Conserved, developmentally regulated mechanism couples chromosomal looping and heterochromatin barrier activity at the homeobox gene A locus

Abstract

Establishment and segregation of distinct chromatin domains are essential for proper genome function. The insulator protein CCCTC-binding factor (CTCF) is involved in creating boundaries that segregate chromatin and functional domains and in organizing higher-order chromatin structures by promoting chromosomal loops across the vertebrate genome. Here, we investigate the insulation properties of CTCF at the human and mouse homeobox gene A (HOXA) loci. Although cohesin loading at the CTCF binding site is required for looping, we found that cohesin is dispensable for chromatin barrier activity at that site. Using mouse embryonic stem cells in both a pluripotent and differentiated neuronal progenitor state, we determined that embryonic stem cell pluripotency factor OCT4 antagonizes cohesin loading at the CTCF binding site. Loss of OCT4 in the committed and differentiated neuronal progenitor cells results in loading of cohesin and chromosome looping, which contributes to heterochromatin partitioning and selective gene activation across the HOXA locus. Our analysis reveals that chromatin barrier activity of CTCF is evolutionarily conserved and is responsible for the coordinated establishment of chromatin structure, higher-order architecture, and developmental expression of the HOXA locus.

The establishment and segregation of different chromatin domains (heterochromatin and euchromatin) and maintenance of these structures are essential for proper genome function. Segregation of euchromatin and heterochromatin structures in the genome is mediated by noncoding DNA elements known as insulators (1), which are bound by CCCTC-binding factor (CTCF). CTCF binding sites (CBSs) across the genome define the boundaries of silent heterochromatin domains exhibiting trimethylated lysine residue 27 on histone H3 (H3K27me3) (2, 3). Potential CTCF cofactors have been identified (4–8); among these, suppressor of zeste 12 (SUZ12), a component of the polycomb repressive complex (PRC), seems highly relevant to the establishment of heterochromatin barrier activity (9).

CTCF has also been implicated in mediating long-range inter- and intrachromosomal interactions (10–12). DNA looping has been identified as a potential mechanism of transcriptional insulation at the apolipoprotein cluster and interferon gamma locus (13, 14). At these loci and numerous other sites in the genome (15), CTCF partners with cohesin to mediate looping, enhancer-blocking, and establishment of barrier sites; although cohesin has been implicated as a regulator in these studies, both CTCF and cohesin are present at these sites. In fact, cohesin is present at a significant majority of CTCF sites, and vice versa, making the exact role of each protein in barrier activity or the formation of chromatin loops unclear. In Drosophila, cohesin was recently identified as a member of the trithorax group (TRX), a family of transcriptional regulators that generally promote gene expression through chromatin remodeling (16). TRX proteins were discovered because of their ability to regulate the homeobox (HOX) genes, where they were usually associated with establishment of domains characterized by trimethylated lysine 4 on histone H3 (H3K4me3) (17). Coordination of CTCF and cohesin in establishing expression patterns and chromatin domains at HOX genes is obviously significant to development.

The HOX genes are arranged into four clusters (HOXA, B, C, and D) in vertebrates. Each locus spans several hundred thousand base pairs of exceptionally high evolutionary conservation in both coding and noncoding intergenic regions. The HOX genes encode master transcription factors responsible for patterning the anterior-posterior body axis during development. Colinearity between the order of genes in the cluster and their expression pattern along the anterior–posterior body axis has been observed across the animal phyla (18). In human lung fibroblasts, the upstream half of the HOXA locus lacks H3K27 trimethylation as well as association with repressive PRC component SUZ12, whereas the downstream half of the locus exhibits extensive H3K27 trimethylation and SUZ12 association (9). In foot fibroblasts, this pattern is completely reversed (19). In either tissue location, the absence of heterochromatin is marked by extended deposition of dimethylation (H3K4me2) and trimethylation (H3K4me3) of the lysine residue 4 of histone H3 at the promoters of the HOXA genes. The developmentally crucial HOXA locus demonstrates well-delineated chromatin domains, and we have utilized this property of the locus to elucidate the specific function of CTCF and cohesin in the establishment of chromatin domains, chromosomal looping, and gene expression across a large genomic region.

Unlike normal fibroblast cells, embryonic stem cells have a bivalent chromatin structure composed of both euchromatin and heterochromatin, exhibiting no segregation in the HOXA locus (20, 21). The mechanistic basis of this cell-type specificity in chromatin structure is unclear. According to a recent study, induced pluripotent stem cells can be generated by expressing four reprogramming factors: octamer binding factor 4 (OCT3/4), sex determining region Y-box 2 (SOX2), avian myelocytomatosis viral oncogene homolog (MYC), and Krueppel-like factor 4 (KLF4) (22). Among these factors, only OCT4 has been shown to interact with cohesin at enhancer and promoter sites to activate gene expression (23) and also interact with CTCF in X chromosome inactivation (24). Therefore, we hypothesized that OCT4 might be responsible for the bivalent chromatin structure observed in embryonic stem cells (ES cells). In this study, we use normal human lung fibroblasts (IMR90 cells) as well as mouse ES cells, which can further be differentiated into neural progenitor cells (NPC). Using this system, we are able to discriminate the specific functions of CTCF and cohesin in maintenance of chromatin domains and secondary higher-order structure. Through the use of ES cells, we are able to further connect the activities of both CTCF and cohesin to the role of pluripotency factor OCT4, which we demonstrate can disrupt chromatin looping, an activity which has not been previously described for this factor. This model system allows us to offer evidence that this integrated mechanism of chromatin domain formation and characterization is both conserved and developmentally regulated.

Results

CTCF Binding Site 5 (CBS5) Is a Conserved Mammalian Chromatin Barrier.

We used available chromatin immunoprecipitation coupled to deep sequencing (ChIP-Seq) data (21, 25, 26) to investigate the chromatin structure and organization of the HOXA locus and to gain insight into the role of CTCF in establishing large-scale chromatin and expression patterns. We had previously identified seven CTCF binding sites at the HOXA locus in the IMR90 cell line (27). One of the seven CTCF binding sites, CBS5, demarcates an extended H3K27me3 domain (Fig. 1A and Fig. S1) that encompasses all downstream HOX genes (HOXA9-13). In the absence of heterochromatin, HOXA genes upstream of CBS5 (HOXA1-7) are marked by dimethylation and trimethylation of histone H3K4 (H3K4me2, H3K4me3) at the promoters and trimethylation of histone H3K36 (H3K36me3) at the gene bodies, indicating expression of these genes in IMR90 cells (Fig. S2). Additionally, examination of genomic alignments (28) across vertebrates reveals that conservation of the sequence of CBS5 is limited largely to mammals, from human and mouse to platypus (Fig. 1 B and C). Therefore, at this locus, heterochromatin and euchromatin domains are arranged around a conserved CBS, suggesting potential developmentally regulated segregation of chromatin domains.

Fig. 1.

CBS5 functions as a chromatin barrier in the HOXA locus. A represents the HOXA gene expression status and CTCF binding sites in ES and differentiated cells. B is the result of CBS5 sequence alignment; the red box is the core 20 bp of CBS5 and the blue box is a candidate site for OCT4 binding in mouse HOXA locus. C is a phylogenetic tree indicating conservation of the CBS5 sequence primarily in mammals. D is the percentage of EGFP expression levels normalized to the gamma actin (ACTG1) gene expression, determined by RT-qPCR, upon expression of LacI or LacI-EMD proteins, for cells containing either a control reporter or a reporter containing the CBS5 element between LacO and the EGFP gene. Schematics of the CBS5 and control reporters are shown in E.

To confirm the chromatin barrier function of the CBS5 DNA element, we used a previously developed reporter system containing 256 Lac operator (LacO) sites (29). This LacO/LacI reporter system was recently utilized to demonstrate that tethering of the reporter to the nuclear periphery results in silencing of the reporter gene by de novo heterochromatin deposition and DNA methylation (30). We cloned CBS5 between the LacO sites and the EGFP reporter gene. We generated single copy stably integrated clones of HEK293 cells that contain either the control or the CBS5-containing reporter. Using these stably integrated clones, we transfected LacI-Emerin (EMD) fusion protein, previously shown to repress the reporter by localizing it to the nuclear periphery (30). As expected, the control reporter was efficiently repressed by expression of LacI-EMD, whereas CBS5 preserved EGFP reporter expression (Fig. 1D). This result suggests that CBS5 can function as a barrier element in a synthetic reporter system, likely by inhibiting the intrusion of heterochromatin.

Barrier Site CBS5 Is Developmentally Regulated.

We further examined the available ChIP-Seq data for evidence that establishment of this barrier at the HOXA locus is appropriately developmentally regulated. Both mouse and human embryonic stem (mES and hES, respectively) cells exhibit bivalent chromatin, composed of both euchromatin (H3K4me3) and heterochromatin (H3K27me3) marks across the entire HOXA locus (Fig. 1A and Fig. S1), as previously demonstrated (20, 21). Upon differentiation of mouse ES cells to NPC, a complete reorganization of the HOXA chromatin structure is observed, with CBS5 now segregating the large extended H3K27me3 heterochromatin structure comparable to that seen in human lung fibroblasts, providing direct evidence that these chromatin domains are developmentally regulated.

CTCF Is Required for Barrier Activity at CBS5.

Recent studies have found that cohesin colocalizes at a majority of CTCF binding sites in the human and mouse genomes (15, 31). We first investigated the individual role of CTCF in chromatin barrier activity at CBS5 by reducing the levels of CTCF using lentiviral vectors expressing specific shRNA. We verified CTCF binding at CBS5 using ChIP analyzed by quantitative real-time PCR (ChIP-qPCR, Table S1), and found that both CTCF and radiation repair protein 21 homog (RAD21, cohesin subunit) bound this site in IMR90 human fibroblasts (Fig. 2A, Left). Upon CTCF knockdown using the lentivirus-delivered shRNA in IMR90 human fibroblasts, CTCF binding at CBS5 was reduced to background levels and RAD21 binding at CBS5 was also reduced (Fig. 2A, Right). Our ChIP-qPCR experiments confirm the heterochromatin (H3K27me3) domain was bounded by CBS5 in IMR90 cells as observed in the ChIP-Seq data. Upon CTCF knockdown, H3K27me3 levels between CBS4 and CBS5 significantly increased (Fig. 2B), indicating an upstream spread of heterochromatin. We also analyzed expression of HOXA genes under these conditions by reverse transcription quantitative real-time PCR (RT-qPCR, Table S2). Normally, genes downstream of CBS5 are repressed; after knockdown of CTCF, these genes (HOXA9–10) significantly increased, while upstream genes (HOXA6 and 7) decreased in expression (Fig. 2C). These results suggest a loss of chromatin integrity at CBS5 as a result of loss of CTCF and, indirectly, cohesin.

Fig. 2.

CTCF function in chromatin barrier activity and looping. CTCF knockdown was performed in IMR90 cells to investigate the function of CTCF in chromatin barrier activity and looping. A shows qRT-PCR results of CTCF and RAD21 (cohesin subunit) ChIP analysis of chromatin isolated from IMR90 cells infected with either a control virus, pGIPz (IMR90), or a CTCF knockdown virus (CTCF KD). Dark-gray bars represent enrichment level of target protein at CBS5, whereas light-gray bars represent the enrichment at a random control site. Inset is Western blot analysis showing the efficacy of CTCF KD in IMR90 cells. B is the ChIP-qPCR results using H3K27me3 antibody, and shows the H3K27me3 levels at CTCF binding sites (CBS4 to CBS7) including CBS5 in the HOXA locus. C shows the relative expression level of HOXA genes near CBS5 (two upstream genes, HOXA6 and HOXA7, and downstream genes, HOXA9 and HOXA10), in IMR90 cells infected with CTCF KD and control (pGIPz) virus, respectively. The y axes represent the cDNA copy number of HOXA genes normalized by the cDNA copy number of the control ACTG1 gene (HOXA copy no./ACTG1 copy no.  × 10⁸).

Cohesin Is Required for Appropriate Gene Expression, but Not Barrier Activity.

Because loss of CTCF binding resulted in concomitant loss of RAD21 binding at CBS5, we knocked down RAD21 in IMR90 lung fibroblasts using the same lentiviral shRNA system. Accordingly, RAD21 was no longer bound at CBS5, but CTCF binding at this site was not perturbed, confirming previous observations that CTCF binding is independent of the presence of cohesin (15, 31) (Fig. 3A). ChIP-qPCR analysis of H3K27me3 indicated no significant change in the heterochromatin at and around CBS5 (Fig. 3B), indicating that barrier activity remained intact. Finally, mRNA levels of the euchromatin HOXA genes adjacent to CBS5 (HOXA6 and 7) were uniformly decreased after cohesin knockdown, whereas HOXA9 and HOXA10 genes residing within heterochromatin were not significantly affected (Fig. 3C). Our results indicate that cohesin is not required for the maintenance of heterochromatin barrier sites, but is necessary for appropriate gene expression at this locus.

Fig. 3.

Cohesin function in chromatin barrier activity and looping. RAD21 knockdown was performed in IMR90 to determine the role of cohesin in chromatin barrier activity and looping independent of CTCF. A and B are ChIP-qPCR results using CTCF, RAD21, and H3K27me3 antibody in IMR90 cells infected with RAD21 knockdown virus (RAD21 KD). Inset is Western blot analysis showing the efficacy of RAD21 KD in IMR90 cells. In B, the H3K27me3 levels at CTCF binding sites (CBS4 to CBS7) in the HOXA locus were determined by ChIP-qPCR. C shows the mRNA expression level (relative copy number) of HOXA genes (HOXA6–HOXA13) in control (pGIPz) and RAD21 KD cells. The y axes represent the cDNA copy number of HOXA genes normalized by the cDNA copy number of the control gamma actin gene, ACTG1 (HOXA copy no./ACTG1 copy no.  × 10⁸).

CBS5 Is Directly Involved in Forming Heterochromatin Loops.

Previous studies have implicated that CTCF can mediate formation of long extended loops that can segregate distinct chromatin environments (11). We investigated whether detectable looping organized at the CBS5 barrier site is present across the HOXA locus using chromosome conformation capture (3C) assays (32–34). We designed a series of primers positioned at all unique EcoRI restriction digestion sites across the entire locus (Fig. S3 and Table S3). Using an anchor primer located at CBS5, we performed qPCR assays to determine the proximity of different regions of the HOXA locus to CBS5 in IMR90 cells. We identified two sites along the HOXA locus that interact with CBS5. The major interaction site is located directly over the heterochromatin boundary downstream of the EVX1 gene (p40) (Fig. 4A). Additionally, we detected a weak interaction upstream near the end of the euchromatin boundary of the HOXA locus. We further confirmed these results by performing 3C using a different restriction enzyme (MseI) (Fig. S4). The higher-order architecture of this locus in IMR90 cells appears to be one upstream looped region composed of euchromatin and one downstream looped region of heterochromatin, organized around CBS5.

Fig. 4.

Chromosome architecture of the human and mouse HOXA locus. Interaction frequencies between the anchor primer (noted by a yellow circle) and other primers in the locus were determined by real-time PCR and normalized to a random ligation library generated from EcoRI restriction digestion fragments from the bacterial artificial clones (BAC) covering the HOXA locus (CTD-3054H22 and CTD-2536K9) of this locus. A shows the interaction frequencies observed with the anchor primer located at CBS5 (blue vertical bar) with the all the other primers in the locus in the top panel. The middle panel shows the interaction frequencies observed with a control anchor primer located 12-Kb downstream from CBS5 with all the other primers in the locus. The bottom panel shows the corresponding H3K27me3 ChIP-seq density across the HOXA locus in IMR90 cells. B is the result of the 3C assay in IMR90 cells infected with either CTCF or RAD21 knockdown (KD) viruses, or control pGIPz virus, tested to determine the interaction between CBS5 and the heterochromatin boundary (p40), which was detected as the strongest specific long-range interaction site in A. C and D show the interaction frequency of CBS5 or control primer (noted by yellow circles) with all the other 3C primers in mES cells and mNPC across the mouse HOXA locus. The interaction frequencies were calculated as in A. The control random ligation library for the mouse HOXA locus was generated using the BAC, RP23-33N14. The H3K27me3 ChIP-Seq densities across the HOXA locus from mES and mNPC are shown in the bottom panel. A strong interaction frequency observed between the close proximal site (within 10-Kb downstream to CBS5) and the anchor site at CBS5 likely results from self-ligation and thus the strong signal may not indicate long-range interaction between the two sites. E–G are the results of ChIP-qPCR at CBS5 performed using CTCF, RAD21, and OCT4 antibodies, respectively, and using mES and mNPC chromatin. H shows the results of ReChIP performed with CTCF and RAD21 antibodies following a primary OCT4 ChIP in ES cells.

Both CTCF and Cohesin Are Required for Heterochromatin Loop Formation.

To determine the specific roles of CTCF and cohesin in formation of the loops at the HOXA locus, we again utilized CTCF and RAD21 shRNA lentiviruses in IMR90 cells. We performed 3C assays to detect the interaction between CBS5 and the downstream heterochromatin barrier (p40) after separate CTCF and RAD21 knockdown. We found that independent knockdown of CTCF and RAD21 both resulted in loss of looping between CBS5 and the downstream heterochromatin barrier site p40 (Fig. 4B). Our results are consistent with the hypothesis that CTCF is required to load cohesin, which would be the major mediator of looping at this site.

Heterochromatin Looping Mediated by CBS5 Is Conserved and Developmentally Regulated in Mouse Cells.

We investigated whether the heterochromatin architecture of the HOXA locus is conserved between human and mouse. We performed 3C analysis of CBS5 interactions in mES cells and mNPC (Fig. S3B and Table S4). Interestingly, except for a strong proximal ligation signal most likely due to self-ligation, which did not change in its intensity upon differentiation of mES cells to mNPC, we observed that mES cells display very weak long-range interactions across the HOXA locus at CBS5 (Fig. 4C). However, upon differentiation into mNPC, we detected strong induced interactions, indicating two large loops corresponding to euchromatin and heterochromatin domain boundaries and centered around CBS5 (Fig. 4D). Except for the strong proximal interaction, looping of the heterochromatin domain in the HOXA locus in mNPC resembles closely the architecture observed in IMR90 cells.

OCT4 and Cohesin Bind at CBS5.

Our results demonstrate that perturbation of insulator function causes the architecture of the HOXA locus in IMR90 cells to closely resemble that of ES cells. We hypothesized that mES cell-specific factors might regulate loading of cohesin at CBS5. We found an experimentally determined binding site for OCT4 within 20 bp of CBS5 in the mouse HOXA locus (35) (Fig. 1B). We first used ChIP-qPCR to verify and further analyze CTCF, RAD21, and OCT4 binding at CBS5 in mES and mNPC cells. We found that, although CTCF was bound at CBS5 in both mES cells and mNPC, RAD21 was bound at CBS5 only in NPC (Fig. 4 E and F). By ChIP-qPCR, we determined that OCT4 was bound at CBS5 only in mES but not in mNPC (Fig. 4G). To verify this interaction, we performed sequential ChIP assays (ReChIP) with CTCF and RAD21 antibodies after the primary OCT4 ChIP in ES cells (Fig. 4H). Again, OCT4 interacted with CTCF but not RAD21. Therefore, it appears that binding of cohesin and OCT4 at CBS5 is mutually exclusive at CBS5.

Exogenous OCT4 Antagonizes Cohesin Binding and Loop Formation at CBS5.

To analyze the role of OCT4 in higher-order chromatin structure, we overexpressed OCT4 in human IMR90 fibroblasts by lentiviral infection and performed 3C, ChIP, and gene expression analyses in parallel. Ectopic OCT4 bound at CBS5 and resulted in significant loss of RAD21, but not CTCF binding (Fig. 5 A–C). ChIP-qPCR using H3K27me3 antibody revealed that the heterochromatin domain also remained intact after OCT4 overexpression (Fig. 5E). However, 3C analysis of OCT4-overexpressing cells showed loss of looping across the major heterochromatin domain (Fig. 5D). RT-qPCR analysis of HOXA transcripts showed expression of adjacent genes HOXA6 and HOXA7 is reduced (Fig. 5F). Exogenous expression of OCT4 seems to replace cohesin at CBS5 and result in chromatin looping and gene expression changes similar to those observed as a result of RAD21 knockdown. Combined with our 3C and ChIP data from mES and mNPC, our results suggest that OCT4 prevents chromatin looping at the HOXA locus by antagonizing cohesin loading at CBS5.

Fig. 5.

Effect of OCT4 overexpression on chromatin structure and architecture. A–C are the results of ChIP-qPCR analysis of CTCF, OCT4, and RAD21, respectively, at the CBS5 site using chromatin isolated from IMR90 cells overexpressing OCT4 (OCT4 OE) or a control protein (LacZ). Western blot analysis showing the efficacy of OCT4 OE in IMR90 cells is displayed in the inset of B. D shows the interaction frequency for major looping between CBS5 and the heterochromatin boundary site (p40) in IMR90 cells overexpressing OCT4 and LacZ. In E, H3K27me3 ChIP-qPCR analysis of chromatin isolated from IMR90 cells overexpressing OCT4 (OCT4 OE) was carried out at four CTCF binding sites, CBS4, 5, 6, and 7 in the HOXA locus. F is the mRNA expression analysis on the HOXA genes (HOXA6, 7, 9, and 10) after OCT4 overexpression in IMR90 cells. Values of the y axis represent the relative copy number of HOXA cDNAs normalized to ACTG1 cDNA. G is the proposed model of regulated chromatin looping at HOXA locus.

Looping Reinforces the Heterochromatin Stability.

We expanded our analyses of HOXA mRNA expression to other genes within the heterochromatin domain (HOXA9–13) to determine whether changes in looping across the heterochromatin domain have long-range effects on expression of genes within the heterochromatin domain. CTCF knockdown in IMR90 resulted in domain-wide activation of HOXA9, HOXA10, HOXA11, and HOXA13 (Fig. S5A). In contrast, RAD21 knockdown resulted in HOXA13 up-regulation only (Fig. S5B), although other silent genes were not affected. Similarly, in the IMR90 cells overexpressing OCT4, only HOXA13 was derepressed (Fig. S5C). Because OCT4 overexpression and RAD21 knockdown affects only the looping and not the chromatin barrier activity, activation of HOXA13 is most likely due to loss of looping. These results suggest that looping across the heterochromatin domain provides an additional mechanism for stable repression of HOXA genes.

Discussion

We have elucidated the mechanism of a functionally conserved mammalian chromatin barrier at CBS5 in the HOXA locus of both human and mouse. Our combined study of chromatin structure, architecture, and gene expression has defined distinct roles for CTCF and cohesin at barrier sites and implicates chromosomal looping in the stable maintenance of heterochromatin. CTCF is necessary for restricting the heterochromatin domain of the HOXA locus, and reduction of CTCF levels results in loss of chromatin barrier activity (Fig. S6). In addition, CTCF provides a docking site for the cohesin complex, which results in the formation of higher-order loops. Alternately, cohesin is responsible for formation of chromosomal loops and proper regulation of expression at the HOXA locus, but displays a limited role in chromatin barrier activity. In addition, chromosomal looping mediated by cohesin may contribute to a more stable heterochromatin domain, as indicated by modest derepression of one of the HOXA genes in the silent heterochromatin domain as a result of the reduction in cohesin levels. In contrast to its role in looping of the heterochromatin domain, cohesin seems to have a direct effect on transcriptional activation, independently of CTCF, of genes that are present within the euchromatin domain.

Previously, the mechanisms governed by CTCF and cohesin that contribute to chromatin state have been investigated independently (10–14, 36). Our study, however, investigates how these functions (barrier activity, transcriptional regulation, and looping) are accomplished in parallel at a conserved locus through the dissection of each responsible components (CTCF and cohesin). Thus, we provide a conserved regulatory mechanism that links gene expression patterns with higher-order chromatin state.

Our work with ES cells demonstrates that this model is also developmentally regulated and provides a context for the establishment of these expression domains. We identified OCT4, pluripotency factor and the principal regulator of ES cell state (22), as a negative regulator of loop formation mediated by cohesin at a CTCF binding site in mouse ES cells. OCT4 has been shown recently to interact separately with cohesin at enhancer and promoter sites where cohesin and mediator, a transcriptional coactivator, interact to promote gene activation by looping the intervening DNA sequences between these elements (23). OCT4 has also been shown to interact with CTCF when regulating X chromosome inactivation (24), We observe that OCT4 colocalizes with CTCF at CBS5, and reduction of OCT4 and loss of its binding during mNPC differentiation allows cohesin to bind to CBS5, which facilitates looping and heterochromatin segregation. Additionally, our experiments show that ectopic OCT4 expression in differentiated cells causes the deregulation of HOXA and other developmental genes by antagonizing cohesin and looping. Detailed mechanisms underlying mutually exclusive binding of OCT4 and cohesin at CBS5 remain to be elucidated. A possible mechanism may involve the epigenetic and sequence features of the CBS5 chromatin barrier that is distinct from OCT4 binding sites that function as enhancer elements. Alternatively, the previously demonstrated direct interaction between CTCF and OCT4 (24) may mask the domains on both proteins that promote cohesin loading at CBS5. This function of OCT4 is likely representative of the mechanism of maintaining cells in a pluripotent state by regulation of developmentally regulated loci.

Based on our observations and previous studies, we propose a three-dimensional model for the heterochromatin folding and gene expression in HOXA locus (Fig. 5G). We have investigated the functions of two important epigenetic regulators, CTCF and cohesin, comprehensively and in parallel, to form a definitive model for establishment and maintenance of lineage-associated chromatin domains, which is in turn important for proper gene expression. Combined with previous related studies, our results suggest deregulated expression of the HOXA genes (37, 38) and mutations in the sequence of CTCF or CBSs could result in cancer (39–41) via the mechanisms outlined in this paper.

Methods

Cell Lines and Antibodies.

IMR90 cells and mouse ES cells (AB2.2) were obtained from American Type Culture Collection and cultivated according to the supplier’s instructions. Neural progenitor cells were obtained by in vitro differentiation of the mouse ES cells using the previously published protocol (42). Antibodies of H3K27me3, H3K4me2, H3K4me3, CTCF, and RAD21 were obtained from Millipore. Oct3/4 and pan-actin antibodies were obtained from Santa Cruz Biotechnology.

LacO Reporter Assay.

In order to analyze the chromatin barrier functions at CBS5, we constructed EGFP reporter plasmids containing the 50 bp CBS5 element (including the core 20 bp CBS) flanking the EGFP expression cassette and the LacO256 repeat elements located upstream (Fig. 1E). Stable HEK293 clones harboring a single copy reporter were identified and used in the LacI transfection experiments. To accelerate the EGFP transgene silencing, we transfected a LacI-EMD fusion protein expression vector (30) and compared the resulting EGFP expression levels against the control LacI expression vector without EMD. EGFP expression was determined by RT-qPCR using the following primer pair: forward 5′-ACGACGGCAACTACAAGACC-3′ and reverse 5′-ACCTTGATGCCGTTCTTCTG-3′.

Chromatin Immunoprecipitation.

ChIP was performed as described previously (27). The DNA concentration of the recovered ChIP samples were determined using Quant-iT PicoGreen reagent (Invitrogen) using a set of DNA concentration standards generated from the total chromatin sample. ReChIP assays were performed as previously described (20).

RNA Preparation and Reverse Transcription.

Total RNA (extracted using Trizol reagent, Invitrogen) was treated with DNase I (Roche) for 30 min at 37 °C and further extracted with acidic phenol:chloroform and precipitated with ammonium acetate and ethanol. The RNA was reverse transcribed using SuperScript III (Invitrogen). The resulting cDNA was incubated with 10 μg of RNaseA for 30 min at 37 °C and purified using the QIAquick PCR purification kit (Qiagen).

Chromosome Conformation Capture.

The 3C assays were performed as described previously (33, 34, 43). One million formaldehyde cross-linked nuclei were digested with either EcoRI or MseI (New England Biolabs) overnight at 37 °C. The subsequent steps were carried out as previously described. Quantitative real-time PCR was performed to confirm the specific ligation between two DNA fragments in the test and control 3C libraries. Interaction frequencies were calculated by dividing the amount of PCR product obtained with the test 3C library constructed from nuclei by the amount of PCR product obtained with the control library DNA generated from ligating EcoRI or MseI fragments from the corresponding bacterial artificial clones. All 3C analyses were performed, at a minimum, in triplicate. The position of primers designed for 3C assay is represented in Fig. S3 and their sequence information is in Tables S3 and S4.

Lentiviral shRNA and cDNA Expression.

Plasmids containing shRNAs targeting either RAD21 or CTCF were obtained from Open Biosystems. Each shRNA plasmid was cotransfected into 293T cells with Trans-Lentiviral Packaging mix (Open Biosystems) according to the manufacturer’s protocol. The resultant viruses were titered and used to infect IMR90 cells at a multiplicity of infection MOI = 5 (for CTCF knockdown) or MOI = 1 (for RAD21 knockdown) for 24 h in the presence of 10 μg/mL polybrene (Specialty Media Products), and infected cells were harvested for RNA extraction, chromatin extraction, or 3C analysis at 48 h after infection. For OCT4 expression, we cloned full-length human OCT4 (POU5F1) into the pLenti6.3/V5-TOPO lentiviral expression vector (Invitrogen) and transfected into 293T cells according to the protocol of the ViraPower HiPerform Lentiviral Expression System (Invitrogen). A control virus carrying LacZ was also generated according to this protocol. IMR90 cells were infected with either OCT4 or LacZ lentivirus at an MOI = 2 for 24 h and harvested for RNA extraction, chromatin extraction, or 3C analysis at 72 h after infection.