Our attention is focusing on the behavior of the chromatin fiber during transcription and replication. To understand how these two processes are coordinated during the -S-phase, we analyze the chromatin structure of replicative intermediates of S. cerevisiae, Xenopus
Regulation of polymerase I transcription
We have demonstrated that from yeast to human, active and inactive ribosomal chromatin coexist as two distinct structures. The coding region of one type contains nucleosomes and represents the inactive copies, whereas the other type is devoid of nucleosomes and corresponds to the active genes (Conconi et al., 1989, Dammann et al., 1993). In yeast cells, a structural link between transcription units and the adjacent 3' flanking enhancer has been demonstrated. Each transcriptionally active gene is flanked by a non-nucleosomal enhancer, whereas inactive, nucleosome-packed gene copies are followed by an enhancer organized in nucleosomes (Dammann et al., 1995). In order to determine whether the enhancer acts on the upstream or downstream transcription units, or either of them, chromatin studies and analyses of expression of contiguous but differently tagged rRNA genes have been done. Enhancer deletion mutants of the tagged rDNA repeats are used to measure the enhancer function during transcription and replication of adjacent rRNA gene copies.
Chromatin structure and methylation on rat rRNA genes
By using formaldehyde cross-linking of histones to DNA and gel retardation assay we show that formaldehyde fixation, similar to previously established psoralen photocross-linking, discriminates between nucleosome-packed (inactive) and nucleosome-free (active) fractions of ribosomal RNA genes. By both cross-linking techniques we were able to purify from agarose gels fragments corresponding to coding, enhancer or promoter sequences of rRNA genes, which were further investigated with respect to DNA methylation. This approach allows us to analyze independently and in detail methylation patterns of active and inactive rRNA gene copies by the combination of HpaII and MspI restriction enzymes. We found CpG methylation mainly present in enhancer and promoter regions of inactive rRNA gene copies. The methylation of one single HpaII site, located in the promoter region, showed particularly strong correlation with the transcriptional activity (Stancheva et al., 1997). Direct determination of the extent and the rate of remethylation from rDNA sequences on newly replicated DNA is in progress.
Activation of replication origins with the rRNA gene locus
In the yeast S. cerevisiae, ARS elements located in the intergenic spacers of the rRNA gene locus, are infrequently activated as origins of replication. This unexplained characteristic has been addressed by using the intercalating drug psoralen which in vivo specifically marks the transcribing gene copies, or by taking advantage of the selective accessibility of restriction sites from transcriptionally active genes in combination with 2D gels which reveals replication initiation sites. We found that initiation of replication preferentially, if not exclusively, starts at the ARS placed immediately downstream from transcribing rRNA genes . This clear correlation between transcription and replication is consistent with the presence of open, nucleosome free enhancers at the 3’end of each transcriptionally active gene copy suggesting the implication of the Abf1p in the mechanism of replication initiation at the ARS in the rRNA gene locus.
Replication fork barrier in the rDNA locus
For dissecting the molecular mechanisms involved in the action of the replication fork barrier (RFB) at the 3' end of transcriptionally active rRNA genes (Lucchini and Sogo, 1994), we are developing an in vitro assay with a functional RFB. This in vitro approach will be complemented with in vivo studies using yeast mutants.
"In vivo" mapping of replicating chromatin reveals positioned nucleosomes at the replication fork
By the use of psoralen-crosslinking and primer extension, a method has been developed which allows the analysis of chromatin structure “in vivo” (Wellinger and Sogo, 1998). The fidelity of the technique was initially tested by mapping the positions of the nucleosomes in the well known TRURAP yeast minichromosome and in the rRNA intergenic spacers of yeast cells.
Since the advance of the replication machinery transiently destabilizes the nucleosomal organization of the chromatin fiber and since the first nucleosome on daughter strands is detected at a distance of about 300 nucleotides from the elongation point, we wanted to adress the question of nucleosome positoning on newly replicated chromatin. Analysis of sites of psoralen intercalation (which coincide with linkers and non-nucleosomal DNA) in the “in vivo” crosslinked rRNA intergenic spacers excised from the replicated branches of replications intermediates purified from preparative two-dimensional gels, reveals that: nucleosomes mapping between the 5S gene and the 5’ end of the rDNA transcription unit are precisely positioned inmediately after the pasage of the replication machinery.
By using the same aproach, the chromatin structure of the newly replicated ARS sequences at the rRNA gene locus is as well under study. and rodent cell lines. As tools we preferentially use the psoralen cross-linking technique in combination with to-dimensional agarose gel electrophoresis and electron microscopy.