CbfA, the C-module DNA-binding factor, plays an essential role in the initiation of Dictyostelium discoideum development

We recently isolated from Dictyostelium discoideum cells a DNA-binding protein, CbfA, that interacts in vitro with a regulatory element in retrotransposon TRE5-A. We have generated a mutant strain that expresses CbfA at <5% of the wild-type level to characterize the consequences for D. discoideum cell physiology. We found that the multicellular development program leading to fruiting body formation is highly compromised in the mutant. The cells cannot aggregate and stay as a monolayer almost indefinitely. The cells respond properly to prestarvation conditions by expressing discoidin in a cell density-dependent manner. A genomewide microarray-assisted expression analysis combined with Northern blot analyses revealed a failure of CbfA-depleted cells to induce the gene encoding aggregation-specific adenylyl cyclase ACA and other genes required for cyclic AMP (cAMP) signal relay, which is necessary for aggregation and subsequent multicellular development. However, the cbfA mutant aggregated efficiently when mixed with as few as 5% wild-type cells. Moreover, pulsing cbfA mutant cells developing in suspension with nanomolar levels of cAMP resulted in induction of acaA and other early developmental genes. Although the response was less efficient and slower than in wild-type cells, it showed that cells depleted of CbfA are able to initiate development if given exogenous cAMP signals. Ectopic expression of the gene encoding the catalytic subunit of protein kinase A restored multicellular development of the mutant. We conclude that sensing of cell density and starvation are independent of CbfA, whereas CbfA is essential for the pattern of gene expression which establishes the genetic network leading to aggregation and multicellular development of D. discoideum.     

Non-LTR retrotransposons with unique integration preferences downstream of Dictyostelium discoideum tRNA genes

Retrotransposable elements are genetic entities which move and replicate within host cell genomes. We have previously reported on the structures and genomic distributions of two non-long terminal repeat (non-LTR) retrotransposons, DRE and Tdd-3, in the eukaryotic microorganism Dictyostelium discoideum. DRE elements are found inserted upstream, and Tdd-3 elements downstream, of transfer RNA (tRNA) genes with remarkable position and orientation specificities. The data set currently available from the Dictyostelium Genome Project led to the characterisation of two repetitive DNA elements which are related to the D. discoideum non-LTR retrotransposon Tdd-3 in both their structural properties and genomic distributions. It appears from our data that in the D. discoideum genome tRNA genes are major targets for the insertion of mobilised non-LTR retrotransposons. This may be interpreted as the consequence of a process of coevolution, allowing a viable population of retroelements to transpose without being deleterious to the small microbial host genome which carries only short intergenic DNA sequences. A new nomenclature is introduced to designate all tRNA gene-targeted non-LTR retrotransposons (TREs) in the D. discoideum genome. TREs inserted 5′ and 3′ of tRNA genes are named TRE5 and TRE3, respectively. According to this nomenclature DRE and Tdd-3 are renamed TRE5-A and TRE3-A, respectively. The new retroelements described in this study are named TRE3-B (formerly RED) and TRE3-C. Received: 27 May 1999 / Accepted: 23 July 1999     

On the problem of establishing the subcellular localization of Dictyostelium retrotransposon TRE5-A proteins by biochemical analysis of nuclear extracts

At first sight a protein that is enriched in extracts prepared from nuclei by means of biochemical methods can be considered to be a nuclear protein in vivo. Although this assumption will hold true for most of the analyzed proteins, it could also lead to false interpretations. We analyzed the subcellular distribution of endogenous and plasmid-borne proteins derived from the retrotransposon TRE5-A of Dictyostelium discoideum. In biochemical fractionation experiments the proteins encoded by TRE5-A open reading frame 1 (ORF1p) and the putative endonuclease encoded in ORF2 (ENp) were found in the detergent-insoluble material containing the nuclei. However, salt extraction of isolated nuclei did not considerably release the TRE5-A proteins. Instead, the TRE5-A proteins were strongly enriched in a fraction that contained the chromosomal DNA after removal of most cytoskeletal and histone proteins. These observations implied that ORF1p and ENp were both attached to chromatin in vivo, but this conclusion was disproved by the expression of genetic fusions of green fluorescent protein with either ORF1p or ENp. We show conclusive evidence that both fusion proteins were located as large aggregates of native protein in the cytoplasm of D. discoideum cells.     

Selective inhibition of Alu retrotransposition by APOBEC3G

The non-LTR retrotransposon LINE-1 (L1) comprises  17% of the human genome, and the L1-encoded proteins can function in trans to mediate the retrotransposition of non-autonomous retrotransposons (i.e., Alu and probably SVA elements) and cellular mRNAs to generate processed pseudogenes. Here, we have examined the effect of APOBEC3G and APOBEC3F, cytidine deaminases that inhibit Vif-deficient HIV-1 replication, on Alu retrotransposition and other L1-mediated retrotransposition processes. We demonstrate that APOBEC3G selectively inhibits Alu retrotransposition in an ORF1p-independent manner. An active cytidine deaminase site is not required for the inhibition of Alu retrotransposition and the resultant integration events lack G to A or C to T hypermutation. These data demonstrate a differential restriction of L1 and Alu retrotransposition by APOBEC3G, and suggest that the Alu ribonucleoprotein complex may be targeted by APOBEC3G.     

A computational study of the dynamics of LTR retrotransposons in the Populus trichocarpa genome

Retrotransposons are an ubiquitous component of plant genomes, especially abundant in species with large genomes. Populus trichocarpa has a relatively small genome, which was entirely sequenced; however, studies focused on poplar retrotransposons dynamics are rare. With the aim to study the retrotransposon component of the poplar genome, we have scanned the complete genome sequence searching full-length long-terminal repeat (LTR) retrotransposons, i.e., characterised by two long terminal repeats at the 5′ and 3′ ends. A computational approach based on detection of conserved structural features, on building multiple alignments, and on similarity searches was used to identify 1,479 putative full-length LTR retrotransposons. Ty1-copia elements were more numerous than Ty3-gypsy. However, many LTR retroelements were not assigned to any superfamily because lacking of diagnostic features and non-autonomous. LTR retrotransposon remnants were by far more numerous than full-length elements, indicating that during the evolution of poplar, large amplification of these elements was followed by DNA loss. Within superfamilies, Ty3-gypsy families are made of more members than Ty1-copia ones. Retrotransposition occurred with increasing frequency following the separation of Populus sections, with different waves of retrotransposition activity between Ty3-gypsy and Ty1-copia elements. Recently inserted elements appear more frequently expressed than older ones. Finally, different levels of activity of retrotransposons were observed according to their position and their density in the linkage groups. On the whole, the results support the view of retrotransposons as a community of different organisms in the genome, whose activity (both retrotransposition and DNA loss) has heavily impacted and probably continues to impact poplar genome structure and size.