Large-scale chromatin organization: the good, the surprising, and the still perplexing

Abstract

Traditionally large-scale chromatin structure has been studied by microscopic approaches, providing direct spatial information but limited sequence context. In contrast, newer 3C (chromosome capture conformation) methods provide rich sequence context but uncertain spatial context. Recent demonstration of large, topologically linked DNA domains, hundreds to thousands of kb in size, may now link 3C data to actual chromosome physical structures, as visualized directly by microscopic methods. Yet, new data suggesting that 3C may measure cytological rather than molecular proximity prompts a renewed focus on understanding the origin of 3C interactions and dissecting the biological significance of long-range genomic interactions.

Fig. 1. Large-scale chromatin domains in search of genomic context- genomic interactions in search of structural context.

(A-C) Three staining methods demonstrate large-scale chromatin domains in thin-section TEM of freeze-substitution (A) or conventional (B-C) glutaraldehyde fixed samples: (A) DNA post-embedding immunostaining (dark, positive staining) of TEM thin section reveals most nuclear DNA is present in large-scale, frequently fiber-like structures. Scale bar = 1 μm. Reprinted from Fig. 4a, reference [7], Copyright 1994 J Histochem Cytochem. (B) DNA post-embedding staining by osmium ammine of rat liver nucleus shows no "euchromatin" texture. Large regions of nucleus are DNA free. Staining sensitivity is shown by visualization of decondensed chromatin within nucleolus (arrow). Reprinted from Fig. 1, reference [9], Copyright 1989 J Histochem Cytochem. (C) Uranyl and lead staining of detergent permeabilized Hela nucleus in buffer preserving large-scale chromatin structure. Staining is white in negative image. Extraction of nucleoplasmic protein results in absence of "euchromatin" texture after fixation and reveals large-scale chromatin structures. (D-G) Thin section, serial sections of CHO nuclei reveal (D-E) fiber-like, large-scale structures (small arrows) consisting of ~Mbp size segments punctuated by less condensed chromatin (large arrows). (F-G) Abrupt transitions from thick (large arrows) (~100-130 nm) to thinner (~60-80 nm) diameter size are seen, with local discontinuities by decondensed chromatin (thin arrows). Reprinted from Fig. 6 (D-E) and Fig. 7 (F-G), reference [16] , Copyright JCB 1994. Scale bars = 1 μm (A,B) and 500 nm (C), 120 nm (F-G). (H) Hi-C scatterplot showing long-distance DNA interactions in Drosophila embryonic nuclei over region of chromosome 3R. Off-diagonal, box-like, yellow regions define topological domains within which 3C interactions are favored over neighboring regions. X-Y axes show regions of 3R (bp) with associated chromatin average states. Reprinted from Fig. 3F, reference [37], Copyright 2012, Cell. (I) Cartoon illustrating that topological domains detected by 3C methods correspond to local regions of compact, but irregular chromatin folding separated by extended chromatin. (J) Boundary between topological domains (grey bar) is defined by asymmetry in DNA interactions up versus downstream of boundary region, with DNA within a topological domain interacting preferentially. (I-J) reprinted from Fig. 1b, reference [38], Copyright 2012 Nature.

Fig. 2. An incomplete list of important biological features of 3C defined topological domains.

(A) Conservation of topological domains- comparison of mouse versus human domain organization over syntenic region. Reprinted from Fig. 3g-h, reference [38], Copyright 2012 Nature. (B) Preservation of topological domain organization during differentiation- comparison of domain organization in undifferentiated ES cells versus neuronal progenitor cells. Topological domains are fixed, while lamin associated domains (LADs) change organization. However, topological boundaries appear to mark boundaries of LADs and other epigenetic domains. (C) In topological domains identified in one 5C study, genes within topological domains show higher correlation of gene expression than genes lying in different domains. Reprinted from Fig. 3 (B) and Fig. 4 (C) from reference [39], Copyright 2012 Nature. (D) Topological boundaries are enriched in marks for active chromatin and transcription. Reprinted from Fig. 4a, reference [38], Copyright 2012 Nature.

Fig. 3. 3C Interactions predominately derive from insoluble fraction corresponding to swollen nuclei.

(A) Comparison of 3C interactions between beta-globin promoter region and flanking regulatory sequences in beta-globin locus from soluble (dotted lines) versus insoluble (solid lines) fractions. Data is normalized for DNA amount in each fraction. Red lines correspond to fetal liver where gene is active, while blue lines correspond to brain where gene is inactive. 3C peaks from known long-distance interactions in beta-globin locus derive from insoluble fraction containing swollen nuclei. (B) Nuclear structure immediately after fixation (top) versus just prior to 3C ligation step (previously assumed to act on soluble, small, DNA-protein molecular complexes). Nuclei enlarge with SDS and other treatments prior to ligation step, chromosome territories swell, but nuclear bodies such as nucleoli, SC35 domains, and patches of epigenetically marked chromatin domains are still recognizable. DNA (DAPI) staining blue, immunofluorescence or FISH signals red. (C,D) TEM images of nuclei after fixation (C) or after fixation but just prior to ligation step of 3C procedure (D)- 3C procedures result in obvious disruption of condensed chromatin masses in fetal liver nuclei. Scale bars = 5000 nm (B), 1000 nm in (C-D). Reprinted from Fig. 2C (A), Fig. 3 (B), and Fig. 4 (C-D) from reference [50], Copyright 2013 Nucleic Acids Research.

Fig. 4. Do long-range cis and trans 3C interactions imply molecular versus cytological proximity?

(A-C) Conventional models interpret 3C interactions as demonstrating molecular proximity. Top: After fixation (A), chromosomes (dark blue) within nuclei (light green) are digested and solubilized by restriction enzyme digestion (B), followed by dilution of soluble fraction prior to ligation to favor intramolecular ligation events (C). Bottom: DNA strands from distant chromosome sites (blue or red) are brought into proximity through specific binding of trans factors (green and purple) binding to different cis elements; this proximity is fixed by formaldehyde cross-linking of trans and cis factors (A). Restriction digest cuts restriction sites (black lines) (B) which then ligate DNA fragments together from different genomic regions (C). (D-F) Alternative model suggests 3C interactions correspond instead to proximity over cytological rather than distances. Top: Interphase chromosome structures (blue) after fixation (D) are disrupted progressively during 3C procedure. Restriction digest (E) results in fragments that remain cross-linked to insoluble, cross-linked nuclear structures and ligation (F) occurs within these partially disrupted chromosome residual structures. Bottom: Chromosome regions (red and blue) adjacent to nuclear bodies (brown) are densely cross-linked by formaldehyde fixation (D). SDS treatment and heat prior to restriction digest denatures histone and nonhistone proteins (purple) that act as linkers creating a protein-DNA network anchoring restriction fragments after restriction digest (E). Ligation events (black lines) connect fragments over large distances within this disordered network (F). In this model, long-range 3C interactions might derive for instance from co-association of two gene loci to the same nuclear body, even if they are separated in the live cell by cytological (100-1000 nm) distances.