Abstract
CCCTC-binding factor (CTCF) is a conserved transcription factor that performs diverse roles in transcriptional regulation and chromatin architecture. Cancer genome sequencing reveals diverse acquired mutations in CTCF, which we have shown functions as a tumour suppressor gene. While CTCF is essential for embryonic development, little is known of its absolute requirement in somatic cells and the consequences of CTCF haploinsufficiency. We examined the consequences of CTCF depletion in immortalised human and mouse cells using shRNA knockdown and CRISPR/Cas9 genome editing as well as examined the growth and development of heterozygous Ctcf (Ctcf+/−) mice. We also analysed the impact of CTCF haploinsufficiency by examining gene expression changes in CTCF-altered endometrial carcinoma. Knockdown and CRISPR/Cas9-mediated editing of CTCF reduced the cellular growth and colony-forming ability of K562 cells. CTCF knockdown also induced cell cycle arrest and a pro-survival response to apoptotic insult. However, in p53 shRNA-immortalised Ctcf+/− MEFs we observed the opposite: increased cellular proliferation, colony formation, cell cycle progression, and decreased survival after apoptotic insult compared to wild-type MEFs. CRISPR/Cas9-mediated targeting in Ctcf+/− MEFs revealed a predominance of in-frame microdeletions in Ctcf in surviving clones, however protein expression could not be ablated. Examination of CTCF mutations in endometrial cancers showed locus-specific alterations in gene expression due to CTCF haploinsufficiency, in concert with downregulation of tumour suppressor genes and upregulation of estrogen-responsive genes. Depletion of CTCF expression imparts a dramatic negative effect on normal cell function. However, CTCF haploinsufficiency can have growth-promoting effects consistent with known cancer hallmarks in the presence of additional genetic hits. Our results confirm the absolute requirement for CTCF expression in somatic cells and provide definitive evidence of CTCF’s role as a haploinsufficient tumour suppressor gene. CTCF genetic alterations in endometrial cancer indicate that gene dysregulation is a likely consequence of CTCF loss, contributing to, but not solely driving cancer growth.
Highlights
CTCF is a conserved multivalent transcription factor with diverse roles in transcriptional regulation and three-dimensional genome organisation such that it has been called the ‘master weaver’protein [1]
We observed a significant reduction in sh.CTCF K562 colony number compared to non dox treatment and to sh.control
We examined the response of sh.CTCF K562 cells after UV insult and observed CTCF knockdown in K562 cells resulted in an increase in cell viability after recovery from UV exposure (p = 0.004) and a decrease in apoptotic (Annexin V-positive) cells (p = 0.0006, Figure 1E)
Summary
CTCF is a conserved multivalent transcription factor with diverse roles in transcriptional regulation and three-dimensional genome organisation such that it has been called the ‘master weaver’protein [1]. CTCF is a conserved multivalent transcription factor with diverse roles in transcriptional regulation and three-dimensional genome organisation such that it has been called the ‘master weaver’. Tissue-specific deletion of this ubiquitous factor in mice using conditional Ctcf alleles has highlighted the importance of CTCF availability in somatic tissues. Conditional deletion of Ctcf in the limb mesenchyme induces extensive apoptosis during limb development highlighting CTCF’s pro-survival role [4]. Deletion of Ctcf during early mouse brain development, led to PUMA upregulation and subsequent massive apoptosis [5]. Of relevance for our studies, Ctcf heterozygous mice, are more prone to the formation of spontaneous cancers, as well those induced by radiation and chemical means [6]
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