Imre Cserpán

Two Different Drosophila ADA2 Homologues Are Present in Distinct GCN5 Histone Acetyltransferase-Containing Complexes

ABSTRACT We have isolated a novel Drosophila (d) gene coding for two distinct proteins via alternative splicing: a homologue of the yeast adaptor protein ADA2, dADA2a, and a subunit of RNA polymerase II (Pol II), dRPB4. Moreover, we have identified another gene in the Drosophila genome encoding a second ADA2 homologue (dADA2b). The two dADA2 homologues, as well as many putative ADA2 homologues from different species, all contain, in addition to the ZZ and SANT domains, several evolutionarily conserved domains. The dada2a/rpb4 and dada2b genes are differentially expressed at various stages of Drosophila development. Both dADA2a and dADA2b interacted with the GCN5 histone acetyltransferase (HAT) in a yeast two-hybrid assay, and dADA2b, but not dADA2a, also interacted with Drosophila ADA3. Both dADA2s further potentiate transcriptional activation in insect and mammalian cells. Antibodies raised either against dADA2a or dADA2b both immunoprecipitated GCN5 as well as several Drosophila TATA binding protein-associated factors (TAFs). Moreover, following glycerol gradient sedimentation or chromatographic purification combined with gel filtration of Drosophila nuclear extracts, dADA2a and dGCN5 were detected in fractions with an apparent molecular mass of about 0.8 MDa whereas dADA2b was found in fractions corresponding to masses of at least 2 MDa, together with GCN5 and several Drosophila TAFs. Furthermore, in vivo the two dADA2 proteins showed different localizations on polytene X chromosomes. These results, taken together, suggest that the two Drosophila ADA2 homologues are present in distinct GCN5-containing HAT complexes.

De novo chromosome formations by large-scale amplification of the centromeric region of mouse chromosomes

Chromosomes formedde novo which originated from the centromeric region of mouse chromosome 7, have been analysed. These new chromosomes were formed by apparently similar large-scale amplification processes, and are organized into amplicons of ∼30 Mb. Centromeric satellite DNA was found to be the constant component of all amplicons. Satellite DNA sequences either bordered the large euchromatic amplicons (E-type amplification), or made up the bulk of the constitutive heterochromatic amplicons (H-type amplification). Detailed analysis of a heterochromatic megachromosome formedde novo by an H-type amplification revealed that it is composed of a tandem array of 10–12 large (∼30 Mb) amplicons each marked with integrated ‘foreign’ DNA sequences at both ends. Each amplicon is a giant palindrome, consisting of two inverted doublets of ∼7.5-Mb blocks of satellite DNA. Our results indicate that the building units of the pericentric heterochromatin of mouse chromosomes are ∼7.5-Mb blocks of satellite DNA flanked by nonsatellite sequences. We suggest that the formationde novo of various chromosome segments and chromosomes seen in different cell lines may be the result of large-scale E- and H-type amplification initiated in the pericentric region of chromosomes.

Evidence for a megareplicon covering megabases of centromeric chromosome segments.

We have analysed the replication of the heterochromatic megachromosome that was formedde novo by a large-scale amplification process initiated in the centromeric region of mouse chromosome 7. The megachromosome is organized into amplicons ∼30 Mb in size, and each amplicon consists of two large inverted repeats delimited by a primary replication initiation site. Our results suggest that these segments represent a higher order replication unit (megareplicon) of the centromeric region of mouse chromosomes. Analysis of the replication of the megareplicons indicates that the pericentric heterochromatin and the centromere of mouse chromosomes begin to replicate early, and that their replication continues through approximately three-quarters of the S-phase. We suggest that a replication-directed mechanism may account for the initiation of large-scale amplification in the centromeric regions of mouse chromosomes, and may also explain the formation of new, stable chromosome segments and chromosomes.

Different isoforms of PRIP-interacting protein with methyltransferase domain/trimethylguanosine synthase localizes to the cytoplasm and nucleus

A protein family including the recently identified PIMT/Tgs1 (PRIP-interacting protein with methyltransferase domain/trimethylguanosine synthase) was identified by searching databases for homologues of a newly identified Drosophila protein with RNA-binding activity and methyltransferase domain. Antibodies raised against a short peptide of the mammalian homologue show a 90-kDa isoform expressed specifically in rat brain and testis and a 55-kDa form expressed ubiquitously. In HeLa cells, the larger isoform of the protein is nuclear and associated with a 600-kDa complex, while the smaller isoform is mainly cytoplasmic and co-localizes to the tubulin network. Inhibition of PIMT/Tgs1 expression by siRNA in HeLa cells resulted in an increase in the percentage of cells in G2/M phases. In yeast two-hybrid and in vitro GST pull down experiments, the conserved C-terminal region of PIMT/Tgs1 interacted with the WD domain containing EED/WAIT-1 that acts as a polycomb-type repressor in the nucleus and also binds to integrins in the cytoplasm. Our experiments, together with earlier data, indicate that isoforms of the PIMT/Tgs1 protein with an RNA methyltransferase domain function both in the nucleus and in the cytoplasm and associate with both elements of the cytoskeletal network and nuclear factors known to be involved in gene regulation.

Physical relationship between satellite I and II DNA in centromeric regions of sheep chromosomes

Fluorescence in situ hybridization (FISH) with probes representing sheep satellite I and satellite II DNAs shows a different distribution of the two repetitive DNA families in the centromeric region of most chromosomes. The single signal per chromosome produced by the satellite I probe suggests close proximity of this DNA family to the primary constriction. Satellite II produces two separate signals on the sister chromatids, and large blocks of satellite II DNA constitute most of the short arm of all acrocentric chromosomes. We have isolated and sequenced a phage clone containing a junction between discrete blocks of satellite I and satellite II sequences. The junction is characterized by an abrupt juxtaposition of arrays of the two satellites. The possibility that the peculiar structural features of this junction could have a functional significance is discussed.

A combined artificial chromosome-stem cell therapy method in a model experiment aimed at the treatment of Krabbe’s disease in the Twitcher mouse

Abstract.Mammalian artificial chromosomes (MACs) are safe, stable, non-integrating genetic vectors with almost unlimited therapeutic transgene-carrying capacity. The combination of MAC and stem cell technologies offers a new strategy for stem cell-based therapy, the efficacy of which was confirmed and validated by using a mouse model of a devastating monogenic disease, galactocerebrosidase deficiency (Krabbe’s disease). Therapeutic MACs were generated by sequence-specific loading of galactocerebrosidase transgenes into a platform MAC, and stable, pluripotent mouse embryonic stem cell lines were established with these chromosomes. The transgenic stem cells were thoroughly characterized and used to produce chimeric mice on the mutant genetic background. The lifespan of these chimeras was increased twofold, verifying the feasibility of the development of MAC-stem cell systems for the delivery of therapeutic genes in stem cells to treat genetic diseases and cancers, and to produce cell types for cell replacement therapies.

The chAB4 and NF1-related long-range multisequence DNA families are contiguous in the centromeric heterochromatin of several human chromosomes

We have investigated the large-scale organization of the human chAB4-related long-range multisequence family, a low copy-number repetitive DNA located in the pericentromeric heterochromatin of several human chromosomes. Analysis of genomic clones revealed large-scale ( approximately 100 kb or more) sequence conservation in the region flanking the prototype chAB4 element. We demonstrated that this low copy-number family is connected to another long-range repeat, the NF1-related (PsiNF1) multisequence. The two DNA types are joined by an approximately 2 kb-long tandem repeat of a 48-bp satellite. Although the chAB4- and NF1-like sequences were known to have essentially the same chromosomal localization, their close association is reported here for the first time. It indicates that they are not two independent long-range DNA families, but are parts of a single element spanning approximately 200 kb or more. This view is consistent both with their similar chromosomal localizations and the high levels of sequence conservation among copies found on different chromosomes. We suggest that the master copy of the linked chAB4-PsiNF1 DNA segment appeared first on the ancestor of human chromosome 17.

Generation of induced pluripotent stem cells by using a mammalian artificial chromosome expression system

Direct reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPS) was achieved recently by overexpression of four transcription factors encoded by retroviral vectors. Most of the virus vectors, however, may cause insertional mutagenesis in the host genome and may also induce tumor formation. Therefore, it is very important to discover novel and safer, non-viral reprogramming methods. Here we describe the reprogramming of somatic cells into iPS cells by a novel protein-based technique. Engineered Oct4, Sox2 and Klf4 transcription factors carrying an N-terminal Flag-tag and a C-terminal polyarginine tail were synthesized by a recently described mammalian artificial chromosome expression system (ACEs). This system is suitable for the high-level production of recombinant proteins in mammalian tissue culture cells. Recombinant proteins produced in this system contain all the post-translational modifications essential for the stability and the authentic function of the proteins. The engineered Oct4, Sox2 and Klf4 proteins efficiently induced the reprogramming of mouse embryonic fibroblasts by means of protein transduction. This novel method allows for the generation of iPS cells, which may be suitable for therapeutic applications in the future.

Cloning, characterization and localization of Chinese hamster HP1 isoforms.

The Chinese hamster is one of the few mammalian species that are characterized by relatively poor heterochromatin content. It was intriguing to test whether or not the lack of large blocks of heterochromatin in the hamster chromosomes could be correlated with the absence or species-specific differences of the HP1 proteins, the main structural components of heterochromatin. To address this, we attempted to clone HP1 from the Chinese hamster. It is shown here that all three isoforms of HP1 known in mammals are present in hamster, and the amino acid sequences deduced from the cDNAs of the isoforms are 97–100% identical to those of the known mammalian homologues. All three isoforms are localized mainly in heterochromatic regions in the native chromosomes and nuclei. The hamster HP1α gene was cloned, sequenced and mapped to the short arm of hamster chromosome 2.These data indicate that the Chinese hamster has all the HP1 components necessary for the establishment of heterochromatin. The limited amount of heterochromatin in hamster cells may probably be attributed to the unusual satellite DNA content of the hamster genome.

Variation in sequence-specific repair of UV damage in human pericentromeric heterochromatin of different cell lines

Damaged nucleotides are removed from the condensed non-coding, or transcriptionally inactive regions of the genome by the relatively slow global genome repair system. Since few data are available for the repair of the pericentric heterochromatin region our aim was to study the repair of a specific sequence, known to be located in this region. We applied a PCR based method to monitor UV damage and repair in chAB4, a human pericentromeric heterochromatin sequence in 10 human cell lines. We here present evidence that excision repair of a sequence in the pericentromeric heterochomatin also varies between cell lines in a manner inconsistent with the canonical model. In some cell lines repair rates were efficient in heterochromatin, comparable to transcription coupled repair, but in some tumour-derived and repair-deficient cell lines we have detected deficient repair.