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. 2004 May 15;18(10):1119-30.
doi: 10.1101/gad.292104.

Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription

Séverine Chambeyron  1 , Wendy A Bickmore
Affiliations

Chromatin decondensation and nuclear reorganization of the HoxB locus upon induction of transcription

Séverine Chambeyron et al. Genes Dev. .

Abstract

The colinearity of genes in Hox clusters suggests a role for chromosome structure in gene regulation. We reveal programmed changes in chromatin structure and nuclear organization upon induction of Hoxb expression by retinoic acid. There is an early increase in the histone modifications that are marks of active chromatin at both the early expressed gene Hoxb1, and also at Hoxb9 that is not expressed until much later. There is also a visible decondensation of the chromatin between Hoxb1 and Hoxb9 at this early stage. However, a further change in higher-order chromatin structure, looping out of genes from the chromosome territory, occurs in synchrony with the execution of the gene expression program. We suggest that higher-order chromatin structure regulates the expression of the HoxB cluster at several levels. Locus-wide changes in chromatin structure (histone modification and chromatin decondensation) may establish a transcriptionally poised state but are not sufficient for the temporal program of gene expression. The choreographed looping out of decondensed chromatin from chromosome territories may then allow for activation of high levels of transcription from the sequence of genes along the cluster.

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Figures

Figure 1.
Figure 1.
Organization and differential activation of the Hoxb locus in ES cells. (A) Chromosomal organization of genes (open boxes) in the murine Hoxb complex. 5′ and 3′ indicate the direction of the transcription. The distance between genes is indicated below the map, as is the position of FISH probes (gray bars) for Hoxb1 and Hoxb9. The arrow indicates the colinear properties of the Hoxb cluster with respect to spatial expression (anterior to posterior), time of expression (early to late), and sensitivity to RA. Modified from Hunt and Krumlauf (1992). (B) Differential expression of Hoxb genes in OS25 ES cells. RT–PCR analysis of Hoxb1, 9, and 13 in undifferentiated (UN) cells and in cells induced to differentiate with RA for 2 to 12 d. Differentiation was confirmed by the loss of Oct4 expression. Total RNA from E11.5 embryo (E) and analysis of Gapdh were used as positive controls.
Figure 2.
Figure 2.
Histone H3 modifications upon induction of HoxB in ES cells. (A) Map of Hoxb and the elements analysed by ChIP. Exons of Hoxb1 and Hoxb9 are represented by two open bars. The arrows denote the transcription start sites of Hoxb1 and Hoxb9. The 3′ RARE (3′DR2; Marshall et al. 1994) and the r4 enhancer (Popperl et al. 1995) are indicated by the filled bars. The position of the PCR products detected by ChIP are indicated by black bars underneath. (B) Quantification of ChIP by real-time PCR. The graphs show the mean levels of product amplified from samples after ChIP with antibodies recognizing AcH3-K9, met2H3-K4, and met2H3-K9 (after subtraction of mock IP levels) normalized relative to those obtained by ChIP with antibody recognizing the C terminus of H3. The analysis was carried out on chromatin prepared from undifferentiated ES cells (open bars) and from cells induced to differentiate with RA for 4 d (hatched bars) and 10 d (filled bars).
Figure 3.
Figure 3.
Decondensation of Hoxb. (A) FISHof Hoxb1 (red) and Hoxb9 (green) to MAA-fixed nuclei from undifferentiated (UN) cells, from cells at 2 or 10 d of differentiation, and from undifferentiated cells treated with TSA. Nuclei were counterstained with DAPI (blue). Bar, 5μm. Below it, each image the mean-square interprobe distance—d2 (μm2) ± S.E.M.—is shown (n = 50 cells). The value for cells at 2 d, fixed with pFa, is also shown. (B) FISH of Pax6 (red) and Rcn (green) probes on cells before and after (2 d) differentiation.
Figure 4.
Figure 4.
Progressive looping of the HoxB cluster from 3′ to 5′. (A) FISH with MMU11 chromosome paint (green) and gene-specific probes (red) for Hoxb1 (top) or Hoxb9 (bottom) on MAA-fixed nuclei from undifferentiated (UN) cells, and from cells at 4 or 10 d of differentiation. Nuclei were counterstained with DAPI (blue). Bars, 5 μm. (B) Histograms showing the distribution of hybridization signals for Hoxb1 (open bars) or Hoxb9 (filled bars) relative to the MMU11 territory edge (μm). Negative distances indicate signals located beyond the visible limits of the detectable CT. n = 100 territories. (C) FISH with MMU11 chromosome paint (red) and gene-specific probes for Hoxb1 (green/red, 1:1) or Hoxb9 (green) on nuclei from cells at 10 d of differentiation. Nuclei were counter-stained with DAPI (blue). Bar, 5 μm. (D) Mean position (μm, ± S.E.M.) of Hoxb1 and Hoxb9 relative to the MMU11 territory edge during 12 d of differentiation. The position of Rcn in the MMU2 territory is shown as control (Mahy et al. 2002a). n = 100 territories. Normalization of the data to the size of the nucleus showed that the Hoxb movements are not due to the increased size of the nucleus and CT upon differentiation (data not shown).
Figure 5.
Figure 5.
Movement of the Hoxb1 toward the center of the nucleus. The localization of Hoxb1 and Hoxb9 hybridization signals within erosion shells from the edge (shells 1 and 2) toward the center (shells 4 and 5) of the nucleus. Analysis was performed on MAA-fixed nuclei from undifferentiated ES cells (open bars) and from cells after 4 and 10 d of differentiation (hatched and filled bars, respectively). n = 50 cells.
Figure 6.
Figure 6.
Changes in chromatin structure at HoxB. In undifferentiated ES cells, the entire Hoxb locus (black) is condensed with the MMU11 CT (gray). However, the 3′ Hoxb1 gene is at the territory surface, poised to respond to retinoic acid (RA). The more 5′ Hoxb9 is further inside the territory. After 2 d of induction with RA, the Hoxb chromatin fiber decondenses, and the 3′ end of the locus, including Hoxb1, is extruded from the CT. By 10 d of differentiation, Hoxb1 is reeled in toward the CT, and Hoxb9 has now left the CT.
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