3D chromatin remodeling potentiates transcriptional programs driving cell invasion.

The contribution of deregulated chromatin architecture, including topologically associated domains (TADs), to cancer progression remains ambiguous. CCCTC-binding factor (CTCF) is a central regulator of higher-order chromatin structure that undergoes copy number loss in over half of all breast cancers, but the impact of this defect on epigenetic programming and chromatin architecture remains unclear. We find that under physiological conditions, CTCF organizes subTADs to limit the expression of oncogenic pathways, including phosphatidylinositol 3-kinase (PI3K) and cell adhesion networks. Loss of a single CTCF allele potentiates cell invasion through compromised chromatin insulation and a reorganization of chromatin architecture and histone programming that facilitates de novo promoter-enhancer contacts. However, this change in the higher-order chromatin landscape leads to a vulnerability to inhibitors of mTOR. These data support a model whereby subTAD reorganization drives both modification of histones at de novo enhancer-promoter contacts and transcriptional up-regulation of oncogenic transcriptional networks.

Single base-pair resolution analysis of DNA binding motif with MoMotif reveals an oncogenic function of CTCF zinc-finger 1 mutation.

Defining the impact of missense mutations on the recognition of DNA motifs is highly dependent on bioinformatic tools that define DNA binding elements. However, classical motif analysis tools remain limited in their capacity to identify subtle changes in complex binding motifs between distinct conditions. To overcome this limitation, we developed a new tool, MoMotif, that facilitates a sensitive identification, at the single base-pair resolution, of complex, or subtle, alterations to core binding motifs, discerned from ChIP-seq data. We employed MoMotif to define the previously uncharacterized recognition motif of CTCF zinc-finger 1 (ZF1), and to further define the impact of CTCF ZF1 mutation on its association with chromatin. Mutations of CTCF ZF1 are exclusive to breast cancer and are associated with metastasis and therapeutic resistance, but the underlying mechanisms are unclear. Using MoMotif, we identified an extension of the CTCF core binding motif, necessitating a functional ZF1 to bind appropriately. Using a combination of ChIP-Seq and RNA-Seq, we discover that the inability to bind this extended motif drives an altered transcriptional program associated with the oncogenic phenotypes observed clinically. Our study demonstrates that MoMotif is a powerful new tool for comparative ChIP-seq analysis and characterising DNA-protein contacts.

Beyond EZH2: is the polycomb protein CBX2 an emerging target for anti-cancer therapy?

Introduction: Epigenetic modifications are important regulators of transcription and appropriate gene expression answering an environmental stimulus. In cancer, these epigenetic modifications are altered, which impact the transcriptome, promoting initiation and cancer progression. Thus, targeting epigenetic machinery has proven to be an efficient cancer therapy. Areas covered: We review CBX2 as a therapeutic target. CBX2 is a polycomb protein, responsible for polycomb-repressive complex 1 (PRC1) targeting to chromatin via recognition of the repressive mark H3K27me3. Mechanistically, CBX2 overexpression may be implicated in poor survival by maintaining cancer stem cells in an undifferentiated state and via repression of tumor suppressors. We discuss strategies used to target CBX proteins and provide insights into biomarker considerations that may be important when targeting CBX family members for anti-cancer therapy. Expert opinion: CBX2 inhibition is a promising approach for the targeting of polycomb complexes in the cancer stem cell niche. However, extensive optimization of the current field of small molecules targeting CBX family proteins will be critical to reach in vivo, or clinical, utility.

Oncogenic activity of poly (ADP-ribose) glycohydrolase.

Poly (ADP-ribosylation), known as PARylation, is a post-translational modification catalyzed by poly (ADP-ribose) polymerases (PARP) and primarily removed by the enzyme poly (ADP-ribose) glycohydrolase (PARG). While the aberrant removal of post-translation modifications including phosphorylation and methylation has known tumorigenic effects, deregulation of PARylation has not been widely studied. Increased hydrolysis of PARylation chains facilitates cancer growth through enhancing estrogen receptor (ER)-driven proliferation, but oncogenic transformation has not been linked to increased PARG expression. In this study, we find that elevated PARG levels are associated with a poor prognosis in breast cancers, especially in HER2-positive and triple-negative subtypes. Using both in vitro and in vivo models, we demonstrate that heightened expression of catalytically active PARG facilitates cell transformation and invasion of normal mammary epithelial cells. Catalytically inactive PARG mutants did not recapitulate these phenotypes. Consistent with clinical data showing elevated PARG predicts poor outcomes in HER2+ patients, we observed that PARG acts in synergy with HER2 to promote neoplastic growth of immortalized mammary cells. In contrast, PARG depletion significantly impairs the growth and metastasis of triple-negative breast tumors. Mechanistically, we find that PARG interacts with SMAD2/3 and significantly decreases their PARylation in non-transformed cells, leading to enhanced expression of SMAD target genes. Further linking SMAD-mediated transcription to the oncogenicity of PARG, we show that PARG-mediated anchorage-independent growth and invasion are dependent, at least in part, on SMAD expression. Overall, our study underscores the oncogenic impact of aberrant protein PARylation and highlights the therapeutic potential of PARG inhibition in breast cancer.

Peroxisomes and cancer: The role of a metabolic specialist in a disease of aberrant metabolism.

Cancer is irrevocably linked to aberrant metabolic processes. While once considered a vestigial organelle, we now know that peroxisomes play a central role in the metabolism of reactive oxygen species, bile acids, ether phospholipids (e.g. plasmalogens), very-long chain, and branched-chain fatty acids. Immune system evasion is a hallmark of cancer, and peroxisomes have an emerging role in the regulation of cellular immune responses. Investigations of individual peroxisome proteins and metabolites support their pro-tumorigenic functions. However, a significant knowledge gap remains regarding how individual functions of proteins and metabolites of the peroxisome orchestrate its potential role as a pro-tumorigenic organelle. This review highlights new advances in our understanding of biogenesis, enzymatic functions, and autophagic degradation of peroxisomes (pexophagy), and provides evidence linking these activities to tumorigenesis. Finally, we propose avenues that may be exploited to target peroxisome-related processes as a mode of combatting cancer.

Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide.

In the human genome, translation initiation from non-AUG codons plays an important role in various gene regulation programs. However, mechanisms regulating the non-AUG initiation rate remain poorly understood. Here, we show that the non-AUG initiation rate is nearly consistent under a fixed nucleotide context in various human and insect cells. Yet, it ranges from <1% to nearly 100% compared to AUG translation, depending on surrounding sequences, including Kozak, and possibly additional nucleotide contexts. Mechanistically, this range of non-AUG initiation is controlled in part, by the eIF5-mimic protein (5MP). 5MP represses non-AUG translation by competing with eIF5 for the Met-tRNAi-binding factor eIF2. Consistently, eIF5 increases, whereas 5MP decreases translation of NAT1/EIF4G2/DAP5, whose sole start codon is GUG. By modulating eIF5 and 5MP1 expression in combination with ribosome profiling we identified a handful of previously unknown non-AUG initiation sites, some of which serve as the exclusive start codons. If the initiation rate for these codons is low, then an AUG-initiated downstream ORF prevents the generation of shorter, AUG-initiated isoforms. We propose that the homeostasis of the non-AUG translatome is maintained through balanced expression of eIF5 and 5MP.

CTCF facilitates DNA double-strand break repair by enhancing homologous recombination repair.

The repair of DNA double-strand breaks (DSBs) is mediated via two major pathways, nonhomologous end joining (NHEJ) and homologous recombination (HR) repair. DSB repair is vital for cell survival, genome stability, and tumor suppression. In contrast to NHEJ, HR relies on extensive homology and templated DNA synthesis to restore the sequence surrounding the break site. We report a new role for the multifunctional protein CCCTC-binding factor (CTCF) in facilitating HR-mediated DSB repair. CTCF is recruited to DSB through its zinc finger domain independently of poly(ADP-ribose) polymers, known as PARylation, catalyzed by poly(ADP-ribose) polymerase 1 (PARP-1). CTCF ensures proper DSB repair kinetics in response to γ-irradiation, and the loss of CTCF compromises HR-mediated repair. Consistent with its role in HR, loss of CTCF results in hypersensitivity to DNA damage, inducing agents and inhibitors of PARP. Mechanistically, CTCF acts downstream of BRCA1 in the HR pathway and associates with BRCA2 in a PARylation-dependent manner, enhancing BRCA2 recruitment to DSB. In contrast, CTCF does not influence the recruitment of the NHEJ protein 53BP1 or LIGIV to DSB. Together, our findings establish for the first time that CTCF is an important regulator of the HR pathway.

Proximal and distal regulation of the HYAL1 gene cluster by the estrogen receptor α in breast cancer cells.

Chromosomal and genome abnormalities at the 3p21.3 locus are frequent events linked to epithelial cancers, including ovarian and breast cancers. Genes encoded in the 3p21.3 cluster include HYAL1, HYAL2 and HYAL3 members of hyaluronidases involved in the breakdown of hyaluronan, an abundant component of the vertebrate extracellular matrix. However, the transcriptional regulation of HYAL genes is poorly defined. Here, we identified the estrogen receptor ERα as a negative regulator of HYAL1 expression in breast cancer cells. Integrative data mining using METABRIC dataset revealed a significant inverse correlation between ERα and HYAL1 gene expression in human breast tumors. ChIP-Seq analysis identified several ERα binding sites within the 3p21.3 locus, supporting the role of estrogen as an upstream signal that diversely regulates the expression of 3p21.3 genes at both proximal and distal locations. Of these, HYAL1 was repressed by estrogen through ERα binding to a consensus estrogen response element (ERE) located in the proximal promoter of HYAL1 and flanked by an Sp1 binding site, required to achieve optimal estrogen repression. The repressive chromatin mark H3K27me3 was increased at the proximal HYAL1 ERE but not at other EREs contained in the cluster, providing a mechanism to selectively downregulate HYAL1. The HYAL1 repression was also specific to ERα and not to ERß, whose expression did not correlate with HYAL1 in human breast tumors. This study identifies HYAL1 as an ERα target gene and provides a functional framework for the direct effect of estrogen on 3p21.3 genes in breast cancer cells.

The transcriptional co-repressor TLE3 suppresses basal signaling on a subset of estrogen receptor α target genes.

Chromatin constitutes a repressive barrier to the process of ligand-dependent transcriptional activity of nuclear receptors. Nucleosomes prevent the binding of estrogen receptor α (ERα) in absence of ligand and thus represent an important level of transcriptional regulation. Here, we show that in breast cancer MCF-7 cells, TLE3, a co-repressor of the Groucho/Grg/TLE family, interacts with FoxA1 and is detected at regulatory elements of ERα target genes in absence of estrogen. As a result, the chromatin is maintained in a basal state of acetylation, thus preventing ligand-independent activation of transcription. In absence of TLE3, the basal expression of ERα target genes induced by E2 is increased. At the TFF1 gene, the recruitment of TLE3 to the chromatin is FoxA1-dependent and prevents ERα and RNA polymerase II recruitment to TFF1 gene regulatory elements. Moreover, the interaction of TLE3 with HDAC2 results in the maintenance of acetylation at a basal level. We also provide evidence that TLE3 is recruited at several other regulatory elements of ERα target genes and is probably an important co-regulator of the E2 signaling pathway. In sum, our results describe a mechanism by which TLE3 affects ligand dependency in ERα-regulated gene expression via its binding restricting function and its role in gene regulation by histone acetylation.

LRH-1 governs vital transcriptional programs in endocrine-sensitive and -resistant breast cancer cells.

Tumor characteristics are decisive in the determination of treatment strategy for patients with breast cancer. Patients with estrogen receptor α (ERα)-positive breast cancer can benefit from long-term hormonal treatment. Nonetheless, the majority of patients will develop resistance to these therapies. Here, we investigated the role of the nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) in antiestrogen-sensitive and -resistant breast cancer cells. We identified genome-wide LRH-1-binding sites using ChIP-seq (chromatin immunoprecipitation sequencing), uncovering preferential binding to regions distal to transcriptional start sites. We further characterized these LRH-1-binding sites by integrating overlapping layers of specific chromatin marks, revealing that many LRH-1-binding sites are active and could be involved in long-range enhancer-promoter looping. Combined with transcriptome analysis of LRH-1-depleted cells, these results show that LRH-1 regulates specific subsets of genes involved in cell proliferation in antiestrogen-sensitive and antiestrogen-resistant breast cancer cells. Furthermore, the LRH-1 transcriptional program is highly associated with a signature of poor outcome and high-grade breast cancer tumors in vivo. Herein, we report the genome-wide location and molecular function of LRH-1 in breast cancer cells and reveal its therapeutic potential for the treatment of breast cancers, notably for tumors resistant to treatments currently used in therapies.