Karin Ackermann

Genome-wide expression screens indicate a global role for protein kinase CK2 in chromatin remodeling.

Protein kinase CK2, a vital, pleiotropic and highly conserved serine/threonine phosphotransferase is involved in transcription-directed signaling, gene control and cell cycle regulation and is suspected to play a role in global processes. Searching for these global roles, we analyzed the involvement of CK2 in gene expression at cell cycle entry by using genome-wide screens. Comparing expression profiles of Saccharomyces cerevisiae wild-type strains with strains with regulatory or catalytic subunits of CK2 deleted, we found significant alterations in the expression of genes at all cell cycle phases and often in a subunit- and isoform-specific manner. Roughly a quarter of the genes known to be regulated by the cell cycle are affected. Functionally, the genes are involved with cell cycle entry, progression and exit, including spindle pole body formation and dynamics. Strikingly, most CK2-affected genes exhibit no common transcriptional control features, and a considerable proportion of temporarily altered genes encodes proteins involved in chromatin remodeling and modification, including chromatin assembly, (anti-)silencing and histone (de-)acetylation. In addition, various metabolic pathway and nutritional supply genes are affected. Our data are compatible with the idea that CK2 acts at different levels of cellular organization and that CK2 has a global role in transcription-related chromatin remodeling.

Human Primary Osteocyte Differentiation in a 3D Culture System

Studies on primary osteocytes, which compose >90–95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 3D culture method of primary human osteoblast differentiation into osteocytes. These 3D‐differentiated osteocytes were compared with 2D‐cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times, as well as by quantitative RT‐PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Histological analysis showed, after only 1 wk, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte‐like cells. Real‐time PCR expression of a set of bone‐related genes confirmed their osteocyte phenotype. Comparison with plastic‐cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid‐osteocytes. This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast‐osteocyte differentiation pathway, the osteocyte interaction with the other bone cells, and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the cross‐talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.

Bone metastasis: Osteoblasts affect growth and adhesion regulons in prostate tumor cells and provoke osteomimicry.

Bone metastasis is the primary cause of death in human prostate cancer. Disseminated from primary tumor and distributed via the bloodstream, a proportion of prostate carcinoma cells eventually reach the skeleton and develop into metastases, requiring adhesion to inner bone surfaces lined by osteoblasts. The crosstalk of tumor cells with osteoblasts is a critical but poorly characterized step in the metastatic process. Using an in vitro metastasis model system, we have been examining effects of osteoblast‐released factors on gene expression of prostate carcinoma cells. Here, we show by large‐scale transcript profiling and quantitative RT‐PCR that osteoblast‐released factors target in particular the proliferation and adhesion regulons of tumor cells. Genes encoding components of the cell‐cycle control machinery and connected pathways are predominantly repressed and cell proliferation is slowed down, resembling in vivo observations assumed to render commonly used chemotherapeutic measures ineffective. Genes encoding anchoring junction components are predominantly elevated, and the adhesion properties of tumor cells are altered. Moreover, prostate carcinoma cells are provoked to undergo osteomimicry, i.e., to express bone cell–related genes. The data indicate that the crosstalk with osteoblasts induces expressional changes in prostate carcinoma cells favoring the bone colonization process.

Cystic Duct Dilatations and Proliferative Epithelial Lesions in Mouse Mammary Glands upon Keratin 5 Promoter-Driven Overexpression of Cyclooxygenase-2

Expression and pharmacological studies support a contribution of cyclooxygenase (COX)-2 to mammary gland tumorigenesis. In a recent transgenic study, mouse mammary tumor virus promoter-driven COX-2 expression in mouse mammary glands was shown to result in alveolar hyperplasia, dysplasia, and carcinomas after multiple rounds of pregnancy and lactation. In the study presented here, the effects of constitutive COX-2 overexpression in keratin 5-positive myoepithelial and luminal cells, driven by the keratin 5 promoter in a hormone-independent manner, was investigated. In nulliparous female mice, aberrant COX-2 overexpression correlated with increased prostaglandin (PG) E(2) levels and caused cystic duct dilatations, adenosis, and fibrosis whereas carcinomas developed rarely. This phenotype depended on COX-2-mediated PGE(2) synthesis and correlated with increased expression of proliferation-associated Ki67 in epithelial cells. No changes in the expression of apoptosis-related Bcl-2, caspase 3, or p53 were observed. Hyperproliferation of the mammary gland epithelial cells was associated with increased aromatase mRNA levels in this tissue. The spontaneous pathologies bear analogies to the human breast with fibrocystic changes. Intriguingly, strong COX-2 expression was observed in fibrocystic changes, as compared to low expression in normal breast epithelium. These results show for the first time that aberrant COX-2 expression contributes to the development of fibrocystic changes (FC), indicating that COX-2 and COX-2-mediated PG synthesis represent potential targets for the therapy of this most frequent benign disorder of the human breast.

The genes encoding human protein kinase CK2 and their functional links.

Abstract Protein kinase CK2 is a Ser⧸Thr phosphotransferase that occurs ubiquitously among eukaryotes. It is pleiotropic, vital, and highly conserved. CK2 is a tetramer composed of two catalytic (α) and two regulatory (β) subunits. Both subunits may occur in isoforms and both may play roles independent of the holoenzymes they form. Humans express α, α′, and β subunits. The human genome contains four CK2 loci at different chromosomes, enclosing three active genes and a pseudogene. This chapter reviews the chromosomal location, structural organization, and expressional control of the genes. It shows that CK2s conservation can also be recognized at the nucleic acid level, that the three active genes have features in common, and that some of these are appropriate for a coordinate transcriptional regulation. In particular, an identical Ets1 double motif that cross-talks to multiple Sp1 (and other) sites is present in the α and β gene promoters, and CK2 holoenzyme but not CK2α phosphorylates Sp1, resulting in a loss of DNA binding. This is compatible with a negative feedback control according to which expression of α and β genes leads to an increased holoenzyme level and thus phosphorylation, which, in turn, decreases transcription. As a consequence, constant transcript levels of both genes are expected to adjust. In human cultured cells, this is indeed the case, independent of their respective proliferation or differentiation status. The chapter also provides an overview of functional links of the CK2 genes to cell-cycle-regulated genes. Based on comparative genome-wide transcript profiles of Saccharomyces cerevisiae wild-type and CK2 mutant strains, CK2 is shown to be involved in transcription regulation of various genes related to cell-cycle control, including genes encoding cyclins and components of spindle pole body formation and dynamics. Strikingly, most of the affected genes lack common elements in their promoters and expression of a large group of genes encoding chromatin remodeling factors is altered, compatible with the idea that CK2 plays a role in the global process of transcription-related chromatin remodeling. In addition, functional links of CK2 are seen to diverse metabolic and nutritional supply pathways, including MET genes responsible for methionine synthesis, and the PHO gene group responsible for phosphate maintenance, which, interestingly, is uncoupled from its central cyclin-Cdk control upon CK2 perturbation.

Serum‐stimulated cell cycle entry of fibroblasts requires undisturbed phosphorylation and non‐phosphorylation interactions of the catalytic subunits of protein kinase CK2

Protein kinase CK2 is a pleiotropic Ser/Thr kinase occurring as α2β2, α′2β2, or αα′β2 tetramers. A requirement in serum‐stimulated cell cycle entry in both the cytoplasm and the nucleus of human fibroblasts for phosphorylation(s) by CK2 has been concluded from stimulation inhibition by microinjected antibodies against the regulatory subunit (β). We have now examined this idea more directly by microinjection‐mediated perturbation of phosphorylation and non‐phosphorylation interactions of the catalytic subunits (α and α′), and by verifying the supposed matching of the cellular partition of CK2 subunits in the fibroblasts employed. While immunostaining and cell fractionation indicate that the partitions of subunits indeed match each other (with their predominant location in the nucleus in both quiescent and serum‐stimulated cells), microinjection of substrate or pseudosubstrate peptides competing for the CK2‐mediated phosphorylation in vitro resulted in significant inhibition of serum stimulation when placed into the nucleus but not when placed into the cytoplasm. Also inhibitory were nuclear but not cytoplasmic injections of antibodies against α and α′ that affect neither their kinase activity in vitro nor their complexing to β. The data indicate that the role played by CK2 in serum‐stimulated cell cycle entry is predominantly nuclear and more complex than previously assumed, involving not only phosphorylation but also experimentally separable non‐phosphorylation interactions by the catalytic subunits.

Genes targeted by protein kinase CK2: a genome-wide expression array analysis in yeast.

Protein kinase CK2, a tetramer composed of two catalytically active (CK2α isoforms) and two regulatory (CK2β isoforms) subunits, is suspected to have, among others, a role in gene transcription. To identify the genes targeted by CK2, the transcriptional effect of silencing the CK2 subunit genes in Saccharomyces cerevisiae (CK2α isoform genes: CKA1 and CKA2; CK2β isoform genes: CKB1 and CKB2) was examined using genome-wide expression array analysis (oligonucleotide array chips). Silencing did not influence the overwhelming majority (5801) of the over six thousand open reading frames composing the yeast genome. Cells knocked-out for both CKA1 and CKA2 and plasmid-rescued by Ckal affected specifically at 2-fold discrimination level the transcription of 57 genes, and when rescued by Cka2, the transcription of 118 genes. In CKB1/CKB2 double knock-outs, transcription of 54 genes was specifically altered. Interestingly, aside overlaps between the gene spectra affected by CKA1 and CKA2 silencing, there were overlaps also between those influenced by CK2α and CK2β isoform silencing. The data indicate a distinct role of CK2 in gene transcription control, identify specific functional differences between the two catalytic subunits in gene targeting, and reveal independent effects by the regulatory subunits. (Mol Cell Biochem 227: 59–66, 2001)

The catalytic subunit α′ gene of human protein kinase CK2 (CSNK2A2): Genomic organization, promoter identification and determination of Ets1 as a key regulator

The human genome contains four protein kinase CK2 loci, enclosing three active genes coding for the catalytic subunits α and α′ and the regulatory subunit β, and a processed α subunit pseudogene. Extensive structure and transcriptional control data of the genes are available, except for the CK2α′ gene (CSNK2A2). Using in silico and experimental approaches, we find CSNK2A2 to be located on the long arm of chromosome 16 (in contrast to published data), to span 40 kb and to consist of 12 exons, with the translational start in Exon 1 and the stop in Exon 11. Exon/intron boundaries conform to the gt/ag rule, and various potential polyadenylation signals determine transcript species with lengths of 1.7–5.7 kb. The upstream region of the gene displays housekeeping characteristics, lacking a TATA box and possessing several GC boxes as well as a CpG island around Exon 1. According to reporter gene assay results, the promoter activity ranges from −1308 to 197 with the highest activity in region −396 to −129. This region contains binding motifs for various transcription factors, including NFκB, Sp and Ets family members. Site-directed mutagenesis indicates that the Ets motifs play, in cooperation with Sp motif clusters, a central role in regulating CK2α′ gene transcription. A similar control has been described for the transcription of the CK2α and CK2β genes so that the presented data are compatible with the assumption of a coordinate transcriptional regulation of all three active human CK2 genes decisively determined by Ets family members.

Transcriptional coordination of the genes encoding catalytic (CK2α) and regulatory (CK2β) subunits of human protein kinase CK2

Little is known of how protein kinase CK2 genes are regulated, and it is unclear whether there are mechanisms of transcriptional coordination. Response elements present in the promoter sequences of the human catalytic (CK2α) and regulatory (CK2β) subunit genes have been examined for the significance in transcriptional control using reporter gene assays, electrophoretic mobility shift assays, site-directed mutagenesis, ectopic protein expressions, and transcript assessments. Most strikingly, in both promoters the regions of highest transcriptional activity contain two adjoining, completely identical and conserved Ets1 response elements, and both the mutation of motifs and the overexpression of Ets1 affect significantly transcriptional activity. Also in common are Sp1 response elements that cooperate with Ets1, and Sp1 is phosphorylatable by CK2 holoenzyme but not by individual CK2α, the phosphorylation negatively affecting DNA binding. CK2α and CK2β transcript levels and stoichiometries of mRNA species turned out quite constant in cultured cells despite progressing through various stages of proliferation and differentiation. The data seem to indicate transcriptional coordination of the human genes encoding CK2α and CK2β based on an Ets1 double motif common to both genes cooperating with Sp1 motifs and amenable to negative feedback control by the gene products which, following complexation into CK2 holoenzyme, could phosphorylate Sp1 (and Ets1?) and thus downregulate transcription and contribute to the observed constant cellular CK2α and CK2β transcripts situation.

Protein kinase CK2 in gene control at cell cycle entry.

Protein kinase CK2 has diverse links to gene control and cell cycle. Comparative genome-wide expression profiling of CK2 mutants of the budding yeast Saccharomyces cerevisiae at cell cycle entry has revealed that a significant proportion of cell-cycle genes are affected by CK2. Here, we examine how CK2 realizes this effect. We show that the CK2 action may be directed to gene promoters causing genes with promoter homologies to respond comparably to CK2 perturbation. Examples are metabolic pathway and nutrition supply genes such as the PHO and MET regulon genes, responsible for phosphate maintenance and methionine biosynthesis, respectively. CK2 perturbation affects both regulons permanently and both via repression of a central transcription factor, but with different mechanisms: In the PHO regulon, the gene encoding the central transcription factor Pho4 is repressed and, in addition, Pho4 and/or the cyclin-dependent kinase of the regulon’s control complex may be affected by CK2 phosphorylation. In the MET regulon, the repression of the central transcription factor Met4 occurs not by expression inhibition, but rather by availability tuning via a CK2-mediated phosphorylation of a degradation complex. On the other hand, the CK2 action may be directed to the chromatin regulon, thus affecting globally the expression of genes, i.e., the CK2 perturbation results either in comparable responses of genes which have no promoter homologies or in deviating responses despite promoter homologies. The effect is rather transient and concerns aside various cell cycle control genes a notable number of genes encoding chromatin remodeling and modification proteins with functions in chromatin assembly and (anti-)silencing as well as in histone (de-)acetylation, and frequently are also substrates of CK2, suggesting additional tuning at protein level. In line with these findings, we observe in human cells sequence-independent but cell-cycle-dependent CK2 associations with promoters of cell-cycle-regulated genes at periods of extensive gene expression alterations, including cell cycle entry. Our observations are compatible with the idea that the gene control by CK2 is achieved via different mechanisms and at different levels of organization and includes a global role in transcription-related chromatin remodelling and modification.