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2002

A heterochromatin protein 1 homologue in Caenorhabditis elegans acts in germline and vulval development.

Author(s) : Couteau F, Guerry F, Muller F, Palladino F,
Journal : EMBO Rep
2002
Proteins of the highly conserved heterochromatin protein 1 (HP1) family have been found to function in the dynamic organization of nuclear architecture and in gene regulation throughout the eukaryotic kingdom. In addition to being key players in heterochromatin-mediated gene silencing, HP1 proteins may also contribute to thetranscriptional repression of euchromatic genes via the recruitment to specific promoters. To investigate the role played by these different activities in specific developmental pathways, we identified HP1 homologues in the genome of Caenorhabditis elegans and used RNA-mediated interference to study their function. We show that one of the homologues, HPL-2, is required for the formation of a functional germline and for the development of the vulva by acting in an Rb-related pathway. We suggest that, by acting as repressors of gene expression, HP1 proteins may fulfil specific functions in both somatic and germline differentiation processes throughout development.

Centromeres become unstuck without heterochromatin.

Author(s) : Bernard P, Allshire R,
Journal : Trends Cell Biol
2002
In most if not all eukaryotes, sister-chromatid cohesion, which is mediated by the chromosomal complex Cohesin, is destroyed by proteolysis at the transition from metaphase to anaphase. In metazoans, Cohesin is removed from chromosomes intwo steps, and the centromere and its associated pericentric heterochromatin constitute the last point of linkage between sister chromatids at metaphase. Mechanistic insight is now emerging on the way in which cells distinguish cohesion at the centromere from cohesion along chromosome arms. We discuss recent advances in our understanding of the role of centromeric heterochromatin in sister-chromatid cohesion and propose a causal relationship between this specialized type of chromatin and the removal by proteolysis of Cohesins that are associated with it.

Organizer activity of the polar cells during Drosophila oogenesis.

Author(s) : Grammont M, Irvine K,
Journal : Development
2002
Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and arespecified by the activation of Notch. We have investigated their role in follicle patterning by creating clones of cells mutant for the Notch modulator fringe. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells, the immediately adjacent follicle cell fate, are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. Our observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis.

Phosphorylation of the RNA polymerase II carboxyl-terminal domain by CDK9 is directly responsible for human immunodeficiency virus type 1 Tat-activated transcriptional elongation.

Author(s) : Kim Y, Bourgeois C, Isel C, Churcher M, Karn J,
Journal : Mol Cell Biol
2002
Stimulation of transcriptional elongation by the human immunodeficiency virus type 1 Tat protein is mediated by CDK9, a kinase that phosphorylates the RNA polymerase II carboxyl-terminal domain (CTD). In order to obtain direct evidencethat this phosphorylation event can alter RNA polymerase processivity, we prepared transcription elongation complexes that were arrested by the lac repressor. The CTD was then dephosphorylated by treatment with protein phosphatase 1. The dephosphorylated transcription complexes were able to resume the transcription elongation when IPTG (isopropyl-beta-D-thiogalactopyranoside) and nucleotides were added to the reaction. Under these chase conditions, efficient rephosphorylation of the CTD was observed in complexes containing the Tat protein but not in transcription complexes prepared in the absence of Tat protein. Immunoblots and kinase assays with synthetic peptides showed that Tat activated CDK9 directly since the enzyme and its cyclin partner, cyclin T1, werepresent at equivalent levels in transcription complexes prepared in the presenceor absence of Tat. Chase experiments with the dephosphorylated elongation transcription complexes were performed in the presence of the CDK9 kinase inhibitor DRB (5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole). Under these conditions there was no rephosphorylation of the CTD during elongation, and transcription through either a stem-loop terminator or bent DNA arrest sequence was strongly inhibited. In experiments in which the CTD was phosphorylated priorto elongation, the amount of readthrough of the terminator sequences was proportional to the extent of the CTD modification. The change in processivity is due to CTD phosphorylation alone, since even after the removal of Spt5, the second substrate for CDK9, RNA polymerase elongation is enhanced by Tat-activated CDK9 activity. We conclude that phosphorylation of the RNA polymerase II CTD by CDK9 enhances transcription elongation directly.

Properties of a chimeric simian-human immunodeficiency virus expressing an hybrid HIV-1 Nef/SIVmac Nef protein.

Author(s) : Bertsch C, Cluet D, Beyer C, Gloeckler L, Cecile A, Gut J, Aubertin A,
Journal : Arch Virol
2002
The nef genes of human and simian immunodeficiency viruses code for a membrane associated protein critical for AIDS development. SIVmac Nef presents C-terminala 27 amino acid extension absent of HIV-1 Nef. To estimate the influence of thisC-terminal domain on virus properties, we constructed viruses derived from SIVmac239 by replacing SIV nef with HIV-1 Lai nef gene (SHIV NefLai4) or with a sequence encoding a Nef fusion protein: HIV-1 Lai Nef/SIV Nef-Cterm (SHIV-Cterm). The recombinant viruses replicated efficiently in vitro in CEMx174 cells and in activated macaque PBMCs. The addition of SIV Nef C-terminal domain to HIV-1 Nef in SHIVNefLai4 did not change the in vitro properties of the chimeric virus, both viruses being more infectious than a nef deleted virus. Although the half-life of Nef fusion protein was augmented, SHIV-Cterm remained slightly less infectious than SIVmac239.

Spt5 cooperates with human immunodeficiency virus type 1 Tat by preventing premature RNA release at terminator sequences.

Author(s) : Bourgeois C, Kim Y, Churcher M, West M, Karn J,
Journal : Mol Cell Biol
2002
The human immunodeficiency virus type 1 (HIV-1) Tat protein activates transcription elongation by stimulating the Tat-activated kinase (TAK/p-TEFb), aprotein kinase composed of CDK9 and its cyclin partner, cyclin T1. CDK9 is able to hyperphosphorylate the carboxyl-terminal domain (CTD) of the large subunit ofRNA polymerase during elongation. In addition to TAK, the transcription elongation factor Spt5 is required for the efficient activation of transcriptional elongation by Tat. To study the role of Spt5 in HIV transcription in more detail, we have developed a three-stage Tat-dependent transcription assay that permits the isolation of active preinitiation complexes, early-stage elongation complexes, and Tat-activated elongation complexes. Spt5 is recruited in the transcription complex shortly after initiation. After recruitment of Tat during elongation through the transactivation response element RNA, CDK9 is activated and induces hyperphosphorylation of Spt5 in parallel to the hyperphosphorylation of the CTD of RNA polymerase II. However, immunodepletion experiments demonstrate that Spt5 is not required for Tat-dependent activation of the kinase. Chase experiments using the Spt5-depleted extracts demonstrate that Spt5 is not required for early elongation. However, Spt5 plays an important rolein late elongation by preventing the premature dissociation of RNA from the transcription complex at terminator sequences and reducing the amount of polymerase pausing at arrest sites, including bent DNA sequences. This novel biochemical function of Spt5 is analogous to the function of NusG, an elongationfactor found in Escherichia coli that enhances RNA polymerase stability on templates and shows sequence similarity to Spt5.

Strong-association-rule mining for large-scale gene-expression data analysis: a case study on human SAGE data.

Author(s) : Becquet C, Blachon S, Jeudy B, Boulicaut J, Gandrillon O,
Journal : Genome Biol
2002
BACKGROUND: The association-rules discovery (ARD) technique has yet to be applied to gene-expression data analysis. Even in the absence of previous biological knowledge, it should identify sets of genes whose expression is correlated. The first association-rule miners appeared six years ago and proved efficient at dealing with sparse and weakly correlated data. A huge international research effort has led to new algorithms for tackling difficult contexts and these are particularly suited to analysis of large gene-expression matrices. To validate the ARD technique we have applied it to freely available human serial analysis of gene expression (SAGE) data. RESULTS: The approach described here enables us to designate sets of strong association rules. We normalized the SAGE data before applying our association rule miner. Depending on the discretization algorithm used, different properties of the data were highlighted. Both common and specific interpretations could be made from the extracted rules. In each and every case the extracted collections of rules indicated that a very strong co-regulation ofmRNA encoding ribosomal proteins occurs in the dataset. Several rules associating proteins involved in signal transduction were obtained and analyzed, some pointing to yet-unexplored directions. Furthermore, by examining a subset of these rules, we were able both to reassign a wrongly labeled tag, and to proposea function for an expressed sequence tag encoding a protein of unknown function.CONCLUSIONS: We show that ARD is a promising technique that turns out to be complementary to existing gene-expression clustering techniques.

The v-erbA oncogene blocks expression of alpha2/beta1 integrin a normal inhibitor of erythroid progenitor proliferation.

Author(s) : Mey A, Gandrillon O, McNagny K, Clegg D, Samarut J,
Journal : Oncogene
2002
T2EC are chicken erythrocytic progenitors that balance between self-renewal and differentiation as a function of response to specific growth factors. Their transformation by the v-erbA oncogene locks them into the self-renewal program. We show here that the expression of the VLA-2 integrin alpha2 subunit mRNA is downregulated by v-erbA and that VLA-2 engagement and clustering, brought about by treatment with an alpha2-specific antibody or by culture on the VLA-2 ligand collagen I, inhibits T2EC proliferation. From competition studies using antibodies, VLA-2 was shown to be involved in the collagen-induced response. While engagement of VLA-2 inhibited proliferation, it was not sufficient to induce differentiation. The transformation of T2EC by v-erbA decreased their interaction with collagen I and the VLA-2 brake on cell proliferation, which mayaccount for the increased proliferation potential of transformed erythrocytic progenitors and for their shedding into the blood of infected chickens. Our datasuggest that the interaction between erythroid progenitors and collagen, mediated by VLA-2, play a major role in the control of erythropoiesis in vitro and that this pathway is a target of the v-erbA oncogene.

The v-erbA oncogene. Assessing its differentiation-blocking ability using normal chicken erythrocytic progenitor cells.

Author(s) : Gandrillon O,
Journal : Methods Mol Biol
2002