Interphase cytofission maintains genomic integrity of human cells after failed cytokinesis.

In cell division, cytokinesis is tightly coupled with mitosis to maintain genomic integrity. Failed cytokinesis in humans can result in tetraploid cells that can become aneuploid and promote cancer. However, the likelihood of aneuploidy and cancer after a failed cytokinesis event is unknown. Here we evaluated cell fate after failed cytokinesis. We interrupted cytokinesis by brief chemical treatments in cell populations of human epithelial lines. Surprisingly, up to 50% of the resulting binucleate cells generated colonies. In RPE1 cells, 90% of colonies obtained from binucleate founders had a karyotype that matched the parental cell type. Time-lapse videomicroscopy demonstrated that binucleate cells are delayed in the first growth phase of the cell cycle (G1) and undergo interphase cellular fission (cytofission) that distributes nuclei into separate daughters. The fission is not compatible with delayed cytokinesis because events occur in the absence of polymerized microtubules and without canonical components of the cytokinetic machinery. However, the cytofission can be interrupted by inhibiting function of actin or myosin II. Fission events occur in both two- and three-dimensional culture. Our data demonstrate that cytofission can preserve genomic integrity after failed cytokinesis. Thus, traction-mediated cytofission, originally observed in Dictyostelium, is relevant to human biology--where it seems to be an evolutionarily conserved mechanism that can preserve genomic integrity.

The mouse RANKL gene locus is defined by a broad pattern of histone H4 acetylation and regulated through distinct distal enhancers.

RANKL is a stromal cell-derived tumor necrosis factor (TNF)-like factor that plays a primary role in osteoclast formation and function. Recent studies suggest that 1,25(OH)(2) D(3) induces Rankl expression via vitamin D receptor (VDR) interaction at several enhancers located up to 76 kb upstream of the gene's transcriptional start site (TSS). In the current studies, we explored these interactions further using ChIP-chip and RNA analysis. We confirm VDR and RXR binding to the five enhancers described previously and identify two additional sites, one located within the Rankl coding region. We also show that RNA polymerase II is recruited to these enhancers, most likely through transcription factors TBP, TFIIB, and TAF(II) 250. Interestingly, the recruitment of these factors leads to the production of RNA transcripts, although their role at present is unknown. We also discovered that histone H4 acetylation (H4ac) marks many upstream Rankl enhancers under basal conditions and that H4ac is increased upon 1,25(OH)(2) D(3) treatment. Surprisingly, the hormone also induces C/EBPß binding across the Rankl locus. C/EBPß binding correlates directly with increased H4ac activity following 1,25(OH)(2) D(3) treatment. Finally, elevated H4ac is restricted to an extended region located between two potential insulator sites occupied by CTCF and Rad21. These data suggest a mechanism whereby 1,25(OH)(2) D(3) functions via the VDR and C/EBPß to upregulate Rankl expression.

Distinct functions of dispersed GATA factor complexes at an endogenous gene locus.

The reciprocal expression of GATA-1 and GATA-2 during hematopoiesis is an important determinant of red blood cell development. Whereas Gata2 is preferentially transcribed early in hematopoiesis, elevated GATA-1 levels result in GATA-1 occupancy at sites upstream of the Gata2 locus and transcriptional repression. GATA-2 occupies these sites in the transcriptionally active locus, suggesting that a "GATA switch" abrogates GATA-2-mediated positive autoregulation. Chromatin immunoprecipitation (ChIP) coupled with genomic microarray analysis and quantitative ChIP analysis with GATA-1-null cells expressing an estrogen receptor ligand binding domain fusion to GATA-1 revealed additional GATA switches 77 kb upstream of Gata2 and within intron 4 at +9.5 kb. Despite indistinguishable GATA-1 occupancy at -77 kb and +9.5 kb versus other GATA switch sites, GATA-1 functioned uniquely at the different regions. GATA-1 induced histone deacetylation at and near Gata2 but not at the -77 kb region. The -77 kb region, which was DNase I hypersensitive in both active and inactive states, conferred equivalent enhancer activities in GATA-1- and GATA-2-expressing cells. By contrast, the +9.5 kb region exhibited considerably stronger enhancer activity in GATA-2- than in GATA-1-expressing cells, and other GATA switch sites were active only in GATA-1- or GATA-2-expressing cells. Chromosome conformation capture analysis demonstrated higher-order interactions between the -77 kb region and Gata2 in the active and repressed states. These results indicate that dispersed GATA factor complexes function via long-range chromatin interactions and qualitatively distinct activities to regulate Gata2 transcription.

An enhancer 20 kilobases upstream of the human receptor activator of nuclear factor-kappaB ligand gene mediates dominant activation by 1,25-dihydroxyvitamin D3.

Receptor activator of nuclear factor-kappaB ligand (RANKL) is a TNF-like factor that is both produced by osteoblasts, mesenchymal cells, and activated T cells and required for osteoclast maturation and survival. The gene is up-regulated by the two primary calcemic hormones, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] and PTH. Previous studies have indicated that five enhancer regions located significantly upstream of the mouse Rankl transcriptional start site mediate up-regulation by 1,25(OH)2D3 and PTH. The most distal of these, termed mRLD5, is highly conserved in the human gene at -96 kb where it was also shown to be functionally active. Four additional mouse Rankl upstream enhancers are also highly conserved in the human gene at -20, -25, -75, and -87 kb. In the present studies, we characterized the activity of these regions, explored their capacity to mediate the actions of 1,25(OH)2D3, and identified the vitamin D response elements contained within the two most proximal segments. Interestingly, whereas the most distal of the five enhancers is the dominant mediator of 1,25(OH)2D3 activity in the mouse Rankl gene, that role in the human gene is manifested by the most proximal element at -20 kb. Importantly, activity at this region in response to 1,25(OH)2D3 was associated with a significant increase in histone acetylation as well as the enhanced recruitment of RNA polymerase II. Both likely reflect the primary role of this enhancer in human RANKL gene expression. Our studies confirm the complex nature of RANKL regulation and indicate that although the five enhancers are evolutionarily conserved across several species, their relative contributions to RANKL expression in response to 1,25(OH)2D3 may be different.

Multifunctional enhancers regulate mouse and human vitamin D receptor gene transcription.

The vitamin D receptor (VDR) mediates the endocrine actions of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] and autoregulates the expression of its own gene in target cells. In studies herein, we used chromatin immunoprecipitation-chip analyses to examine further the activities of 1,25(OH)(2)D(3) and to assess the consequences of VDR/retinoid X receptor heterodimer binding at the VDR gene locus. We also explored mechanisms underlying the ability of retinoic acid, dexamethasone, and the protein kinase A activator forskolin to induce VDR up-regulation as well. We confirmed two previously identified intronic 1,25(OH)(2)D(3)-inducible enhancers and discovered two additional regions, one located 6 kb upstream of the VDR transcription start site. Although RNA polymerase II was present at the transcription start site in the absence of 1,25(OH)(2)D(3), it was strikingly up-regulated at both this site and at individual enhancers in its presence. 1,25(OH)(2)D(3) also increased basal levels of H4 acetylation at these enhancers as well. Surprisingly, many of these enhancers were targets for CCAAT enhancer-binding protein-beta and runt-related transcription factor 2; a subset also bound cAMP response element binding protein, retinoic acid receptor, and glucocorticoid receptor. Unexpectedly, many of these factors were resident at the Vdr gene locus in the absence of inducer, suggesting that they might contribute to basal Vdr gene expression. Indeed, small interfering RNA down-regulation of CCAAT enhancer-binding protein-beta suppressed basal VDR expression. These regulatory activities of 1,25(OH)(2)D(3), forskolin, and dexamethasone were recapitulated in MC3T3-E1 cells stably transfected with a full-length VDR bacterial artificial chromosome (BAC) clone-luciferase reporter gene. Finally, 1,25(OH)(2)D(3) also induced accumulation of VDR and up-regulated H4 acetylation at conserved regions in the human VDR gene. These data provide important new insights into VDR gene regulation in bone cells.

Emerging regulatory paradigms for control of gene expression by 1,25-dihydroxyvitamin D3.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) functions as a steroid hormone to modulate the expression of genes. Its actions are mediated by the vitamin D receptor (VDR) which binds to target genes and functions to recruit coregulatory complexes that are essential for transcriptional modulation. ChIP analysis coupled to tiled DNA microarray hybridization (ChIP-chip) or massively parallel DNA sequencing (ChIP-seq) is now providing critical new insight into how genes are regulated. In studies herein, we utilized these techniques as well as gene expression analysis to explore the actions of 1,25(OH)2D3 at the genome-wide and individual target gene levels in cells. We identify a series of overarching principles that likely define the actions of 1,25(OH)2D3 at most target genes. We discover that while VDR binding to target sites is ligand-dependent, RXR binding is ligand-independent. We also show that while VDR/RXR binding can localize to promoters, it occurs more frequently at multiple sites many kilobases from target gene promoters. We then describe a new method whereby the regulatory regions of complex genes can be evaluated using large recombineered bacterial artificial chromosomes. We conclude that these new approaches are likely to replace many of the traditional methods used to explore the regulation of transcription.

GATA-1-mediated transcriptional repression yields persistent transcription factor IIB-chromatin complexes.

The hematopoietic GATA factors GATA-1 and GATA-2, which have distinct and overlapping roles to regulate blood cell development, are reciprocally expressed during erythropoiesis. GATA-1 directly represses Gata2 transcription, and reduced GATA-2 synthesis promotes red blood cell development. Gata2 repression involves "GATA switches" in which GATA-1 displaces GATA-2 from Gata2 regulatory regions. We show that extragenic GATA switch sites occupied by GATA-2 associate with as much RNA polymerase II (Pol II) and basal transcription factors as present at the active Gata2 promoters. Pol II bound to GATA switch sites in the active locus was phosphorylated on serine 5 of the carboxyl-terminal domain, indicative of elongation competence. GATA-1-mediated displacement of GATA-2 from GATA switch sites reduced Pol II recruitment to all sites except the far upstream -77-kb region. Surprisingly, TFIIB occupancy persisted at most sites upon repression. These results indicate that GATA-2-bound extragenic regulatory elements recruit Pol II, GATA-1 binding expels Pol II, and despite the persistent TFIIB-chromatin complexes, Pol II recruitment is blocked.

Developmental control via GATA factor interplay at chromatin domains.

Despite the extraordinary task of packaging mammalian DNA within the constraints of a cell nucleus, individual genes assemble into cell type-specific chromatin structures with high fidelity. This chromatin architecture is a crucial determinant of gene expression signatures that distinguish specific cell types. Whereas extensive progress has been made on defining biochemical and molecular mechanisms of chromatin modification and remodeling, many questions remain unanswered about how cell type-specific chromatin domains assemble and are regulated. This mini-review will discuss emerging studies on how interplay among members of the GATA family of transcription factors establishes and regulates chromatin domains. Dissecting mechanisms underlying the function of hematopoietic GATA factors has revealed fundamental insights into the control of blood cell development from hematopoietic stem cells and the etiology of pathological states in which hematopoiesis is perturbed.

Dynamic GATA factor interplay at a multicomponent regulatory region of the GATA-2 locus.

Given the simplicity of the DNA sequence that mediates binding of GATA transcription factors, GATA motifs reside throughout chromosomal DNA. However, chromatin immunoprecipitation analysis has revealed that GATA-1 discriminates exquisitely among these sites. GATA-2 selectively occupies the -2.8-kilobase (kb) region of the GATA-2 locus in the active state despite there being numerous GATA motifs throughout the locus. The GATA-1-mediated displacement of GATA-2 is tightly coupled to repression of GATA-2 transcription. We have used high resolution chromatin immunoprecipitation to show that GATA-1 and GATA-2 occupy two additional regions, -3.9 and -1.8 kb of the GATA-2 locus. GATA-1 and GATA-2 had distinct preferences for occupancy at these regions, with GATA-1 and GATA-2 occupancy highest at the -3.9- and -1.8-kb regions, respectively. Activation of an estrogen receptor fusion to GATA-1 (ER-GATA-1) induced similar kinetics of ER-GATA-1 occupancy and GATA-2 displacement at the sites. In the transcriptionally active state, DNase I hypersensitive sites (HSs) were detected at the -3.9- and -1.8-kb regions, with a weak HS at the -2.8-kb region. Whereas ER-GATA-1-instigated repression abolished the -1.8-kb HS, the -3.9-kb HS persisted in the repressed state. Transient transfection analysis provided evidence that the -3.9-kb region functions distinctly from the -2.8- and -1.8-kb regions. We propose that GATA-2 transcription is regulated via the collective actions of complexes assembled at the -2.8- and -1.8-kb regions, which share similar properties, and through a qualitatively distinct activity of the -3.9-kb complex.

Biochemical stabilization enhances red blood cell recovery and stability following cryopreservation.

Glycerolized red blood cells (RBC) are approved for long-term cryopreservation. However, the need to remove the glycerol cryoprotectant prior to transfusion has limited the usefulness of this cryopreservation method. This report describes using non-cryoprotectant biochemical stabilization techniques to substitute for the standard glycerol cryoprotectant. The glycerolized RBC method was compared to a newly developed LC-V method that combines transfusable cryoprotectants (hydroxyethyl starch and dextran) and specific non-cryoprotectant biochemical stabilizers (nicotinamide, nifedipine, and flurbiprofen). Results demonstrate that the biochemical stabilizers significantly reduce cryopreservation-induced hemolysis compared to cryopreservation in their absence and that thaw hemolysis levels approach those of standard 40% (w/v) glycerolized RBC (3.1+/-0.2% for 40% glycerol compared to 8.7+/-0.9% for the LC-V protocol). Furthermore, LC-V cryopreserved RBC exhibit a significantly enhanced post-thaw stability compared to glycerolized RBC as determined by osmotic fragility index (0.557+/-0.034 for 40% glycerol compared to 0.478+/-0.016 for the LC-V protocol). Analysis of biochemically stabilized RBC proteins revealed a transient translocation of carbonic anhydrase to the membrane fraction. However, the enhanced RBC recovery and stability could not be attributed to this event. Finally, DSC analysis demonstrated that the biochemical stabilizers of the LC-V process were not functioning as surrogate cryoprotectants in that they did not affect the quantity or quality of ice formed. Overall, this work demonstrates that cryopreservation-induced RBC damage may be corrected or prevented through specific biochemical stabilization and represents a significant step toward a directly transfusable cryopreserved RBC product.