CTCF Controls HOXA Cluster Silencing and Mediates PRC2-Repressive Higher-Order Chromatin Structure in NT2/D1 Cells

Abstract

dHOX cluster genes are activated sequentially in their positional order along the chromosome during vertebrate development. This phenomenon, known as temporal colinearity, depends on transcriptional silencing of 5= HOX genes. Chromatin looping was recently identified as a conserved feature of silent HOX clusters, with CCCTC-binding factor (CTCF) binding sites located at the loop bases. However, the potential contribution of CTCF to HOX cluster silencing and the underlying mechanism have not been established. Here, we demonstrate that the HOXA locus is organized by CTCF into chromatin loops and that CTCF depletion causes significantly enhanced activation of HOXA3 to -A7, -A9 to -A11, and -A13 in response to retinoic acid, with the highest effect observed for HOXA9. Our subsequent analyses revealed that CTCF facilitates the stabilization of Polycomb repressive complex 2 (PRC2) and trimethylated lysine 27 of histone H3 (H3K27me3) at the human HOXA locus. Our results reveal that CTCF functions as a controller of HOXA cluster silencing and mediates PRC2-repressive higher-order chromatin structure. HOX genes are organized in clusters in vertebrate genomes and are essential for the patterning of the anterior-posterior body axis. These genes are activated in a temporal-spatial manner according to their position along the chromosome in vertebrates. This phenomenon, temporal-spatial colinearity, is pivotal for the correct patterning of animal bodies and depends on the proper silencing of 5= HOX genes (1). In a cellular model, a progressive transition from a repressed to an active chromatin state along the cluster from 3= to 5= has been proposed to mediate HOX colinearity (2-5), accompanied by nuclear reorganization (6-9) and changes in the higher-order chromatin structure of the clusters (10). A recent study revealed a transition in HOX cluster architecture from an initial single 3-dimensional (3-D) structure to a bimodal state that separates the active and inactive genes during colinear activation of HOX genes in mouse embryos (11). Whether spatial chromatin organization is the cause or a consequence of colinear activation and 5= silencing remains unknown. In addition, the factors responsible for the organization of the higher-order chromatin structure of HOX clusters remain to be identified.Recent findings have indicated that CCCTC-binding factor (CTCF) acts as a "master weaver" of the genome (12). Approximately 20,000 CTCF-binding sites (CBSs) have been identified in the human genome (13-15). These sites are frequently associated with cohesin complexes, which mediate sister chromatid cohesion during mitosis and gene regulation in postmitotic cells (16)(17)(18)(19). Multiple highly conserved CBSs have been identified in the human HOXA cluster. Bioinformatics analyses have suggested that CTCF mediates 3-D structure and thereby regulates gene expression in HOX clusters (10). CTCF-binding site 5 in the HOXA cluster (CBS5) has been reported to function as a developmental stage-specific insulator that regulates the expressio...

Exploring CTCF and cohesin related chromatin architecture at HOXA gene cluster in primary human fibroblasts

“…Specifically, in both types of cells, the leader fragment CBSA56/A5P (F8) interacts extensively to the examined DNA fragments throughout the HOXA cluster, including the most CBSs in the cluster and the HOXA1-HOXA4 gene promoter elements, suggesting that this region might be located at the center of higher-order chromatin structure of the HOXA cluster in primary cells. This observation is consistent with our previous results in NT2/D1 cells [18], and suggesting that CBSA56 may be one of the key CBSs for chromatin structure organization of the HOXA cluster. The interactions between CBSA56/A5 promoter and other fragments were also significantly decreased in foreskin than that in fetal lung fibroblasts.…”

“…The ligation frequency of a total of 18 BglII fragments containing the nine predicted CTCF-binding sites (CBSs), HOXA gene promoters or retinoic acid response elements (RAREs) were measured. Five leader fragments were chosen to explore the chromatin interactions between the leader fragments and others, these include three CBSs-containing fragments (CBSA56/A5P F8; CBSA79/A7P F11; CBSA10b/A10bP F14) which exhibited strong interactions with other fragments in our previous work [18], and the two border fragments (RARE1 F1 and CBSA13E2/E3 F18). For the first time, we observed strong interactions between fragments containing CBSs in primary human fibroblasts (Figure 2), suggesting that CTCF and its binding sites may also play a critical structural role in the HOXA cluster organization in primary human fibroblasts.…”

“…The 3C assay was performed as previously described [18]. Briefly, restricted enzyme BglII (NEB, R0144M) was used to digest the 1.7% formaldehyde-cross-linked nuclei from FsF or HLF cells.…”

“…Primer sets used in RT-PCR, chromosome conformation capture (3C) and chromatin immunoprecipitation (ChIP) assays were described previously [18].…”

“…Multiple CTCF binding sites (CBSs) were identified in human HOX clusters, most of which belong to the highly conserved noncoding elements. Our previous work revealed that CTCF functions as a controller of HOXA cluster silencing and mediates polycomb repressive complex 2 (PRC2)-repressive higher-order chromatin structure in NTERA-2 cl.D1 human teratocarcinoma (NT2/D1) cells [18]. Fibroblasts are principal component of human body and have been considered to be very important in wound healing [19] and reprogramming [20].…”