Chromatin insulators: linking genome organization to cellular function

Abstract

A growing body of evidence suggests that insulators have a primary role in orchestrating the topological arrangement of higher-order chromatin architecture. Insulator-mediated long-range interactions can influence the epigenetic status of the genome and, in certain contexts, may have important effects on gene expression. Here we discuss higher-order chromatin organization as a unifying mechanism for diverse insulator actions across the genome.

Figure 1. Experimental paradigms for enhancer blocking and barrier insulation

(A) To test barrier insulator activity, a transgene flanked by insulators is randomly integrated into the genome. Multiple integration sites are considered to control for position-effect variegation. If sequences are true barrier insulators, reporter expression over time in culture should remain constant, whereas a control transgene that does not contain insulators should eventually be silenced by encroachment of heterochromatin. (B) To test enhancer blocking insulator activity, transgene constructs are designed by placing a putative insulator sequence in various positions with respect to an enhancer driving a reporter gene. The degree of ‘insulation’ (or ability to abrogate the enhancer) is assayed as the level of reporter gene expression after transient transfection, or integration of the vector, into target cells. To rule out effects of position-independent silencing, results are compared to control constructs in which insulators are placed adjacent to, but not in between, linked enhancer-promoter sequences. Limitations of these assays to be considered during data interpretation, include: the spacing between elements which does not mimic the endogenous locus, the integration of the reporter into multiple ectopic genomic locations, and the often use of heterologous enhancer-promoter sequences that also do not represent the genomic context of insulator sequences.

Figure 2. Higher-order genome organization as a unifying mechanism for insulator function

(A) Model for CTCF in mediating TAD boundaries and intra-TAD genomic organization via long-range interactions in wild type cells. (B) Blurring of TAD boundaries after deletion of a 50–80 kb boundary between TADs that contains CTCF and active genes (adapted from (Nora et al., 2012)). In principle, the TAD boundary would also be responsible for demarcating the border of the repressive H3K27me3 mark. Thus, we hypothesize that deletion of the boundary would lead to aberrant heterochromatin spreading. TADs are defined by the genome-wide 3-D mapping technology Hi-C. Counts are directly proportional to the frequency in which genomic fragments interact, with deep red pixels depicting high frequency interactions and light pink pixels depicting low frequency interactions. Point-to-point looping interactions at kb resolution can be mapped by Chromosome-conformation-capture-carbon-copy (5C) and are depicted by black bars connecting two genomic segments. CTCF ChIP-seq track in purple. Active enhancer depicted as a blue ball. Active genes depicted as green arrows. ChIP-seq track for H3K27me3 repressive chromain mark in orange.

Figure 3. Role of CTCF in higher-order chromatin architecture at the mouse HoxD locus

(A) 2-D organization of the HoxD locus. The HoxD gene cluster is a developmentally regulated locus that must be partitioned into discrete regulatory landscapes. 3′ Hox genes (HoxD9-HoxD1) are activated during early limb bud development via enhancers in a gene desert region on the 3′ side of the cluster toward the telomeres. 5′ Hox genes (HoxD13-HoxD10), as well as adjacent Lnp and Evx2 genes, are activated later in development during patterning of digits, and this wave of transcription is controlled by different enhancers in a gene desert region on the 5′ side of the cluster toward the centromeres. Centromeric enhancers have been well characterized: there is a distal GCR (global control region) 180 kb upstream of HoxD13 that contains multiple enhancers, as well as a proximal enhancer 50 kb upstream from HoxD13. (B) 3-D organization of the HoxD locus. Topological domains identified with HiC analyses by Dixon et al. in ES cells (top) and mouse cortex (bottom) with counts ranging from low (white) to high (deep red). Genome-browser tracks from Dixon et al. are also displayed for CTCF, mark for active genes H3K4me3, and enhancer marks H3K4me1 and p300 in ES cells (Dixon et al., 2012).

Figure 4. Role for CTCF in higher-order chromatin architecture at the human Pcdh locus

Pcdh genes encode a large number of calcium-dependent cell-cell adhesion molecules important in establishing neural diversity. (A) 2-D organization of the human Pcdh locus. Two enhancer elements downstream of the 13 alternative isoforms (1–13) and 2 c-type ubiquitous isoforms (c1, c2) have been identified as necessary for appropriate tissue-specific expression: HS7 in the intron between constant exons 2 and 3 (light green) and HS5-1 downstream of constant exon 3 (dark green) (Ribich et al., 2006). In the diploid human neuroblastoma cell line SK-N-SH, CTCF and cohesin occupied sites have been mapped with ChIP-seq (adapted from (Guo et al., 2012)). (B) Model for the role of enhancer-promoter looping interactions in variable exon expression in the diploid human neuroblastoma cell line SK-H-SH (adapted from (Guo et al., 2012)). (C) Model for neural diversity created by alternative Pcdh isoform expression through looping interactions between alternative promoters and the downstream HS5-1 enhancer.