Structures In Human Cells Research Articles

Rif1 regulates the replication timing domains on the human genome

DNA replication is spatially and temporally regulated during S-phase. DNA replication timing is established in early-G1-phase at a point referred to as timing decision point. However, how the genome-wide replication timing domains are established is unknown. Here, we show that Rif1 (Rap1-interacting-factor-1), originally identified as a telomere-binding factor in yeast, is a critical determinant of the replication timing programme in human cells. Depletion of Rif1 results in specific loss of mid-S replication foci profiles, stimulation of initiation events in early-S-phase and changes in long-range replication timing domain structures. Analyses of replication timing show replication of sequences normally replicating early is delayed, whereas that normally replicating late is advanced, suggesting that replication timing regulation is abrogated in the absence of Rif1. Rif1 tightly binds to nuclear-insoluble structures at late-M-to-early-G1 and regulates chromatin-loop sizes. Furthermore, Rif1 colocalizes specifically with the mid-S replication foci. Thus, Rif1 establishes the mid-S replication domains that are restrained from being activated at early-S-phase. Our results indicate that Rif1 plays crucial roles in determining the replication timing domain structures in human cells through regulating higher-order chromatin architecture.

H2B monoubiquitylation is a 5′-enriched active transcription mark and correlates with exon–intron structure in human cells

H2B monoubiquitylation (H2Bub1), which is required for multiple methylations of both H3K4 and H3K79, has been implicated in gene expression in numerous organisms ranging from yeast to human. However, the molecular crosstalk between H2Bub1 and other modifications, especially the methylations of H3K4 and H3K79, remains unclear in vertebrates. To better understand the functional role of H2Bub1, we measured genome-wide histone modification patterns in human cells. Our results suggest that H2Bub1 has dual roles, one that is H3 methylation dependent, and another that is H3 methylation independent. First, H2Bub1 is a 5′-enriched active transcription mark and co-occupies with H3K79 methylations in actively transcribed regions. Second, this study shows for the first time that H2Bub1 plays a histone H3 methylations-independent role in chromatin architecture. Furthermore, the results of this work indicate that H2Bub1 is largely positioned at the exon–intron boundaries of highly expressed exons, and it demonstrates increased occupancy in skipped exons compared with flanking exons in the human and mouse genomes. Our findings collectively suggest that a potentiating mechanism links H2Bub1 to both H3K79 methylations in actively transcribed regions and the exon–intron structure of highly expressed exons via the regulation of nucleosome dynamics during transcription elongation.

Inhibition of Translation by Small RNA-Stabilized mRNA Structures in Human Cells

RNA-mediated gene regulation and expression are critically dependent on both nucleic acid architecture and recognition. We present a novel mechanism for the regulation of gene expression through direct RNA-RNA interactions between small RNA and mRNA in human cells. Using mRNA reporters containing G-rich sequences in the 5'-untranslated region (5'-UTR), in the coding region, or both, we showed that G-rich small RNAs bind to the reporter mRNAs and form an intermolecular RNA G-quadruplex that can inhibit gene translation in living cells. Using a combination of circular dichroism (CD) and RNase footprinting in vitro, we found that the intermolecular G-quadruplexes show a parallel G-quadruplex structure. We next investigated whether the intermolecular G-quadruplex is present in living cells. Employing the fluorophore-labeled probes, we found that two G-rich RNA molecules form an intermolecular G-quadruplex structure in living cells. These results extend the concept of small RNA-mediated expression and suggest an important role for such RNA structures in the inhibition of mRNA translation.

Race and NationAmerica Becoming: Racial Trends and Their Consequences. Edited by Neil J. Smelser, William Julius Wilson, and Faith Mitchell. Washington, D.C.: National Academy Press, 2001. 2 vols. Pp. 1084. $49.94 (cloth); $24.95 (paper) each volume.

The sociologist W. E. B. DuBois was surely prescient in his projection at the beginning of the twentieth century that this would be “the century of the color line” (DuBois 1903, p. vii). He foresaw the coming of a heightened struggle for social justice on the part of the people of color, not only in the United States but in Latin America, Asia, and Africa. Before his death in Africa in 1963 at the age of 95, in the midst of the historic Civil Rights March on Washington that year, DuBois could see profound changes in the relations between the “colonized” people of color and the dominant European people. Since that time race and ethnic relations as well as race and ethnic diversity have exhibited unanticipated patterns of conflict and accommodation, progress and retrogression. Since the seventeenth century, through American slavery, emancipation, segregation, and the modern civil rights movement, no issue has been more persistently salient in the United States than the issue of race and ethnic relations. For generations, scientists have urged that the concept of race be abandoned in our public discourse because it has no scientific or biological meaning. In recent years, using DNA samples to study human cell structures, laboratory scientists working

Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability.

We have reconstituted wild type or mutant telomerase activity in two human cell lines that lack constitutive expression of both core subunits of the enzyme and maintain telomeres by a telomerase-independent mechanism (ALT cells). Wild type telomerase RNA and four telomerase RNAs with single point mutations in their template domain were used to express enzymes specifying different telomeric DNA sequences. Expression of wild type telomerase for up to 32 days had no detectable effect on cell growth or viability. In contrast, cells expressing mutant telomerases had slower growth rate, abnormal cell cycle and reduced viability. Dramatically aberrant nuclei, typical of cells undergoing mitotic catastrophe, and large numbers of fused chromosomes were also characteristic of these populations. Notably, all phenotypes were apparent within the first few cell divisions after expression of the enzymes. Unlike wild type, mutant telomerase activity was progressively selected against with cell culturing, and this correlated with the disappearance of cells with aberrant phenotypes. Our results suggest that even very limited synthesis of mutated sequences can affect telomere structure in human cells, and that the toxicity of mutant telomerases is due to telomere malfunction.

The 2G2 antibody recognizes an acidic 110-kDa human mitochondrial protein.

Using fluorescence microscopy, the mouse monoclonal antibody 2G2 was found to label mitochondria in human cells, as assessed by double staining with either Rhodamine 123 or a polyclonal antibody to mitochondrial matrix HSP-60 proteins. No reactivity to the 2G2 antibody was detected in cells from mouse, rat and chicken. Immunoblotting analysis demonstrated that the 2G2 antigen corresponds to a human protein with a relative mobility of 110kDa and an approximate isoelectric point of 6.5 that co-partitions with HSP-60 proteins during isolation of mitochondria from HeLa cells. Close examination of the 2G2 staining pattern in HeLa and Fanconi's anaemia cells revealed differences in the morphology and organization of mitochondria in these two cell types. In HeLa cells, mitochondria appear as individual tubular compartments of variable length and are closely associated with vimentin filaments, particularly at the periphery of the nucleus. In Fanconi's anaemia cells, mitochondria have a filamentous shape and form an interconnected cytoplasmic reticulum running in parallel with both vimentin filaments and microtubules. After stabilization with aldehyde- or alcohol-based fixation protocols that optimize the preservation of cytoskeletal components, the epitope targeted by the 2G2 antibody may serve as a valuable marker in the investigation of relationships between mitochondria and other cellular structures in human cells.

Sequences Attaching Loops of Nuclear and Mitochondrial DNA to Underlying Structures in Human Cells: The Role of Transcription Units

DNA sequences attaching loops of nuclear and mitochondrial DNA to underlying structures in HeLa cells have been cloned and 106 representative clones sequenced; 10 clones containing random genomic fragments served as controls. As chromatin is prone to rearrangement, care was taken to isolate sequences using 'physiological' conditions that did not create additional attachments. Comparison (by Southern blotting) of the concentration of each cloned sequence in 'total' and 'attached' fractions of DNA showed that most clones did contain attached sequences, but even highly-attached sequences were not attached in all cells in the population. Results demonstrated that 28% of clones were derived from three specific parts of the mitochondrial genome and 22% from different parts of the alu repeat. In addition, 41% of clones contained unique nuclear sequences; these contained no more of the motifs found attached to nuclear scaffolds or matrices (ie SARs or MARs) than would be expected from their base composition. No other attachment motif(s) could be identified by sequence analysis. However, Northern blotting showed that all the mitochondrial clones and 76% of clones containing unique sequences were transcribed; the degree of attachment correlated with transcriptional activity. These results are consistent with transcription being responsible for ever-changing attachments in both nuclei and mitochondria.

Effects of nitrilotriacetic acid (NTA) on chromosome replication and structure in human cells

Effects of nitrilotriacetic acid (NTA) on chromosome replication and structure in human cells