General, negative feedback mechanism for regulation of Trithorax-like gene expression in vivo: new roles for GAGA factor in flies

Expression of every gene is first regulated at the transcriptional level. While some genes show acute and discrete periods of expression others show a rather steady expression level throughout development. An example of the latter is Trithorax-like (Trl) a member of the Trithorax group that encodes GAGA factor in Drosophila. Among other functions, GAGA factor has been described to stimulate transcription of several genes, including some homeotic genes. Here we show that GAGA factor is continuously down-regulating the expression of its own promoter using a negative feedback mechanism in vivo. Like its expression, repression by GAGA factor is ubiquitous, prevents its accumulation, and takes place throughout development. Experimental alteration of GAGA factor dosage results in several unexpected phenotypes, not related to alteration of homeotic gene expression, but rather to functions that take place later during development and affect different morphogenetic processes. The results suggest that GAGA factor is essential during development, even after homeotic gene expression is established, and indicate the existence of an upper limit for GAGA factor dosage that should not be exceeded

Identification of functionally important acidic residues in transducin by group-specific labeling

Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the a-subunit (Ta) and the bg-complex (Tbg). The role of the carboxyl groups in T was evaluated by labeling with N, N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of b, g-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when Ta was combined with DCCD-treated Tbg. However, the binding of guanine nucleotides to the reconstituted T was ~50% inhibited when DCCD-labeled Ta was incubated with Tbg. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified Ta was combined with Tbg, and when EDC-treated Tbg was incubated with T

GAGA factor repression of transcription is a rare event but the negative regulation of Trl is conserved in Drosophila species

GAGA is a highly conserved Drosophila transcription factor encoded by the Trithorax-like (. Trl) gene. While GAGA usually activates transcription, it represses its own promoter. Here we show that GAGA-mediated repression of Trl is conserved between two distant Drosophila species. A detailed promoter study showed that GAGA repressive activity can't be attributed to any discrete element in the Trl promoter. Genome-wide analysis of the transcriptome in S2 cells indicated that repression of Trl is very likely unique, being GAGA factor a transactivator for all the other promoters. Taken together, our results suggest a new mechanism to explain GAGA-mediated repression that involves a dose-dependent change in the architecture of the Trl promoter. © 2013 Elsevier B.V.This work was supported by grants of the Ministerio de Educación y Ciencia of the Spanish Government[BFU-2007-64395/BMC], the MICINN (CSD2006-49 and BFU2009-07111) and the Generalitat de Catalunya (SGR2009-1023). D. Piñeyro was supported by FPU fellowship from the Spanish Government. M. Blanch was supported by I3P CPG_06_0034 contract from CSIC, and grant BFU-2007-64395/BMC contract of the Spanish Government. M. Badal was supported by grant BFU-2007-64395/BMC contract of the Spanish Government. This work was carried out in the frame of the “Centre de Referència en Biotecnologia” of the Generalitat de Catalunya.Peer Reviewe

Resisting arrest: Recovery from checkpoint arrest through dephosphorylation of Chk1 by PP1

The G DNA damage checkpoint prevents mitotic entry in the presence of damaged DNA, and thus is essential for cells to replicate with stable genetic inheritance. Whilst significant progress has been made in the past 10 years on the mechanism of checkpoint activation, little attention has been paid to how the DNA damage checkpoint is switched off to allow cell cycle re-entry. Insight into the mechanism of cell cycle re-entry was recently provided by our finding that the Schizosaccharomyces pombe type 1 phosphatase (PP1] Dis2 dephosphorylates the checkpoint effector kinase Chk1. This occurs on a site phosphorylated by the ATR homologue Rad3 in response to DNA damage, and results in Chk1 inactivation and checkpoint release. Here we discuss the implications of this finding on DNA damage checkpoint signaling, and speculate on models for checkpoint maintenance and release

Repression by TTK69 of GAGA-mediated Activation Occurs in the Absence of TTK69 Binding to DNA and Solely Requires the Contribution of the POZ/BTB Domain of TTK69

tramtrack 69 (TTK69) is known to repress GAGA-mediated activation of the eve promoter in S2 cells. Here, we show that repression by TTK69 occurs in the absence of bona fide TTK69-binding sites on the template, indicating that it does not require the binding of TTK69 to DNA. Consistent with this interpretation, the POZ/BTB domain of TTK69, which does not bind DNA, is sufficient for repression. Moreover, a fusion protein in which the POZ/BTB domain of GAGA is replaced by that of TTK69 is not capable of activating the eve promoter but efficiently represses GAGA-dependent activation. Repression involves GAGA-TTK69 interaction because TTK69 is not capable of repressing basal transcription. Most probably, GAGA-TTK69 interaction occurs at the promoter because GAGA·TTK69 complexes are fully competent in binding DNA in vitro. Our results also show that repression by TTK69 of GAGA-dependent activation of the eve promoter is not mediated by any of the co-repressors known to interact with TTK69 (dMi2 or C-terminal binding protein) or by trichostatin A-sensitive histone deacetylases. Altogether, these observations strongly suggest that the binding of TTK69 prevents the interaction of GAGA with the transcription machinery and, therefore, compromises its activation potentialThis work was financed by grants from the Ministerio de Ciencia y Tecnología (BMC2000-878 and BMC2000-898) and the Comissió Interdepartamental de Recerca i Innovació Tecnològica (CIRIT) (2001SGR-00344). This work was carried out within the framework of the Centre de Referència en Biotecnologia de la Generalitat de CatalunyaPeer reviewe

GAGA factor down-regulates its own promoter

GAGA factor is involved in many nuclear transactions, notably in transcription as an activator in Drosophila. The genomic region corresponding to the Trl promoter has been obtained, and a minimal version of a fully active Trl promoter has been defined using transient transfection assays in S2 cells. DNase I footprinting analysis has shown that this region contains multiple GAGA binding sites, suggesting a potential regulatory role of GAGA on its own promoter. The study shows that GAGA down-regulates Trl expression. The repression does not depend on the GAGA isoform, but binding to DNA is absolutely required. A fragment of the Trl promoter can mediate repression to a heterologous promoter only upon GAGA overexpression in transiently transfected S2 cells. Chromatin immunoprecipitation analysis of S2 cells confirmed that GAGA factors are bound to the Trl promoter over a region of 1.4 kbp. Using a double-stranded RNA interference approach, we show that endogenous GAGA factors limit Trl expression in S2 cells. Our results open the possibility of observing similar GAGA repressive effects on other promoters.This work was supported by Grants BMC2000-0898 from the Spanish “Ministerio de Ciencia y Tecnologı́a” and SGR97-55 from the “Comissió Interdepartamental de Recerca i Innovació Tecnològica” (CIRIT) of the Generalitat de Catalunya. This work was carried out in the context of the “Centre de Referència en Biotecnologia” of CIRIT, the Generalitat de CatalunyaPeer Reviewe

Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas

We have performed cDNA microarray analyses to identify gene expression differences between highly invasive glioblastoma multiforme (GBM) and typically benign pilocytic astrocytomas (PA). Despite the significant clinical and pathological differences between the 2 tumor types, only 63 genes were found to exhibit 2-fold or greater overexpression in GBM as compared to PA. Forty percent of these genes are related to the regulation of the cell cycle and mitosis. QT-PCR validation of 6 overexpressed genes: MELK, AUKB, ASPM, PRC1, IL13RA2 and KIAA0101 confirmed at least a 5-fold increase in the average expression levels in GBM. Maternal embryonic leucine zipper kinase (MELK) exhibited the most statistically significant difference. A more detailed investigation of MELK expression was undertaken to study its oncogenic relevance. In the examination of more than 100 tumors of the central nervous system, we found progressively higher expression of MELK with astrocytoma grade and a noteworthy uniformity of high level expression in GBM. Similar level of overexpression was also observed in medulloblastoma. We found neither gene promoter hypomethylation nor amplification to be a factor in MELK expression, but were able to demonstrate that MELK knockdown in malignant astrocytoma cell lines caused a reduction in proliferation and anchorage-independent growth in in vitro assays. Our results indicate that GBM and PA differ by the expression of surprisingly few genes. Among them, MELK correlated with malignancy grade in astrocytomas and represents a therapeutic target for the management of the most frequent brain tumors in adult and children. (C) 2007 Wiley-Liss, Inc