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

Abstract

BackgroundCTCF 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.ResultsUsing 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.ConclusionsOur 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.

High-Resolution Mapping of Chromatin Packaging in Mouse Embryonic Stem Cells and Sperm

“…Despite the presence of this protein in sperm, to date we have been unable to successfully recover ChIP material, likely due to the unique crosslinking and fragmentation challenges presented by the highly compact sperm nucleus. As an indirect approach to the question of the CTCF footprinting protein, we focused on CTCF-specific and BORIS-specific binding events recently described in ES cells (Sleutels et al, 2012) and asked whether these sites were occupied by short MNase DNA fragments from our sperm dataset. We find that <60bp MNase footprints over CTCF motifs in sperm preferentially occur over sites bound exclusively (in ES cells) to CTCF, while they are completely absent over known CTCFL-only binding sites (Figure S5).…”

The Role of CCCTC-Binding Factor (CTCF) in Genomic Imprinting, Development, and Reproduction1

“…BORIS/Boris is present in testis [94,95], indicating a likely role in male germ line establishment [96] and absence of expression results in subfertility [97]. Knockout mice have defects in spermatogenesis with dramatically reduced expression of the cerebroside sulfotransferase (Cst) gene, which is known to play a crucial role in meiosis [98].…”

Initiation of Meiotic Homologous Recombination: Flexibility, Impact of Histone Modifications, and Chromatin Remodeling

“…Other genetic variants have been identified that alter the genetic map in humans: (1) CTCFL, the testis-specific paralog of CTCF, a Zn-finger protein organizing chromatin loops with cohesins (Sleutels et al 2012), (2) the meiosis-specific cohesin RAD21L, which plays a role in the formation of meiotic chromosome axis and synapsis, (3) the SUMO ligase RNF212, ubiquitin ligase HEI10 (CCNB1IP1), and MutS homolog MSH4, all related to members of the ZMM class of pro-CO factors identified in S. cerevisiae, although with different synapsis defects (Reynolds et al 2013;Kong et al 2014;Qiao et al 2014). Strikingly, significant differences are seen among these genetic variants, as those having a large impact on recombination rates have no effect on hotspot usage.…”