Gene knockdown studies revealed CCDC50 as a candidate gene in mantle cell lymphoma and chronic lymphocytic leukemia.

The two B-cell non-Hodgkin's lymphoma entities, chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), show recurrent chromosomal gains of 3q25-q29, 12q13-q14 and 18q21-q22. The pathomechanisms affected by these aberrations are not understood. The aim of this study was to identify genes, located within these gained regions, which control cell death and cell survival of MCL and CLL cancer cells. Blood samples collected from 18 patients with CLL and 6 patients with MCL, as well as 6 cell lines representing both malignancies were analyzed by gene expression profiling. By a comparison of genomic DNA and gene expression, 72 candidate genes were identified. We performed a limited RNA interference screening with these candidates to identify genes affecting cell survival. CCDC50 (coiled coil domain containing protein 50), SERPINI2 and SMARCC2 mediated a reduction of cell viability in primary CLL cells as well as in cell lines. Gene knockdown and a nuclear factor kappa B (NFkappaB) reporter gene assay revealed that CCDC50 is required for survival in MCL and CLL cells and controls NFkappaB signaling.

FHL2, a novel tissue-specific coactivator of the androgen receptor.

The control of target gene expression by nuclear receptors requires the recruitment of multiple cofactors. However, the exact mechanisms by which nuclear receptor-cofactor interactions result in tissue-specific gene regulation are unclear. Here we characterize a novel tissue-specific coactivator for the androgen receptor (AR), which is identical to a previously reported protein FHL2/DRAL with unknown function. In the adult, FHL2 is expressed in the myocardium of the heart and in the epithelial cells of the prostate, where it colocalizes with the AR in the nucleus. FHL2 contains a strong, autonomous transactivation function and binds specifically to the AR in vitro and in vivo. In an agonist- and AF-2-dependent manner FHL2 selectively increases the transcriptional activity of the AR, but not that of any other nuclear receptor. In addition, the transcription of the prostate-specific AR target gene probasin is coactivated by FHL2. Taken together, our data demonstrate that FHL2 is the first LIM-only coactivator of the AR with a unique tissue-specific expression pattern.

Activation of somatostatin receptor II expression by transcription factors MIBP1 and SEF-2 in the murine brain.

Somatostatin receptor type II expression in the mammalian brain displays a spatially and temporally very restricted pattern. In an investigation of the molecular mechanisms controlling these patterns, we have recently shown that binding of the transcription factor SEF-2 to a novel initiator element in the SSTR-2 promoter is essential for SSTR-2 gene expression. Further characterization of the promoter identified a species-conserved TC-rich enhancer element. By screening a mouse brain cDNA expression library, we cloned a cDNA encoding the transcription factor MIBP1. MIBP1 interacts specifically with both the TC box in the SSTR-2 promoter and with the SEF-2 initiator-binding protein to enhance transcription from the basal SSTR-2 promoter. We then investigated SSTR-2, SEF-2, and MIBP1 mRNA expression patterns in the developing and adult murine brain by Northern blotting and in situ hybridization. While SEF-2 is widely expressed in many neuronal and nonneuronal tissues, MIBP1 expression overlapped precisely with expression of SSTR-2 in the frontal cortex and hippocampus. In summary, our data for the first time define a regulatory role for the transcription factor MIBP1 in mediating spatially and temporally regulated SSTR-2 expression in the brain.

Enhanced apoptotic cell death of renal epithelial cells in mice lacking transcription factor AP-2beta.

Expression of AP-2 transcription factors has been detected previously in embryonic renal tissues. We show here that AP-2beta -/- mice complete embryonic development and die at postnatal days 1 and 2 because of polycystic kidney disease. Analyses of kidney development revealed that induction of epithelial conversion, mesenchyme condensation, and further glomerular and tubular differentiation occur normally in AP-2beta-deficient mice. At the end of embryonic development expression of bcl-X(L), bcl-w, and bcl-2 is down-regulated in parallel to massive apoptotic death of collecting duct and distal tubular epithelia. Addressing the molecular mechanism we show that transfection of AP-2 into cell lines in vitro strongly suppresses c-myc-induced apoptosis pointing to a function of AP-2 in programming cell survival during embryogenesis. The position of the human AP-2beta gene was identified at chromosome 6p12-p21.1, within a region that has been mapped for autosomal recessive polycystic kidney disease (ARPKD). Sequence analyses of ARPKD patients and linkage analyses using intragenic polymorphic markers indicate that the AP-2beta gene is located in close proximity to but distinct from the ARPKD gene.

The helix-loop-helix transcription factor SEF-2 regulates the activity of a novel initiator element in the promoter of the human somatostatin receptor II gene.

The effects of somatostatin hormones are mediated by a family of five different seven-helix transmembrane spanning receptors (SSTR1-5). The expression of the five different SSTR subtypes displays a complex temporal- and tissue-specific pattern. To investigate the molecular mechanisms controlling the different expression patterns of the SSTRs, we cloned the 5'-flanking region of the human SSTR2 gene. Characterization of the SSTR2 promoter resulted in the identification of a novel initiator element (SSTR2inr). Transcriptional activity of the SSTR2inr is dependent on the presence of a binding site (E-box) for basic helix-loop-helix (bHLH) transcription factors. By screening a mouse brain cDNA expression library we isolated a cDNA coding for the bHLH transcription factor SEF-2. SEF-2 binds to the E-box present in the SSTR2inr, both in vitro and in vivo and activates transcription from the SSTR2inr. A single point mutation within the E-box eliminates binding of SEF-2 and results in a complete loss of transcriptional activity of the SSTR2inr. Furthermore, DNA binding studies demonstrate that the basal transcription factor TFIIB can be tethered to the SSTR2inr through physical interaction with SEF-2. In summary, the SSTR2inr represents a novel type of initiator element that confers gene expression in the absence of a TATA-box or binding sites for other known initiator factors, like YY-1 or USF.

Cloning and characterization of a second AP-2 transcription factor: AP-2 beta.

AP-2 has been characterized previously as a unique 52 x 10(3) M(r) transcription activator encoded by a single gene that is expressed in a restricted pattern during embryonic morphogenesis of the peripheral nervous system, face, skin and nephric tissues. Here we report the isolation of genomic and cDNA clones encoding for a second AP-2 related transcription factor, designated AP-2 beta. AP-2 beta binds specifically to a series of well-characterized AP-2 binding sites, consensus to the sequence G/CCCN3GGC, and transactivates transcription from a reporter plasmid under the control of an AP-2-dependent promoter. A C-terminal domain known to mediate homodimerization of the previously cloned AP-2 alpha transcription activator is highly conserved and sufficient to mediate interaction between the two proteins. Northern blot and in situ hybridizations revealed that the two genes are expressed in murine embryos between days 9.5 and 19.5 p.c. Coexpression of both mRNAs was detected in many tissues at day 13.5 and 15.5 of embryogenesis but some regions of the developing brain and face including the primordium of midbrain and the facial mesenchyme differed in their expression pattern of AP-2 genes. AP-2 alpha and AP-2 beta signals in the central and peripheral nervous system overlapped with regions of developing sensory neurons. In adult tissues AP-2 alpha expression was found mainly in the skin, eye and prostate and AP-2 beta expression in the kidney. In summary, our analyses of embryonic and adult mice demonstrate that two different AP-2 transcription factors are specifically expressed during differentiation of many neural, epidermal and urogenital tissues.

The genomic structure of the human AP-2 transcription factor.

The transcription factor AP-2 is encoded by a gene located on chromosome 6 near the HLA locus. Here we describe the genomic organization of the AP-2 gene including an initial characterization of the promoter. We have mapped two mRNA initiation sites, the entire exon-intron structure and located two polyadenylation sites. The mature AP-2 mRNA is spliced from 7 exons distributed over a region of 18 kb genomic DNA. A recently cloned inhibitory AP-2 protein is generated by alternative usage of a C-terminal exon. The proline-rich transactivation motif is encoded by a single exon within the N-terminal region in contrast to the complex DNA binding and dimerization motif which involves amino acid residues located on four different exons. The sites of mRNA initiation are located 220 and 271 bases upstream from the ATG translation start site. Although the promoter contains no canonical sequence motifs for basal transcription factors, such as TATA-, CCAAT- or SP-1 boxes, it mediates cell-type-specific expression of a CAT reporter gene in PA-1 human teratocarcinoma cells and is inactive in murine F9 teratocarcinoma cells. We demonstrate that the promoter of the AP-2 gene is subject to positive autoregulation by its own gene product. A consensus AP-2 binding site is located at position -622 with respect to the ATG. This site binds specifically to bacterially expressed AP-2 as well as to multiple proteins, including AP-2, present in PA-1 and HeLa cell nuclear extracts. A partial AP-2 promoter fragment including the AP-2 consensus binding site is approximately 5-fold transactivated by cotransfection of an AP-2 expression plasmid.

[Molecular cloning of a new AP-2 transcription factor, AP-2beta, and its function in cell differentiation]. / Molekulare Klonierung eines neuen AP-2 Transkriptionsfaktors, AP-2 beta, und seine Funktion in der Zelldifferenzierung.

Transcription factor AP-2 has been previously shown to play an important function in embryonal development and cell differentiation. We have investigated the possibility that AP-2 function in embryonic development is exerted by a multigene family of AP-2 related transcription factors. Here we describe the molecular cloning of such an AP-2 related gene, AP-2 beta, and prove that it encodes for a functional transcription factor. In situ hybridizations of murine embryo sections revealed a temporally restricted and tissue-specific expression pattern that indicates a function of AP-2 beta in the development of the midbrain in the differentiation of sensory neurons for taste, olfaction and palpation.