CTCF roles and functions

CTCF is a versatile nuclear factor, involved in various roles such as transcriptional activations/repression, insulation, regulation of genetic imprinting, developmental programme modulation, structural domains organization and guarding genomic fidelity[337] (Figure 1.6). The mechanisms underlying the diverse functions of CTCF in genome biology derive from its function in mediating long-range interactions between two or more DNA sequences. 4C analyses of the mouse imprinted maternal H19–insulin-like growth factor 2 (Igf2) locus demonstrated that the H19 imprinting control region (ICR) is involved in extensive inter-chromosomal and intra- chromosomal interactions across the genome that require the CTCF binding within the ICR[338]. CTCF binding at several DNase I hypersensitive sites is central to preserving the unique chromatin architecture at the murine haemoglobin subunit beta (Hbb) locus[339].CTCF-mediated interactions modulate facets of genome function in a context-dependent manner. The functional consequences of these interactions rely on the sequences flanking CTCF- binding sites and the presence of other specific architectural proteins[80].

Furthermore, CTCF promotes transcriptional activation of some genes, such as the case of CTCF binding to the amyloid precursor protein (APP) promoter[340] (Figure 1.6b). The structural domain of 107 amino acids in the N-terminal tail of CTCF regulates transcriptional activation and chromatin de-condensation, and upregulates its expression as it approaches the promoter location[341]. Conversely, CTCF may also play a role in transcriptional inhibition, by combining promoter and upstream silencer together. CTCF was originally identified as a transcriptional repressor of chicken c-Myc gene[266, 342]. CTCF binding along with thyroid hormone

receptor to the isogenous locus forms a repressor complex that lead to c-Myc reduced expression[343]. CTCF-mediated transcription repression could be achieved via recruitment of histone deacetylase and deacetylation of CTCF via binding of SIN3 transcription regulator family member A[344]. Recent genome-wide studies indicate that CTCF can additionally act as an enhancer blocker at particular loci. 15,000 CTCF binding sites were identified in human genome-wide search for conserved regulatory motif. These sites appeared to demarcate adjacent genes which show notably conversed correlation in gene expression compared with genes that are in a similar architecture, but that are not separated by CTCF-binding sites[299]. CTCF also works as an insulator bounding factor inhibiting interactions between promoter, enhancer, and silencer, provided that the CTCF binding site resides in-between regulatory elements that fail to properly function[345]. A study identified a 42-bp insulator sequence that could block the promoter activity of β-globulin, and equally works as a binding site of CTCF in humans[346].

Despite being originally thought off as an insulator and blocker of gene activity(Figure 1.6b), recent studies have identified CTCF as an important factor in tethering distant enhancers to their promoters. 79% of long-range interactions between promoters and their regulatory sequences were shown not to be blocked by the presence of one or more intervening CTCF-bound sites[39]. Strikingly, a subset of these long- range interactions are significantly enriched for CTCF and/ or histone modifications that are marked for active enhancers such as H3K27ac, H3K4me1 and H3K4me2. These results propose an alternative role for CTCF in genome biology may be to facilitate the communication between regulatory elements and promoters. Further support for this hypothesis came by finding a significant overlap between tissue-specific CTCF occupancy and enhancer elements, in addition to similar studies at several other loci[242]. For example, activation of major histocompatibility complex class II (MHC- II) gene expression by treatment with interferon-γ (IFNγ) requires CTCF-mediated looping of the XL9 enhancer element and its core promoters, MHC class II transactivator (CIITA) and specific transcription factors[347]. Thus, CTCF-mediated topological organization precedes transcriptional activation[348].

CTCF is also involved in regulating transcriptional pausing and modulating alternative mRNA splicing. For example, the first intron and upstream regulatory sequence in the mouse myeloblastosis oncogene (Myb) locus are bound by CTCF. CTCF-mediated looping between the first intron, promoter and upstream enhancer elements, along with its associated erythroid transcription and elongation factors is necessary for RNAP II to mediate transcriptional elongation and the upregulation of the Myb gene during erythroid differentiation[325]. The genome-wide distribution of

with high pausing indexes suggesting that the effect of CTCF on Pol II elongation may be more common than previously thought[349]. Recent studies have shown that disruption of mRNA elongation by RNAP II by CTCF may cause the inclusion/exclusion of particular exons in the mature mRNA[350, 351]. CTCF binding to exon 5 of CD45 gene promotes its alternative splicing in the mRNA, whereas blocking CTCF binding results in removal of this exon from the final edited mRNA[351].

An additional role of CTCF is in chromosome X inactivation. During development in mammals, one copy of the X chromosome's pair in females undergoes a process of inactivation as a measure of gene-dosage control. The process relies on the expression of the inactive x-specific transcript (Xist) and is inhibits by the antisense gene Tsix[352]. The imprinting centre of the X chromosome harbours a battery of CTCF binding sites with methylation-sensitive enhancer blocking activity. CTCF in association with the inhibitory Tsix regulates the epigenetic switch of X chromosome inactivation by stimulating Tsix transcription or blocking Xist from interacting with its enhancer. Expression of Tsix prevents Xist mRNA accumulation[352].