing the fold-change and sensitivity of repression by assisting LacR-mediated DNA looping.

Targeting a Dosage Compensation Complex to X Chromosomes (Cancelled)

Barbara J. Meyer, HHMI, Dept. of Molecular & Cell Biology, U.C. Berkeley, CA 94720

Chromosomes must be properly expressed, resolved, compacted, and segregated for genome stability. These diverse processes are controlled by an interacting set of proteins and complexes. On set of such proteins, the condensin complex, is essential for chromosome resolution and compaction during mitosis and meiosis. A homologous set of proteins is essential for the X-chromosome-wide process of dosage compensation, which ensures that males (XO) and hermaphrodites (XX) express equal levels of X-linked gene products, despite their difference in X chromosome dose. This dosage compensation complex (DCC) binds the entirety of both hermaphrodite X chromosomes to achieve chromosome-wide reduction in gene expression. The DCC not only resembles mitotic condensin, it shares a component with condensin, and DCC components also participate in other aspects of chromosome segregation, for example the regulation of crossover interference during meiosis. This talk will focus on the connection between dosage compensation and chromosome segregation and on the mechanism by which the DCC specifically recognizes and binds X chromosomes.

A novel ‘Roll and Slide’ mechanism of DNA folding in chromatin: implications for nucleosome positioning

Michael Y. Tolstorukov

V. Karazin Kharkov National University, Kharkov, Ukraine

Victor B. Zhurkin

National Cancer Institute, National Institutes of Health, Bethesda, Maryland

Andrew V. Colasanti, David McCandlish, Wilma K. Olson

Rutgers, the State University of New Jersey, Piscataway, New Jersey

The bending of DNA in high-resolution nucleosome structures is accompanied by large changes in Slide (the shearing of adjacent base pairs along their long axes). These shear deformations play a much more important role in the formation of nucleosomal DNA than heretofore thought. Specifically, if Slide is set to zero at all base-pair steps, the crystallographically observed DNA superhelical pathway is flattened into a circle. Moreover, the large Slide deformations imposed on DNA by the histone proteins at sites of sharp local bending into the minor groove (negative Roll) appear to govern nucleosome positioning. The computed cost of deforming DNA on the nucleosome increases substantially if the crystallized sequence is displaced relative to its observed positioning. Furthermore, this positioning preference disappears if the contribution from Slide is omitted from the calculations. Other DNA sequences, which are experimentally known to position nucleosomes in solution, show similar dips in the total deformation energy close to the observed “natural” settings (1-3 bp). The computed scores are lowest when the sequences are positioned such that the most easily deformed base-pair steps (TA and CA:TG) occur at sites of large positive Slide and negative Roll.

Physics of Genome Management: from Viruses to Nucleosomes

Rob Phillips, Caltech

Tightly bent DNA is a fact of life. Whether we consider how genomes are packaged into their hosts or how genes are transcribed, there are many examples where the properties of DNA as a physical object play a critical role in dictating biological function. In this talk, I will describe several case studies in which the mechanics of DNA plays an intriguing role in biological function. One case study will center on how bacterial viruses both package and deliver their genomes and will focus on recent experiments which measured these properties at the single-molecule level. The second case study in DNA packaging will consider the accessibility of nucleosomal DNA. In particular, I will describe our recent attempts to model the dynamics of site accessibility in nucleosomes. As a final related example in which the mechanics of tightly bent DNA plays a role, I will describe our efforts to export what we have learned about sequence-dependent elasticity from nucleosomal studies to DNA looping in transcriptional regulation.

Polycomb complexes, Polycomb silencing and its genomic targets

Vincenzo Pirrotta

Department of Molecular Biology & Biochemistry, Rutgers University, Piscataway, NJ.

Polycomb Group (PcG) silencing mechanisms, first known for their role in homeotic gene regulation in Drosophila, have now been found to have a fundamental role in the epigenetic programming of genomic information controlling growth, differentiation and self-renewal in Drosophila and in vertebrates. PcG proteins are recruited to regulatory regions called Polycomb Response Elements (PREs) by multiple DNA-binding proteins. Chromatin immunopreciptiation experiments analysed by hybridization to Drosophila genomic tiling microarrays reveal that such presumptive PREs are found at several hundred genomic loci, corresponding to key genes controlling most differentiation, growth and pattern-formation processes. The PREs bind PRC2 complex with histone H3 methyltransferase activity, as well as a PRC1 complex containing a histone H2A ubiquitylating activity and a chromodomain protein, PC. Although found only at the PRE, the PRC2 complex methylates a large domain including the regulatory and transcriptional unit of the target gene. The PRE itself is not methylated because its core region is nucleosome free. We present models for the long-distance action of the PRE.

Genome-scale studies on static and dynamics aspects of chromatin structure in yeast

Oliver Rando, Bauer Center, Harvard

We have designed a tiled oligonucleotide microarray to sequence the primary structure of half a megabase of yeast chromatin. We have found that yeast promoters exhibit remarkably stereotyped chromatin architecture, with upstream regulatory DNA sequence found in a long nucleosome-free region surrounded by two well-positioned nucleosomes with a characteristic modification pattern. These modifications associated with promoter nucleosomes comprise one of two groups of correlated histone modifications, with the other group consisting of histone modifications that occur over coding regions and whose levels vary with transcription levels. In yeast, histone modifications occur in few combinations, suggesting that much less information is carried in

histone modifications than is theoretically possible. Altogether, we suggest that the dominant role of histone modification in transcriptional control is permissive, rather than instructive, and hypothesize that one of the reasons for the plethora of histone modifications observed is to allow for analog control of gene expression. Finally, I will discuss recent results on dynamic aspects of nucleosome occupancy and histone modification.

On the establishment, containment and evolution of silencing in Saccharomyces

J. Babiarz J. Halley, J. Gallagher, E. Osborn, B. Ozydin, L. Teytelman, O. Zill, and J. Rine

Dept. Molec. Cell Biologym UC Berkeley, Berkeley Calif. 94720

Silencing at HM loci requires binding of the Sir proteins with proteins at the silencer, and subsequent spreading of Sir proteins to barriers that require histone H2A.Z. We have re-evaluated the cell-cycle requirements for silencing using mating type itself as the benchmark for whether silencing. We inject a single-cell dose of wild-type Sir proteins into sir- mutants using yeast mating, and then monitor the establishment of silencing at the single cell level by the acquisition of sensitivity to mating pheromone. The single-cell studies reveal a lag between phenotype and genotype, reflecting an increasing sub-population of cells that have stochastically switched from On to Off at HML and HMR. The probability of switching from On to Off increases with successive cell divisions, with evidence of patterns to the switches in state, and increases in mutants lacking distinct histone modifications, suggesting that the removal of specific histone modifications increases the probability of switching to the silenced state. Histone variant H2A.Z, conserved from yeast to man. We showed that S. cerevisiae H2A.Z is acetylated on four N-terminal lysines by a combination of NuA4 and SAGA. Additionally, H2A.Z deposition by SWR-Com was a pre-requisite for acetylation. H2A.Z acetylation is critical in H2A.Z’s function as a boundary to silent chromatin at the telomeres. Because bromo domains can bind to acetylated lysines, we tested the contribution of the 10 yeast bromodomains to heterochromatin boundary function. Two bromo domain proteins specifically bind the acetylated H2A.Z amino terminal tail in vitro and in vivo. Synthetic lethal interactions with bromo domain mutants are leading to deeper understanding of the post-translational marks. Curiously, the Rap1 binding site at the silencers is a unusual variant of consensus found at most other locations, and the odd variant is conserved among silencer is sensu stricto strains. Substituting the Rap1 consensus sequence in the silencer creates a conditional silencer, underscoring the importance of the variant binding site.

Sir1 is a target of evolutionary change in Saccharomyces with 4 paralogs is some species. We show that each contribute to silencing in S. bayanus, and have uncovered novel epigenetic phenomena controlled by both SUM1 and by mating type.

Modeling Epigenetic Silencing

Mohammad Sedighi and Anirvan Sengupta, Rutgers

We formulate a mathematical version of the conventional model of maintenance of silencing in S. cerevisiae and analyze the conditions for bistability as well as for formation of stationary boundaries. Although the model is perhaps too simple, the structure of the bifurcation diagram, describing parameter regions with different kinds of qualitative behavior, is likely to be more robust. We can place some of the known mutants in different regions of this diagram. One interesting finding of this study is that, under some conditions, the lowering of acetylation rates might have non-obvious consequences for silencing. Possible improvements of the model are discussed at the end.

Phase diagram of DNA with shorter positive polycations.

Boris Shklovskii,

University of Minnesota

We study a water solution of long polyanions (PA) such as DNA with shorter polycations (PC) such as polylysine which is a model system for gene delivery compounds. We concentrate on the equilibrium phase diagram of this solution in the plane of ratio x of total charges of PC and PA and concentration of monovalent salt. Each PA adsorbs many PCs and makes a complex. At x >1 complexes are positive and at x <1 they are negative. At |x| very close to 1 complexes are weakly charged, their Coulomb repulsion is weak, and their short range attraction leading to condensation into almost neutral macroscopic droplets. At large |x-1| the Coulomb repulsion dominates and makes all complexes stable. At each sign of x, between stable phase and condensed phase there is domain, where PA disproportionate themselves between different complexes. Some complexes become almost neutral and condense into liquid droplets, others become stronger charged and stay stable. We show that with increasing concentration of a monovalent salt the phase of macroscopic liquid widens and the domain of disproprtionation vanishes. We discuss application of this model to chromatine, where the role of PA is played by the net negative chain of DNA with nucleosomes without H1 and the role of shorter PC is played by H1. In this case x < 1, because chromatin is overcharged by DNA even with H1. The stable (large 1-x) phase is identified then with 10 nm fiber and the smaller 1-x condensed phase is thought of as 30 nm fiber and more condensed phases of chromatine.

Mechanisms of communication over a distance

Yury S. Polikanov, Mikhail A. Rubtsov, Vladimir A. Bondarenko, Alexander Vologodskii, Vasily M. Studitsky

Department of Pharmacology, UMDNJ, Piscataway, NJ 08854, USA

Abstract

The majority of eukaryotic genes are regulated by activator-binding DNA sequences (enhancers, E) that can efficiently communicate and directly interact with their targets (promoters, P) over a large distance (1). This interaction is accompanied by formation of large loops including spacer DNA organized into chromatin structure (1). However it is unclear what mechanisms mediate formation of large DNA loops with high efficiency and at a high rate. Previously we have shown that DNA supercoiling greatly facilitates E-P communication over a large (2.5 kb), but not over a short distance (2,3), most likely facilitating sliding (“slithering”) of intertwined DNA double helices (4). Based on the knowledge of the mechanism, a rationally designed insulator-like elements that prevents E-P communication was constructed (4). In more recent studies we have shown that the rate of E-P communication on supercoiled DNA is not limited by diffusion of the DNA sites one to another and that DNA supercoiling strongly increases the probability of E-P juxtaposition. In contrast, the rate of communication on linear or relaxed DNA most likely is diffusion-limited and is not affected by the presence of the insulator-like elements. Action of the enhancer action on DNA assembled in chromatin was also studied. Assembly of linear, communication-deficient templates in chromatin or introduction of negative DNA supercoiling results in efficient, quantitatively similar, but mechanistically distinct enhancer action over a distance. The efficiency of enhancer action over a large distance in chromatin does not depend on the level of unconstrained DNA supercoiling. Thus eukaryotic chromatin structure per se can support highly efficient communication over a distance and functionally mimic the supercoiled state of DNA characteristic for prokaryotes. Mechanisms increasing the efficiency of communication over a distance are likely to be widely utilized in both pro- and eukaryotic enhancer-dependent systems.

References

1. Bondarenko, V. A., Liu, Y. V., Jiang, Y. I., and Studitsky, V. M. (2003) Biochem. Cell. Biol. 81, 241-251.

2. Liu, Y., Bondarenko, V., Ninfa, A., and Studitsky, V. M. (2001) Proc. Natl. Acad. Sci. USA 98, 14883-14888.

3. Bondarenko, V., Liu, Y., Ninfa, A., and Studitsky, V. M. (2002) Nucleic Acids Res. 30, 636-642.

4. Bondarenko, V. A., Jiang, Y. I., and Studitsky, V. M. (2003) EMBO J. 22, 4728-4737.

Nucleosome dynamics and positioning

Jonathan Widom

Dept. of Biochemistry, Molecular Biology and Cell Biology, and Dept. of Chemistry,

Northwestern University, 2153 Sheridan Road, Evanston, IL 60208-3500, USA

j-widom@northwestern.edu

The genomic DNA of eukaryotes is tightly wrapped into chromosomes through a hierarchical series of folding steps. In the lowest level of compaction, short stretches of DNA are wrapped around small octameric protein spools, forming structures known as nucleosomes. The structure of the nucleosome occludes most of the wrapped DNA from interaction with the regulatory proteins and enzymes that must act on it. I will discuss studies using enzymatic and fluorescence probes that show nucleosomes to spontaneously undergo rapid, large-scale, conformational fluctuations that facilitate the invasion of nucleosomes by gene regulatory proteins. I will then discuss studies on DNA sequence motifs that bias where nucleosomes are placed along the DNA and control the stabilities of nucleosomes. I will summarize our progress identifying such nucleosome positioning DNA sequences, and our current understanding of how such sequences function to attract and stabilize nucleosomes. We have used this information in a novel computational approach to construct and experimentally validate a nucleosome-DNA interaction model, and to predict the genome-wide organization of nucleosomes. Our results demonstrate that genomes encode an intrinsic nucleosome organization, and that this intrinsic organization itself can explain ~50% of the in vivo nucleosome positions. This nucleosome positioning code may facilitate specific chromosome functions, including transcription factor binding, transcription initiation, and even remodeling of the nucleosomes themselves.

Macromolecular assembly on looped DNA

Jose Vilar, Memorial Sloan Kettering

The formation of DNA loops by proteins and protein complexes is ubiquitous to many fundamental cellular processes, including transcription, recombination and replication. I will discuss recent advances in understanding the properties of DNA looping in its natural context and how they propagate to the cellular behavior through gene regulation. The results of connecting the molecular properties of DNA looping with cellular physiology measurements indicate that looping of DNA in vivo is much more complex and easier than predicted from current models, and reveals a wealth of previously unappreciated details.