Multiple mechanisms deregulate EZH2 and histone H3 lysine 27 epigenetic changes in myeloid malignancies.

Polycomb repressive complex 2 (PRC2) is involved in trimethylation of histone H3 lysine 27 (H3K27), chromatin condensation and transcriptional repression. The silencing function of PRC2 complex is mostly attributed to its intrinsic activity for methylating H3K27. Unlike in B-cell lymphomas, enhancer of zeste homolog 2 (EZH2) mutations in myeloid malignancies are inactivating/hypomorphic. When we assessed the mutational status in myeloid malignancies (N=469 cases examined), we found EZH2 and EED/SUZ12 mutations in 8% and 3.3% of cases, respectively. In addition to mutant cases, reduced EZH2 expression was also found in 78% cases with hemizygous deletion (-7/del7q cases involving EZH2 locus) and 41% of cases with diploid chromosome 7, most interestingly cases with spliceosomal mutations (U2AF1/SRSF2 mutations; 63% of cases). EZH2 mutations were characterized by decreased H3K27 trimethylation and increased chromatin relaxation at specific gene loci accompanied by higher transcriptional activity. One of the major downstream target is HOX gene family, involved in the regulation of stem cell self-renewal. HOXA9 was found to be overexpressed in cases with decreased EZH2 expression either by EZH2/spliceosomal mutations or because of -7/del7q. In summary, our results suggest that loss of gene repression through a variety of mutations resulting in reduced H3K27 trimethylation may contribute to leukemogenesis.

Purification and molecular characterization of recombinant rat betacellulin.

A method for the large scale expression and purification of rat betacellulin (BTC) from Escherichia coli has been developed using a cleavable fusion protein strategy. Insoluble fusion protein collected as inclusion bodies was dissolved in urea under reducing conditions, re-folded, and purified by gel filtration chromatography and C(4) RP-HPLC. Authentic rat BTC was obtained after proteolytic cleavage of the fusion protein with Factor Xa. Factor Xa cleaved an additional site within the BTC protein, generating a truncated isoform separable from full-length BTC by heparin-affinity chromatography. Recombinant rat BTC stimulated the proliferation of mouse Balb/c 3T3 fibroblasts and competed for binding to the ErbB1 receptor in a dose-dependent manner analogous to that of BTC purified from natural sources.

Measurement of betacellulin levels in bovine serum, colostrum and milk.

Betacellulin, a member of the epidermal growth factor (EGF) family, was originally isolated and identified from the conditioned medium from a murine pancreatic beta-cell carcinoma cell line. Recently, we isolated bovine betacellulin from a growth factor enriched cheese whey extract, but there is no information on the presence of betacellulin in other biological fluids. We have cloned the cDNA for bovine betacellulin, produced recombinant betacellulin and shown that it has a similar potency to the purified native molecule in stimulating the proliferation of Balb/c3T3 fibroblasts. We have produced a polyclonal antiserum to bovine betacellulin which did not cross-react with EGF or transforming growth factor-alpha (TGF-alpha). The antibody was used in a homologous RIA that was able to detect betacellulin in pooled bovine colostrum sampled during the first 3 days after calving (2.30+/-0.11 ng/ml mean+/-s.e.m.; n=6), in bovine milk soluble fraction (1.93+/-0.64 ng/ml mean+/-s.e.m.; n=5) and in bovine cheese whey (2.59+/-0.16 ng/ml mean+/-s.e.m.; n=3). The betacellulin concentration in foetal bovine serum (FBS) (3.68+/-0.59 ng/ml mean+/-s.e.m.; n=6) greatly exceeded that of betacellulin in serum from male calves 1 and 5 weeks of age (0.53+/-0.15 ng/ml and 0.70+/- 0.09 ng/ml respectively; mean+/-s.e.m.; n=9). Betacellulin measured in the serum of these same animals when aged between 27 and 43 weeks was below the detection limits of the RIA. Sera from 10 out of 36 unmated heifers contained betacellulin levels within the detection limits of the assay (0.433+/-0.06 ng/ml mean+/-s.e.m.; n=10). The presence of betacellulin in bovine colostrum and milk suggests that it plays a role in the growth and development of the neonate and/or mammary gland function. The results also show that betacellulin is undetectable in the castrated adult male circulation. Additionally, although present in very low amounts, serum betacellulin could be under hormonal regulation in the female, since betacellulin was detected in sera from 27% of the unmated heifers examined in this study. The high levels of betacellulin detected in FBS relative to newborn and adult serum suggests a possible endocrine role for this growth factor in the bovine foetus.

Structure-function and biological role of betacellulin.

Betacellulin (BTC) belongs to the epidermal growth factor (EGF) family of peptide ligands that are characterised by a six-cysteine consensus motif that forms three intra-molecular disulfide bonds crucial for binding the ErbB receptor family. BTC was initially described, purified and cloned from a mouse insulinoma cell line. BTC is proteolytically processed from a larger membrane-anchored precursor and is a potent mitogen for a wide variety of cell types. BTC binds and activates ErbB-1 and ErbB-4 homodimers and is further characterised by its unique ability to activate all possible heterodimeric ErbB receptors. BTC is widely expressed in most tissues and various body fluids, including milk. Expression is particularly high in the pancreas where it is thought to play a role in the differentiation of pancreatic beta cells. While much is known about the ErbB receptor binding characteristics of BTC and its effect on a variety of cultured cells under different conditions, the challenge that lies ahead is to determine the role of BTC in vivo. This review will focus on the structure of BTC and the various biological effects ascribed to this member of the EGF family.

Identification of an alternatively spliced mRNA transcript of human betacellulin lacking the C-loop of the EGF motif and the transmembrane domain.

This paper describes the cloning and characterization of a novel cDNA encoding a short form of betacellulin (BTC-beta), and reports the expression of this mRNA in a variety of human tissues and cell types. BTC-beta is likely the result of alternative splicing. This splicing event leads to the generation of an mRNA encoding an unusual BTC precursor in which the C-loop of the EGF domain and the transmembrane domain are deleted while the remainder of the mature molecule is fused in-frame to the C-terminal cytoplasmic tail.

Cloning of rat betacellulin and characterization of its expression in the gastrointestinal tract.

Betacellulin (BTC) is relatively a more recently discovered member of the EGF family of growth factors. As a prelude to its expression and functional studies in rat models of gut damage/repair, we have cloned rat BTC and examined its expression in the gastrointestinal tract. Rat BTC was found to be nearly identical to mouse betacellulin. A single 3 kb mRNA species was detected by Northern blotting, and ribonuclease protection analysis showed that its expression was ubiquitous but low in abundance throughout the gut. BTC mRNA and protein were found expressed in the gastric surface and upper pit epithelium as well as in some cells of gastric glands. In the jejunum, BTC mRNA and protein were localised to the crypt epithelium and in villous goblet cells. In the colon, BTC mRNA and protein were found produced in crypt and surface epithelium as well as in goblet cells. Taken together, the wide spread expression in the gut epithelium and in mucous cells in particular suggests an important and unique role for BTC in the gastrointestinal tract.

Identification of betacellulin as a major peptide growth factor in milk: purification, characterization and molecular cloning of bovine betacellulin.

Betacellulin (BTC), a member of the epidermal growth factor (EGF) family of peptide growth factors, was purified from a growth-factor-enriched whey fraction of bovine milk by a combination of ion-exchange chromatography, gel-filtration chromatography, affinity chromatography and reverse-phase HPLC. Bovine BTC (bBTC) had an apparent molecular mass of 21-22 kDa on SDS/PAGE and exists in a glycosylated form. The cDNA encoding bBTC was obtained by a combination of 5' and 3' rapid amplification of cDNA ends ('RACE'). The primary translation product consists of 178 amino acid residues containing a putative signal sequence, a transmembrane domain, the mature BTC domain and a cytoplasmic domain containing a highly hydrophilic Arg-Lys-rich region similar to that of mouse BTC and human BTC. The amino acid sequence of the bBTC precursor was 88% identical with human BTC and 79% identical with mouse BTC. The bBTC gene was found to be expressed in a wide range of tissues, including the mammary gland. The identification of BTC in milk raises the possibility that it has a major role in the growth and development of the neonatal gastrointestinal tract.

The kangaroo cation-independent mannose 6-phosphate receptor binds insulin-like growth factor II with low affinity.

The mammalian cation-independent mannose 6-phosphate receptor (CI-MPR) binds mannose 6-phosphate-bearing glycoproteins and insulin-like growth factor (IGF)-II. However, the CI-MPR from the opossum has been reported to bind bovine IGF-II with low affinity (Dahms, N. M., Brzycki-Wessell, M. A., Ramanujam, K. S., and Seetharam, B. (1993) Endocrinology 133, 440-446). This may reflect the use of a heterologous ligand, or it may represent the intrinsic binding affinity of this receptor. To examine the binding of IGF-II to a marsupial CI-MPR in a homologous system, we have previously purified kangaroo IGF-II (Yandell, C. A., Francis, G. L., Wheldrake, J. F., and Upton, Z. (1998) J. Endocrinol. 156, 195-204), and we now report the purification and characterization of the CI-MPR from kangaroo liver. The interaction of the kangaroo CI-MPR with IGF-II has been examined by ligand blotting, radioreceptor assay, and real-time biomolecular interaction analysis. Using both a heterologous and homologous approach, we have demonstrated that the kangaroo CI-MPR has a lower binding affinity for IGF-II than its eutherian (placental mammal) counterparts. Furthermore, real-time biomolecular interaction analysis revealed that the kangaroo CI-MPR has a higher affinity for kangaroo IGF-II than for human IGF-II. The cDNA sequence of the kangaroo CI-MPR indicates that there is considerable divergence in the area corresponding to the IGF-II binding site of the eutherian receptor. Thus, the acquisition of a high-affinity binding site for regulating IGF-II appears to be a recent event specific to the eutherian lineage.

Analysis of mRNA levels for developmentally regulated prespore specific glutamine synthetase in Dictyostelium discoideum.

The enzyme glutamine synthetase (GS) of Dictyostelium discoideum is developmentally regulated, preferentially localized in prespore cells and is likely to play an important role in controlling the levels of ammonia, a known morphogen, in this organism. To further investigate the regulation of GS, a portion of the GS gene was isolated and used as a probe to examine the changes in GS mRNA throughout development and the level of GS mRNA in the two precursor cell types. The amino acid sequence of the cloned DNA fragment isolated is highly homologous to other eukaryotic GS genes. DNA blot analysis demonstrated that the GS gene exists as a single copy in D. discoideum. Analysis of RNA indicated that there is a single 1.7 kb GS transcript that increased during development to peak at the initial stages of culmination. Furthermore, GS mRNA was highly localized in prespore cells, which is consistent with a proposed source-sink model for ammonia assimilation in this organism.

Effect of the glutamine synthetase inhibitor, methionine sulfoximine, on the growth and differentiation of Dictyostelium discoideum.

To examine further the role of the enzyme glutamine synthetase in Dictyostelium discoideum we report here the effects of a specific glutamine synthetase inhibitor, methionine sulfoximine, on the growth and differentiation of this organism. Vegetative AX3 cells grown in the presence of methionine sulfoximine did not complete culmination in the normal time but were blocked at the finger stage. In these cells glutamine synthetase activity was almost completely abolished. However, methionine sulfoximine did not affect the level of glutamine synthetase mRNA, suggesting that there is no link between glutamine synthetase activity and mRNA transcription. Eventually glutamine synthetase activity reappeared and at the time culmination occurred. These results suggest that glutamine synthetase plays an important role in the assimilation of ammonia during the later stages of development in D. discoideum and that this assimilation is necessary for the completion of culmination.

Purification and properties of glutamine synthetase from the cellular slime mould Dictyostelium discoideum.

Glutamine synthetase (GS) from the cellular slime mould Dictyostelium discoideum was purified to apparent electrophoretic homogeneity with a final yield of 21.7%. The native enzyme appeared to be a GS-II type enzyme. SDS-PAGE of the final preparation revealed a single band of 43.5 kDa. The enzyme has a native molecular mass of 376 kDa, determined using Superose 6, indicating that the enzyme is likely to be an octamer of identical subunits. Dictyostelium discoideum GS has an optimal temperature of 42.5 degrees C, although it is thermolabile in the absence of L-glutamate and (or) Mg(2+)-ATP. The enzyme exhibits a K(m) for L-glutamate, ATP, and NH4Cl of 2.18, 0.18, and 0.11 mM, respectively, in the L-glutamine synthetic reaction with an optimal pH of 7.9. GS from D. discoideum does not appear to be significantly inhibited by various end products of L-glutamine metabolism, although it is potently inhibited by methionine sulphoximine. These properties are those expected for an enzyme for which the primary function is the assimilation of ammonia.

A calcium and calmodulin-dependent protein kinase present in differentiating Dictyostelium discoideum.

A protein kinase from Dictyostelium discoideum which phosphorylates the synthetic peptide, calmodulin-dependent protein kinase substrate (CDPKS, amino acid sequence: PLRRTLSVAA) and is stimulated by Ca2+/calmodulin is described. This is the first report of a protein kinase with these characteristics in D. discoideum. The enzyme was partially purified by Q-Sepharose chromatography. The protein kinase is very labile, and rapidly loses Ca2+/calmodulin-dependence upon standing at 4 degrees C, even in the presence of protease inhibitors, making further purification and characterisation difficult. In the active fractions, a 55 kDa polypeptide is labelled with [gamma-32P]ATP in vitro under conditions in which intramolecular rather than intermolecular reactions are favoured. The phosphorylation of this peptide is stimulated in the presence of Ca2+ and calmodulin but not Ca2+ alone. Ca2+/calmodulin-dependent stimulation is inhibited in the presence of the calmodulin antagonist, trifluoperazine (TFP). It is proposed that the 55 kDa polypeptide may represent the autophosphorylated form of the enzyme.