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Huck-Hui Ng

Publications and source records attributed to Huck-Hui Ng.

10 recordsLinked to original sources
PubMed2006

Application of advanced technologies in ageing research.

Several technologies that emerged in the post-genomic era have been particularly useful in dissecting the molecular mechanisms of complex biological processes through the systems approach. Here, we review how three of these technologies, namely transcriptional profiling, large-scale RNA interference (RNAi) and genome-wide location analysis of protein-DNA interactions, have been used in the study of ageing in metazoans. We also highlight recent developments of these three technologies and how these developments are applicable to ageing research.

Aging↗
PubMed2006

Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1.

Embryonic stem (ES) cells are pluripotent cells that can self-renew or differentiate into many cell types. A unique network of transcription factors and signalling molecules are essential for maintaining this capability. Here, we report that a spalt family member, Sall4, is required for the pluripotency of ES cells. Similarly to Oct4, a reduction in Sall4 levels in mouse ES cells results in respecification, under the appropriate culture conditions, of ES cells to the trophoblast lineage. Sall4 regulates transcription of Pou5f1 which encodes Oct4. Sall4 binds to the highly conserved regulatory region of the Pou5f1 distal enhancer and activates Pou5f1 expression in vivo and in vitro. Microinjection of Sall4 small interfering (si) RNA into mouse zygotes resulted in reduction of Sall4 and Oct4 mRNAs in preimplantation embryos and significant expansion of Cdx2 expression into the inner cell mass. These results demonstrate that Sall4 is a transcriptional activator of Pou5f1 and has a critical role in the maintenance of ES cell pluripotency by modulating Oct4 expression. The data also indicates that Sall4 is important for early embryonic cell-fate decisions.

Animals↗
PubMed2006

Sall4 interacts with Nanog and co-occupies Nanog genomic sites in embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells with self-renewing property. Nanog is a homeobox transcription factor required to maintain ES cells in a non-differentiated state. Using affinity purification coupled to liquid chromatography-tandem mass spectrometry analysis, we identified Sall4 as a Nanog co-purified protein. Co-immunoprecipitation and glutathione S-transferase pulldown experiments confirmed the interaction between Nanog and Sall4. We showed that Nanog and Sall4 co-occupied Nanog and Sall4 enhancer regions in living ES cells. Knockdown of Nanog or Sall4 by RNA interference led to a reduction in Nanog and Sall4 enhancer activities, providing evidence that these factors are positively regulating these enhancers. Importantly, co-transfection of Sall4 with these ES cell-specific enhancers led to transactivation in heterologous somatic cells. Chromatin immunoprecipitation experiments also showed that Sall4 co-occupied many Nanog binding sites in ES cells. Our data implicate Sall4 as an important component of the transcription regulatory networks in ES cells by cooperating with Nanog. We suggest that Sall4 and Nanog form a regulatory circuit similar to that of Oct4 and Sox2. This study highlights the extensive regulatory loops connecting genes, which encode for key transcription factors in ES cells.

Animals↗
PubMed2006

The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells.

Oct4 and Nanog are transcription factors required to maintain the pluripotency and self-renewal of embryonic stem (ES) cells. Using the chromatin immunoprecipitation paired-end ditags method, we mapped the binding sites of these factors in the mouse ES cell genome. We identified 1,083 and 3,006 high-confidence binding sites for Oct4 and Nanog, respectively. Comparative location analyses indicated that Oct4 and Nanog overlap substantially in their targets, and they are bound to genes in different configurations. Using de novo motif discovery algorithms, we defined the cis-acting elements mediating their respective binding to genomic sites. By integrating RNA interference-mediated depletion of Oct4 and Nanog with microarray expression profiling, we demonstrated that these factors can activate or suppress transcription. We further showed that common core downstream targets are important to keep ES cells from differentiating. The emerging picture is one in which Oct4 and Nanog control a cascade of pathways that are intricately connected to govern pluripotency, self-renewal, genome surveillance and cell fate determination.

Animals↗
PubMed2006

A global map of p53 transcription-factor binding sites in the human genome.

The ability to derive a whole-genome map of transcription-factor binding sites (TFBS) is crucial for elucidating gene regulatory networks. Herein, we describe a robust approach that couples chromatin immunoprecipitation (ChIP) with the paired-end ditag (PET) sequencing strategy for unbiased and precise global localization of TFBS. We have applied this strategy to map p53 targets in the human genome. From a saturated sampling of over half a million PET sequences, we characterized 65,572 unique p53 ChIP DNA fragments and established overlapping PET clusters as a readout to define p53 binding loci with remarkable specificity. Based on this information, we refined the consensus p53 binding motif, identified at least 542 binding loci with high confidence, discovered 98 previously unidentified p53 target genes that were implicated in novel aspects of p53 functions, and showed their clinical relevance to p53-dependent tumorigenesis in primary cancer samples.

Binding Sites↗
PubMed2005

p53 functions as a negative regulator of osteoblastogenesis, osteoblast-dependent osteoclastogenesis, and bone remodeling.

p53 is a well known tumor suppressor. We show that p53 also regulates osteoblast differentiation, bone formation, and osteoblast-dependent osteoclast differentiation. Indeed, p53(-/-) mice display a high bone mass phenotype, and p53(-/-) osteoblasts show accelerated differentiation, secondary to an increase in expression of the osteoblast differentiation factor osterix, as a result. Reporter assays indicate that p53 represses osterix transcription by the minimal promoter in a DNA-binding-independent manner. In addition, p53(-/-) osteoblasts have an enhanced ability to favor osteoclast differentiation, in association with an increase in expression of macrophage-colony stimulating factor, which is under the control of osterix. Furthermore, inactivating p53 is sufficient to rescue the osteoblast differentiation defects observed in mice lacking c-Abl, a p53-interacting protein. Thus, these results identify p53 as a novel regulator of osteoblast differentiation, osteoblast-dependent osteoclastogenesis, and bone remodeling.

Animals↗
PubMed2005

Transcriptional regulation of nanog by OCT4 and SOX2.

Nanog, Sox2, and Oct4 are transcription factors all essential to maintaining the pluripotent embryonic stem cell phenotype. Through a cooperative interaction, Sox2 and Oct4 have previously been described to drive pluripotent-specific expression of a number of genes. We now extend the list of Sox2-Oct4 target genes to include Nanog. Within the Nanog proximal promoter, we identify a composite sox-oct cis-regulatory element essential for Nanog pluripotent transcription. This element is conserved over 250 million years of cumulative evolution within the eutherian mammals. A Nanog proximal promoter-EGFP (enhanced green fluorescent protein) reporter transgene recapitulates endogenous Nanog mRNA expression in embryonic stem cells and their differentiated derivatives. Sox2 and Oct4 interaction with the Nanog promoter was confirmed through mutagenesis and in vitro binding assays. Electrophoretic mobility shift assays indicate that the Sox2-Oct4 heterodimer forms more efficiently on the composite element within Nanog than the similar element within Fgf4. Using chromatin immunoprecipitation, we show that Oct4 and Sox2 bind to the Nanog promoter in living mouse and human embryonic stem cells. Furthermore, by specific knockdown of Oct4 and Sox2 mRNA by RNA interference in embryonic stem cells, we provide genetic evidence for a link between Oct4, Sox2, and the Nanog promoter. These studies extend the understanding of the pluripotent genetic regulatory network within which the Sox2-Oct4 complex are at the top of the regulatory hierarchy.

Animals↗
PubMed2005

Reciprocal transcriptional regulation of Pou5f1 and Sox2 via the Oct4/Sox2 complex in embryonic stem cells.

Embryonic stem cells (ESCs) are pluripotent cells that can either self-renew or differentiate into many cell types. Oct4 and Sox2 are transcription factors essential to the pluripotent and self-renewing phenotypes of ESCs. Both factors are upstream in the hierarchy of the transcription regulatory network and are partners in regulating several ESC-specific genes. In ESCs, Sox2 is transcriptionally regulated by an enhancer containing a composite sox-oct element that Oct4 and Sox2 bind in a combinatorial interaction. It has previously been shown that Pou5f1, the Oct4 gene, contains a distal enhancer imparting specific expression in both ESCs and preimplantation embryos. Here, we identify a composite sox-oct element within this enhancer and show that it is involved in Pou5f1 transcriptional activity in ESCs. In vitro experiments with ESC nuclear extracts demonstrate that Oct4 and Sox2 interact specifically with this regulatory element. More importantly, by chromatin immunoprecipitation assay, we establish that both Oct4 and Sox2 bind directly to the composite sox-oct elements in both Pou5f1 and Sox2 in living mouse and human ESCs. Specific knockdown of either Oct4 or Sox2 by RNA interference leads to the reduction of both genes' enhancer activities and endogenous expression levels in addition to ESC differentiation. Our data uncover a positive and potentially self-reinforcing regulatory loop that maintains Pou5f1 and Sox2 expression via the Oct4/Sox2 complex in pluripotent cells.

Animals↗
PubMed2004

p53-regulated transcriptional program associated with genotoxic stress-induced apoptosis.

By using a genome-wide approach, we sought the identification of p53-regulated genes involved in cellular apoptosis. To this end, we assessed the transcriptional response of HCT116 colorectal cancer cells during apoptosis induced by the anticancer drug 5-fluorouracil as the function of p53 status, and we identified 230 potential targets that are regulated by p53. Previously identified p53 targets known to be involved in growth arrest and apoptosis were observed to be induced, thus validating the approach. Strikingly, we found that p53 regulates gene expression primarily through transcriptional repression (n = 189) rather than activation (n = 41), and selective blockade of p53-dependent gene repression resulted in the reduction in 5-fluorouracil-induced apoptosis. Reporter and chromatin immunoprecipitation assays demonstrated that p53 can suppress the promoter activities of three further studied candidate genes PLK, PTTG1, and CHEK1 but would only bind directly to PTTG1 and CHEK1 promoters, revealing that p53 can repress gene expression through both direct and indirect mechanisms. Moreover, RNA(i)-mediated knockdown of PLK and PTTG1 expression was sufficient to induce apoptosis, suggesting that repression of novel anti-apoptotic genes by p53 might contribute to a significant portion of the p53-dependent apoptosis. Our data support the divergent functions of p53 in regulating gene expression that play both synergistic and pleiotropic roles in p53-associated apoptosis.

Apoptosis↗