Differentiation-induced replication-timing changes are restricted to AT-rich/long interspersed nuclear element (LINE)-rich isochores

The replication timing of some genes is developmentally regulated, but the significance of replication timing to cellular differentiation has been difficult to substantiate. Studies have largely been restricted to the comparison of a few genes in established cell lines derived from different tissues, and most of these genes do not change replication timing. Hence, it has not been possible to predict how many or what types of genes might be subject to such control. Here, we have evaluated the replication timing of 54 tissue-specific genes in mouse embryonic stem cells before and after differentiation to neural precursors. Strikingly, genes residing within isochores rich in GC and poor in long interspersed nuclear elements (LINEs) did not change their replication timing, whereas half of genes within isochores rich in AT and long interspersed nuclear elements displayed programmed changes in replication timing that accompanied changes in gene expression. Our results provide direct evidence that differentiation-induced autosomal replication-timing changes are a significant part of mammalian development, provide a means to predict genes subject to such regulation, and suggest that replication timing may be more related to the evolution of metazoan genomes than to gene function or expression pattern.     

A compositional map of human chromosome band Xq28.

The molar fractions of guanine plus cytosine (GC) in DNA were determined for 36 yeast artificial chromosomes (YACs) which almost completely cover human chromosome band Xq28, a terminal reverse band, corresponding to about 8 Mb of DNA. This allowed the construction of the most complete compositional map to date of a chromosomal band; three regions were observed: (i) a proximal 3.5-Mb region formed by GC-poor L and GC-rich H1 isochores; (ii) a middle 2,2-Mb region essentially formed by a GC-rich H2 isochore and a very GC-rich H3 isochore separated by a GC-poor L isochore, YACs from this region being characterized by a striking compositional heterogeneity and instability; and (iii) a distal 1.3-Mb region exclusively formed by GC-poor L isochores. Gene and CpG island concentrations increased with the GC levels of the isochores, as expected. Xq28 exemplifies a subset of reverse bands which are different from the two other subsets, namely from telomeric bands, which are characterized by specific cytogenetic properties and by the predominance of H2 and H3 isochores, and from the majority of reverse bands, which do not contain H2 and H3 isochores.     

Correlations between isochores and chromosomal bands in the human genome.

The human genome is made up of long DNA segments, the isochores, which are compositionally homogeneous and can be subdivided into a small number of families characterized by different G+C levels. Chromosome in situ suppression hybridization (in which excess unlabeled human DNA is added to suppress hybridization of repeated sequences present in the probe, enabling enhanced observation of single-copy sequences) of DNA fractions characterized by an increasing G+C level was carried out to determine the distribution of "single-copy" sequences corresponding to isochore families L1+L2, H1, H2, and H3 on metaphase chromosomes. This produced a banding pattern progressing from a relatively diffuse staining to an R-banding, to a T-banding. More specifically, our results showed that (i) T-bands are formed by the G+C-richest isochores of the H3 family and by part of the G+C-rich isochores of the H1 and H2 families (with a predominance of the latter); (ii) R'-bands (namely, R-bands exclusive of T-bands) are formed to almost equal extents by G+C-rich isochores of the H1 families (with a minor contribution of the H2 and H3 families) and by G+C-poor isochores of the L1+L2 families; (iii) G-bands essentially consist of G+C-poor isochores from the L1+L2 families, with a minor contribution of isochores from the H1 family. These results not only clarify the correlations between DNA base composition and chromosomal bands but also provide information on the distribution of genes in chromosomes, gene concentration increasing with the G+C levels of isochores.     

Asynchronous replication timing of telomeres at opposite arms of mammalian chromosomes

Telomeres are defining structural elements of all linear chromosomes, yet information concerning the timing of their replication in higher eukaryotes is surprisingly limited. We developed an approach that allowed a study of telomere replication patterns of specific mammalian chromosomes. In the Indian muntjac (Muntiacus muntjac), replication timing between respective telomeres of homologous chromosomes was highly coordinated, but no such synchrony was evident for p- and q-arm telomeres of the same chromosome. This finding contrasts with the coordinated timing of both ends of each chromosome in yeast. Also in contrast to yeast, where replication of all telomeres is confined to late S phase, we found specific telomeres in Indian muntjac chromosomes that replicated early in S and other telomeres that replicated later. Finally, replication timing of some but not all telomeres was influenced by telomere length. Knowledge of telomere replication timing represents a first step toward understanding the relationship between telomere replication and telomerase action. The approach, which we call replicative detargeting fluorescence in situ hybridization, is widely applicable to different species and genetic loci.     

Premature condensation induces breaks at the interface of early and late replicating chromosome bands bearing common fragile sites

Various studies suggest a tight relationship between chromosome rearrangements driving tumor progression and breaks at loci called common fragile sites. Most of these sites are induced after perturbation of the replication dynamics, notably by aphidicolin treatment. We have mapped the majority of these sites to the interface of R and G bands, which calls into question the previous assignment of aphidicolin-sensitive sites to R bands. This observation suggests that most of them correspond to loci that ensure the transition between early and late replicating domains. We show that calyculin A, which triggers chromosome condensation at any phase of the cell cycle but does not markedly impair replication, induces damage in the chromosomes of human lymphocytes treated in G(2) but not in G(1) phase. We demonstrate that these lesions colocalize with those induced by aphidicolin treatment. Hence, common fragile site stability is compromised, whether aphidicolin delays replication or calyculin A advances condensation. We also show that, in cells that go through an unperturbed S phase, completion of their replication and/or replication-associated chromatin reorganization occur all along the G(2) phase, which may explain their inability to condense properly after calyculin A treatment during this phase of the cell cycle.     

c-src is consistently conserved in the chromosomal deletion (20q) observed in myeloid disorders.

The proto-oncogene c-src has been mapped to two bands in human chromosomes, 1p36 and 20q13, both of which are involved in rearrangements in human tumors. In particular, deletions (loss of part of a chromosome) of the long arm of chromosome 20, del(20q), are commonly observed in hematologic malignant diseases. By using in situ chromosomal hybridization of a c-src probe to metaphase cells prepared from leukemic bone marrow cells of three patients with a del(20q), we observed specific labeling on the deleted chromosome in each patient, indicating that the c-src locus was conserved. The presence on the rearranged chromosomes of c-src, which is normally located on the most distal band of 20q, indicated that the deletions were not terminal as they appeared to be on the basis of chromosome morphology, but rather that they were interstitial. The location of c-src relative to the distal breakpoint in these deletions is unknown. By using the v-src probe in Southern blot analysis of genomic DNA from three patients with a del(20q), we found that no major genomic rearrangements or amplification of the c-src genes had occurred within the regions homologous to v-src. Our observation that c-src is consistently preserved in these rearranged chromosomes suggests that this gene may play a role in the pathogenesis of some myeloid disorders.     

Phosphorylation of hepatitis C virus nonstructural protein 5A modulates its protein interactions and viral RNA replication

The study of the hepatitis C virus (HCV) has been hindered by the lack of in vitro model systems. The recent development of HCV subgenomic RNA replicons has permitted the study of viral RNA replication in cell culture; however, the requirements for efficient replication of replicons in this system are poorly understood. Many viral isolates do not function as replicons and most require conserved changes, termed adaptive mutations, to replicate efficiently. In this report, we focus on the HCV nonstructural protein 5A (NS5A), a frequent locus for adaptive mutation. We found the interaction between NS5A and human vesicle-associated membrane protein-associated protein A (hVAP-A), a cellular target N-ethylmaleimide-sensitive factor attachment protein receptor, to be required for efficient RNA replication: NS5A mutations that blocked interaction with hVAP-A strongly reduced HCV RNA replication. Further analyses revealed an inverse correlation between NS5A phosphorylation and hVAP-A interaction. A subset of the previously identified adaptive mutations suppressed NS5A hyperphosphorylation and promoted hVAP-A binding. Our results support a model in which NS5A hyperphosphorylation disrupts interaction with hVAP-A and negatively regulates viral RNA replication, suggesting that replicon-adaptive mutations act by preventing the phosphorylation-dependent dissociation of the RNA replication complex.     

Regulation of PKR and IRF-1 during hepatitis C virus RNA replication

The virus-host interactions that influence hepatitis C virus (HCV) replication are largely unknown but are thought to involve those that disrupt components of the innate intracellular antiviral response. Here we examined cellular antiviral pathways that are triggered during HCV RNA replication. We report that (i) RNA replication of HCV subgenomic replicons stimulated double-stranded RNA (dsRNA) signaling pathways within cultured human hepatoma cells, and (ii) viral RNA replication efficiency corresponded with an ability to block a key cellular antiviral effector pathway that is triggered by dsRNA and includes IFN regulatory factor-1 (IRF-1) and protein kinase R (PKR). The block to dsRNA signaling was mapped to the viral nonstructural 5A (NS5A) protein, which colocalized with PKR and suppressed the dsRNA activation of PKR during HCV RNA replication. NS5A alone was sufficient to block both the activation of IRF-1 and the induction of an IRF-1-dependent cellular promoter by dsRNA. Mutations that clustered in or adjacent to the PKR-binding domain of NS5A relieved the blockade to this IRF-1 regulatory pathway, resulting in induction of IRF-1-dependent antiviral effector genes and the concomitant reduction in HCV RNA replication efficiency. Our results provide further evidence to support a role for PKR in dsRNA signaling processes that activate IRF-1 during virus infection and suggest that NS5A may influence HCV persistence by blocking IRF-1 activation and disrupting a host antiviral pathway that plays a role in suppressing virus replication.     

Different genetic pathways in leukemogenesis for patients presenting with therapy-related myelodysplasia and therapy-related acute myeloid leukemia

Development of myelodysplasia (MDS) with subsequent progression to acute myeloid leukemia (AML) is an example of the multistep process of malignant transformation in which each step often relates to genetic abnormalities that can be directly seen as chromosomal aberrations. Therapy-related MDS and AML (t-MDS and t-AML) may serve as an ideal model for a study of the genetic evolution of MDS and AML because chromosomal abnormalities are observed in most cases and because the disease is often diagnosed early due to a close patient follow-up. The cytogenetic characteristics at diagnosis were studied in 137 consecutive cases of t-MDS and t-AML, including 22 new cases, and correlated with the clinical characteristics and the course of the disease. Balanced translocations to chromosome bands 11q23 and 21q22 represent primary steps in pathways leading directly to overt t-AML. Specific chromosomal deletions or losses, on the other hand, represent primary or secondary events in alternative pathways leading to t-MDS with potential for subsequent transformation to overt t-AML. Loss of a whole chromosome 7 (-7) or deletion of its long arm (7q-) and deletion of the long arm of a chromosome 5 (5q-) were the most frequent primary abnormalities significantly related to t-MDS. Loss of a whole chromosome 5 (-5) was also a primary event, but surprisingly, was observed equally in t-MDS and in t-AML. Deletion of chromosome 13, including bands q13q14, was another less common primary aberration of t- MDS. Except for -7 and del(13q), these primary aberrations were most often observed together with secondary abnormalities. These included balanced aberrations involving band 3q26 and various deletions of chromosome 3, a gain of a whole chromosome 8, deletions of the short arm or loss of chromosomes 12 and 17, loss of a whole chromosome 18, and deletions of the short arm of chromosome 21. Deletions or loss or chromosomes 5 and 7 were significantly associated with previous therapy with alkylating agents (P = .002), and balanced translocations to chromosome bands 3q26, 11q23, and 21q22 were significantly associated with previous therapy with drugs targeting DNA-topoisomerase II (P < .00005). Other characteristic aberrations were not related to any specific type of therapy. The molecular changes believed to contribute to the development of t-MDS and t-AML have been identified for many of these chromosomal abnormalities.     

A fourth class of theta-replicating plasmids: the pAM beta 1 family from gram-positive bacteria.

Plasmid pAM beta 1 from Enterococcus faecalis uses a unidirectional theta mode of replication. We show here that this replication (i) is dependent on a plasmid-encoded replication protein (Rep) but not on a DNA structure typical for origins of most Rep-dependent plasmids and (ii) is initiated by DNA polymerase I (PolI). pAM beta 1 minimal replicon shares no homology with highly conserved ColE1-type replicons, which use PolI for initiation but do not encode a Rep, or with ColE2 and ColE3 replicons, which require PolI for replication and encode a Rep. We propose that pAM beta 1 and a number of other naturally occurring and closely related plasmids from a distinct plasmid class.     

Chromosomal localization of the human diazepam binding inhibitor gene.

We have used in situ chromosome hybridization and human-mouse somatic cell hybrids to map the gene(s) for human diazepam binding inhibitor (DBI), an endogenous putative modulator of the gamma-aminobutyric acid receptor acting at the allosteric regulatory center of this receptor that includes the benzodiazepine recognition site. In 784 chromosome spreads hybridized with human DBI cDNA, the distribution of 1476 labeled sites revealed a significant clustering of autoradiographic grains (11.3% of total label) on the long arm of chromosome 2 (2q). Furthermore, 63.5% of the grains found on 2q were located on 2q12-21, suggesting regional mapping of DBI gene(s) to this segment. Secondary hybridization signals were frequently observed on other chromosomes and they were statistically significant mainly for chromosomes 5, 6, 11, and 14. In addition, DNA from 32 human-mouse cell hybrids was digested with BamHI and probed with human DBI cDNA. A 3.5-kilobase band, which probably represents the human DBI gene, was assigned to chromosome 2. Four higher molecular weight bands, also detected in BamHI digests, could not be unequivocally assigned. A chromosome 2 location was excluded for the 27-, 13-, and 10-kilobase bands. These results assign a human DBI gene to chromosome 2 (2q12-21) and indicate that three of the four homologous sequences detected by the human DBI probe are located on three other chromosomes.     

Construction and characterization of genomic libraries from specific human chromosomes.

Highly purified fractions of human chromosomes 21 and 22 were isolated from a suspension of metaphase chromosomes stained with ethidium bromide by using a fluorescence-activated cell sorter (FACS II). Two recombinant DNA libraries, representing chromosomes 21 and 22, were constructed by complete digestion of DNA from these fractions with EcoRI and insertion into the vector lambda gtWES lambda B. Twenty clones selected at random from the chromosome 22 library hybridized to EcoRI-digested human DNA, and five of these clones hybridized to single bands identical in size to the phage inserts. These five single-copy sequences and a clone coding for an 8S RNA isolated by screening the chromosome 22 library for expressed sequences were characterized in detail. Hybridization of all six clones to a panel of sorted chromosomes and hybrid cell lines confirmed the assignment of the sequences to chromosome 22. The sequences were localized to regions of chromosome 22 by hybridization to translocated chromosomes sorted from a cell line having a balanced translocation t(17;22)(p13;q11) and to hybrid cell lines containing the various portions of another translocation t(X;22)(q13;q112). Five clones reside on the long arm of chromosome 22 between q112 and pter, while one clone and an 18S rRNA gene isolated from the chromosome 22 library reside pter and g112. The construction of chromosome-specific libraries by this method has the advantage of being direct and applicable to nearly all human chromosomes and will be important in molecular analysis of human genetic diseases.     

Assignment of human beta-, gamma-, and delta-globin genes to the short arm of chromosome 11 by chromosome sorting and DNA restriction enzyme analysis.

Normal human metaphase chromosomes isolated from fibroblasts were resolved into 14 peaks based on total Hoechst 33258 fluorescence and sorted with the fluorescence-activated cell sorter. The chromosomal DNA was extracted and characterized by EcoRI analysis. As expected, analysis of the peak containing chromosomes 16 and 18 detected the alpha-globin genes and of the peak containing chromosomes 9, 10, 11, and 12 detected the beta-, gamma-, and delta-globin genes. Translocations were then used to localize further the beta-, gamma-, and delta-globin genes. The first translocation t(11;22)(q25;q11), which moved nearly all of chromosome 11 to a different peak, confirmed that the beta-, gamma-, and delta-globin genes are on this chromosome. The second, t(4;11)(q25;q13), which moved the distal portion of the long arm of chromosome 11 to a new peak, showed that the genes are not in this segment. The third, t(X;11)(q11;p13), moved the distal region of the short arm of chromosome 11 to a peak which now contained the beta-, gamma-, and delta-globin genes. Therefore, the beta-, gamma-, and delta-globin genes residue on the distal portion of the chromosome 11 short arm including bands p13, p14, and p15. This sorting method may be used generally to assign other genes to chromosomal segments of the entire chromosome complement.     

A myeloid-related sequence that localizes to human chromosome 8q21.1-22

A myeloid-related sequence (mrs) has previously been identified that is highly expressed in selected subpopulations of myeloid leukocytes. Nucleotide sequence analysis indicates that mrs encodes what is apparently a unique 93-amino acid protein that includes an 18-amino acid leader sequence. Hybridization of an mrs cDNA probe to a Southern blot made from somatic cell hybrid DNAs shows 100% concordance with human chromosome 8, thus indicating that mrs localizes to this chromosome. In situ hybridization to metaphase chromosomes further sublocalizes mrs to bands 8q21.1-23 as 58% of the grains displayed on chromosome 8 were clustered in this region. This area encompasses the translocation breakpoint 8q22, which is rearranged in an estimated 18% of patients diagnosed with the M2 subclassification of acute nonlymphocytic leukemia (M2-ANLL). When Southern blot hybridization was performed by using somatic cell hybrid DNAs harboring either a single 8q- or a single 21q+ chromosome from two different patients with M2-ANLL, a signal was only detected in the hybrid containing the 8q-chromosome.     

Evolutionary conservation of zebrafish linkage group 14 with frequently deleted regions of human chromosome 5 in myeloid malignancies

Recurring interstitial loss of all or part of the long arm of chromosome 5, del(5q), is a hallmark of myelodysplastic syndrome and acute myeloid leukemia. Although the genes affected by these changes have not been identified, two critically deleted regions (CDRs) are well established. We have identified 76 zebrafish cDNAs orthologous to genes located in these 5q CDRs. Radiation hybrid mapping revealed that 33 of the 76 zebrafish orthologs are clustered in a genomic region on linkage group 14 (LG14). Fifteen others are located on LG21, and two on LG10. Although there are large blocks of conserved syntenies, the gene order between human and zebrafish is extensively inverted and transposed. Thus, intrachromosomal rearrangements and inversions appear to have occurred more frequently than translocations during evolution from a common chordate ancestor. Interestingly, of the 33 orthologs located on LG14, three have duplicates on LG21, suggesting that the duplication event occurred early in the evolution of teleosts. Murine orthologs of human 5q CDR genes are distributed among three chromosomes, 18, 11, and 13. The order of genes within the three syntenic mouse chromosomes appears to be more colinear with the human order, suggesting that translocations occurred more frequently than inversions during mammalian evolution. Our comparative map should enhance understanding of the evolution of the del(5q) chromosomal region. Mutant fish harboring deletions affecting the 5q CDR syntenic region may provide useful animal models for investigating the pathogenesis of myelodysplastic syndrome and acute myeloid leukemia.     

Heme oxygenase-1 suppresses hepatitis C virus replication and increases resistance of hepatocytes to oxidant injury

Oxidative injury to hepatocytes occurs as a result of hepatitis C virus (HCV) infection and replication. Modulation of host cell antioxidant enzymes such as heme oxygenase-1 (HO-1) may be useful therapeutically to minimize cellular injury, reduce viral replication, and attenuate liver disease. In this report, we evaluated the effects of HO-1 overexpression on HCV replication and hepatocellular injury. Full-length (FL) (Con1) or nonstructural (NS) replicons (I 389 NS3-3') were transfected with complete human HO-1 sequences or empty vector for control. Cell lines overexpressing HO-1 (twofold to sixfold above basal values) or empty vector were isolated, and their HCV RNA synthesis, pro-oxidant levels, and resistance to oxidative injury were assessed. HO-1 overexpression decreased HCV RNA replication in both FL and NS replicons without affecting cellular growth or DNA synthesis. The attenuation of HCV replication was significantly reversed in both replicon systems with HO-1 small interfering RNA (siRNA) knockdown. Both FL and NS replicons that overexpress HO-1 showed reduced prooxidant levels at baseline and increased resistance to oxidant-induced cytotoxicity. HO-1 induction with hemin also markedly decreased HCV replication in both parental FL and NS replicon cell lines. Conversely, knockdown of HO-1 messenger RNA (mRNA) by siRNA in parental FL or NS replicons did not significantly affect HCV replication, suggesting that less than basal levels of HO-1 had minimal effect on HCV replication. CONCLUSION: Overexpression or induction of HO-1 results in decreased HCV replication as well as protection from oxidative damage. These findings suggest a potential role for HO-1 in antiviral therapy and therapeutic protection against hepatocellular injury in HCV infection.     

Delayed replication timing leads to delayed mitotic chromosome condensation and chromosomal instability of chromosome translocations

Chromosomal rearrangements are found in virtually all types of human cancers. We show that certain chromosome translocations display a delay in mitotic chromosome condensation that is associated with a delay in the mitosis-specific phosphorylation of histone H3. This delay in mitotic condensation is preceded by a delay in both the initiation as well as the completion of chromosome replication. In addition, chromosomes with this phenotype participate in numerous secondary translocations and rearrangements. Chromosomes with this phenotype were detected in five of seven tumor-derived cell lines and in five of thirteen primary tumor samples. These data suggest that certain chromosomal rearrangements found in tumor cells cause a significant delay in replication timing of the entire chromosome that subsequently results in delayed mitotic chromosome condensation and ultimately in chromosomal instability.