Binding of Ikaros to the lambda5 promoter silences transcription through a mechanism that does not require heterochromatin formation.

The Ikaros family of proteins are DNA binding factors required for correct development of B and T lymphocytes. Cytogenetic studies have shown that these proteins form complexes with pericentromeric heterochromatin in B cells, and the colocalization of transcriptionally silent genes with these complexes suggests that Ikaros could silence transcription by recruiting genes to heterochromatin. Here we show that a site in the lambda5 promoter that binds Ikaros and Aiolos is required for silencing of lambda5 expression in activated mature B cells. Analysis of methylation and nuclease accessibility indicates that the silenced lambda5 gene is not heterochromatinized in B cells, despite being associated with pericentromeric heterochromatin clusters. We also found that a promoter mutation, which affects Ikaros-mediated silencing of lambda5 expression, is not rescued in a transgenic line that has the gene integrated into pericentromeric heterochromatin. Our results indicate that the Ikaros proteins initiate silencing of lambda5 expression through a direct effect on the promoter with localization to pericentromeric heterochromatin likely to affect the action of Ikaros on regulatory sequences rather than causing heterochromatinization of the gene.

Transcription factor dosage affects changes in higher order chromatin structure associated with activation of a heterochromatic gene.

The mechanisms of transcriptional activation in heterochromatin were investigated by using FISH to directly visualize changes in chromatin organization during activation of a heterochromatic lambda5 transgene. A DNase I hypersensitive site was shown to relocate the transgene to the outside of the pericentromeric heterochromatin complex in the absence of transcription. Activation of transcription, which is dependent on the transcription factor EBF, occurs in a stochastic manner that resembles telomeric silencing in yeast, with the transcribed gene remaining closely associated with the heterochromatin complex. Reducing the dosage of EBF results in a reduced frequency of localization of the transgene to the outside of the heterochromatin complex and lower levels of transcription. These data provide evidence that transcription factors can initiate changes in higher order chromatin structure during the earliest stages of gene activation.

Functional gene expression domains: defining the functional unit of eukaryotic gene regulation.

The term functional domain is often used to describe the region containing the cis acting sequences that regulate a gene locus. "Strong" domain models propose that the domain is a spatially isolated entity consisting of a region of extended accessible chromatin bordered by insulators that have evolved to act as functional boundaries. However, the observation that independently regulated loci can overlap partially or completely raises questions about functional requirements for physically isolated domain structures. An alternative model, the "weak" domain model, proposes that domain structure is determined by the distribution of binding sites for positively acting factors, without a requirement for functional boundaries. The domain would effectively be the region that contains these factor-binding sites. Specificity of promoter-enhancer interactions would play a major role in maintaining the functional autonomy of adjacent genes. Sequences that interfere with these interactions (frequently characterised as insulators) would be selected against if they occurred within the domain but not at the edges, or in the interdomain regions. As a result, insulators would often be found near the borders of domains without necessarily being selected to act as boundaries.

Analysis of mice with single and multiple copies of transgenes reveals a novel arrangement for the lambda5-VpreB1 locus control region.

The murine lambda5-VpreB1 locus encodes two proteins that form part of the pre-B-cell receptor and play a key role in B-lymphocyte development. We have identified a locus control region (LCR) which is responsible for coordinate activation of both genes in pre-B cells. Analysis of mice with single and multiple copies of transgenes shows a clear difference in the expression behavior of the genes depending on the transgene copy number. While expression of both lambda5 and VpreB1 in single- and two-copy integrations requires the presence of a set of DNase I hypersensitive sites located 3' of the lambda5 gene, small fragments containing the genes have LCR activity when arranged in multiple-copy tandem arrays, indicating that additional components of the LCR are located within or close to the genes. The complete LCR is capable of driving efficient copy-dependent expression of a lambda5 gene in pre-B cells even when it is integrated into centomeric gamma-satellite DNA. The finding that activation of expression of the locus by positively acting factors is fully dominant over the silencing effect of heterochromatin has implications for models for chromatin-mediated gene silencing during B-cell development.

Immunity specificity determinants in the P4-like retronphage phi R73.

Retronphage phi R73 exhibits extensive sequence homology to the satellite bacteriophage P4. Bacteriophage P4 superinfection immunity is elicited by a small RNA (CI RNA) that causes premature transcription termination within the operon coding for the P4 replication functions. This control is exerted via interaction of the CI RNA with two complementary target sites on the untranslated leader RNA of the replication operon. We found that phi R73 is endowed with a similar immunity system but is heteroimmune to P4. The heteroimmunity is due to six base differences in the CI RNA and to compensatory base substitutions in the target sequences. The sequence differences in the CI RNA are all located in single-stranded regions, which appear to play a predominant role in the interaction with the target sites. Analysis of phage carrying a hybrid immunity system indicates that, although two target sequences are required for the establishment of lysogeny, a single site is sufficient to make a phage sensitive to the prophage immunity.

Multiple regulatory mechanisms controlling phage-plasmid P4 propagation.

Bacteriophage P4 autonomous replication may result in the lytic cycle or in plasmid maintenance, depending, respectively, on the presence or absence of the helper phage P2 genome in the Escherichia coli host cell. Alternatively, P4 may lysogenize the bacterial host and be maintained in an immune-integrated condition. A key step in the choice between the lytic/plasmid vs. the lysogenic condition is the regulation of P4 alpha operon. This operon may be transcribed from two promoters, PLE and PLL, and encodes both immunity (promoter proximal) and replication (promoter distal) functions. PLE is a constitutive promoter and transcription of the downstream replication genes is regulated by transcription termination. The trans-acting immunity factor that controls premature transcription termination is a short RNA encoded in the PLE proximal part of the operon. Expression of the replication functions in the lytic/plasmid condition is achieved by activation of the PLL promoter. Transcription from PLL is insensitive to the termination mechanism that acts on transcription starting from PLE.PLL is also negatively regulated by P4 orf88, the first gene downstream of PLL. An additional control on P4 DNA replication is exerted by the P4 cnr gene product.

Immunity determinant of phage-plasmid P4 is a short processed RNA.

In the phage-plasmid P4, both lysogenic and lytic functions are coded by the same operon. Early after infection the whole operon is transcribed from the constitutive promoter PLE. In the lysogenic condition transcription from PLE terminates prematurely and only the immunity functions, which are proximal to the promoter, are thus expressed. Fragments of the P4 immunity region were cloned in an expression vector. A DNA fragment as short as 91 bp was sufficient, when transcribed, to express P4 immunity and to complement P4 immunity deficient mutants. This fragment, like prophage P4, produced a 69 nt long RNA (CI RNA). A shorter P4 fragment neither expressed immunity nor synthesized the CI RNA. Thus the CI RNA is the P4 trans-acting immunity factor. The 5' end of the CI RNA, mapped by primer extension, does not correspond to the transcription initiation point, thus suggesting that the CI RNA is produced by processing of the primary transcript. In an RNase P mutant host the processing of the 5' end and the production of a functional CI RNA were impaired. The requirement of RNase P for the correct processing of CI appears to be related to the predicted secondary structure of the precursor CI RNA. A region (seqB) within the CI RNA shows complementarity with two cis-acting sequences (seqA and seqC) required for P4 immunity, suggesting that transcription termination may be caused by pairing of the CI RNA with the complementary target sequences on the nascent transcript.

Control of transcription termination by an RNA factor in bacteriophage P4 immunity: identification of the target sites.

Prophage P4 immunity is elicited by a short, 69-nucleotide RNA (CI RNA) coded for within the untranslated leader region of the same operon it controls. CI RNA causes termination of transcription that starts at the promoter PLE and prevents the expression of the distal part of the operon that codes for P4 replication functions (alpha operon). In this work, we identify two sequences in the untranslated leader region of the alpha operon, seqA and seqC, that are the targets of the P4 immunity factor. seqA and seqC exhibit complementarity to a sequence internal to the CI RNA (seqB). Mutations in either seqA or seqC that alter its complementarity to seqB abolished or reduced P4 lysogenization proficiency and delayed the shutoff of the long transcripts originating from PLE that cover the entire operon. Both seqA and seqC single mutants were still sensitive to P4 prophage immunity, whereas P4 seqA seqC double mutants showed a virulent phenotype. Thus, both functional sites are necessary to establish immunity upon infection, whereas a single site appears to be sufficient to prevent lytic gene expression when immunity is established. A mutation in seqB that restored complementarity to both seqA and seqC mutations also restored premature termination of PLE transcripts, thus suggesting an important role for RNA-RNA interactions between seqB and seqA or seqC in P4 immunity.

Bacteriophage P4 immunity controlled by small RNAs via transcription termination.

Satellite bacteriophage P4 immunity is encoded within a short DNA region 357 bp long containing the promoter PLE and 275 bp downstream. PLE is active both in the early post-infection phase, when genes necessary for P4 lytic cycle are transcribed from this promoter, and in the lysogenic condition, when expression of the above genes is prevented by prophage immunity. In order to understand how P4 immunity is elicited, we have characterized the transcription pattern during the establishment and the maintenance of the satellite phage P4 lysogenic condition. We found that prophage transcription starting at PLE ends prematurely and the transcripts do not extend beyond 300-400 nucleotides downstream of PLE. Thus P4 immunity acts by causing premature transcription termination rather than by repressing transcription initiation. The P4 immunity region is transcribed in the prophage, but it does not seem to be translated; this region contains two elements (seqA and seqB) of a palindromic sequence. In addition to transcripts about 300 nucleotides long, P4 prophage produces a family of shorter transcripts, about 80 nucleotides long, containing seqA or seqB. Evidence is presented suggesting that SeqB RNA is the trans-acting immunity factor, and that interaction of SeqB RNA with the complementary nascent RNA containing seqA may be involved in bringing about premature transcription termination.