Elena M. Pugacheva

Disruption of three-dimensional genome architecture is a major driver of cancer initiation and progression, frequently arising from genetic and epigenetic alterations at CTCF- and cohesin-bound chromatin anchors. These same chromatin loop anchors can also be occupied by the germ cell-specific CTCF paralog CTCFL (BORIS), which is aberrantly activated in multiple malignancies. Here, we show that in ovarian cancer cells, BORIS activation establishes a distinct transcriptional program that collapses following loss of BORIS chromatin binding and is accompanied by widespread changes in CTCF and cohesin occupancy, histone modifications, and chromatin accessibility. These BORIS-dependent transcriptional alterations occur in long-range genomic clusters, resulting in the coordinated activation or repression of neighboring genes, including hormonally regulated gene families. BORIS loss also increases topologically associating domain (TAD) insulation, strengthens A/B compartment segregation and chromatin loop interactions, and results in a more compact and constrained chromatin architecture. Together, our findings suggest that aberrant BORIS activation promotes transcriptional reprogramming by weakening CTCF-mediated chromatin insulation and relaxing three-dimensional genome organization in ovarian cancer. Significance: High-grade serous ovarian carcinoma, the most common subtype of ovarian cancer, remains one of the deadliest gynecological malignancies because of its late diagnosis, extensive genomic instability, and frequent therapeutic resistance. The lack of reliable biomarkers for early detection underscores the need to identify new molecular drivers of disease initiation and progression. Here, we show that the germline-specific gene CTCFL (BORIS) is aberrantly activated in high-grade serous ovarian carcinoma, where it remodels transcriptional, epigenetic, and three-dimensional genome organization by occupying CTCF-bound chromatin loop anchors and weakening CTCF-mediated chromatin insulation. These findings identify BORIS as a key regulator of transcriptional reprogramming in ovarian cancer and support its further investigation as both a prognostic biomarker and a potential therapeutic target.

Cancer-testis antigens are genes normally restricted to the germline but aberrantly activated in many cancers, where their role in tumorigenesis remains unclear. BORIS (CTCFL), a testis-specific paralog of the chromatin organizer CTCF, is one such antigen that shows limited conservation between humans and mice, complicating in vivo functional studies. Here, we generated humanized mice in which both alleles of the endogenous Boris locus are replaced with the full-length human BORIS gene, including its highly diverged cis-regulatory elements. These fully humanized mice maintain normal fertility, indicating accurate germline expression and preserved BORIS function despite evolutionary divergence. However, unlike the strictly testis-specific expression of mouse Boris, human BORIS escapes complete somatic silencing, producing mosaic expression in a minority of mouse somatic cells. This ectopic expression is associated with reduced survival, increased tumor incidence and a shift of tumor spectrum toward aggressive lymphomas. Transcriptomic and chromatin profiling revealed that ectopic human BORIS reactivates testes-specific genes in the soma, including regulators of meiosis and DNA repair, through direct chromatin binding. This transcriptional reprogramming was consistent across tissues and clonal cell lines, revealing a dominant tissue-independent gene activation program. These findings demonstrate that when human BORIS escapes epigenetic silencing in somatic cells, it connects aberrant germline gene activation with increased cancer susceptibility in vivo.

CTCF binding sites serve as anchors for the 3D chromatin architecture in vertebrates. The functionality of these anchors is influenced by the residence time of CTCF on chromatin, which is determined by its binding affinity and its interactions with nucleosomes and other chromatin-associated factors. In this study, we demonstrate that CTCF occupancy is driven by CTCF motifs, strategically positioned at the entry sides of a well-positioned nucleosome, such that, upon binding, the N-terminus of CTCF is oriented towards the nucleosome. We refer to this nucleosome as the CTCF priming nucleosome (CPN). Our analyses suggest that CTCF can more easily displace the CPN if the nucleosome is not marked by CpG methylation or repressive histone modifications. Under these permissive conditions, the N-terminus of CTCF recruits SMARCA5 to reposition the CPN downstream, thereby creating nucleosome-free regions that enhance CTCF occupancy and cohesin stalling. In contrast, when CPNs carry repressive epigenetic marks, CTCF binding is weaker, with no nucleosome displacement or chromatin opening, and cohesin is retained less effectively at CTCF binding sites. We propose that the epigenetic status of CPNs shapes cell-specific CTCF binding patterns, ensuring the maintenance of chromatin architecture throughout the cell cycle.

CTCF binding sites serve as anchors for the 3D chromatin architecture in vertebrates. The functionality of these anchors is influenced by the residence time of CTCF on chromatin, which is determined by its binding affinity and its interactions with nucleosomes and other chromatin-associated factors. In this study, we demonstrate that CTCF occupancy is driven by CTCF motifs, strategically positioned at the entry sides of a well-positioned nucleosome, such that, upon binding, the N-terminus of CTCF is oriented towards the nucleosome. We refer to this nucleosome as the CTCF priming nucleosome (CPN). CTCF recognizes its binding sites if they are not methylated. It can then displace the CPN, provided the nucleosome is not marked by CpG methylation or repressive histone modifications. Under these permissive conditions, the N-terminus of CTCF recruits SMARCA5 to reposition the CPN downstream, thereby creating nucleosome-free regions that enhance CTCF occupancy and cohesin stalling. In contrast, when CPNs carry repressive epigenetic marks, CTCF binding is transient, without nucleosome displacement or chromatin opening. In such cases, cohesin is not effectively retained at CTCF binding sites. We propose that the epigenetic status of CPNs shapes cell-specific CTCF binding patterns, ensuring the maintenance of chromatin architecture throughout the cell cycle. Graphical

Background: Pervasive usage of alternative promoters leads to the deregulation of gene expression in carcinogenesis and may drive the emergence of new genes in spermatogenesis. However, little is known regarding the mechanisms underpinning the activation of alternative promoters. Results: Here we describe how alternative cancer-testis-specific transcription is activated. We show that intergenic and intronic CTCF binding sites, which are transcriptionally inert in normal somatic cells, could be epigenetically reprogrammed into active de novo promoters in germ and cancer cells. BORIS/CTCFL, the testis-specific paralog of the ubiquitously expressed CTCF, triggers the epigenetic reprogramming of CTCF sites into units of active transcription. BORIS binding initiates the recruitment of the chromatin remodeling factor, SRCAP, followed by the replacement of H2A histone with H2A.Z, resulting in a more relaxed chromatin state in the nucleosomes flanking the CTCF binding sites. The relaxation of chromatin around CTCF binding sites facilitates the recruitment of multiple additional transcription factors, thereby activating transcription from a given binding site. We demonstrate that the epigenetically reprogrammed CTCF binding sites can drive the expression of cancer-testis genes, long noncoding RNAs, retro-pseudogenes, and dormant transposable elements. Conclusions: Thus, BORIS functions as a transcription factor that epigenetically reprograms clustered CTCF binding sites into transcriptional start sites, promoting transcription from alternative promoters in both germ cells and cancer cells.

Many tumors express meiotic genes that could potentially drive somatic chromosome instability. While germline cohesin subunits SMC1B, STAG3, and REC8 are widely expressed in many cancers, messenger RNA and protein for RAD21L subunit are expressed at very low levels. To elucidate the potential of meiotic cohesins to contribute to genome instability, their expression was investigated in human cell lines, predominately in DLD-1. While the induction of the REC8 complex resulted in a mild mitotic phenotype, the expression of the RAD21L complex produced an arrested but viable cell pool, thus providing a source of DNA damage, mitotic chromosome missegregation, sporadic polyteny, and altered gene expression. We also found that genomic binding profiles of ectopically expressed meiotic cohesin complexes were reminiscent of their corresponding specific binding patterns in testis. Furthermore, meiotic cohesins were found to localize to the same sites as BORIS/CTCFL, rather than CTCF sites normally associated with the somatic cohesin complex. These findings highlight the existence of a germline epigenomic memory that is conserved in cells that normally do not express meiotic genes. Our results reveal a mechanism of action by unduly expressed meiotic cohesins that potentially links them to aneuploidy and chromosomal mutations in affected cells.

CTCF is a key organizer of the 3D genome. Its specialized paralog, BORIS, heterodimerizes with CTCF but is expressed only in male germ cells and in cancer states. Unexpectedly, BORIS-null mice have only minimal germ cell defects. To understand the CTCF-BORIS relationship, mouse models with varied CTCF and BORIS levels were generated. Whereas Ctcf^+/+Boris^+/+, Ctcf^+/−Boris^+/+, and Ctcf^+/+Boris^−/− males are fertile, Ctcf^+/−Boris^−/− (Compound Mutant; CM) males are sterile. Testes with combined depletion of both CTCF and BORIS show reduced size, defective meiotic recombination, increased apoptosis, and malformed spermatozoa. Although CM germ cells exhibit only 25% of CTCF WT expression, chromatin binding of CTCF is preferentially lost from CTCF-BORIS heterodimeric sites. Furthermore, CM testes lose the expression of a large number of spermatogenesis genes and gain the expression of developmentally inappropriate genes that are “toxic” to fertility. Thus, a combined action of CTCF and BORIS is required to both repress pre-meiotic genes and activate post-meiotic genes for a complete spermatogenesis program.

CTCF is a key organizer of the 3D genome. Its specialized paralog, BORIS, heterodimerizes with CTCF but is expressed only in male germ cells and in cancer states. Unexpectedly, BORIS-null mice have only minimal germ cell defects. To understand the CTCF-BORIS relationship, mouse models with varied CTCF and BORIS levels were generated. Whereas Ctcf ^{+ / +} Boris ^{+ / +} , Ctcf ^{+ /-} Boris ^{+ / +} , and Ctcf ^{+ / +} Boris ^-/- males are fertile, Ctcf ^{+ /-} Boris ^-/- (Compound Mutant; CM) males are sterile. Testes with combined depletion of both CTCF and BORIS show reduced size, defective meiotic recombination, increased apoptosis, and malformed spermatozoa. Although CM germ cells exhibit only 25% of CTCF WT expression, chromatin binding of CTCF is preferentially lost from CTCF-BORIS heterodimeric sites. Furthermore, CM testes lose the expression of a large number of spermatogenesis genes and gain the expression of developmentally inappropriate genes that are “toxic” to fertility. Thus, a combined action of CTCF and BORIS is required to both repress pre-meiotic genes and activate post-meiotic genes for a complete spermatogenesis program.

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF–BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF–DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.

A common aberration in cancer is the activation of germline-specific proteins. The DNA-binding proteins among them could generate novel chromatin states, not found in normal cells. The germline-specific transcription factor BORIS/CTCFL, a paralog of chromatin architecture protein CTCF, is often erroneously activated in cancers and rewires the epigenome for the germline-like transcription program. Another common feature of malignancies is the changed expression and epigenetic states of genomic repeats, which could alter the transcription of neighboring genes and cause somatic mutations upon transposition. The role of BORIS in transposable elements and other repeats has never been assessed. The investigation of BORIS and CTCF binding to DNA repeats in the K562 cancer cells dependent on BORIS for self-renewal by ChIP-chip and ChIP-seq revealed three classes of occupancy by these proteins: elements cohabited by BORIS and CTCF, CTCF-only bound, or BORIS-only bound. The CTCF-only enrichment is characteristic for evolutionary old and inactive repeat classes, while BORIS and CTCF co-binding predominately occurs at uncharacterized tandem repeats. These repeats form staggered cluster binding sites, which are a prerequisite for CTCF and BORIS co-binding. At the same time, BORIS preferentially occupies a specific subset of the evolutionary young, transcribed, and mobile genomic repeat family, SVA. Unlike CTCF, BORIS prominently binds to the VNTR region of the SVA repeats in vivo. This suggests a role of BORIS in SVA expression regulation. RNA-seq analysis indicates that BORIS largely serves as a repressor of SVA expression, alongside DNA and histone methylation, with the exception of promoter capture by SVA. Thus, BORIS directly binds to, and regulates SVA repeats, which are essentially movable CpG islands, via clusters of BORIS binding sites. This finding uncovers a new function of the global germline-specific transcriptional regulator BORIS in regulating and repressing the newest class of transposable elements that are actively transposed in human genome when activated. This function of BORIS in cancer cells is likely a reflection of its roles in the germline.