Advances in Cell biology, vol. 32, 2005, issue 1

Summary: Retroelements constitute a large fraction of the repetitive DNA of eukaryotic genomes. They include LTR (Long Terminal Repeat) and non-LTR retrotransposons, lacking the long terminal repeats and subdivided into LINEs (Long Interspersed Nuclear Elements) and SINEs (Short Interspersed Nuclear Elements), have been discovered as ubiquitous components of nuclear genomes in many species. LINEs are able to transpose autonomously, while non-autonomous SINEs depend on the reverse transcription machinery of other retrotransposons. Non-LTR retrotransposons were first discovered in mammalian genomes but have also been identified in plants, fungi and invertebrates. SINEs are a moderately to highly amplified sequence class of Eukaryotes which have been most extensively studied in mammalian species (Alu family). SINEs are up to several hundred basepairs in length (>500 bp) and have a composite structure. The 5' region of SINEs is similar to tRNA (the major class), 5S rRNA or to 7SL RNA genes (the minor classes). The 3' region of many SINEs shows similarity to the 3' end of LINEs. SINEs are terminated by a poly(A) tract or A- or T-rich sequences. Two well conserved sequence motifs are found in the tRNA-related part of SINEs. Similar to tRNA genes, these sequences, called box A and box B, serve as an internal promotor for RNA polymerase III. SINEs do not encode their own reverse transcriptase and are therefore unable to transpose autonomously. Similar to LINEs, they move by retrotransposition and generate short target site duplications upon reintegration. Data on possible functions for SINEs are still incomplete and controversial, but it is likely that SINEs have a major impact on their genomes. They have a significant role in genome/gene evolution, structure and transcription levels. The distribution of these elements has been implicated in some genetic diseases and cancers. They are very useful as markers for phylogenetic analysis, because species exhibit variation in the genomic localization of SINE inserts.

Key words: SINEs, short interspersed elements, non-LTR retrotransposons, retroelements, genome

Genetic Control of Self-Perpetuation of The Shoot Apical Meristem in Arabidopsis thaliana

Summary: Two fundamental processes take place at the shoot apical meristem (SAM) the maintenance of its size and shape i.e. self-perpetuation, and the formation of lateral organs, like leaves or lateral shoots. Internally, SAM is organized into cytohistological zones. The central zone, involved in the self-perpetuation, is located most distally. The peripheral zone occupies meristem flanks. The cells here participate in the formation of primordia of lateral organs. In the central zone of Arabidopsis thaliana shoot apical meristem CLAVATA (CLV) and WUSCHEL (WUS) genes are expressed. The self-perpetuation is regulated by a feedback loop in their activities. Mutations in CLV genes lead to excessive proliferation of the central zone cells and as a consequence, to increase in the SAM size. Mutations in WUS lead to premature termination of the SAM growth, after formation of only few primordia. The same effect has been shown in plants overexpressing the CLV3 gene. It has been suggested, that the products of ULTRAPETALA, HANABA TARANU and AGAMOUS genes negatively regulate WUS expression, while SPLAYED and STIMPY regulate positively. Most of these genes products act as transcriptional factors. Thereby the genes are able to influence indirectly the self-perpetuation process. The experiments with laser ablation of the central zone in Lycopersicon esculentum shoot apex led to ectopic expression of the WUSCHEL gene at the peripheral zone, followed by the establishment of a new meristem centre. After the ablation of the central zone the organ formation is not affected. Studies of monocots, like Oryza sativa and Zea mays, led to discovery of genes orthologous to CLV and WUS, which are also involved in the control of the SAM self-perpetuation.

Key words: Arabidopsis thaliana, shoot apical meristem (SAM), stem cells, self-perpetuation, CLAVATA, WUSCHEL

Nucleolin – Characteristics of Protein and its Role in Biology of Cancers and Viral Infections

Summary: Nucleolin is a multifunctional nucleolar protein that has three domains of different structure and functions. This protein is located in nucleoli but is also detected in karyoplasm outside nucleoli, in cytoplasm and in the cell membrane. It shuttles between these structures. Different localization of nucleolin in a cell points to its involvement in different physiologic and pathologic processes. The main function of nucleolin is participation in rRNA processing from rDNA transcription to assembly of preribosomal particles. Nucleolin induces changes of chromatin structure, elongation of primary transcript of rRNA and ribosome maturation. It potentially can stabilize mRNA, play a role in formation of nucleoli or apoptosis. Nucleolin is involved in human papilloma virus-induced carcinogenesis and influences suppressor proteins and transcription factors. Results of studies on expression of nucleolin in breast cancers showed its relation to a histological type, estrogen receptor expression, cell cycle phases and lymph nodes metastases. In last years the localization of nucleolin in the cell membrane brought interest to its participation in viral infections. Nucleolin might be a therapeutic target in HIV infection. It also influences the replication of hepatotrophic viruses. The essential role of nucleolin in viral infection is also supported by its colocalization with many viral antigens. In the present study the structure, regulation of expression and posttranslational modifications as well as main functions of nucleolin in a cell are discussed. Moreover the latest data on the role of nucleolin in biology of cancers and in viral infections, especially HIV and hepatotrophic viruses are presented.

Summary: Experiments on androgenetic and gynogenetic mammalian embryos showed that both maternal and paternal genomes are needed for normal development. The existence of parental genomes non equivalency was proposed and term genomic imprinting was coined. Genomic imprinting is an epigenetic phenomenon that results in monoallelic expression of certain genes in a parent-of-origin-dependent manner. The mechanism by which imprinting is realized is cytosine methylation. DNA sequences that are modified with cytosine methylation are called imprinting control regions (ICRs). ICRs are usually located in CpG-reach regions close to promoters of imprinted genes and can regulate parent-specific gene expression bidirectionally over long distances. Allelic methylation marks are established during gametogenesis, following the erasure of preexisting DNA methylation in the primordial germ cells. In females, gene imprinting timing depends on oogenesis stage and oocyte dimension. For example, Ndn and Snrpn genes are imprinted during the primordial to primary follicle stages, while Peg3 gene in secondary follicle. In males, imprinting of some genes (i.e. H19) is completed at spermatogonial stage of spermatogenesis, thus before meiosis occurs. Mouse model showed that most imprinted genes have non-random location and are clustered within imprinting regions of the genome. The first identified cluster of imprinted genes was that containing paternally expressed Igf2 and maternally expressed H19. Transcription of these genes is regulated by physical contact between differentially methylated regions (DMRs) that contain insulators, activators and silencers. Disruption of IGF2 imprinted expression (and also mutations in closely linked KCNQ1 cluster) leads to congenital growth disorder, Beckwith-Wiedemann syndrome (BWS) in humans. Other well studied examples of neurodevelopmental imprinting disorders are Prader-Willi (PWS) and Angelman (AS) syndromes. Both diseases could be caused by paternal and maternal uniparental disomies, deletions of the entire imprinting domain and mutations in imprinting centre, which leads to the failure of the imprinting mechanism per se. Clusters of imprinted genes contain multiple imprinted mRNA genes and at least one imprinted noncoding RNA. Imprinted genes are involved in various processes in cell. For example, some of them codes for transporters of organic cathion (i.e. Slc22a2), basic and neutral aminoacids (Slc38a4) and potasium ions (Kcnq1). Other protein products of imprinted genes are involved in cell cycle control (i.e. Cdkn1c), intracellular signaling cascades (i.e. Grb10), creatine synthesis (Gatm) or endocrine pathways (i.e. Igf2, Igf2r). More that 25% of imprinted genes code for ncRNA (non-coding RNA). Among them are genes coding for antisense RNA (Igf2r coding for Air), small nucleolar RNA (snoRNA; i.e. SNRPN coding for HBII-52 and HBII-85), microRNA (miRNA; i.e. Rtl1 coding for mir-127 and mir-136). So far, the silencing mechanisms has been determined for only three imprinted clusters and contain insulator- and RNA-mediated silencing. The results from other imprinted clusters are awaited to see whether only two types of basic imprinting mechanisms are present in mammals. The best tool in unravelling this secret will be genome-wide screening and knockout studies of particular imprinted genes in the mouse.

Key words: genomic imprinting, clusters, imprinting control regions, insulators, non-coding RNA

Summary: The centromere is a highly specialized domain on a chromosome that controls the process of faithfull sister chromatid segregation during mitosis and meiosis. The common feature of the centromeric DNA is the presence of long tandem arrays in which the monomers are species-specific. Centromere identity is epigenetically determined by the presence of centromere protein CENP-A and its homologues, conserved histone H3 variants. Neocentromeres are the structures devoid of complete activity characteristic of centromeres. Neocentromeres originate from ectopic, noncentromeric region of a chromosome. It is possible that neocentromere formation depends on the inactivation of an endogenous centromere. Inactivation of endogenous centromere consists in loss of centromeric constitutive proteins, with exception of CENP-B (if present). In dicentric chromosomes formed by fusion of two chromosome arms belonging to the same chromosome, each bearing one centromere, therefore containing the same kind of centromeric DNA, the switching of the centromere activity seems to be due to epigenetic changes, e.g. as a result of histone hyperacetylation. Neocentromeres are formed mainly on acentric chromosome fragments, therefore such a chromosome behaves during mitosis and meiosis as a normal one. Despite the absence of centromeric DNA, neocentromeres are able to assemble all the centromere and kinetochore proteins. Human neocentromeres are formed mainly as a result of chromosome rearrangements, on chromosomal acentric arm fragments, but neocentromeres can also originate on unarranged chromosomes. Till 2004, 70 neocentromeres have been identified on human chromosomes. Non-random distribution of neocentromeres in the human genome has been observed. Disproportional number of neocentromeres is formed on 3q, 13q and 15q. Formation of centromere on 4q21 is not preceded by chromosome rearrangement. Formation of neocentromeres can be induced in vitro on mammalian minichromosomes and human artificial chromosomes (HACs). Upon introduction by transfection or microinjection of centromere DNA repeats into several kinds of cells, functional neocentromeres are formed. Neocentromeres on human minichromosomes and on HACs are formed after transfection of human centromeric alphoid DNA repeats containing CENP-B boxes elements into cells in tissue culture. In plants, true neocentromeres, i. e. devoid of CENH3 protein (homologue of CENP-A) and other centromere and kinetochore proteins, are similar to those described in human chromosomes. They are formed in response to chromosome rearrangements. The term of “neocentromere” is used also to denote the structures being large heterochromatic domains – knobs – that contain two kinds of DNA tandem repeats, 350 bp and 180 bp. Knobs associate with microtubules and move rapidly poleward during 2^nd anaphase in meiosis and function together with an endogenous, true centromere. These structures contain neither CENH3, nor other centromeric proteins and are described in detail only in abnormal chromosome 10 in maize. In endopoliploid nuclei, unable to enter mitosis, in spite of proportional centromere DNA multiplication, the amount of CENH3 does not increase proportionally to the level of centromeric DNA. Human neocentromeres that originate on euchromatic bands of a chromosome, contain some unique sequences of DNA, similary as endogenous centromere of rice chromosome 8. Contrary to canonical endogenous centromeres, transcriptional competence has been demonstrated of both types of centromere, i. e. human neocentromeres and centromere of rice chromosome 8.

Keywords: neocentromeres, knobs, dicentric chromosomes, minichromosomes, artificial chromosomes.

Neocentromeres. II. Molecular Factors Required for Formation of Centromere And Neocentromere

Summary: Contrary to endogenous centromeres containing the species-specific tandem repeated DNA arrays, the neocentromeres are formed on DNA sequences without tandem arrays, therefore DNA does not decide about a neocentromere identity, and epigenetic mechanisms have an essential role in neocentromere assembly. The transposable elements are the most abundant class of DNA in higher Eukaryota. LTR retrotransposons are present both in centromeric and pericentromeric heterochromatin in the endogenous centromeric region. Chromatin immunoprecipitation data indicate thet the key proteins for the centromere assembly, CENP-A/CENH3 interact with retroelements. It is possible that, even single copy, retroelements could be the first mark for the site of a future neocentromere. Overexpression of CID (CENP-A homologue in Drosophila) results its mislocalization, i. e. the presence in normally noncentromeric chromosome region. Several disturbances of mitosis (e. g. chromosome missegregation and aneuploidy) indicate that these ectopic neocentromeres induce active multicentromere chromosomes. Mechanism that prevents deposition of CID out of endogenous centromeres consists in proteolysis of soluble CENP-A, not incorporated into centromeric chromatin nucleosomes. Nucleosomal location of the centromere-specific variant of hitone H3, CENP-A and its homologues is necessary for the assembly of other centromeric and kinetochore proteins. Studies performed recently with fusion protein CENP-A or its homologues – GFP/YFP/EYEP gave precise information concerning the time of synthesis organisms (mammalians and angiosperm plant species), synthesis and loading of CENP-A/CENH3 occur mainly in G2 faze, but even in the same type of cells, HeLa, maximum expression and CENP-A loading were shown to take place, depending on the authors, either in G2 or during telophase and G1 phase. In yeast these processes occur in S and G2 phase, while in Drosophila – during S phase. Therefore, the synthesis and loading of CENP-A and its homologues should take place during interphase before the onset of mitosis with chromosome bearing a neocentromere. In human, Drosophila, yeast and Arabidopsis thaliana cells it has been shown that histone-fold domain is required for centromere-specific deposition of CENP-A and its homologues. The C-terminal parts of these proteins are responsible for the recognition of centromeres. This region, CENP-A targeting domain (CATD) includes the L1 linker and a2 helix of CENP-A and is sufficient to direct this protein to centromeres, even when its N-terminal (species-specific) tail is absent. Heterochromatin is an integral part of the centromere region. It is present at the centromere itself and in the pericentromeric region flanking it. In endogenous centromeres both kinds of heterochromatin display the epigenetic DNA and histone modifications characteristic of condensed and transcriptionally inactive chromatin, i.e. cytosine methylation in DNA, methylation of lysine 9 and 27 in the histone H3, methylation of lysine 20 in the histone H4, absence of acetylation of the latter two. Moreover, heterochromatin of the centromeric region contains HP1 protein. In endogenous centromeres in human cells mutation of DNA methytransferase gene results in decreased methylation of centromeric DNA and in several disturbances in sister chromatid separation during mitosis. The amount of heterochromatin at neocentromeres formed on euchromatin bands in chromosomes is reduced as compared to endogenous centromeres, although some epigenetic marks characteristic of heterochromatin and the presence of HP1 have been observed. These modifications do not occur in those domains in neocentromere region that contain coding DNA sequences. The role of cohesion is to establish sister chromatid cohesion. It is generally admitted that in higher Eukaryota cohesin is preferentially bound by heterochromatic parts of chromatin just after DNA replication and chromatin restitution. During mitosis the last disappearing part of cohesin is that bound to pricentromeric heterochromatin. Reduction of sister chromatids cohesion as a result of diminished amount of pricentromeric heterochromatin in some human neocentromeres has been observed. It has to be noted that recent results suggest possibility of cohesion binding to euchromatic parts of chromatin. Some data obtained from the studies on chromosome evolution can facilitate understanding of neocentromere formation. Chromosome rearrangements are known as mechanisms that contribute to the changes of chromosome shape and of centromere position, e. g. in rare de novo formation of centromeres on acentric chromosome fragments. A particular role in centromere evolution has been attributed to transposable elements, as they have an important role in initiating heterochromatin formation from a single-copy domain at an euchromatic region. The location of a centromere and its inactivation or activation can change during evolution. Centromere repositioning occurred, among others, in mammalian X chromosome. The centromeres of X chromosome in two lemur species and in human are located in different positions and contain different DNA repeats. Therefore, centromere repositioning may due to the endogenous centromere inactivation/neocentromere formation. The presence of active genes in centromere 8 region in rice and in some human neocentromeres may indicate their relatively recent formation. Up to now, there are only few data concerning the question of why a neocentromere originates in a particular – both heterochromatic as euchromatic – location and what, besides endogenous centromere inactivation, triggers their formation.

Key words: neocentromere, transposable elements, CENP-A and homologues, heterochromatin, cohesion, evolution