Decoding the role of chromatin architecture in development: coming closer to the end of the tunnel

Abstract

Form and function in biology are intimately related aspects that are often difficult to untangle. While the structural aspects of chromatin organization were apparent from early cytological observations long before the molecular details of chromatin functions were deciphered, the extent to which genome architecture may impact its output remains unclear. A major roadblock to resolve this issue is the divergent scales, both temporal and spatial, of the experimental approaches for examining these facets of chromatin biology. Recent advances in high-throughput sequencing and informatics to model and monitor genome-wide chromatin contact sites provide the much-needed platform to close this gap. This mini-review will focus on discussing recent efforts applying new technologies to elucidate the roles of genome architecture in coordinating global gene expression output. Our discussion will emphasize the potential roles of differential genome 3-D structure as a driver for cell fate specification of multicellular organisms. An integrated approach that combines multiple new methodologies may finally have the necessary temporal and spatial resolution to provide clarity on the roles of chromatin architecture during development.

Keywords: INTACT method; cell fate specification; chromatin; chromatin beacons; chromatin conformation capture.

FIGURE 1

Chromatin organization and topological domains. Chromosomes occupy distinct territories in the nucleus (shaded with different colors). Long-range chromatin looping, at the sub-megabase level, partitions the chromosomal region into Topologically Associating Domains (TADs; individuals illustrated on the rights. The TADs remain largely unchanged in differentiated cells and stem cells. However, at a finer levH pluripotency factors and chromatin architectural proteins organize higher-order chromatin connectivity during reprogramming. Pluripotency factors co-localize and occupy distinct spatial regions from PcG proteins in stem cells. Such chromatin reorganization induced by pluripotency factors is important for cell-specific gene expression.

FIGURE 2

Nuclear envelope tagging of specific leaf cell types in Arabidopsis. Transgenic plants were created with Inserts that express an NTF marker gene driven by the GL2 (A), CaMV35S (B), or AtMYB60 (C) promoters for trichome, constitutive, or guard cell-specific expression. The NTF marker is a nuclear envelope-anchored GFP fused to a biotinylation epitope from bacteria. For the GL2 promoter driven construct, a separate vector is used to produce the bacterial BirA gene in trans to catalyze the biotinylation of the NTF in order to facilitate rapid nuclei purification (Deal and Henikoff, 2010). To create a more facile labeling system, we have generated a new INTACT vector that contains both the NTF expression cassette as well as the BirA gene for plant expression. The CaMV 35S promoter and a guard cell-specific promoter from the AtMYB60 gene are used to create the constructs shown in panels (B) and (C), respectively. Mature rosette leaves from stable transgenic lines are examined by a stereo epifluorescence microscope fitted with a GFP filter set for images shown. Inset on panel (C) shows a confocal image of a tagged guard cell from a single focal plane to Illustrate the nuclear envelope localization of the expressed NTF protein from the new vector driven by the AtMYB60 promoter.