CTCF regulates cell cycle progression of αβ T cells in the thymus

Abstract

The 11-zinc finger protein CCCTC-binding factor (CTCF) is a highly conserved protein, involved in imprinting, longrange chromatin interactions and transcription. To investigate its function in vivo, we generated mice with a conditional Ctcf knockout allele. Consistent with a previous report, we find that ubiquitous ablation of the Ctcf gene results in early embryonic lethality. Tissue-specific inactivation of CTCF in thymocytes specifically hampers the differentiation of ab T cells and causes accumulation of late double-negative and immature single-positive cells in the thymus of mice. These cells are normally large and actively cycling, and contain elevated amounts of CTCF. In Ctcf knockout animals, however, these cells are small and blocked in the cell cycle due to increased expression of the cyclin-CDK inhibitors p21 and p27. Taken together, our results show that CTCF is required in a dose-dependent manner and is involved in cell cycle progression of ab T cells in the thymus. We propose that CTCF positively regulates cell growth in rapidly dividing thymocytes so that appropriate number of cells are generated before positive and negative selection in the thymus.

The DNA-binding factor Ctcf critically controls gene expression in macrophages

“…Deletion of the Ctcf gene was monitored by the expression of the bacterial b-galactosidase (lacZ) reporter present in the floxed Ctcf allele. 18 As expected, 23 we found lacZ expression, detected by fluorescein-di-b-D-galactopyranoside in conjunction with cell-specific surface markers, in substantial fractions of myeloid cell populations, including granulocytes, monocytes and macrophages of LysM-Cre Ctcf f/f mice (not shown). To assess whether deletion of the Ctcf allele resulted in the lack of Ctcf protein expression, we performed immunohistochemical analyses in the liver.…”

“…24 Genotyping of mice for the presence of transgenic constructs was performed as previously described. 18,23,24 Crosses of LysM-Cre transgenic and Ctcf f/f mice yielded mice with myeloid-specific deletion of the Ctcf gene (LysM-Cre Ctcf f/f ) mice, as well as littermates that did not have the LysM-Cre transgene or a floxed Ctcf gene, both of which were referred to as 'wild-type' mice. Mice were bred and maintained at the Erasmus MC animal facility under specific pathogen-free conditions, and used for experiments at 6-12 weeks of age.…”

“…To obtain mice with conditional knockout of Ctcf in macrophages, mice bearing the Ctcf allele flanked with loxP sites (Ctcf f mice) 18 were crossed with mice expressing Cre recombinase under the LysM promoter (LysM-Cre mice). 23 To investigate the efficiency of Cre-mediated deletion in the various myeloid cell lineages, we used a Cre-reporter strain harboring a targeted insertion of enhanced yellow fluoresencent protein (EYFP) into the ROSA26 locus.…”

“…To examine the role of Ctcf in the myeloid cell lineage in vivo, we crossed mice carrying a Ctcf-floxed allele 18,23 with LysM-Cre transgenic mice, which express the Cre recombinase under the control of the LysM promoter, 18,23 thereby confining Ctcf gene deletion to myeloid cells. First, we confirmed that the LysM-Cre transgene is functionally expressed in various macrophage populations, using a mouse Cre-reporter strain harboring a targeted insertion of EYFP into the ROSA26 locus.…”

“…15 Recently, Ctcf was found to control MHC class II gene expression, 16 and it was reported that enforced overexpression of Ctcf in DC caused increased apoptosis, reduced proliferation and impaired differentiation. 17 Conditional targeting experiments in mice showed that Ctcf controls both T-cell development 18 and differentiation into effector subsets, particularly into type T helper (Th)-2 cells. 19 We previously reported that mice with a Ctcf defect in CD4 1 T cells exhibit reduced Th2 development and production of the Th2 cytokines IL-4, IL-5 and IL-13, despite the expression of normal levels of the key Th2 transcription factors Gata3 and Satb1.…”