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Nucleic Acids Research, 2017, Vol Published online 13 June 2017 Nucleic Acids Research, 2017, Vol. 45, No. 13 7555–7570 doi: 10.1093/nar/gkx531 SURVEY AND SUMMARY Molecular structures guide the engineering of chromatin Stefan J. Tekel and Karmella A. Haynes* School of Biological and Health Systems Engineering, Arizona State University, Tempe, AZ 85287, USA Received March 29, 2017; Revised May 18, 2017; Editorial Decision June 06, 2017; Accepted June 07, 2017 ABSTRACT and flexible control over cohorts of genes that determine cell fate and tissue organization. Chromatin states, i.e. ac- Chromatin is a system of proteins, RNA, and DNA tively transcribed and silenced, can switch from one to the that interact with each other to organize and regulate other. At the same time chromatin-mediated regulation can genetic information within eukaryotic nuclei. Chro- be very stable, persisting over many cycles of DNA replica- matin proteins carry out essential functions: pack- tion and mitosis. The latter property is a mode of epigenetic ing DNA during cell division, partitioning DNA into inheritance, where cellular information that is not encoded sub-regions within the nucleus, and controlling lev- in the DNA sequence is passed from mother to daughter els of gene expression. There is a growing interest cells. The stability of chromatin states allows specific epige- in manipulating chromatin dynamics for applications netic programs to scale with tissue development in multicel- in medicine and agriculture. Progress in this area re- lular organisms. quires the identification of design rules for the chro- Early biochemical and protein structure studies of the nu- cleosome (3) have generated a high resolution model that matin system. Here, we focus on the relationship be- has persisted over time. A single nucleosome includes a tween the physical structure and function of chro- complex of eight histone proteins arranged in a spiral-like matin proteins. We discuss key research that has elu- disc. Each histone contains a C-terminal globular region cidated the intrinsic properties of chromatin proteins composed of helix-turn-helix motifs called the histone fold and how this information informs design rules for domain, and an unfolded N-terminal tail (4)(Figure1A). synthetic systems. Recent work demonstrates that Within each nucleosome, a tetramer of histones H3 and H4 chromatin-derived peptide motifs are portable and in is stacked between two dimers of histones H2A and H2B some cases can be customized to alter their func- (Figure 1B). The stacking model can be viewed as a data- tion. Finally, we present a workflow for fusion protein guided 3D animation, created by D. Berry (5). Roughly 200 design and discuss best practices for engineering bp of DNA is wrapped twice around the histone complex. / chromatin to assist scientists in advancing the field Histones H1 H5 interact with the ‘linker’ DNA at the entry and exit site of the wrapped DNA (6,7). Nucleosome struc- of synthetic epigenetics. tures appear repeatedly along each linear chromosome in eukaryotic cells and support higher-order packaging of the CHROMATIN ENGINEERING: AN IMPORTANT AND entire genome (Figure 1B). CHALLENGING UNDERTAKING In natural systems, the histone octamer is modular and Chromatin is a dynamic nuclear structure that has a central dynamic. There are four natural variants of H2A and H3 role in eukaryotic development. The mechanics of this an- with distinct amino acid sequences (reviewed in 8,9), while cient, highly conserved system (1,2) are primarily driven by histones H4 and H2B are largely invariant (10). Substitu- the physical structure and interactions of its components, tions of H2A and H3 with variants in the octamer com- proteins and nucleic acids. Electrostatic bonds and hy- plex play critical roles in gene regulation, DNA replica- drophobic interactions determine the composition of multi- tion, and chromosome structure (4). Kinetic studies of part subunits such as nucleosomes, transcription initiation fluorescently labelled histones have shown that H3 and complexes, and repressive complexes. Because of its impact H4 turnover is very slow. In contrast, exchange of the / on tissue development, chromatin has great potential for en- H2A H2B dimer occurs within a few minutes to two hours gineering cell populations. Chromatin proteins exert strong (11). Exchange of histone H1 occurs in under two minutes *To whom correspondence should be addressed. Tel: +1 480 965 4636; Email: [email protected] C The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 7556 Nucleic Acids Research, 2017, Vol. 45, No. 13 the first report of the structure of the histone-binding do- main from P/CAF in 1999 (18) a plethora of other 3D struc- tures have become available to the scientific community. In- vestigations of binding pocket specificity support the idea that peptide motifs can distinguish one PTM from another (19–21). These protein structure and interaction data can be used by synthetic biologists to design artificial epigenetic systems and to further confirm or correct aspects of the his- tone code model. Taverna et al. provide an excellent detailed review of lessons learned from the molecular structures of PTM-binding domains (19). In spite of its potential usefulness, chromatin is often per- ceived by biological engineers as an impediment rather than as an enabling tool. Cells are typically engineered by in- tegrating exogenous, recombinant DNA into the chromo- somes of the host cell. These transgenes include regulatory components that are carefully designed to operate with pre- determined kinetics. However, the transgene often becomes subjected to the surrounding chromatin environment and is mis-regulated (silenced or hyper-activated). The molec- ular complexity of chromatin may give scientists the im- pression that chromatin-mediated expression states are im- possible to control. Chromatin complexes are often com- posed of multiple subunits, which have several paralogs in a single organism. For instance, Polycomb Repressive Com- plex 1 (PRC1) appears as six sub-types that occupy different genomic regions (22). Each of the PRC1 subunits may be one of several distinct paralogs. Furthermore, the core sub- unit of chromatin known as the nucleosome contains two copies of four types of histones (H2A, H2B, H3, H4) (4), two of which have multiple variants. Histones H3 and H2A have eight and five known variants, respectively. The vari- Figure 1. The nucleosome, the core subunit of chromatin, is a modular ants differ in primary sequence, genome distribution, and protein complex. (A) 3D model based on K. Luger’s 1997 X-ray crys- tallography data, PDB ID: 1AOI (3). (B) The cartoon abstraction shows expression in different tissues and phases of the cell cycle the general shape of each histone: the Z-shape is the globular region and (23,24). Compared to simpler biological principles such as the thinner line is the unfolded N-terminal tail. A H3/H4 tetramer (green Watson–Crick base-pairing, the complex interactions that and blue) and two H2A/H2B dimers (orange and pink) bind across ∼120 govern the behavior of chromatin may seem less amenable bp DNA and become stacked to form a solenoid, DNA-wrapped struc- to bioengineering. ture (for a data-based animation, see (5)). The lower-right cartoon depicts higher-order packaging of nucleosomes into a metaphase chromosome. Is it worthwhile to attempt to engineer multi-layered sys- tems like chromatin within a complex cellular milieu? Syn- thetic biologists have demonstrated so far that such work (12). Addition and erasure of post-translational modifica- produces valuable new knowledge as well as useful innova- tions (PTMs) is another highly dynamic feature of nucle- tions (25,26). We believe that the current wealth of informa- osomes. Histone-modifying enzymes covalently link or re- tion produced by decades of research in chromatin epige- move small molecules at the side chains of specific amino netics provides a sufficient platform to support engineering acids within each histone. These modifications take place efforts. In this review, we discuss how proteins and nucleic mostly in the unfolded tails, while a few occur in the glob- acids that guide epigenetic regulation in nature have been ular domain. Over 15 known modifications include lysine harnessed for custom-built systems. Specifically, we focus acetylation (Kac), lysine methylation (Kme), serine phos- on the molecular structures of chromatin proteins and how phorylation (Sp), sumoylation (su), ubiquitination (ub) and our understanding of molecular interactions can be lever- crotonylation (cr) (13)(Figure2). In total, over 50 differ- aged for chromatin engineering. We discuss best practices ent amino acid positions are known to be modified. Certain for chromatin engineering endeavors and present a flexible, types of PTMs result in transcriptional silencing of a nearby standard workflow for efficient, high-throughput engineer- gene, while others enable activation. ing of chromatin-derived proteins. Collectively, PTMs make up a rich set of biological in- formation in which single or combinations of molecular ENGINEERING NUCLEOSOMES, THE CORE SUB-
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