Fluorescent riboswitch-controlled biosensors for the genome scale analysis of metabolic pathways.

Fluorescent detection in cells has been tremendously developed over the years and now benefits from a large array of reporters that can provide sensitive and specific detection in real time. However, the intracellular monitoring of metabolite levels still poses great challenges due to the often complex nature of detected metabolites. Here, we provide a systematic analysis of thiamin pyrophosphate (TPP) metabolism in Escherichia coli by using a TPP-sensing riboswitch that controls the expression of the fluorescent gfp reporter. By comparing different combinations of reporter fusions and TPP-sensing riboswitches, we determine key elements that are associated with strong TPP-dependent sensing. Furthermore, by using the Keio collection as a proxy for growth conditions differing in TPP levels, we perform a high-throughput screen analysis using high-density solid agar plates. Our study reveals several genes whose deletion leads to increased or decreased TPP levels. The approach developed here could be applicable to other riboswitches and reporter genes, thus representing a framework onto which further development could lead to highly sophisticated detection platforms allowing metabolic screens and identification of orphan riboswitches.

LRH-1 controls proliferation in breast tumor cells by regulating CDKN1A gene expression.

Liver receptor homolog-1 (LRH-1, NR5A2) is an orphan nuclear receptor that has an essential role in cancer progression, notably in breast cancer. Although its role in promoting cancer cell proliferation and migration is well documented, the molecular basis is not completely established. Here, we report that LRH-1 inhibition affects two- and three-dimensional cell proliferation of different types of breast cancer cells, including estrogen receptor α (ERα)-positive and triple-negative cells. This phenotype is accompanied by the upregulation of the cyclin-dependent kinase inhibitor CDKN1A (aka p21(CIP1/WAF1)) in a p53-independent manner. Chromatin immunoprecipitation analysis shows that LRH-1 cooperates with FOXA1 and binds directly to CDKN1A promoter and a distal regulatory region found at -62 kb from its transcriptional start sites, allowing repression of CDKN1A transcription. LRH-1 or FOXA1 depletion induces CDKN1A upregulation by removing histone deacetylase 2 from the promoter and distal regulatory elements and permitting histone acetylation in these regions. Analysis of breast cancer samples reveals that a high LRH-1 level is inversely correlated with CDKN1A expression in breast cancer patients and is associated with poor prognosis. This study reveals a novel mechanism of control of cell proliferation by LRH-1 regulating CDKN1A transcription in breast cancer cells, independent of ERα and p53 status. Targeting LRH-1 may provide an attractive prospect for treatment of tumors that are resistant to hormonal and targeted therapy.

Functional analysis of patient-derived mutations in the Fanconi anemia gene, FANCG/XRCC9.

OBJECTIVE: Fanconi anemia (FA) is an autosomal-recessive cancer susceptibility syndrome with seven complementation groups. Six of the FA genes have been cloned (corresponding to subtypes A, C, D2, E, F, and G) and the encoded proteins interact in a common pathway. Patient-derived mutations in FA genes have been helpful in delineating functional domains of FA proteins. The purpose of this work was to subtype FA patient-derived cell lines in our repository and to identify FA gene mutations. METHODS: We subtyped 62 FA patients as type A, G, C, or non-ACG by using a combination of retroviral gene transfer and immunoblot analysis. Among these FA patients, we identified six FA-G patients for further analysis. We used a strategy involving amplification of FANCG/XRCC9 exons and direct sequencing to identify novel FANCG mutations in cell lines derived from these FA-G patients. We functionally analyzed FANCG mutant alleles by transducing the corresponding cDNAs into a known FA-G indicator cell line and scoring correction of MMC sensitivity. RESULTS: Our results demonstrate a wide range of mutations in the FANCG gene (splice, nonsense, and missense mutations). Based on this mutational screen, a carboxy terminal functional domain of the FANCG protein appears to be required for complementation of FA-G cells and for normal assembly of the FANCA/FANCG/FANCC protein complex. CONCLUSION: The identification of patient-derived mutant alleles of FA genes can provide important insights to the function of FA proteins. FA subtyping is also a necessary precondition for gene therapy.