Regeneration, repair and remembering identity: the three Rs of Hox gene expression

Abstract

Hox genes encode transcription factors that specify embryonic positional identity in cells and guide tissue differentiation. Recent advances have greatly increased our understanding of the epigenetic mechanisms that ensure the faithful expression of Hox genes in adult cells and which involve the interplay of histone methylation, demethylation and intergenic transcription of long non-coding RNAs. The transcriptional memory of Hox genes poses both an opportunity and a challenge for regenerative medicine. Matching the positional identity of transplanted stem cells with that of the host environment, as reflected by their respective Hox profiles, is likely to be required to achieve regenerative healing. Strategies to manipulate the plasticity of Hox gene expression will probably become a major focus in regenerative medicine.

Figure 1

Hox genes and positional identity. (a) Hox genes belong in the family of homeodomain-containing transcription factors. The domain organization of HOXA5 and the crystal structure of the homologous fly Scr Hox protein (brown) in complex with Exd (light blue), binding to DNA (green) are shown. Exd is a Hox cofactor that acts as an accessory DNA-binding factor. Homeodomains are helix-turn-helix motifs (represented by α1, α2 and α3) that bind specific DNA sequences. Sequences N-terminal of the homeodomain, such as the YPWM motif, are involved in protein–protein interaction with accessory DNA-binding factors such as Exd. Part (a) adapted, with permission, from Ref. [44]. (b) Expression of three HoxD genes in developing mouse embryos by in situ hybridization. The schematic above the in situ pictures represents the HoxD locus – the individual numbers refer to different HoxD genes. The white arrows indicate that HoxD-13, -11 and -9 are shown in the three panels, from left to right. The orange and blue dotted lines represent the neural tube and paraxial mesoderm, respectively – the lines are there to highlight the tissue-specific mechanisms for colinear gene regulation: Hox genes tend to have a very distinctive organization in the genome, being arranged in gene clusters in which the order of the genes within the cluster corresponds to (or is ‘colinear with’) some aspect of the gene expression. In addition to spatial colinearity, in vertebrates there is also a temporal colinearity, where the 3′–5′ arrangement of genes also reflects the temporal order in which they are activated during development. The expression domains of Hox genes towards the 3′ UTR seem closer to the head and closer to the trunk (dotted lines), whereas the Hox genes towards the 5′ UTR are expressed closer to the tail and closer to fingers or toes on the limbs (pins). For example, the domains of expression of HoxD13 in both the neural tube (marker by the orange line) and the paraxial mesoderm (the blue line) are quite similar, whereas in HoxD11 and to a greater extent HoxD9, one can really appreciate the variation in the transcript domains of the same gene in different tissues. The pins represent nested domains of expression, from the highest level (black) to lowest (white), again illustrating the colinearity principle. Part (b) reproduced, with permission, from Ref. [45]. (c) HOX genes as the address code of the human body. As illustrated by the decision tree, differential expression of HOX genes reflects the anatomic origin of adult human fibroblasts along the anterior (A)–posterior (P), dorso (D)–ventral (V), and proximal (Px)–distal (Ds) axes, in addition to their origin from cutaneous versus internal organs. Red indicates high expression; green indicates low expression.

Figure 2

Epigenetic landscape of Hox genes. (a) Chromatin state map across~100 kilobases of the human HOXA locus (X axis) obtained from chromatin IP on microarray (ChIP-chip) experiments, a technique for isolation and identification of the DNA sequences occupied by specific DNA-binding proteins in cells. Y axes indicate the occupancy of the indicated proteins (represented by the log₂ ratio of ChIP over input) or expression of RNAs (represented by a linear scale of hybridization intensity). Suz12 is a component of the PRC2 that mediates histone H3 Lys27 methylation (H3K27me3). RNA polymerase (Pol) II broadly occupies the regions that are also transcribed and occupied by H3K4me3 (not shown). Note that fibroblasts from two different anatomic origins (lung and foot) can program the HOXA locus in diametrically opposite ways but along the same boundary. The HOXA locus is depicted at the bottom of the map, with HoxA13 at the distal 5′ end and HoxA1 at the promixal 3′ end. The dashed line highlights the boundary of opposite configurations of chromatin modification and intergenic transcription. (b) Model of chromatin state regulation by lncRNAs. lncRNAs can affect chromatin state in cis or in trans, and in gene activation or silencing, via histone-modification enzymes. (i) lncRNAs might increase the accessibility of Trithorax group proteins such as ASH1 (absent, small, or homeotic discs 1) or MLL or directly recruit them, leading to trimethylation of histone H3K4 and transcriptional activation of downstream targets such as the Hox genes. (ii) By contrast, lncRNAs might also target the PRC2 proteins to trimethylate H3K27 at a distance and render the target genes transcriptionally silent.

Figure 3

Hox status and bone regeneration in mouse. Cells expressing Hox genes (for example Hoxa11) are in light blue and those not expressing Hox are indicated in light grey. Hox⁺ skeletal stem cells can only heal orthotopic Hox⁺ bone injury site (tibia). (a) Hox⁺ skeletal stem cells cannot repair a Hox⁻ injury site (mandible). (b) Conversely, Hox⁻ skeletal stem cells will express the ectopic Hox gene when transplanted into a Hox⁺ injury site and regenerate the ectopic bone.