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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. 21478681

    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. 19544450

    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

  • A CD8+ T cell immune evasion protein specific to Epstein-Barr virus and its close relatives in Old World primates. 17620360

    gamma 1-Herpesviruses such as Epstein-Barr virus (EBV) have a unique ability to amplify virus loads in vivo through latent growth-transforming infection. Whether they, like alpha- and beta-herpesviruses, have been driven to actively evade immune detection of replicative (lytic) infection remains a moot point. We were prompted to readdress this question by recent work (Pudney, V.A., A.M. Leese, A.B. Rickinson, and A.D. Hislop. 2005. J. Exp. Med. 201:349-360; Ressing, M.E., S.E. Keating, D. van Leeuwen, D. Koppers-Lalic, I.Y. Pappworth, E.J.H.J. Wiertz, and M. Rowe. 2005. J. Immunol. 174:6829-6838) showing that, as EBV-infected cells move through the lytic cycle, their susceptibility to EBV-specific CD8(+) T cell recognition falls dramatically, concomitant with a reductions in transporter associated with antigen processing (TAP) function and surface human histocompatibility leukocyte antigen (HLA) class I expression. Screening of genes that are unique to EBV and closely related gamma 1-herpesviruses of Old World primates identified an early EBV lytic cycle gene, BNLF2a, which efficiently blocks antigen-specific CD8(+) T cell recognition through HLA-A-, HLA-B-, and HLA-C-restricting alleles when expressed in target cells in vitro. The small (60-amino acid) BNLF2a protein mediated its effects through interacting with the TAP complex and inhibiting both its peptide- and ATP-binding functions. Furthermore, this targeting of the major histocompatibility complex class I pathway appears to be conserved among the BNLF2a homologues of Old World primate gamma 1-herpesviruses. Thus, even the acquisition of latent cycle genes endowing unique growth-transforming ability has not liberated these agents from evolutionary pressure to evade CD8(+) T cell control over virus replicative foci.
    Document Type:
    Reference
    Product Catalog Number:
    MABF249
    Product Catalog Name:
    Anti-Tapasin Antibody, clone 7F6

  • A cell cycle role for the epigenetic factor CTCF-L/BORIS. 22724006

    CTCF is a ubiquitous epigenetic regulator that has been proposed as a master keeper of chromatin organisation. CTCF-like, or BORIS, is thought to antagonise CTCF and has been found in normal testis, ovary and a large variety of tumour cells. The cellular function of BORIS remains intriguing although it might be involved in developmental reprogramming of gene expression patterns. We here unravel the expression of CTCF and BORIS proteins throughout human epidermis. While CTCF is widely distributed within the nucleus, BORIS is confined to the nucleolus and other euchromatin domains. Nascent RNA experiments in primary keratinocytes revealed that endogenous BORIS is present in active transcription sites. Interestingly, BORIS also localises to interphase centrosomes suggesting a role in the cell cycle. Blocking the cell cycle at S phase or mitosis, or causing DNA damage, produced a striking accumulation of BORIS. Consistently, ectopic expression of wild type or GFP- BORIS provoked a higher rate of S phase cells as well as genomic instability by mitosis failure. Furthermore, down-regulation of endogenous BORIS by specific shRNAs inhibited both RNA transcription and cell cycle progression. The results altogether suggest a role for BORIS in coordinating S phase events with mitosis.
    Document Type:
    Reference
    Product Catalog Number:
    07-729
    Product Catalog Name:
    Anti-CTCF Antibody

  • Host cell factor-1 recruitment to E2F-bound and cell-cycle-control genes is mediated by THAP11 and ZNF143. 25437553

    Host cell factor-1 (HCF-1) is a metazoan transcriptional coregulator essential for cell-cycle progression and cell proliferation. Current models suggest a mechanism whereby HCF-1 functions as a direct coregulator of E2F proteins, facilitating the expression of genes necessary for cell proliferation. In this report, we show that HCF-1 recruitment to numerous E2F-bound promoters is mediated by the concerted action of zinc finger transcription factors THAP11 and ZNF143, rather than E2F proteins directly. THAP11, ZNF143, and HCF-1 form a mutually dependent complex on chromatin, which is independent of E2F occupancy. Disruption of the THAP11/ZNF143/HCF-1 complex results in altered expression of cell-cycle control genes and leads to reduced cell proliferation, cell-cycle progression, and cell viability. These data establish a model in which a THAP11/ZNF143/HCF-1 complex is a critical component of the transcriptional regulatory network governing cell proliferation.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Single cell analysis of RNA-mediated histone H3.3 recruitment to a cytomegalovirus promoter-regulated transcription site. 23689370

    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. 23295675

    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-dependent recruitment of polycomb proteins to the ASNS promoter counteracts C/ebp-mediated transcriptional activation in Bombyx mori. 23382816

    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 lineage identification and stem cell culture in a porcine model for the study of intestinal epithelial regeneration. 23840480

    Significant advances in intestinal stem cell biology have been made in murine models; however, anatomical and physiological differences between mice and humans limit mice as a translational model for stem cell based research. The pig has been an effective translational model, and represents a candidate species to study intestinal epithelial stem cell (IESC) driven regeneration. The lack of validated reagents and epithelial culture methods is an obstacle to investigating IESC driven regeneration in a pig model. In this study, antibodies against Epithelial Adhesion Molecule 1 (EpCAM) and Villin marked cells of epithelial origin. Antibodies against Proliferative Cell Nuclear Antigen (PCNA), Minichromosome Maintenance Complex 2 (MCM2), Bromodeoxyuridine (BrdU) and phosphorylated Histone H3 (pH3) distinguished proliferating cells at various stages of the cell cycle. SOX9, localized to the stem/progenitor cells zone, while HOPX was restricted to the +4/'reserve' stem cell zone. Immunostaining also identified major differentiated lineages. Goblet cells were identified by Mucin 2 (MUC2); enteroendocrine cells by Chromogranin A (CGA), Gastrin and Somatostatin; and absorptive enterocytes by carbonic anhydrase II (CAII) and sucrase isomaltase (SIM). Transmission electron microscopy demonstrated morphologic and sub-cellular characteristics of stem cell and differentiated intestinal epithelial cell types. Quantitative PCR gene expression analysis enabled identification of stem/progenitor cells, post mitotic cell lineages, and important growth and differentiation pathways. Additionally, a method for long-term culture of porcine crypts was developed. Biomarker characterization and development of IESC culture in the porcine model represents a foundation for translational studies of IESC-driven regeneration of the intestinal epithelium in physiology and disease.
    Document Type:
    Reference
    Product Catalog Number:
    AB5977
    Product Catalog Name:
    Anti-Musashi-1 Antibody

  • Cell cycle arrest at the initiation step of human chromosomal DNA replication causes DNA damage. 15456844

    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