Hox in motion: tracking HoxA cluster conformation during differentiation

Abstract

Three-dimensional genome organization is an important higher order transcription regulation mechanism that can be studied with the chromosome conformation capture techniques. Here, we combined chromatin organization analysis by chromosome conformation capture-carbon copy, computational modeling and epigenomics to achieve the first integrated view, through time, of a connection between chromatin state and its architecture. We used this approach to examine the chromatin dynamics of the HoxA cluster in a human myeloid leukemia cell line at various stages of differentiation. We found that cellular differentiation involves a transient activation of the 5'-end HoxA genes coinciding with a loss of contacts throughout the cluster, and by specific silencing at the 3'-end with H3K27 methylation. The 3D modeling of the data revealed an extensive reorganization of the cluster between the two previously reported topologically associated domains in differentiated cells. Our results support a model whereby silencing by polycomb group proteins and reconfiguration of CTCF interactions at a topologically associated domain boundary participate in changing the HoxA cluster topology, which compartmentalizes the genes following differentiation.

Figure 1.

The 5′-end HoxA gene expression fluctuates and chromatin conformation varies during THP-1 differentiation. (A) Linear schematic representation of the human HoxA cluster region is characterized in this study. Genes are shown as left-facing arrows to illustrate transcription direction and to highlight the 3′–5′-end orientation of the cluster. Paralog groups are color-coded and identified above each gene. The color code shown here was used throughout the study. The predicted BglII restriction fragments of the HoxA region characterized in this study are shown below and identified by numbers from left to right. (B) THP-1 differentiation time points examined in this study. (C) The 5′-end HoxA gene expression fluctuates throughout the cellular differentiation time course. The expression of HoxA genes was measured by RT-qPCR and normalized relative to actin. The number below each histogram bar identifies the paralog group. The expression of macrophage-specific ApoE and CD14 markers was measured to monitor differentiation. For comparison, the ApoE and CD14 expression levels were divided by 4 and 20, respectively. Each histogram value is the average of at least three PCRs, and error bars represent the standard deviation. (D) Chromatin looping changes with gene expression at the HoxA 5′-end. Interactions between HoxA9 and the region containing other transcriptionally regulated 5′-end genes were measured by conventional 3C. The genomic region shown above each graph is to scale. The ‘fixed’ 3C region is highlighted in orange and the position of known looping contacts is indicated with green vertical lines. Fragments probed were 71 (fixed), 72, 73, 74, 76, 79 and 80. The y-axis shows normalized IFs and the x-axis indicates the end-point distance from the fixed 3C region. Each contact was measured at least three times in cellular and control libraries, and error bars represent the standard error of the mean.

Figure 2.

Higher 5′-end HoxA expression correlates with less chromatin contacts that differ on macrophage differentiation. (A) Pairwise IFs at the HoxA cluster vary throughout the differentiation time course. Heatmap representation of chromatin contacts measured with the 5C technology (8 kb bins, 8 kb smoothing). A linear diagram of the HoxA region characterized is shown to scale below each heatmap and is according to Figure 1A. DNA contacts are color-coded based on frequency, from low (white) to high (red) as indicated by the color scale below each row. Interaction frequencies were derived from at least three measurements as described in the ‘Materials and Methods’ section. Heatmaps were produced using the ‘my5C’ visualization tool (31). (B) The 5′-end DNA contacts decrease with activation of 5′-end HoxA genes and the interaction profile changes on macrophage differentiation. Changes in chromatin contacts associated with gene activation (‘upper panels’) and on macrophage differentiation (‘lower panels’) are shown in heatmap form. Heatmap values (8 kb bins, 8 kb smoothing) represent the difference of IFs between the time points indicated on the right of each panel. Reduced IFs are shown in red, and greater contacts appear in green as indicated by the color scales on the bottom left.

Figure 3.

The 3D modeling reveals the transient unfolding of the HoxA cluster with gene activation and its reorganization on macrophage differentiation. (A) Spatial modeling of HoxA 5C datasets produces similar 3D HoxA models at individual differentiation time points. Overlay of five 3D HoxA models selected randomly from a pool of 200 variants generated with the MCMC5C program and visualized using PyMOL (37,38). Models are color-coded as shown in the linear HoxA diagram above, and aligned in the same 3′–5′-end orientation. The average model sizes estimated in (B) are highlighted with transparent yellow spheres. (B) The average HoxA cluster size is greater when 5′-end genes are activated. The average HoxA and gene desert model sizes were calculated at each time point across an ensemble of 200 models generated with the MCMC5C program. Model sizes were calculated as the average distance between all pairs of restriction fragments. Error bars represent the standard error of the mean.

Figure 4.

Complete cluster unfolding on 5′-end gene activation precedes spatial reconfiguration of the HoxA middle domain in monocytes/macrophages. (A) The HoxA 3′- and 5′-ends show similar changes of average IFs throughout cellular differentiation. The average 3′- and 5′-end IFs were calculated from the normalized pairwise IFs of the main time course (biological 1) shown in Figure 2A. HoxA cluster 3′-end values include all interactions between fragments 47 and 64, and the 5′-end measurements encompass contacts between fragments 65 and 88. The 5′-end region is highlighted in yellow in (D). (B) The HoxA cluster 3′- and 5′-ends show similar changes in size throughout the differentiation time course. The HoxA cluster 3′- and 5′-end regions are as described in (A). The 3′- and 5′-end sizes were calculated from 200 models. (C) The spatial distance between transcriptionally regulated 5′-end genes correlates with gene expression changes. HoxA1 to A7 represent the 3′-end genes, and 5′-end genes include HoxA9 to A13. Average distances were derived from 200 models. Error bars in (B) and (C) represent the standard error of the mean. (D) The average HoxA local base density decreases across the cluster when 5′-end gene expression increases, and adopts a different profile on macrophage differentiation. Local base densities (y-axis) were estimated every 100 bp along the HoxA cluster region (x-axis) in 200 models with the Microcosm 2.0 program (19). The width of the trace corresponds to the standard deviation with sharper areas corresponding to smaller deviations. The green box highlights the middle HoxA region showing greatest base density changes throughout differentiation. Red dashed lines are used as guides to facilitate comparison.

Figure 5.

The 3′ enrichment and 5′ depletion of H3K27me2/3 at the HoxA cluster correlates with selective 5′-end gene activation. (A) Selective 5′-end gene activation correlates with the local depletion of H3K27me2/3 and its enrichment at the cluster 3′-end. H3K27me2/3 distribution along the HoxA cluster throughout the THP-1 differentiation time course. ChIPed material and input were hybridized onto custom tilling microarrays and normalized as described in ‘Materials and Methods’ section. (B) H3K27me2/3 marks cover the repressed HoxA cluster 3′-end and are depleted from the transcriptionally induced 5′-region in 3D models. The annotated 3D HoxA models are shown for each time point as described in Figure 3. Models were generated with MCMC5C, annotated with the HoxA TSSs (TSSs) and repression H3K27me2/3 marks and visualized using PyMOL. Red and yellow spheres represent TSSs and H3K27me2/3 marks, respectively. Only H3K27me2/3 signals above a threshold with a cutoff = 0.8 are shown.

Figure 6.

Spatial proximity changes between CTCF binding sites are consistent with a role for insulator looping in regulating the HoxA cluster architecture. (A) CTCF binds at the HoxA cluster 5′-end in myelomonocytes and macrophages. CTCF was ChIPed before (0 h) and after (96 h) differentiation of THP-1 cells. ChIPed material was sequenced and analyzed as described in ‘Materials and Methods’ section. The y-axis shows the number of CTCF ‘Tags per 10 millions’ after normalization against input, across the region characterized (x-axis). CTCF peaks are numbered from left to right (CTCF1 to 7). CTCF7 lacks a consensus binding sequence and is highlighted in red. The two orange arrows point to two minor peaks without predicted consensus binding sites. The green box indicates the middle HoxA region with greatest base density changes. (B) HoxA local base density scans at the 0 and 96-h time points from Figure 4 are shown to situate CTCF binding and base density. (C) The predicted spatial proximity of neighboring CTCF binding sites in the HoxA middle domain correlates with the base density changes observed on cellular differentiation. Predicted pairwise spatial distances between the seven CTCF binding sites identified in (A) were estimated from 200 structures, converted into proximity (1/distance), and represented in heatmap form. Values of spatial proximity are color-coded as indicated at the bottom left of the heatmap set. (D) The four CTCF binding sites at the HoxA middle domain are spatially clustered in macrophages. Annotated 3D HoxA models show the predicted position of CTCF binding sites during the differentiation time course. Red spheres represent CTCF binding sites.