Saltatory remodeling of Hox chromatin in response to rostrocaudal patterning signals

Abstract

Hox genes controlling motor neuron subtype identity are expressed in rostrocaudal patterns that are spatially and temporally collinear with their chromosomal organization. Here we demonstrate that Hox chromatin is subdivided into discrete domains that are controlled by rostrocaudal patterning signals that trigger rapid, domain-wide clearance of repressive histone H3 Lys27 trimethylation (H3K27me3) polycomb modifications. Treatment of differentiating mouse neural progenitors with retinoic acid leads to activation and binding of retinoic acid receptors (RARs) to the Hox1-Hox5 chromatin domains, which is followed by a rapid domain-wide removal of H3K27me3 and acquisition of cervical spinal identity. Wnt and fibroblast growth factor (FGF) signals induce expression of the Cdx2 transcription factor that binds and clears H3K27me3 from the Hox1-Hox9 chromatin domains, leading to specification of brachial or thoracic spinal identity. We propose that rapid clearance of repressive modifications in response to transient patterning signals encodes global rostrocaudal neural identity and that maintenance of these chromatin domains ensures the transmission of positional identity to postmitotic motor neurons later in development.

Figure 1. Domain-wide clearance of repressive histone modifications and changes in Hox gene expression in response to RA

A) Expression profiling of ESCs and day 7 RA/Hh treated embryoid bodies differentiated into motor neurons. RA treatment induces rostral Hox1-5 gene expression (yellow). The color indicates log₂ fold change in signal intensity between ESCs and Day 7 differentiated cells. Genes in gray are not represented on GeneChip arrays. B) HoxA chromatin is parceled during motor neuron differentiation into two domains, distinguished by different densities of H3K4me3 and H3K27me3 histone modifications. The red tracks represent H3K27me3 enrichment over 150 kb genomic region spanning HoxA cluster at Day 0 (ESCs) and Day 4 (progenitors). Blue peaks represent RAR binding 8 hours after RA/Hh treatment on Day 2. The color of the genes indicates log₂ fold change in signal intensity between ESCs and Day 7. C) Rapid and synchronous removal of H3K27me3 modifications from Hoxa1-a5 domain. The heat map represents enrichment of H3K27me3 and H3K79me2 histone modifications along HoxA cluster at five time points during motor neuron differentiation. D) Temporal changes in H3K79me2 and H3K27me3 modifications over Hoxa1 and Hoxa5 genes. The values reflect average enrichment of modifications over 1kb genomic regions flanking transcription start sites. Note the delayed accumulation of H3K79me2 modifications in Hoxa5 gene (marking actively transcribed chromatin), compared to the rapid clearance of repressive H3K27me3 modifications over the same chromatin territory. (n=2, ± SD).

Figure 2. Patterns of Hox gene expression in Suz12^βgal/βgal cells differentiated to motor neurons

A) Decreased density of H3K27me3 histone modification over 150 kb region spanning HoxA cluster in Suz12^βgal/βgal ESCs. Positive relative enrichment in H3K27me3 levels in Suz12^βgal/βgal over control cells are colored in red and negative relative enrichment in green. B) A fraction of Suz12^βgal/βgal motor neurons express Hoxc6 and Foxp1 proteins, markers of brachial spinal motor neurons. Although differentiation of Suz12^βgal/βgal cells is compromised, cells that acquired motor neuron identity (Hb9⁺) are detected at Day 7 of differentiation. Some of Suz12^βgal/βgal motor neurons acquired expression of a brachial marker Hoxc6 and a marker of brachial limb innervating motor neurons Foxp1. Scale bar = 50μm C) Upregulation of brachial Hox genes in Suz12^βgal/βgal motor neurons. Quantitative PCR analysis of Hoxa5, Hoxa7, Hoxc6 and Hoxa10 mRNA levels on Day 7. The Hox mRNA levels in Suz12 mutant cells are normalized to control cells. (n=3, p<0.05). D) Suz12^βgal/βgal motor neurons acquire limb innervating identity. Deregulation of Hox gene expression in Suz12^βgal/βgal cells results in a significant increase in the number of Hb9+ motor neurons expressing Foxp1, downregulation of Lhx3 and an increase of Isl1+ motor neurons expressing Raldh2. (Mean ± sem. n=3, p<0.05). E) Quantitative RT-PCR analysis of dynamic changes in Hoxa1, Hoxa5, Hoxa7, Hoxb1, Hoxb4 and Hoxb8 transcripts relative to their maximal levels. Analysis of RNA samples collected at five time points during differentiation of Suz12^βgal/βgal cells revealed a collinear expression profile of Hox genes.

Figure 3. Wnt3A and FGF2 induce Cdx2 and caudalize differentiating motor neurons

A) Wnt3A and FGF2 signaling induce caudal Hox gene expression and LMC character. motor neurons from dissociated cell cultures derived from Hh/RA/Wnt/FGF differentiation are labeled by the Hb9::GFP transgene (gray) and stained with antibodies against Hoxa5, Hoxc6 or Foxp1 (red) and Hoxc8, Hoxc9 and Lhx3 (green). Scale bar = 25μm B) Quantification of Hox gene expression in motor neurons (% of Hb9::GFP⁺ cells). C) Quantification of the columnar markers Foxp1 and Lhx3 in motor neurons (% of Hb9::GFP⁺ cells). (Mean ± sem. n=3). D) Hh/RA/Wnt/FGF caudalization protocol induces Cdx2. Cdx2 mRNA levels normalized to control cultures treated only with RA/Hh 24 hours after the addition of patterning signals (Day 3 of differentiation). E) Cdx2 protein expression in differentiating cells 24 (Day 3) and 48 hours (Day 4) after Hh/RA/Wnt/FGF treatment. Scale bar = 20μm (Day 3), 50μm (Day 4)

Figure 4. Cdx2 induces caudal Hox gene expression during motor neuron differentiation

A) Diagram of an inducible V5 tagged Cdx2 ESC line (iCdx2). Control and induced iCdx2 ESCs stained with anti Cdx2 (red) and Oct4 (green) antibodies demonstrate that tagged Cdx2 is able to repress Oct4 expression. TetOP: Tetracycline Operator. V5: V5 epitope tag. pA: Polyadenylation signal. Scale bar = 20μm B) The expression of Cdx2 in combination with FGF2 treatment induces caudal Hox gene expression during motor neuron differentiation. Day 7 cultures from the Hh/RA (RA Hh), Hh/RA/Dox/FGF (Cdx2 FGF) and Hh/RA/FGF (FGF) were stained with anti Hoxc4, Hoxa5, Hoxc6, Hoxc8 or Hoxc9 (red) and Hb9 (green). Scale bar = 20μm C) Quantification of B). D) Expression of Hox genes at Day 5 of differentiation during Hh/RA/Dox/FGF motor neuron differentiation. The color of the boxes indicates log₂ fold change in signal intensity between ES cells and Day 5. Gray boxes indicate genes that are not represented on the GeneChip arrays.

Figure 5. Cdx2 directly controls Hox gene expression and chromatin modifications

A) Cdx2 protein associates with Hox regulatory regions. The track represents the read count of Cdx2 ChIP-seq experiment over the 35 kb region of HoxC cluster. Blue peaks are significantly enriched over control (p<0.001) suggesting Cdx2 binding. Blue arrows under the ChIP-seq track represent locations of matches to the Cdx2 motif in B). B) The primary motif enriched under Cdx2 ChIP-seq peaks. C) HoxA chromatin boundary is established between Hoxa9 and Hoxa10 after Cdx2 induction. The red tracks represent H3K27me3 enrichment over 150 kb genomic region including the HoxA cluster at Day 0 (ESC stage) and Day 5. The center track represents Cdx2 binding on Day4. Blue peaks denote Cdx2 ChIP-seq signal significantly enriched above background (p<0.001). The color of the genes indicates log₂ fold change in signal intensity between ES cells and Day 5.

Figure 6. The sequential activity of RAR and Cdx2 establishes two distinct chromatin states

A) Relative changes in K27me3 chromatin modifications in HoxA cluster (150 kb) during cervical and brachial/thoracic motor neuron differentiation. The tracks represent the H3K27me3 difference between Day 0 and Day 4 (Hh/RA) or Day 5 (Hh/RA/Cdx2/FGF). Positive relative enrichment levels are colored in red and negative relative enrichment in green. The color of the genes indicates log₂ fold change in mRNA levels between the caudal (Hh/RA/Cdx2/FGF) and rostral (Hh/RA) protocols. Gray boxes indicate genes that are not represented on the GeneChip arrays. Note the repression (blue color) of rostral Hox genes concomitant with the upregulation of Hoxa9 (yellow color). B) RAR and Cdx2 associate with local and putative distal regulatory regions. The tracks represent ChIP-seq enrichment levels for RAR and Cdx2, top and bottom respectively over 500 kb region spanning HoxB cluster and distal binding sites within Scap1 locus. Blue peaks are significantly enriched over control (p<0.001).