Chromatin regulation at the frontier of synthetic biology

Abstract

As synthetic biology approaches are extended to diverse applications throughout medicine, biotechnology and basic biological research, there is an increasing need to engineer yeast, plant and mammalian cells. Eukaryotic genomes are regulated by the diverse biochemical and biophysical states of chromatin, which brings distinct challenges, as well as opportunities, over applications in bacteria. Recent synthetic approaches, including 'epigenome editing', have allowed the direct and functional dissection of many aspects of physiological chromatin regulation. These studies lay the foundation for biomedical and biotechnological engineering applications that could take advantage of the unique combinatorial and spatiotemporal layers of chromatin regulation to create synthetic systems of unprecedented sophistication.

Figure 1. Regulatory features of chromatin at multiple length scales

a | The amino termini of histone proteins have numerous amino acid residues that can be biochemically modified, such as by the addition of methyl (Me), acetyl (Ac), ubiquitin (Ub) and phosphate (P) groups. These modifications influence the binding of DNA and regulatory proteins. b | Genomic DNA, which itself can be methylated on cytosine residues, is wound around 4 pairs of histone proteins, which collectively comprise a nucleosome. c | The positioning of nucleosomes on DNA influences the accessibility of transcription factors to regions such as the promoter. Regulatory proteins (orange, blue, red and purple) bind to nucleosomes, DNA and transcribed non-coding RNA (ncRNA). Histone marks (red circles) often appear in large spatial domains; their occupancy as a function of genomic position (red histogram) can be quantified using chromatin immunoprecipitation followed by DNA sequencing (ChIP–seq)^,. d | Chromosomes exist in spatial territories in the nucleus. There are interactions within and between chromosomes, as well as between chromosomes and nuclear structures such as the nuclear pore, inner nuclear membrane and nuclear lamina.

Figure 2. Synthetic control of biochemical chromatin modifications

Synthetic approaches provide two types of specificity: specificity in the histone residues that are modified (part A) and specificity in the genomic locations of histone modifications (part B). Aa | Synthetic histones can be created by ligating chemically defined peptides (red) to the amino termini of partial histone proteins that are produced recombinantly (grey)^–. Ab | Artificial amino acids that are already biochemically modified can be incorporated into specific residues of histones that are recombinantly produced in bacteria^–. This approach requires expression of an aminoacyl tRNA synthetase and an acetyl-lysine transporter to import exogenously supplied acetyl-lysine. Ac | Genetic mutations can change specific residues into ones that partially mimic the charge and shape of a modified histone residue^–. Histones that are chemically synthesized or that contain artificial amino acids can be used in in vitro assays to measure changes in binding affinities to regulatory proteins or altered reactivity of specific histone residues due to the presence of modifications on other residues. Histones that were genetically mutated can be used in cellular assays to measure the effects of specific residues on global gene expression profiles. Ba | Chromatin-modifying proteins with catalytic properties such as acetyltransferase or methyltransferase activities can be fused to DNA-binding proteins^–,,,–. Bb–c | The fusion proteins bind to specific locations in the genome, altering gene expression of a downstream gene (part Bb) or genes that are targeted by a bound enhancer (part Bc). Bd | Chromatin modifiers can also be dynamically recruited to a genomic locus, allowing temporal measurements of changes in chromatin modifications^,,. TALE, transcription activator-like effector.

Figure 3. Synthetic spatial control of chromatin

A | Several different types of barrier elements (red) can block the spreading of repressive heterochromatin. These include recruitment of transcriptional activators, tethering to a nuclear pore complex, insertion of actively transcribed tRNA genes or insertion of nucleosome-disfavouring DNA sequences^,. B | The recruitment of a self-dimerizing protein can induce chromatin looping and localization of an enhancer or a locus control region (LCR) to a promoter. This could localize factors that phosphorylate (P) and activate RNA polymerase II (Pol II). C | DNA-binding proteins can be used to answer biological questions, for example, whether the spindle checkpoint senses displacement between sister chromatids or within kinetochores (part Ca). Fusing DNA-binding domains to nuclear envelope proteins can localize (and often silence) specific genomic regions (part Cb). Part Ca adapted from REF. 116.

Figure 4. Potential applications of synthetic chromatin biology

A | Eukaryotic organisms such as yeast are widely used in industry to produce diverse molecules that range from food flavourings to pharmaceuticals. Often, multiple biosynthetic genes need to be introduced into an organism's genome. Regulating many genes can be difficult, especially with many distinct promoters and other regulatory elements. Aa | In chromatin-based regulation, chromatin states such as heterochromatin can exert regulation over several kilobases of DNA through the simple recruitment of a repressor to a single location in the genome^,. Barrier elements can be used to prevent spreading of repressive regulation into endogenous genes^–. Ab | Repression of biosynthetic genes when cell densities are low promotes cell growth and fitness by avoiding taxing of cell resources. When cell densities in a bioreactor are optimal, the repressor can be degraded or diluted out, thus turning on expression of the biosynthetic genes. B | Chromatin has been widely implicated in many areas relevant to human health, including cell differentiation, cell proliferation, cell survival in different environmental conditions, epithelial-to-mesenchymal transition (EMT, which is relevant to cancer metastasis) and oncogenic potential^–. Functional tools are lacking to directly perturb chromatin states at specific locations in the genome and to test hypotheses of their roles in these biomedical processes. Type II CRISPR–Cas9 DNA targeting technologies^, could be used to edit chromatin states at multiple genomic locations by fusing chromatin regulators to the Cas9 protein and expressing a library of guide RNAs that are complementary to genomic target sites.