Canonical WNT signaling-dependent gating of MYC requires a noncanonical CTCF function at a distal binding site

Abnormal WNT signaling increases MYC expression in colon cancer cells in part via oncogenic super-enhancer-(OSE)-mediated gating of the active MYC to the nuclear pore in a poorly understood process. We show here that the principal tenet of the WNT-regulated MYC gating, facilitating nuclear export of the MYC mRNA, is regulated by a CTCF binding site (CTCFBS) within the OSE to confer growth advantage in HCT-116 cells. To achieve this, the CTCFBS directs the WNT-dependent trafficking of the OSE to the nuclear pore from intra-nucleoplasmic positions in a stepwise manner. Once the OSE reaches a peripheral position, which is triggered by a CTCFBS-mediated CCAT1 eRNA activation, its final stretch (≤0.7 μm) to the nuclear pore requires the recruitment of AHCTF1, a key nucleoporin, to the CTCFBS. Thus, a WNT/ß-catenin-AHCTF1-CTCF-eRNA circuit enables the OSE to promote pathological cell growth by coordinating the trafficking of the active MYC gene within the 3D nuclear architecture.

CTCF Expression and Dynamic Motif Accessibility Modulates Epithelial–Mesenchymal Gene Expression

Epithelial–mesenchymal transition (EMT) and its reversal, mesenchymal–epithelial transition (MET) drive tissue reorganization critical for early development. In carcinomas, processing through EMT, MET, or partial states promotes migration, invasion, dormancy, and metastatic colonization. As a reversible process, EMT is inherently regulated at epigenetic and epigenomic levels. To understand the epigenomic nature of reversible EMT and its partial states, we characterized chromatin accessibility dynamics, transcriptomic output, protein expression, and cellular phenotypes during stepwise reversible EMT. We find that the chromatin insulating protein machinery, including CTCF, is suppressed and re-expressed, coincident with broad alterations in chromatin accessibility, during EMT/MET, and is lower in triple-negative breast cancer cell lines with EMT features. Through an analysis of chromatin accessibility using ATAC-seq, we identify that early phases of EMT are characterized by enrichment for AP-1 family member binding motifs, but also by a diminished enrichment for CTCF binding motifs. Through a loss-of-function analysis, we demonstrate that the suppression of CTCF alters cellular plasticity, strengthening the epithelial phenotype via the upregulation of epithelial markers E-cadherin/CDH1 and downregulation of N-cadherin/CDH2. Conversely, the upregulation of CTCF leads to the upregulation of EMT gene expression and an increase in mesenchymal traits. These findings are indicative of a role of CTCF in regulating epithelial–mesenchymal plasticity and gene expression.

RedChIP identifies noncoding RNAs associated with genomic sites occupied by Polycomb and CTCF proteins

Nuclear noncoding RNAs (ncRNAs) are key regulators of gene expression and chromatin organization. The progress in studying nuclear ncRNAs depends on the ability to identify the genome-wide spectrum of contacts of ncRNAs with chromatin. To address this question, a panel of RNA–DNA proximity ligation techniques has been developed. However, neither of these techniques examines proteins involved in RNA–chromatin interactions. Here, we introduce RedChIP, a technique combining RNA–DNA proximity ligation and chromatin immunoprecipitation for identifying RNA–chromatin interactions mediated by a particular protein. Using antibodies against architectural protein CTCF and the EZH2 subunit of the Polycomb repressive complex 2, we identify a spectrum of cis- and trans-acting ncRNAs enriched at Polycomb- and CTCF-binding sites in human cells, which may be involved in Polycomb-mediated gene repression and CTCF-dependent chromatin looping. By providing a protein-centric view of RNA–DNA interactions, RedChIP represents an important tool for studies of nuclear ncRNAs.

Deeply hidden genome organization directly mediated by SATB1

Mammalian genomes are organized by multi-layered chromatin folding. Whether and how three-dimensional genome organization contributes to cell-type specific transcription remains unclear. Here we uncover genome architecture formed by specialized sequences, base-unpairing regions (BURs), bound to a nuclear architectural protein, SATB1. SATB1 regulates cell-type specific transcription that underlies changes in cellular phenotypes. We developed a modified ChIP-seq protocol that stringently purifies genomic DNA only with its directly-associated proteins and unmasked previously-hidden BURs as direct SATB1 targets genome-wide. These SATB1-bound BURs are mutually exclusive from CTCF binding sites, and SATB1 is dispensable for CTCF/cohesion-mediated topologically associated domains (TADs). Instead, BURs largely overlap with lamina associated domains (LADs), and the fraction of BURs tethered to the SATB1 protein network in the nuclear interior is cell type-dependent. Our results reveal TAD-independent chromatin folding mediated by BUR sequences, which serve as genome architecture landmarks targeted by SATB1, to regulate cell-type specific gene expression.

High-throughput single-cell epigenomic profiling by targeted insertion of promoters (TIP-seq)

Chromatin profiling in single cells has been extremely challenging and almost exclusively limited to histone proteins. In cases where single-cell methods have shown promise, many require highly specialized equipment or cell type–specific protocols and are relatively low throughput. Here, we combine the advantages of tagmentation, linear amplification, and combinatorial indexing to produce a high-throughput single-cell DNA binding site mapping method that is simple, inexpensive, and capable of multiplexing several independent samples per experiment. Targeted insertion of promoters sequencing (TIP-seq) uses Tn5 fused to proteinA to insert a T7 RNA polymerase promoter adjacent to a chromatin protein of interest. Linear amplification of flanking DNA with T7 polymerase before sequencing library preparation provides ∼10-fold higher unique reads per single cell compared with other methods. We applied TIP-seq to map histone modifications, RNA polymerase II (RNAPII), and transcription factor CTCF binding sites in single human and mouse cells.

Loss of imprinting control of the lncRNA H19-fetal mitogen IGF2 gene cluster in the decidual microenvironment of patients with idiopathic spontaneous miscarriages

Miscarriage, the spontaneous loss of a pregnancy before the fetus achieves viability, is a common complication of pregnancy. Decidualization plays a critical role in the implantation of the embryo. To search for molecular factors underlying miscarriage, we explored the role of long noncoding RNAs (lncRNAs) in the decidual microenvironment, where the molecular crosstalk at the feto–maternal interface occurs. By integrating RNA-seq data from recurrent miscarriage patients and decidualized endometrial stromal cells, we identified H19 , a noncoding RNA that exhibits paternally imprinted monoallelic expression in normal tissues, as the most upregulated lncRNA associated with miscarriage. Aberrant upregulation of H19 lncRNA was observed in decidual tissues derived from patients with spontaneous miscarriage as well as decidualized endometrial stromal cells. The maternally imprinted fetal mitogen Igf2, which is usually reciprocally co-regulated with H19 in the same imprinting cluster, was also upregulated. Notably, both genes underwent loss of imprinting, as H19 and IGF2 were actively transcribed from both parental alleles in decidual tissues. Mechanistically, this loss of imprinting in decidual tissues was associated with the loss of the H3K27m3 suppression marker in the IGF2 promoter, CpG hypomethylation at the central CTCF binding site in the imprinting control center (ICR) that is located between IGF2 and H19 , and the loss of CTCF-mediated intrachromosomal looping. These data provide the first evidence that aberrant control of the ICR epigenotype-intrachromosomal looping- H19/IGF2 imprinting pathway may be a critical epigenetic risk factor in the abnormal decidualization related to miscarriage.

CBIO-05. SOX2 CHROMATIN ARCHITECTURE AND ENHANCER ACTIVITY IN THE MAINTENANCE AND DEVELOPMENT OF NEURAL STEM CELLS

Gain of function mutations in isocitrate dehydrogenase I (IDH1) result in the formation of the oncometabolite 2-hydroxyglutarate (2HG) in adult lower grade gliomas. To gain insight into mechanisms of gliomagenesis, our lab previously created a tractable human cellular model of low grade astrocytoma (LGA) using the putative cell-of-origin, human neural stem cells (NSCs), engineered to express mutant IDH1 and knockdown constructs against TP53 and ATRX, the two other genetic changes that accompany the IDH mutation in these tumors. We found that transcription factor (sex determining region Y)-box 2 SOX2, which is essential to NSC multipotency, the ability to differentiate to neuroglial lineages, behaves as a tumor suppressor during glioma initiation. In this context, we showed SOX2 is transcriptionally downregulated to impair NSC multipotency, thus locking NSCs in an undifferentiated state to initiate gliomagenesis. This downregulation occurs secondary to dynamic reorganization of the topologically associating domain (TAD) of SOX2 and the loss of contact with several genomic loci with histone modifications and chromatin accessibility suggestive of being enhancers. Here we show that those putative enhancers acquire enhancer-like features simultaneous to tje TAD organizing in a way that facilitates interaction with the SOX2 promoter during the process of pluripotent stem cell differentiation into neuroectodermal lineages, suggesting a developmental role. Preliminary data suggests that disruption of the SOX2 TAD by preventing binding of the genome organizer CTCF downregulates SOX2 expression in NSCs. Targeted silencing of several regions of a putative enhancer with CRISPRi also downregulates SOX2. In human embryonic stem cells (hESCs), interfering with these CTCF binding sites biases their differentiation away from the neuroectoderm. We are currently performing CRISPRi screen against all putative enhancer loci, teratoma formation assays on hESCs lacking relevant CTCF binding, and CRISPR mediated deletion of putative enhancers. Understanding this developmental process may reveal underlying vulnerabilities in LGA.

Dissecting CTCF site function in a tense HoxD locus

In this issue of Genes & Development, Amândio and colleagues (pp. 1490–1509) dissect the function of a cluster of several CTCF binding sites in the HoxD cluster by iterative deletions in mice. They found additive functions for some, and intriguingly found that some sites function as insulators, while others function as anchors for enhancer–promoter interactions. These functions vary depending on developmental context. The work provides new insights into the roles played by CTCF in regulating developmental patterns and 3D chromatin organization.