BORIS (for brother of the regulator of imprinted sites), a paralogue of the transcription factor, CTCF, is a novel member of the cancer-testis antigen family. The aims of the present study were as follows: (1) to investigate BORIS expression in breast cells and tumours using immunohistochemical staining, western and real-time RT -PCR analyses and (2) assess potential correlation between BORIS levels in tumours with clinical/pathological parameters. BORIS was detected in all 18 inspected breast cell lines, but not in a primary normal breast cell culture. In 70.7% (41 of 58 cases) BORIS was observed in breast tumours. High levels of BORIS correlated with high levels of progesterone receptor (PR) and oestrogen receptor (ER). The link between BORIS and PR/ER was further confirmed by the ability of BORIS to activate the promoters of the PR and ER genes in the reporter assays. Detection of BORIS in a high proportion of breast cancer patients implies potential practical applications of BORIS as a molecular biomarker of breast cancer. This may be important for diagnosis of the condition and for the therapeutic use of BORIS. The ability of BORIS to activate promoters of the RP and ER genes points towards possible involvement of BORIS in the establishment, progression and maintenance of breast tumours.

CTCF is an evolutionarily conserved and ubiquitously expressed architectural protein regulating a plethora of cellular functions via different molecular mechanisms. CTCF can undergo a number of post-translational modifications which change its properties and functions. One such modifications linked to cancer is poly(ADP-ribosyl)ation (PARylation). The highly PARylated CTCF form has an apparent molecular mass of 180 kDa (referred to as CTCF180), which can be distinguished from hypo- and non-PARylated CTCF with the apparent molecular mass of 130 kDa (referred to as CTCF130). The existing data accumulated so far have been mainly related to CTCF130. However, the properties of CTCF180 are not well understood despite its abundance in a number of primary tissues. In this study we performed ChIP-seq and RNA-seq analyses in human breast cells 226LDM, which display predominantly CTCF130 when proliferating, but CTCF180 upon cell cycle arrest. We observed that in the arrested cells the majority of sites lost CTCF, whereas fewer sites gained CTCF or remain bound (i.e. common sites). The classical CTCF binding motif was found in the lost and common, but not in the gained sites. The changes in CTCF occupancies in the lost and common sites were associated with increased chromatin densities and altered expression from the neighboring genes. Based on these results we propose a model integrating the CTCF130/180 transition with CTCF-DNA binding and gene expression changes. This study also issues an important cautionary note concerning the design and interpretation of any experiments using cells and tissues where CTCF180 may be present.

CTCF is an evolutionary conserved and ubiquitously expressed architectural protein, which regulates a plethora of cellular functions using different molecular mechanisms. The main form of CTCF with an apparent molecular mass of 130 kDa (CTCF130) has been extensively studied, however the properties of CTCF180, highly modified by poly(ADP-ribosyl)ation (PARylation) are not well understood. In this study we performed ChIP-seq and RNA-seq analyses in breast cells 226LDM, proliferating (with CTCF130 as the most abundant CTCF form) and arrested in the cell cycle (with only CTCF180). A dramatic reorganization of CTCF binding was observed during this transition, whereby CTCF was evacuated from many sites ("lost" group), although some sites retained modified CTCF ("common") and others acquired CTCF180 ("gained"). The classic CTCF binding motifs were identified for the former two groups, whereas the latter had no CTCF binding motif. Changes in CTCF occupancies in lost/common (but not gained) sites were associated with increased chromatin densities and altered expression from the neighboring genes. We propose a model integrating CTCF130/180 transition with CTCF-DNA binding and gene expression patterns, and functional outcomes. This study issues an important cautionary note concerning design and interpretation of any experiments using cells and tissues where CTCF180 may be present.

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