Micro- and nanoscale devices for the investigation of epigenetics and chromatin dynamics

Abstract

Deoxyribonucleic acid (DNA) is the blueprint on which life is based and transmitted, but the way in which chromatin - a dynamic complex of nucleic acids and proteins - is packaged and behaves in the cellular nucleus has only begun to be investigated. Epigenetic modifications sit 'on top of' the genome and affect how DNA is compacted into chromatin and transcribed into ribonucleic acid (RNA). The packaging and modifications around the genome have been shown to exert significant influence on cellular behaviour and, in turn, human development and disease. However, conventional techniques for studying epigenetic or conformational modifications of chromosomes have inherent limitations and, therefore, new methods based on micro- and nanoscale devices have been sought. Here, we review the development of these devices and explore their use in the study of DNA modifications, chromatin modifications and higher-order chromatin structures.

Figure 1

Overview of epigenetic layers and corresponding size scales. The root layer is the DNA sequence and covalent modifications such as cytosine methylation (5mC) and hydroxymethylcytosine (5hmC). The DNA is then wrapped around octameric histone proteins into nucleosomes and into chromatin. The nucleosomal histones H2A, H2B, H3 and H4 form pairs with one H3-H4 tetramer and two H2A-H2B dimers and can be exchanged with variants or chemically modified on their protruding tails such as histone 3 - lysine 27 - trimethylation (me3): H3K27me3. The structure of the chromatin is mediated by the nucleosome packing with open / euchromatin having less nucleosomes positioned than closed / heterochromatin. The condensed heterochromatin has been shown to possess a unique solenoid structure and higher-order loops and folds also exist to further compact the chromatin into chromosomes. The various layers and modifications establish whether the gene and the regulatory components (promoter, enhancer) are accessible and transcribed or inactive. DNA cytosine methylation and histone modifications such as H3K27me3 are broadly associated with inactive genes as to where hydroxymethylated cytosine bases and histone modifications such as H3K4me3 are nominally associated with active genes and regulatory elements.

Figure 2

Micro and nano methods for mapping DNA covalent modifications. a) Detection of DNA covalent modifications using single molecule sequencing in a nanophotonic structure. The right panels demonstrate detection of a methylated adenine base compared to a typical adenine. b) The top panel shows the capillary assembly protocol for optical mapping of large-scale arrays of single molecules. The bottom panel shows fluorescently tagged methylation sites (green) on elongated single DNA molecules (red) using capillary assembly. c) Optical mapping fluorescently-tagged methylation sites (red) on single DNA molecules (green) moving through a nanochannel. The inset shows detection of a methylated region along a single molecule. d) Electrical differentiation of DNA with labeled cytosine modifications using a solid-state nanopore (MBDs – methyl binding domains). The bottom panel shows current blockades and translocation times for unmodified DNA molecules (black circles) compared to DNA labeled with varied amounts of cytosine methylation-specific labels (red circles). e) Electrical detection of DNA base modifications using a protein nanopore (α-hemolysin) where the residual current through the nanopore (bottom panel) is unique for each modification.

Figure 3

Micro and nano methods for mapping histone modifications and nucleosome arrays. a) Microfluidic device to perform automated chromatin immunoprecipitation (ChIP) reactions with higher efficiencies than conventional protocols (bottom panel). (b) Optical mapping of fluorescently tagged histone modifications on single unfolded chromatin molecules in nanochannels. c) DNA curtains assay where single DNA molecules are anchored at one end and stretched by fluid flow to image DNA (green) and position quantum dot labeled nucleosomes (magenta). (d) Histone oligomers and nucleosomes moving through a solid-state nanopore block different amounts of current (ΔG corresponds to conductance change) and translocate through the nanopore with different times (Δτ).

Figure 4

Micro and nano methods for understanding chromatin dynamics and chromosome organization. a) Optical tweezers experiment on a single nucleosome and force extension curves of the nucleosome in different salt concentrations. The lower panel shows the nucleosome flipping between a wrapped and unwrapped state. b) Magnetic tweezers setup (left panel), where a single heterochromatin fibre can be pulled apart into different conformations (right panel). The different points on the force-extension curve show as the pulling force is increased, the fibre begins to unravel in a manner similar to a Hookian spring (solenoid shape), which keeps the DNA both condensed and accessible.

Figure 5

Possible hybrid micro and nanodevice architecture to perform multiplexed epigenomic measurements from a single cell. In the first stage of the device (blue channels), specific cells of interest are microfluidically isolated from a population and a single cell is trapped in a small-volume chamber. Next, in-tact chromosomes are extracted from the cell^,, and partitioned (green channels) into chambers where each is profiled for multiple epigenetic modifications using one or several of the micro/nano-based techniques (orange channels). Once profiled, specific molecules of interest can then be sorted^, and recovered (white channels) for amplification or sequencing. The integrated architecture can also include a stimulus inlet (purple channel) to introduce factors such as chromatin remodelers to track how different molecules interact with and modulate the chromosome architecture.