An automated high-content screening image analysis pipeline for the identification of selective autophagic inducers in human cancer cell lines.

Automated image processing is a critical and often rate-limiting step in high-content screening (HCS) workflows. The authors describe an open-source imaging-statistical framework with emphasis on segmentation to identify novel selective pharmacological inducers of autophagy. They screened a human alveolar cancer cell line and evaluated images by both local adaptive and global segmentation. At an individual cell level, region-growing segmentation was compared with histogram-derived segmentation. The histogram approach allowed segmentation of a sporadic-pattern foreground and hence the attainment of pixel-level precision. Single-cell phenotypic features were measured and reduced after assessing assay quality control. Hit compounds selected by machine learning corresponded well to the subjective threshold-based hits determined by expert analysis. Histogram-derived segmentation displayed robustness against image noise, a factor adversely affecting region growing segmentation.

A core Klf circuitry regulates self-renewal of embryonic stem cells.

Embryonic stem (ES) cells are unique in their ability to self-renew indefinitely and maintain pluripotency. These properties require transcription factors that specify the gene expression programme of ES cells. It has been possible to reverse the highly differentiated state of somatic cells back to a pluripotent state with a combination of four transcription factors: Klf4 is one of the reprogramming factors required, in conjunction with Oct4, Sox2 and c-Myc. Maintenance of self-renewal and pluripotency of ES cells requires Oct4, Sox2 and c-Myc, but Klf4 is dispensable. Here, we show that Krüppel-like factors are required for the self-renewal of ES cells. Simultaneous depletion of Klf2, Klf4 and Klf5 lead to ES cell differentiation. Chromatin immunoprecipitation coupled to microarray assay reveals that these Klf proteins share many common targets of Nanog, suggesting a close functional relationship between these factors. Expression analysis after triple RNA interference (RNAi) of the Klfs shows that they regulate key pluripotency genes, such as Nanog. Taken together, our study provides new insight into how the core Klf circuitry integrates into the Nanog transcriptional network to specify gene expression that is unique to ES cells.

Genome-wide mapping of RELA(p65) binding identifies E2F1 as a transcriptional activator recruited by NF-kappaB upon TLR4 activation.

NF-kappaB is a key mediator of inflammation. Here, we mapped the genome-wide loci bound by the RELA subunit of NF-kappaB in lipopolysaccharide (LPS)-stimulated human monocytic cells, and together with global gene expression profiling, found an overrepresentation of the E2F1-binding motif among RELA-bound loci associated with NF-kappaB target genes. Knockdown of endogenous E2F1 impaired the LPS inducibility of the proinflammatory cytokines CCL3(MIP-1alpha), IL23A(p19), TNF-alpha, and IL1-beta. Upon LPS stimulation, E2F1 is rapidly recruited to the promoters of these genes along with p50/RELA heterodimer via a mechanism that is dependent on NF-kappaB activation. Together with the observation that E2F1 physically interacts with p50/RELA in LPS-stimulated cells, our findings suggest that NF-kappaB recruits E2F1 to fully activate the transcription of NF-kappaB target genes. Global gene expression profiling subsequently revealed a spectrum of NF-kappaB target genes that are positively regulated by E2F1, further demonstrating the critical role of E2F1 in the Toll-like receptor 4 pathway.

BLIMP1 regulates cell growth through repression of p53 transcription.

Tight regulation of p53 is essential for maintaining normal cell growth. Here we report that BLIMP1 acts in an autoregulatory feedback loop that controls p53 activity through repression of p53 transcription. p53 binds to and positively regulates BLIMP1, which encodes for a known B cell transcriptional repressor. Knockdown of BLIMP1 by siRNA results in both apoptosis and growth arrest in human colon cancer cells and cell-cycle arrest in primary human fibroblasts. Interestingly, the levels of both p53 mRNA and protein are substantially increased after BLIMP1 depletion, which is accompanied by the induction of p53 target genes. Importantly, the apoptosis induced by BLIMP1 depletion in HCT116 cells is largely abrogated in cells lacking p53 or in cells depleted in p53 by siRNA. We further demonstrate that BLIMP1 binds to the p53 promoter and represses p53 transcription, and this provides a mechanistic explanation for the induction of p53 response in cells depleted of BLIMP1. Hence, suppression of p53 transcription is a crucial function of endogenous BLIMP1 and is essential for normal cell growth.

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.