A novel, high stringency selection system allows screening of few clones for high protein expression.

To obtain highly productive mammalian cell lines, often large numbers of clones need to be screened. This is largely due to low selection stringencies, creating many, but low protein producing clones. To remedy this problem, a novel, very stringent selection system was designed, to create few, but high protein producing clones. In essence, a selection marker with a startcodon that confers attenuated translation initiation frequency was placed upstream of the gene of interest with a startcodon that confers optimal translation initiation. From the transcribed bicistronic mRNA, the selection marker is translated at a low frequency, and the protein of interest at a high frequency. This selection system is so stringent that clones form only rarely. However, application of anti-repressor elements, which increase promoter activity, did induce the formation of clones that expressed proteins at high levels. When combined with anti-repressor elements, this novel selection system can be a valuable tool to rapidly create few, but highly productive mammalian cell lines.

Expression of the p16(INK4a) gene product, methylation of the p16(INK4a) promoter region and expression of the polycomb-group gene BMI-1 in squamous cell lung carcinoma and premalignant endobronchial lesions.

It is generally assumed that squamous cell carcinoma develops in a stepwise manner from normal bronchial epithelium towards cancer by the accumulation of (epi)genetic alterations. Several mechanisms including mutations and homozygous deletions or hypermethylation of the p16(INK4a) promoter region can cause loss of p16 expression. Recent studies suggest overexpression of the polycomb-group gene BMI-1 might also down-regulate p16 expression. In this study, we analyzed the p16 expression in relation to the methylation status of the p16 promoter region of the p16(INK4a) gene and the expression of BMI-1 in bronchial squamous cell carcinomas (SCC) and its premalignant lesions. Nine (69%) SCC showed loss of p16 expression and 10 (77%) showed expression of BMI-1. Of four p16 positive samples two (50%) were BMI-1 positive, whereas among nine p16 negative samples, eight (89%) revealed BMI-1 staining. Four (44%) p16 negative samples were hypermethylated at the p16(INK4a) promoter region; the other p16 negative tumors that showed no hypermethylation revealed BMI-1 staining. Only two premalignant lesions showed absence of p16 expression, of which one (carcinoma in situ) was hypermethylated at the p16(INK4a) promoter region and the other (severe dysplasia) showed BMI-1 expression. In total, 11 precursor lesions (48%) revealed BMI-1 expression. In conclusion, the results of this study suggest that loss of p16 expression by promoter hypermethylation is inconsistently and occurs late in the carcinogenic process at the level of severe dysplasia. To what extent overexpression of the polycomb-group protein BMI-1 attributes to down regulating of p16 expression remains unclear.

Targeting of a histone acetyltransferase domain to a promoter enhances protein expression levels in mammalian cells.

Silencing of transfected genes in mammalian cells is a fundamental problem that probably involves the (in)accessibility status of chromatin. A potential solution to this problem is to provide a cell with protein factors that make the chromatin of a promoter more open or accessible for transcription. We tested this by targeting such proteins to different promoters. We found that targeting the p300 histone acetyltransferase (HAT) domain to strong viral or cellular promoters is sufficient to result in higher expression levels of a reporter protein. In contrast, targeting the chromatin-remodeling factor Brahma does not result in stable, higher protein expression levels. The long-term effects of the targeted p300HAT domain on protein expression levels are positively reinforced, when also anti-repressor elements are applied to flank the reporter construct. These elements were previously shown to be potent blockers of chromatin-associated repressors. The simultaneous application of the targeted p300HAT domain and anti-repressor elements conveys long-term stability to protein expression. Whereas no copy number dependency is achieved by targeting of the p300HAT domain alone, copy number dependency is improved when anti-repressor elements are included. We conclude that targeting of protein domains such as HAT domains helps to facilitate expression of transfected genes in mammalian cells. However, the simultaneous application of other genomic elements such as the anti-repressor elements prevents silencing more efficiently.

Transcriptional repression mediated by polycomb group proteins and other chromatin-associated repressors is selectively blocked by insulators.

Polycomb group (PcG) proteins repress gene activity over a considerable distance, possibly by spreading along the chromatin fiber. Insulators or boundary elements, genetic elements within the chromatin, may serve to terminate the repressing action of PcG proteins. We studied the ability of insulators to block the action of chromatin-associated repressors such as PcG proteins, HP1, and MeCP2. We found that the Drosophila special chromatin structure insulator completely blocks transcriptional repression mediated by all of the repressors we tested. The Drosophila gypsy insulator was able to block the repression mediated by the PcG proteins Su(z)2 and RING1, as well as mHP1, but not the repression mediated by MeCP2 and the PcG protein HPC2. The 5'-located DNase I-hypersensitive site in the chicken beta-globin locus displayed a limited ability to block repression, and a matrix or scaffold attachment region element was entirely unable to block repression mediated by any repressor tested. Our results indicate that insulators can block repression mediated by PcG proteins and other chromatin-associated repressors, but with a high level of selectivity. This high degree of specificity may provide a useful assay to define and characterize distinct classes of insulators.

A bipartite sequence element associated with matrix/scaffold attachment regions.

We have identified a MAR/SAR recognition signature (MRS) which is common to a large group of matrix and scaffold attachment regions. The MRS is composed of two degenerate sequences (AATAAYAA and AWWRTAANNWWGNNNC) within close proximity. Analysis of >300 kb of genomic sequence from a variety of eukaryotic organisms shows that the MRS faithfully predicts 80% of MARs and SARs. In each case where we find a MRS, the corresponding DNA region binds specifically to the nuclear scaffold. Although all MRSs are associated with a SAR, not all known SARs and MARs contain a MRS, suggesting that at least two classes exist, one containing a MRS, the other not. Evidence is presented that the two sequence elements of the bipartite MRS occupy a position on the nucleosome near the dyad axis, together creating a putative protein binding site. The identification of a MAR- and SAR-associated DNA element is an important step forward towards understanding the molecular mechanisms of these elements. It will allow: (i) analysis of the genomic location of SARs, e.g. in relationship to genes, based on sequence information alone, rather than on the basis of an elaborate biochemical assay; (ii) identification and analysis of proteins that specifically bind to the MRS.

C-Terminal binding protein is a transcriptional repressor that interacts with a specific class of vertebrate Polycomb proteins.

Polycomb (Pc) is part of a Pc group (PcG) protein complex that is involved in repression of gene activity during Drosophila and vertebrate development. To identify proteins that interact with vertebrate Pc homologs, we performed two-hybrid screens with Xenopus Pc (XPc) and human Pc 2 (HPC2). We find that the C-terminal binding protein (CtBP) interacts with XPc and HPC2, that CtBP and HPC2 coimmunoprecipitate, and that CtBP and HPC2 partially colocalize in large PcG domains in interphase nuclei. CtBP is a protein with unknown function that binds to a conserved 6-amino-acid motif in the C terminus of the adenovirus E1A protein. Also, the Drosophila CtBP homolog interacts, through this conserved amino acid motif, with several segmentation proteins that act as repressors. Similarly, we find that CtBP binds with HPC2 and XPc through the conserved 6-amino-acid motif. Importantly, CtBP does not interact with another vertebrate Pc homolog, M33, which lacks this amino acid motif, indicating specificity among vertebrate Pc homologs. Finally, we show that CtBP is a transcriptional repressor. The results are discussed in terms of a model that brings together PcG-mediated repression and repression systems that require corepressors such as CtBP.

Characterization of interactions between the mammalian polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian polycomb-group protein complexes.

In Drosophila melanogaster, the Polycomb-group (PcG) and trithorax-group (trxG) genes have been identified as repressors and activators, respectively, of gene expression. Both groups of genes are required for the stable transmission of gene expression patterns to progeny cells throughout development. Several lines of evidence suggest a functional interaction between the PcG and trxG proteins. For example, genetic evidence indicates that the enhancer of zeste [E(z)] gene can be considered both a PcG and a trxG gene. To better understand the molecular interactions in which the E(z) protein is involved, we performed a two-hybrid screen with Enx1/EZH2, a mammalian homolog of E(z), as the target. We report the identification of the human EED protein, which interacts with Enx1/EZH2. EED is the human homolog of eed, a murine PcG gene which has extensive homology with the Drosophila PcG gene extra sex combs (esc). Enx1/EZH2 and EED coimmunoprecipitate, indicating that they also interact in vivo. However, Enx1/EZH2 and EED do not coimmunoprecipitate with other human PcG proteins, such as HPC2 and BMI1. Furthermore, unlike HPC2 and BMI1, which colocalize in nuclear domains of U-2 OS osteosarcoma cells, Enx1/EZH2 and EED do not colocalize with HPC2 or BMI1. Our findings indicate that Enx1/EZH2 and EED are members of a class of PcG proteins that is distinct from previously described human PcG proteins.

Interference with the expression of a novel human polycomb protein, hPc2, results in cellular transformation and apoptosis.

Polycomb (Pc) is involved in the stable and heritable repression of homeotic gene activity during Drosophila development. Here, we report the identification of a novel human Pc homolog, hPc2. This gene is more closely related to a Xenopus Pc homolog, XPc, than to a previously described human Pc homolog, CBX2 (hPc1). However, the hPc2 and CBX2/hPc1 proteins colocalize in interphase nuclei of human U-2 OS osteosarcoma cells, suggesting that the proteins are part of a common protein complex. To study the functions of the novel human Pc homolog, we generated a mutant protein, delta hPc2, which lacks an evolutionarily conserved C-terminal domain. This C-terminal domain is important for hPc2 function, since the delta hPc2 mutant protein which lacks the C-terminal domain is unable to repress gene activity. Expression of the delta hPc2 protein, but not of the wild-type hPc2 protein, results in cellular transformation of mammalian cell lines as judged by phenotypic changes, altered marker gene expression, and anchorage-independent growth. Specifically in delta hPc2-transformed cells, the expression of the c-myc proto-oncogene is strongly enhanced and serum deprivation results in apoptosis. In contrast, overexpression of the wild-type hPc2 protein results in decreased c-myc expression. Our data suggest that hPc2 is a repressor of proto-oncogene activity and that interference with hPc2 function can lead to derepression of proto-oncogene transcription and subsequently to cellular transformation.