Composite CTCF Binding Sites Table 1. Ten T3 regulated genes harbor CTCF/TR composite elements. Name COMT SCD PTPRF NR2F1 ESRRA PER1 LRP1 BCL3 PCK1 LMNA Characteristics Catechol-O-methyltransferase is found in two forms, a soluble form (S-COMT) and a membrane-bound form (MB-COMT). Four Stearoyl-CoA desaturase isoforms, Scd1 through Scd4, have been identified in mouse. 2 SCD isoforms, SCD1 and SCD5, have been identified in human. Protein tyrosine phosphatase receptor type F possesses an extracellular region, a transmembrane region, and two tandem intracytoplasmic catalytic domains. Nuclear receptor subfamily 2, group F (COUP-TF) is a nuclear receptor and binds to both direct repeats and palindromes of the 59-AGGTCA-39 motif. Estrogen-related receptor alpha is a nuclear receptor that is closely related to the estrogen receptor. Function COMT catalyzes the transfer of a methyl group from S-adenosylmethionine to catecholamines, including the neurotransmitters dopamine, epinephrine, and norepinephrine. SCD is an iron-containing enzyme that catalyzes a rate-limiting step in the synthesis of unsaturated fatty acids. PTPs are signaling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. Regulation Down regulated upon T3 treatment. Up regulated upon T3 treatment. Up regulated upon T3 treatment. NR2F1 transcription factor binds to the ovalbumin promoter and, in conjunction with another protein (S300-II) stimulates initiation of transcription. Down regulated upon T3 treatment. Slightly induced after CTCF knockdown. ESRRa function has been demonstrated in the regulation Down regulated upon T3 of a variety of genes including lactoferrin, osteopontin, treatment. medium-chain acyl coenzyme A dehydrogenase (MCAD) and thyroid hormone receptor genes. Period1 is expressed in a circadian pattern in the suprachiasmatic nucleus, the primary circadian pacemaker in the mammalian brain. PER1 Influences clock function by interacting with other circadian regulatory proteins and transporting them to the nucleus. Down regulated upon T3 treatment. B-cell lymphoma 3 is a proto-oncogene BCL3 could be a transcriptional activating factor. Inhibits candidate translocated into the immunoglobulin translocation of NF-kappa-B p50 subunit to the nucleus. alpha-locus in some cases of B-cell leukemia. Up regulated upon T3 treatment. Repressed after CTCF knockdown. Low density lipoprotein-related protein 1 is required for early embryonic development and involved in cellular lipid homeostasis. Phosphoenolpyruvate carboxykinase 1 is a main control point for the regulation of gluconeogenesis. Lamin A is a family member of matrix proteins that are highly conserved in evolution. LRP1 is an endocytic receptor involved in endocytosis and in phagocytosis of apoptotic cells. Down regulated upon T3 treatment. PCK1, along with GTP, catalyzes the formation of phosphoenolpyruvate from oxaloacetate, with the release of carbon dioxide and GDP. During mitosis, the lamina matrix is reversibly disassembled as the lamin proteins are phosphorylated. Lamin proteins are thought to be involved in nuclear stability, chromatin structure and gene expression. Up regulated upon T3 treatment. Down regulated upon T3 treatment. Slightly repressed upon CTCF knockdown. Ref. [29] [29] [27] [27] [29] [28] [27,33] [28,33] [30] [28,33] doi:10.1371/journal.pone.0010119.t001 be performed because of the lack of suitable antibodies. The gene products are involved in neurotransmission, physiology, signalling, transcriptional regulation and cell cycle. Four genes are up- regulated and six are down-regulated by T3. CTCF knockdown [33] affects three of these genes, with two of them being repressed and one being induced (Table 1). Hormone sensitive enhancer blocking on episomes In order to functionally analyze hormone regulated enhancer blocking, we used a test system based on episomal vectors. The episomal system has the advantage that the reporter DNA is not integrated into the genome, and furthermore that the DNA is packaged into chromatin [34]. Therefore, this system allows the study of transcriptional regulation on properly formed chromatin independent of the integration locus in the genome. Using stably integrated DNA for the CTCF/TR composite element of the chicken lysozyme gene (F1F2 element), we have previously shown that CTCF mediated enhancer blocking is T3 sensitive [8]. We wondered whether we could use the F1F2 element as a crucial test system to study composite CTCF/TR elements with episomes. Enhancer function and enhancer blocking is quantified by measuring the activity of the luciferase reporter gene. Transfection of a series of episomes into N2ab cells, which express TRb, revealed that F1F2 mediates enhancer blocking, which is dependent on both the presence of the F1F2 element and enhancer (Fig. 4A, B). A single F1F2 element mediates weak enhancer blocking, whereas a five times multimerized element (5xF1F2) mediates a more than five-fold robust enhancer blocking. The backbone vectors pR or pR E do not change the luciferase activity after the addition of thyroid hormone, whereas in contrast the pR F1F2 E or pR 5xF1F2 E constructs fully abolish enhancer blocking. Again, this effect depends on the presence of the enhancer, since the enhancer-less constructs show only a weak T3 response. It is known that the CTCF-mediated repression function is partially sensitive to the HDAC-inhibitor Trichostatin A (TSA) [35]. Furthermore, it has been shown that CTCF is able to recruit HDAC activity from HeLa extract and interacts in vitro with Sin3A [35]. We analyzed whether TSA has an influence not only on repression, but also on the enhancer blocking function of CTCF. Incubation of the cells transfected with the enhancer blocking F1F2 episome with TSA resulted in a reduction of enhancer blocking activity to about 25% as compared to untreated cells (Fig. 4C). This indicates that HDACs are not only involved in the repression function of CTCF, but also in enhancer blocking. Another modification of CTCF, poly (ADP)-ribose (PAR) binding, has been shown to be essential for mediating enhancer blocking and insulation [36,37]. If indeed the episomal enhancer PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e10119