CTCF binding site sequence differences are associated with unique regulatory and functional trends during embryonic stem cell differentiation

CTCF (CCCTC-binding factor) is a highly conserved multifunctional DNA-binding protein with thousands of binding sites genome-wide. Our previous work suggested that differences in CTCF’s binding site sequence may affect the regulation of CTCF recruitment and its function. To investigate this possibility, we characterized changes in genome-wide CTCF binding and gene expression during differentiation of mouse embryonic stem cells. After separating CTCF sites into three classes (LowOc, MedOc and HighOc) based on similarity to the consensus motif, we found that developmentally regulated CTCF binding occurs preferentially at LowOc sites, which have lower similarity to the consensus. By measuring the affinity of CTCF for selected sites, we show that sites lost during differentiation are enriched in motifs associated with weaker CTCF binding in vitro. Specifically, enrichment for T at the 18^th position of the CTCF binding site is associated with regulated binding in the LowOc class and can predictably reduce CTCF affinity for binding sites. Finally, by comparing changes in CTCF binding with changes in gene expression during differentiation, we show that LowOc and HighOc sites are associated with distinct regulatory functions. Our results suggest that the regulatory control of CTCF is dependent in part on specific motifs within its binding site.     

Preferential binding of insulin-like growth factor-II (IGF-II) to a putative alpha 2 beta 2 IGF-II receptor type in C2 myoblasts.

We have studied insulin-like-growth-factor (IGF) binding in two subclones of the C2 myogenic cell line. In the permissive parental subclone, myoblasts differentiate spontaneously into myotubes in medium supplemented with fetal calf serum. Unlike permissive myoblasts, inducible myoblasts require high concentrations of insulin (1.6 microM) or lower concentrations of IGF-I (25 nM) to differentiate, and expression of MyoD1 is not constitutive. IGF receptors were studied in microsomal membranes of proliferating and quiescent myoblasts and myotubes. IGF-II binding was also studied in inducible myoblasts transfected with the MyoD1 cDNA (clone EP5). Both inducible and permissive cells exhibited a single class of binding sites with similar affinity for IGF-I (Kd 0.8-1.2 nM). Affinity cross-linking of [125I]IGF-I to microsomal membranes, under reducing conditions, revealed a binding moiety with an apparent molecular mass of 130 kDa in permissive cells and 140 kDa in inducible cells, which corresponded to the alpha subunit of the IGF-I receptor. In permissive quiescent myoblasts, linear Scatchard plots suggested that [125I]IGF-II bound to a single class of binding sites (Kd 0.6 nM) compatible with binding to the IGF-II/M6P receptor. This was confirmed by affinity cross-linking experiments showing a labeled complex with an apparent molecular mass of 260 kDa and 220 kDa when studied under reducing and non-reducing conditions, respectively. In contrast, competitive inhibition of [125I]IGF-II binding to inducible quiescent myoblasts generated curvilinear Scatchard plots which could be resolved into two single classes of binding sites. One of them corresponded to the IGF-II/M6P receptor (Kd 0.2 nM) as evidenced by cross-linking experiments. The second was the binding site of highest affinity (Kd 0.04 nM) which was less inhibited by IGF-I than by IGF-II and was not inhibited by insulin. It migrated in SDS/PAGE at a position equivalent a molecular mass of 140 kDa, under reducing conditions, and at approximately 300 kDa, under non-reducing conditions. The labeling of this atypical binding moiety was not inhibited by anti(IGF-II/M6P-receptor) immunoglobulin. It was also observed in permissive and inducible myoblasts at proliferating stage. It was absent for permissive quiescent myoblasts and from permissive and inducible myotubes. Forced expression of MyoD1 in inducible cells (EP5 cells) dramatically reduced [125I]IGF-II binding to this atypical receptor. It emerges from these experiments that C2 cells express a putative alpha 2 beta 2 IGF-II receptor structurally related to the insulin/IGF-I receptor family. It is present in myoblasts but not in myotubes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Visualization of several binding sites for basic fibroblast growth factor (FGF-2) on fibroblasts by photoaffinity labeling: Evidence for intracellular complexes

The internalization of basic fibroblast growth factor (FGF-2) was studied in Chinese hamster lung fibroblasts (CCL39). Recombinant FGF-2 was derivatized with a photoactivable agent, N-hydroxysuccinimidyl-4-azido-benzoate (HSAB), iodinated, and used to visualize intracellular FGF-2-affinity-labeled molecules after internalization at 37°C. Iodinated HSAB-FGF-2 maintained the properties of natural FGF-2 such as affinity for heparin, binding to Bek and Flg receptors, interaction with high- and low-affinity binding sites, and reinitiating of DNA synthesis in CCL39 cells. Affinity-labeling experiments at 4°C with ¹²⁵I-HSAB-FGF-2 led to the detection of several FGF-cell surface complexes with apparent molecular mass of 80, 100, 125, 150, 170–180, 220, 260, and about 320 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), whereas two specific bands at 80 and 130–160 kDa were obtained using the homobifunctional cross-linking reagent, disuccinimidyl suberate. When the cells, preincubated with ¹²⁵I-HSAB-FGF-2 at 4°C and then washed, were shifted to 37°C, irradiation of the internalized labeled FGF-2 led to detection of a similar but fainted profile with one major specific band at 80 kDa. Heparitinase II treatment of the cells reduced binding of ¹²⁵I-HSAB-FGF-2 to its cell surface sites by 80% and internalization by 55%, indicating the involvement of heparan sulfate proteoglycans in these processes. Among the heparitinase-sensitive bands was the 80-kDa complex. © 1996 Wiley-Liss, Inc.     

Adaptive Evolution and the Birth of CTCF Binding Sites in the Drosophila Genome

Changes in the physical interaction between cis-regulatory DNA sequences and proteins drive the evolution of gene expression. However, it has proven difficult to accurately quantify evolutionary rates of such binding change or to estimate the relative effects of selection and drift in shaping the binding evolution. Here we examine the genome-wide binding of CTCF in four species of Drosophila separated by between ∼2.5 and 25 million years. CTCF is a highly conserved protein known to be associated with insulator sequences in the genomes of human and Drosophila. Although the binding preference for CTCF is highly conserved, we find that CTCF binding itself is highly evolutionarily dynamic and has adaptively evolved. Between species, binding divergence increased linearly with evolutionary distance, and CTCF binding profiles are diverging rapidly at the rate of 2.22% per million years (Myr). At least 89 new CTCF binding sites have originated in the Drosophila melanogaster genome since the most recent common ancestor with Drosophila simulans. Comparing these data to genome sequence data from 37 different strains of Drosophila melanogaster, we detected signatures of selection in both newly gained and evolutionarily conserved binding sites. Newly evolved CTCF binding sites show a significantly stronger signature for positive selection than older sites. Comparative gene expression profiling revealed that expression divergence of genes adjacent to CTCF binding site is significantly associated with the gain and loss of CTCF binding. Further, the birth of new genes is associated with the birth of new CTCF binding sites. Our data indicate that binding of Drosophila CTCF protein has evolved under natural selection, and CTCF binding evolution has shaped both the evolution of gene expression and genome evolution during the birth of new genes.     

The tumor suppressor role of CTCF

CTCF is an evolutionary conserved and ubiquitously expressed protein that binds thousands of sites in the human genome. Ectopic expression of CTCF in various normal and tumoral human cell lines inhibits cell division and clonogenicity, with the consequence to consider CTCF a potential tumor-suppressor factor. In this review article, we focused on the molecular mechanisms engaged by CTCF to modulate the expression of several key-regulators of differentiation, cellular senescence, cell cycle control and progression, whose expression is frequently altered in tumors. Moreover, we discussed common features of CTCF at each tumor-related DNA-binding sequence, such as protein-partners, post-translational modifications, and distinctive epigenetic marks establishment. The investigation of the molecular mechanisms engaged by CTCF to modulate tumor-related genes emphasizes the cell-type dependency of its tumor suppressor role. Indeed, the ability of CTCF to bind their promoters strictly depends by cell-type features as DNA methylation, BORIS-binding and post-translational modifications as PARYlation.     

Isolation and characterization of a transferrin binding protein from rat plasma

A transferrin binding protein was isolated from normal rat placenta and from iron-deficient rat plasma using a human transferrin affinity column. The yield of the isolated pure protein from iron-deficient rat plasma was about 0.5 micrograms/ml plasma. The major protein had a molecular mass of 85 kDa and contained carbohydrate. Reduction with mercaptoethanol did not change the molecular mass of the plasma transferrin binding protein whereas the native placental transferrin receptor of 180 kDa was reduced to 90 kDa. The transferrin binding protein reacted with both monoclonal and polyclonal antibodies raised against rat transferrin receptor. Immunoblotting of both normal and iron deficient rat plasma showed that the transferrin binding protein had a molecular mass of 85 kDa. In vitro digestion of purified rat placental transferrin receptor and red blood cells with trypsin provided an identical peptide profile, suggesting that the transferrin binding protein in rat plasma is derived from proteolysis of the extracellular portion of the transferrin receptor of the erythroid tissues.     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

Characterization of erythropoietin receptor on erythropoietin-unresponsive mouse erythroleukemia cells

A membrane receptor for erythropoietin was identified in various erythropoietin-unresponsive mouse erythroleukemia cells. Scatchard analyses of the binding of human 125I-labeled erythropoietin to T3C1-2-0, K-1, GM86 and 707 cells showed the presence of a single class of binding sites with apparent Kd values of 0.27-0.78 nM, which are slightly higher than those of erythropoietin-responsive cells. The number of binding sites varied from 110 to 930 per cell. Crosslinking of 125I-erythropoietin to its binding sites with disuccinimidyl suberate revealed the existence of a single binding protein with molecular mass of 63 kDa. No binding sites with higher molecular mass, as observed in erythropoietin-responsive cells, were detected, nor was any specific binding observed to the non-erythroid hematopoietic cell or to the human erythroleukemia cells examined.     

SID1 transmembrane family, member 2 (Sidt2): a novel lysosomal membrane protein

In a recent proteomic study of lysosomal proteins [10], we identified SID1 transmembrane family, member 2 (Sidt2) as a novel lysosomal membrane protein candidate. The Sidt2 gene encodes an 832-amino acid residues protein with a calculated molecular mass of 94.5 kDa. Bioinformatic analysis showed that Sidt2 is a multipass transmembrane protein that contains 10 putative N-glycosylation sites (NxS/T) and two potential tyrosine-based sorting signals (YGSF and YDTL). Using specific anti-Sidt2 antibody and lysosomal markers, the lysosomal localization of Sidt2 was determined by immunofluorescence. Furthermore, using subcellular fractionation techniques, we demonstrated that Sidt2 is a lysosomal integral membrane protein. Endogenous Sidt2 was detected in multiple tissues of mouse and rat with approximately 120-130 kDa molecular weights due to extensive glycosylation. After digestion with PNGase F, the apparent molecular mass of Sidt2 decreased to the predicted value of 95 kDa. In rats, Sidt2 was highly expressed in the liver, brain, and kidney, whereas no or little expression was found in the skeletal muscles, heart, and other tissues. In summary, Sidt2 is a highly glycosylated lysosomal integral membrane protein that shows tissue-specific expression.     

Coordinate Regulation of the Gel-forming Mucin Genes at Chromosome 11p15.5

Four of the genes that encode gel-forming mucins, which are major components of the mucus layer protecting many epithelial surfaces, are clustered at chromosome 11p15.5 and show both cell- and tissue-specific expression patterns. We aimed to determine whether the individual genes were coordinately regulated by mechanisms involving higher order chromatin structure. CCCTC-binding factor (CTCF) sites were predicted in silico and CTCF occupancy then evaluated by chromatin immunoprecipitation. CTCF was found at many sites across the gene cluster, and its binding was correlated with mucin gene expression. Next, siRNA-mediated depletion of CTCF was shown to increase MUC2 expression in A549 lung carcinoma cells and both MUC6 and MUC5AC expression in LS180 colon carcinoma cells. These changes correlated with loss of CTCF binding at multiple sites, although others retained occupancy. In cells actively expressing the mucins, the gene cluster was shown by chromosome conformation capture to form looped three-dimensional structures with direct interactions between the MUC2 promoter region, regions 30 kb 5′ to it, close to the MUC6 promoter and others near the 3′ end of MUC5AC, >170 kb away. Finally, to demonstrate the importance of CTCF binding to mucin gene expression, Calu-3 lung carcinoma cells were exposed to lipopolysaccharide (LPS). LPS increased the expression of MUC2 and MUC5AC and reduced MUC5B. CTCF occupancy was concurrently depleted at specific binding sites close to these genes. These data suggest that CTCF binding and cell type-specific long-range interactions across the 11p15.5 gene cluster are critical mechanisms for coordinating gel-forming mucin gene expression.     

Characterization of interleukin-13 receptor in carcinoma cell lines and human blood cells and comparison with the interleukin-4 receptor

Abstract

The interleukin-13 receptor is characterized by ligand-binding and crosslinking studies and compared with the interleukin-4 receptor. Crosslinking of radio-labeled hIL-4 and hIL-13 to the receptors on human carcinoma and mast cell lines demonstrated a predominant subunit at 130 kDa with two other minor bands of lower molecular mass (75 kDa and 65 kDa) in autoradiography. All binding of ¹²⁵I-IL-13 was specifically blocked when the carcinoma cell suspensions were incubated with an excess of unlabeled hIL-4. However, unlabeled hIL-13 was unable to completely displace ¹²⁵I-hIL-4 from the 130 kDa protein. In addition, ¹²⁵I-hIL-13 showed no binding to mouse fibroblast cells transfected with human 130 kDa hIL-4 receptor c-DNA. Using weighted nonlinear computer modeling of the data from several equilibrium binding studies with human mast cells, a model of two binding sites for IL-4 (K_d = 50 and 190 pmol/L) and one site for IL-13 (K_d=100 pmol/L) fitted better than a one site model with a very high level of significance (F = 10.66, P < 0.0001). It can be concluded that human IL-4R and hIL-13R are similar but distinct. This conclusion is supported here for the first time by a strong statistical criterion.