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The cell cycle inhibitor p27Kip¹ controls self-renewal and pluripotency of human embryonic stem cells by regulating the cell cycle, Brachyury and Twist.
The continued turn over of human embryonic stem cells (hESC) while maintaining an undifferentiated state is dependent on the regulation of the cell cycle. Here we asked the question if a single cell cycle gene could regulate the self-renewal or pluripotency properties of hESC. We identified that the protein expression of the p27(Kip)¹ cell cycle inhibitor is low in hESC cells and increased with differentiation. By adopting a gain and loss of function strategy we forced or reduced its expression in undifferentiating conditions to define its functional role in self-renewal and pluripotency. Using undifferentiation conditions, overexpression of p27(Kip)¹ in hESC lead to a G₁phase arrest with an enlarged and flattened hESC morphology and consequent loss of self-renewal ability. Loss of p27(Kip)¹ caused an elongated/scatter cell-like phenotype involving up-regulation of Brachyury and Twist gene expression. We demonstrate the novel finding that p27(Kip)¹ protein occupies the Twist1 gene promoter and manipulation of p27(Kip)¹ by gain and loss of function is associated with Twist gene expression changes. These results define p27(Kip)¹ expression levels as critical for self-renewal and pluripotency in hESC and suggest a role for p27(Kip)¹ in controlling an epithelial to mesenchymal transition (EMT) in hESC.
Document Type:

Reference

Product Catalog Number:

AB5922
ES cell cycle progression and differentiation require the action of the histone methyltransferase Dot1L.
Mouse embryonic stem cells (ESCs) proliferate with rapid cell cycle kinetics but without loss of pluripotency. The histone methyltransferase Dot1L is responsible for methylation of histone H3 at lysine 79 (H3K79me). We investigated whether ESCs require Dot1L for proper stem cell behavior. ESCs deficient in Dot1L tolerate a nearly complete loss of H3K79 methylation without a substantial impact on proliferation or morphology. However, shortly after differentiation is induced, Dot1L-deficient cells cease proliferating and arrest in G2/M-phase of the cell cycle, with increased levels of aneuploidy. In addition, many aberrant mitotic spindles occur in Dot1L-deficient cells. Surprisingly, these mitotic and cell cycle defects fail to trigger apoptosis, indicating that mouse ESCs lack stringent cell cycle checkpoint control during initial stages of differentiation. Transcriptome analysis indicates that Dot1L deficiency causes the misregulation of a select set of genes, including many with known roles in cell cycle control and cellular proliferation as well as markers of endoderm differentiation. The data indicate a requirement for Dot1L function for early stages of ESC differentiation where Dot1L is necessary for faithful execution of mitosis and proper transcription of many genes throughout the genome.
Document Type:

Reference

Product Catalog Number:

17-295
Product Catalog Name:

Chromatin Immunoprecipitation (ChIP) Assay Kit
Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site.
Unlike the core histones, which are incorporated into nucleosomes concomitant with DNA replication, histone H3.3 is synthesized throughout the cell cycle and utilized for replication-independent (RI) chromatin assembly. The RI incorporation of H3.3 into nucleosomes is highly conserved and occurs at both euchromatin and heterochromatin. However, neither the mechanism of H3.3 recruitment nor its essential function is well understood. Several different chaperones regulate H3.3 assembly at distinct sites. The H3.3 chaperone, Daxx, and the chromatin-remodeling factor, ATRX, are required for H3.3 incorporation and heterochromatic silencing at telomeres, pericentromeres, and the cytomegalovirus (CMV) promoter. By evaluating H3.3 dynamics at a CMV promoter-regulated transcription site in a genetic background in which RI chromatin assembly is blocked, we have been able to decipher the regulatory events upstream of RI nucleosomal deposition. We find that at the activated transcription site, H3.3 accumulates with sense and antisense RNA, suggesting that it is recruited through an RNA-mediated mechanism. Sense and antisense transcription also increases after H3.3 knockdown, suggesting that the RNA signal is amplified when chromatin assembly is blocked and attenuated by nucleosomal deposition. Additionally, we find that H3.3 is still recruited after Daxx knockdown, supporting a chaperone-independent recruitment mechanism. Sequences in the H3.3 N-terminal tail and αN helix mediate both its recruitment to RNA at the activated transcription site and its interaction with double-stranded RNA in vitro. Interestingly, the H3.3 gain-of-function pediatric glioblastoma mutations, G34R and K27M, differentially affect H3.3 affinity in these assays, suggesting that disruption of an RNA-mediated regulatory event could drive malignant transformation.
Document Type:

Reference

Product Catalog Number:

09-838
Product Catalog Name:

Anti-Histone H3.3 Antibody
Cell cycle stage-specific roles of Rad18 in tolerance and repair of oxidative DNA damage.
The E3 ubiquitin ligase Rad18 mediates tolerance of replication fork-stalling bulky DNA lesions, but whether Rad18 mediates tolerance of bulky DNA lesions acquired outside S-phase is unclear. Using synchronized cultures of primary human cells, we defined cell cycle stage-specific contributions of Rad18 to genome maintenance in response to ultraviolet C (UVC) and H(2)O(2)-induced DNA damage. UVC and H(2)O(2) treatments both induced Rad18-mediated proliferating cell nuclear antigen mono-ubiquitination during G(0), G(1) and S-phase. Rad18 was important for repressing H(2)O(2)-induced (but not ultraviolet-induced) double strand break (DSB) accumulation and ATM S1981 phosphorylation only during G(1), indicating a specific role for Rad18 in processing of oxidative DNA lesions outside S-phase. However, H(2)O(2)-induced DSB formation in Rad18-depleted G1 cells was not associated with increased genotoxin sensitivity, indicating that back-up DSB repair mechanisms compensate for Rad18 deficiency. Indeed, in DNA LigIV-deficient cells Rad18-depletion conferred H(2)O(2)-sensitivity, demonstrating functional redundancy between Rad18 and non-homologous end joining for tolerance of oxidative DNA damage acquired during G(1). In contrast with G(1)-synchronized cultures, S-phase cells were H(2)O(2)-sensitive following Rad18-depletion. We conclude that although Rad18 pathway activation by oxidative lesions is not restricted to S-phase, Rad18-mediated trans-lesion synthesis by Polη is dispensable for damage-tolerance in G(1) (because of back-up non-homologous end joining-mediated DSB repair), yet Rad18 is necessary for damage tolerance during S-phase.
Document Type:

Reference

Product Catalog Number:

05-636
Product Catalog Name:

Anti-phospho-Histone H2A.X (Ser139) Antibody, clone JBW301
Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase (mOGG1): mOGG1 associates with microtubules during interphase and mitosis.
8-Oxoguanine DNA glycosylase (OGG1) is a major DNA repair enzyme in mammalian cells. OGG1 participates in the repair of 8-oxoG, the most abundant known DNA lesion induced by endogenous reactive oxygen species in aerobic organisms. In this study, antibodies directed against purified recombinant human OGG1 (hOGG1) or murine (mOGG1) protein were chemically conjugated to either the photosensitizer Rose Bengal or the fluorescent dye Texas red. These dye-protein conjugates, in combination with binding assays, were used to identify associations between mOGG1 and the cytoskeleton of NIH3T3 fibroblasts. Results from these binding studies showed that mOGG1 associates with the cytoskeleton by specifically binding to the centriole and microtubules radiating from the centrosome at interphase and the spindle assembly at mitosis. Similar results were obtained with hOGG1. Together results reported in this study suggest that OGG1 is a microtubule-associated protein itself or that OGG1 utilizes yet to be identified motor proteins to ride on microtubules as tracks facilitating the movement and redistribution of cytoplasmic OGG1 pools during interphase and mitosis and in response to oxidative DNA damage.
Document Type:

Reference

Product Catalog Number:

MAB1636
Cell cycle-dependent recruitment of polycomb proteins to the ASNS promoter counteracts C/ebp-mediated transcriptional activation in Bombyx mori.
Epigenetic modifiers and transcription factors contribute to developmentally programmed gene expression. Here, we establish a functional link between epigenetic regulation by Polycomb group (PcG) proteins and transcriptional regulation by C/ebp that orchestrates the correct expression of Bombyx mori asparagine synthetase (BmASNS), a gene involved in the biosynthesis of asparagine. We show that the cis-regulatory elements of YY1-binding motifs and the CpG island present on the BmASNS promoter are required for the recruitment of PcG proteins and the subsequent deposition of the epigenetic repression mark H3K27me3. RNAi-mediated knockdown of PcG genes leads to derepression of the BmASNS gene via the recruitment of activators, including BmC/ebp, to the promoter. Intriguingly, we find that PcG proteins and BmC/ebp can dynamically modulate the transcriptional output of the BmASNS target in a cell cycle-dependent manner. It will be essential to suppress BmASNS expression by PcG proteins at the G2/M phase of the cell cycle in the presence of BmC/ebp activator. Thus, our results provide a novel insight into the molecular mechanism underlying the recruitment and regulation of the PcG system at a discrete gene locus in Bombyx mori.
Document Type:

Reference

Product Catalog Number:

17-622
Product Catalog Name:

ChIPAb+ Trimethyl-Histone H3 (Lys27) - ChIP Validated Antibody and Primer Set
Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage.
Cell cycle arrest in response to environmental effects can lead to DNA breaks. We investigated whether inhibition of DNA replication during the initiation step can lead to DNA damage and characterised a cell-cycle-arrest point at the replication initiation step before the establishment of active replication forks. This arrest can be elicited by the iron chelators mimosine, ciclopirox olamine or 2,2'-bipyridyl, and can be reversed by the removal of the drugs or the addition of excess iron. Iron depletion induces DNA double-strand breaks in treated cells, and activates a DNA damage response that results in focal phosphorylation of histone H2AX, focal accumulation of replication protein A (RPA) and ATR (ATM and Rad3-related kinase), and activation of CHK1 kinase. Abrogation of the checkpoint response does not abolish the cell cycle arrest before the establishment of active DNA replication forks. DNA breaks appear concomitantly with the arrival of cells at the arrest point and persist upon release from the cell cycle block. We conclude that DNA double-strand breaks are the consequence, and not the cause, of cell cycle arrest during the initiation step of DNA replication by iron chelation.
Document Type:

Reference

Product Catalog Number:

07-164
Product Catalog Name:

Anti-phospho-H2A.X (Ser139) Antibody
Cell cycle regulated phosphorylation of the telomere-associated protein TIN2.
The protein TIN2 is a member of telomere-binding protein complex that serves to cap and protect mammalian chromosome ends. As a number of proteins in this complex are phosphorylated in a cell cycle-dependent manner, we investigated whether TIN2 is modified by phosphorylation as well. We performed phospho-proteomic analysis of human TIN2, and identified two phosphorylated residues, serines 295 and 330. We demonstrated that both these sites were phosphorylated during mitosis in human cells, as detected by Phos-tag reagent and phosphorylation-specific antibodies. Phosphorylation of serines 295 and 330 appeared to be mediated, at least in part, by the mitotic kinase RSK2. Specifically, phosphorylation of TIN2 at both these residues was increased upon expression of RSK2 and reduced by an inhibitor of the RSK family of kinases. Moreover, RSK2 phosphorylated TIN2 in vitro. The identification of these specifically timed post-translational events during the cell cycle suggests a potential mitotic regulation of TIN2 by phosphorylation.
Document Type:

Reference

Product Catalog Number:

ABE1314
Product Catalog Name:

Anti-phospho TIN2 (Ser295) Antibody
Cell cycle-dependent turnover of 5-hydroxymethyl cytosine in mouse embryonic stem cells.
Hydroxymethylcytosine in the genome is reported to be an intermediate of demethylation. In the present study, we demonstrated that maintenance methyltransferase Dnmt1 scarcely catalyzed hemi-hydroxymethylated DNA and that the hemi-hydroxymethylated DNA was not selectively recognized by the SRA domain of Uhrf1, indicating that hydroxymethylcytosine is diluted in a replication-dependent manner. A high level of 5-hydroxymethylcytosine in mouse embryonic stem cells was produced from the methylcytosine supplied mainly by de novo-type DNA methyltransferases Dnmt3a and Dnmt3b. The promoter regions of the HoxA gene cluster showed a high hydroxymethylation level whilst the methylcytosine level was quite low, suggesting that methylated CpG is actively hydroxylated during proliferation. All the results indicate that removal and production of hydroxymethylcytosine are regulated in replication-dependent manners in mouse embryonic stem cells.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple
Cell cycle-dependent activation of Ras.
BACKGROUND: Ras proteins play an essential role in the transduction of signals from a wide range of cell-surface receptors to the nucleus. These signals may promote cellular proliferation or differentiation, depending on the cell background. It is well established that Ras plays an important role in the transduction of mitogenic signals from activated growth-factor receptors, leading to cell-cycle entry. However, important questions remain as to whether Ras controls signalling events during cell-cycle progression and, if so, at which point in the cell-cycle it is activated. RESULTS: To address these questions we have developed a novel, functional assay for the detection of cellular activated Ras. Using this assay, we found that Ras was activated in HeLa cells, following release from mitosis, and in NIH 3T3 fibroblasts, following serum-stimulated cell-cycle entry. In each case, peak Ras activation occurred in mid-G1 phase. Ras activation in HeLa cells at mid-G1 phase was dependent on RNA and protein synthesis and was not associated with tyrosine phosphorylation of Shc proteins and their binding to Grb2. Significantly, activation of Ras and the extracellular-signal regulated (ERK) sub-group of mitogen-activated protein kinases were not temporally correlated during G1-phase progression. CONCLUSIONS: Activation of Ras during mid-G1 phase appears to differ in many respects from its rapid activation by growth factors, suggesting a novel mechanism of regulation that may be intrinsic to cell-cycle progression. Furthermore, the temporal dissociation between Ras and ERK activation suggests that Ras targets alternate effector pathways during G1-phase progression.
Document Type:

Reference

Product Catalog Number:

Multiple
Product Catalog Name:

Multiple