The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner

Epigenetics & Chromatin The male germ cell gene regulator CTCFL is functionally different from CTCF and binds CTCF-like consensus sites in a nucleosome composition-dependent manner Frank Sleutels 0 Widia Soochit 0 Marek Bartkuhn 3 Helen Heath 0 Sven Dienstbach 3 Philipp Bergmaier 3 Vedran Franke 2 Manuel Rosa-Garrido 6 7 Suzanne van de Nobelen 0 Lisa Caesar 0 Michael van der Reijden 0 Jan Christian Bryne 2 Wilfred van IJcken 5 J Anton Grootegoed 4 M Dolores Delgado 7 Boris Lenhard 2 Rainer Renkawitz 3 Frank Grosveld 0 1 Niels Galjart 0 1 0 Department of Cell Biology Erasmus Medical Center , Rotterdam , The Netherlands 1 Cancer Genomics Center, NGI , Rotterdam , The Netherlands 2 Computational Biology Unit, Bergen Center for Computational Science, University of Bergen , Thormhlensgate 55, N-5008, Bergen , Norway 3 Institut fur Genetik, Justus-Liebig-Universitat , Giessen, Heinrich-Buff-Ring 58-62, 35392, Giessen , Germany 4 Department of Reproduction and Development Erasmus 5 Center for Biomics Erasmus Medical Center , Rotterdam , The Netherlands 6 Cell Cycle, Stem Cell Fate and Cancer Laboratory, Fundacion Marques de Valdecilla (IFIMAV) , Santander , Spain 7 Department of Molecular Biology, Instituto de Biomedicina y Biotecnologia de Cantabria, IBBTEC Universidad de Cantabria-CSIC-IDICAN , Santander , Spain Background: CTCF is a highly conserved and essential zinc finger protein expressed in virtually all cell types. In conjunction with cohesin, it organizes chromatin into loops, thereby regulating gene expression and epigenetic events. The function of CTCFL or BORIS, the testis-specific paralog of CTCF, is less clear. Results: Using immunohistochemistry on testis sections and fluorescence-based microscopy on intact live seminiferous tubules, we show that CTCFL is only transiently present during spermatogenesis, prior to the onset of meiosis, when the protein co-localizes in nuclei with ubiquitously expressed CTCF. CTCFL distribution overlaps completely with that of Stra8, a retinoic acid-inducible protein essential for the propagation of meiosis. We find that absence of CTCFL in mice causes sub-fertility because of a partially penetrant testicular atrophy. CTCFL deficiency affects the expression of a number of testis-specific genes, including Gal3st1 and Prss50. Combined, these data indicate that CTCFL has a unique role in spermatogenesis. Genome-wide RNA expression studies in ES cells expressing a V5- and GFP-tagged form of CTCFL show that genes that are downregulated in CTCFL-deficient testis are upregulated in ES cells. These data indicate that CTCFL is a male germ cell gene regulator. Furthermore, genome-wide DNA-binding analysis shows that CTCFL binds a consensus sequence that is very similar to that of CTCF. However, only ~3,700 out of the ~5,700 CTCFL- and ~31,000 CTCF-binding sites overlap. CTCFL binds promoters with loosely assembled nucleosomes, whereas CTCF favors consensus sites surrounded by phased nucleosomes. Finally, an ES cell-based rescue assay shows that CTCFL is functionally different from CTCF. Conclusions: Our data suggest that nucleosome composition specifies the genome-wide binding of CTCFL and CTCF. We propose that the transient expression of CTCFL in spermatogonia and preleptotene spermatocytes serves to occupy a subset of promoters and maintain the expression of male germ cell genes. CTCF; CTCFL; Gametogenesis; Genome-wide binding; Nucleosome - Background Three-dimensional folding of the eukaryotic genome occurs in a highly organized manner so as to compact chromatin while allowing temporal and spatial expression of genes. The genome contains regulatory elements, such as promoters, enhancers, locus control regions, insulators and enhancer blockers, that can orchestrate chromatin folding and gene activity over short and long distances, both in cis and in trans [1]. CTCF is a key coordinator of three-dimensional chromatin structure, allowing loop formation and specific chromatin compositions [2,3]. Gene activity is controlled in a positive or negative manner depending on the regulatory sequences present in the loops that are formed. The importance of CTCF in chromatin organization is emphasized by its evolutionary conservation, its ubiquitous expression, and its essential role in virtually all cells and tissues examined [4,5]. Hence, CTCF has been termed the master weaver of the genome [3]. The genome-wide binding by CTCF has been studied by different groups (see, for example, [6-10]). This has revealed ~35,000 CTCF-binding sites in the mammalian genome, of which more than 70% are shared between cell types. A relatively long consensus-binding motif for CTCF has been determined, which displays variability when compared to sites of transcription factors like KLF4, SOX2 and MYC [7]. The majority of CTCF binding-sites are found near genes, and ~8% is in the vicinity of transcription start sites (TSSs). Arrays of positioned (or phased) nucleosomes are found surrounding the nucleosome-free CTCF-binding sites [11-13], suggesting that CTCF binding promotes the ordered positioning of histones in its vicinity. CTCF has also been proposed to regulate the positioning of variant histones, such as H2A. Z [6,14]. Interestingly, the cohesin complex binds at the same position as CTCF in a CTCF-dependent manner. Together with CTCF, cohesin is essential for a proper three-dimensional chromatin structure and correct gene regulation [15-17]. CTCF-dependent loop formation is of crucial importance at imprinted loci. A well-studied example is the imprinted Igf2-H19 locus, in which Igf2 is expressed from the paternal and H19 from the maternal allele [18]. The imprinting control region (ICR) located in between the Igf2 and H19 genes is methylated on the paternal allele, preventing CTCF binding. As a result the enhancer downstream of the H19 gene can interact with the Igf2 promoter and drive expression of this gene. On the nonmethylated maternally derived ICR, CTCF does bind, thereby preventing enhancer-Igf2 interaction, resulting in a chromatin loop that allows enhancer-H19 association and H19 expression. By binding the ICR, CTCF therefore acts as a regulator of imprinted sites. The CTCF-like (CTCFL) protein, or Brother Of the Regulator of Imprinted Sites (BORIS) [19], has a central domain of 11 zinc fingers (ZFs) that is very similar to that of CTCF and that is essential for DNA binding. The N- and C-terminal domains of CTCF and CTCFL are not homologous. CTCFL is less conserved across species, and the protein arose later in evolution, as it is detected in amniotes only [20]. Furthermore, expression of CTCFL is restricted to testis, several types of cancers and a number of cell lines [21-23]. Studies of CTCF and CTCFL protein distribution in the testis have yielded contradictory results. Initially, a mutually exclusive expression pattern of CTCFL and CTCF was described [19], with CTCF being present in round spermatids (i.e. after meiosis) and CTCFL in prima (...truncated)