Two recent, large whole-genome association studies (GWAS) in European populations have associated a ϳ47-kb region that contains part of the FTO gene with high body mass index (BMI). The functions of FTO and adjacent FTM in human biology are not clear. We examined expression of these genes in organs of mice segregating for monogenic obesity mutations, exposed to underfeeding/overfeeding, and to 4°C. Fto/Ftm expression was reduced in mesenteric adipose tissue of mice segregating for the A y , Lep ob , Lepr db , Cpe fat , or tub mutations, and there was a similar trend in other tissues. These effects were not due to adiposity per se. Hypothalamic Fto and Ftm expression were decreased by fasting in lean and obese animals and by cold exposure in lean mice. The fact that responses of Fto and Ftm expression to these manipulations were almost indistinguishable suggested that the genes might be coregulated. The putative overlapping regulatory region contains at least two canonical CUTL1 binding sites. One of these nominal CUTL1 sites includes rs8050136, a SNP associated with high body mass. The A allele of rs8050136 associated with lower body mass than the C allele preferentially bound CUTL1 in human fibroblast DNA. 70% knockdown of CUTL1 expression in human fibroblasts decreased FTO and FTM expression by 90 and 65%, respectively. Animals and humans with various genetic interruptions of FTO or FTM have phenotypes reminiscent of aspects of the Bardet-Biedl obesity syndrome, a confirmed "ciliopathy." FTM has recently been shown to be a ciliary basal body protein.obesity; hypothalamus; adipose tissue; CUTL1 HERITABILITY OF ADIPOSITY, which reflects genetic contribution to the phenotype within a specific environment, is high, and it is variously estimated at 40 -60% (28, 49). The search for the underlying genes for obesity-using conventional linkage, association, and candidate gene approaches-has generated a large number of positive findings, many of which have not been replicated (e.g., 19, 25, 32, 37, 55, 67). Among the reasons for lack of consistent replication may be the relatively small population sizes, few markers genotyped, and blunt phenotypes. The recent generation of high-density single nucleotide polymorphism (SNP) and haplotype maps (International HapMap project; http://www.hapmap.org/) has revolutionized the field of human quantitative genetics. Applied to large, suitably phenotyped groups of subjects, whole-genome association studies (GWAS) are implicating novel genes not previously considered based on extant understanding of the molecular physiology of specific phenotypes. The discovery of the "Fat Mass and Obesity Associated gene" (FTO) as a potentially important contributor to human adiposity is such an example.In two GWAS involving a total of ϳ42,000 obese and nonobese subjects, dose-dependent highly significant effects of specific SNPs on chr. 16 have been associated with increased body mass index (BMI) (14, 52). In agreement with these results, Dina et al. (8) identified an association between rs1121980 ...

Summary Common polymorphisms in the first intron of FTO are associated with a increased body weight in adults. Previous studies have suggested that a CUX1 regulatory element within the implicated FTO region controls expression of FTO and the nearby ciliary gene, RPGRIP1L. Given the role of ciliary genes in energy homeostasis, we hypothesized that mice hypomorphic for Rpgrip1l would display increased adiposity. We find that Rpgrip1l+/− mice are hyperphagic, fatter, and display diminished suppression of food intake in response to leptin administration. In the hypothalamus of Rpgrip1l+/− mice, and in human fibroblasts with hypomorphic mutations in RPGRIP1L, the number of AcIII-positive ciliais diminished, accompanied by impaired convening of the leptin receptor to the vicinity of the cilium, and diminished pStat3 in response to leptin. These findings suggest that RPGRIP1L may be partly or exclusively responsible for the obesity susceptibility signal at the FTO locus.

The first intron of FTO contains common single nucleotide polymorphisms associated with body weight and adiposity in humans. In an effort to identify the molecular basis for this association, we discovered that FTO and RPGRIP1L (a ciliary gene located in close proximity to the transcriptional start site of FTO) are regulated by isoforms P200 and P110 of the transcription factor, CUX1. This regulation occurs via a single AATAAATA regulatory site (conserved in the mouse) within the FTO intronic region associated with adiposity in humans. Single nucleotide polymorphism rs8050136 (located in this regulatory site) affects binding affinities of P200 and P110. Promoter-probe analysis revealed that binding of P200 to this site represses FTO, whereas binding of P110 increases transcriptional activity from the FTO as well as RPGRIP1L minimal promoters. Reduced expression of Fto or Rpgrip1l affects leptin receptor isoform b trafficking and leptin signaling in N41 mouse hypothalamic or N2a neuroblastoma cells in vitro. Leptin receptor clusters in the vicinity of the cilium of arcuate hypothalamic neurons in C57BL/6J mice treated with leptin, but not in fasted mice, suggesting a potentially important role of the cilium in leptin signaling that is, in part, regulated by FTO and RPGRIP1L. Decreased Fto/Rpgrip1l expression in the arcuate hypothalamus coincides with decreased nuclear enzymatic activity of a protease (cathepsin L) that has been shown to cleave full-length CUX1 (P200) to P110. P200 disrupts (whereas P110 promotes) leptin receptor isoform b clustering in the vicinity of the cilium in vitro. Clustering of the receptor coincides with increased leptin signaling as reflected in protein levels of phosphorylated Stat3 (p-Stat3). Association of the FTO locus with adiposity in humans may reflect functional consequences of A/C alleles at rs8050136. The obesity-risk (A) allele shows reduced affinity for the FTO and RPGRIP1L transcriptional activator P110, leading to the following: 1) decreased FTO and RPGRIP1L mRNA levels; 2) reduced LEPR trafficking to the cilium; and, as a consequence, 3) a diminished cellular response to leptin.

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SummaryThe tylosin biosynthetic gene cluster of Streptomyces fradiae is remarkable in harbouring at least five regulatory genes, two of which (tylS and tylT) encode proteins of the Streptomyces antibiotic regulatory protein (SARP) family. The aim of the present work was to assess the respective contributions of TylS and TylT to tylosin production. A combination of targeted gene disruption, fermentation studies and gene expression analysis via reverse transcriptase-polymerase chain reaction (RT-PCR) suggests that tylS is essential for tylosin production and controls the expression of tylR (previously shown to be a global activator of the biosynthetic pathway) plus at least one other gene involved in polyketide metabolism or regulation thereof. This is the first demonstration of a SARP acting to control another regulatory gene during antibiotic biosynthesis. In contrast, tylT is not essential for tylosin production.

SummaryControl of tylosin production in Streptomyces fradiae features interplay between a repressor, TylQ, and an activator, TylS, during regulation of tylR . The latter encodes a pathway-specific activator that controls most of the tylosin-biosynthetic ( tyl ) genes that are subject to regulation. This was established by targeted gene disruption applied separately to tylR and tylS together with transcript analysis involving reverse transcription polymerase chain reaction (RT-PCR). TylR controls multiple genes that encode the synthesis or addition of all three tylosin sugars, plus polyketide ring oxidation, and at least one of the polyketide synthase (PKS) megagenes, tylGI . (Expression of a few tyl genes, plus the resistance determinants tlrB and tlrD , together with some ancillary or unassigned genes, is not apparently regulated during fermentation, consistent with constitutive expression.) In contrast, the only gene known for sure to be directly controlled by TylS is tylR , and there are very few additional candidates. These include the mycinose-biosynthetic gene, tylJ , and two previously unassigned genes, ORF12* ( tylU ) plus ORF11* ( tylV ). TylS also controls the PKS genes [ tylGIII-tylGIV-tylGV ] although not in obligatory fashion. These genes can be transcribed (i.e. tylosin can be produced) in a tylS -KO strain by forcing overexpression of tylR using a foreign promoter. We therefore suspect that TylS might control the PKS genes indirectly, although this remains to be established unequivocally. Conceivably, the direct effects of TylS are exerted exclusively on other regulators. Tylosin production levels were elevated when tylS or (especially) tylR was overexpressed in S. fradiae wild-type and yield increments of industrial significance were generated by similar manipulation of an enhanced production strain.

Expression analysis by RT-PCR, applied to the entire tyl cluster, revealed that the pattern of transcription is more complex than expected. For example, the five tylG polyketide synthase genes are not necessarily cotranscribed or even coregulated. Among the regulatory genes, tylQ has emerged as a key factor. Although several genes (including the positive regulator, tylS) were possibly expressed constitutively, only tylQ was silent during secondary metabolism. Analysis of engineered strains, in which tylQ was disrupted or overexpressed, showed that the TylQ protein is a transcriptional repressor that blocks tylosin biosynthesis by controlling expression of the activator, tylR. Before tylosin production can be triggered, tylQ must be switched off, or at least downregulated.

Regulation of tylosin production and morphological differentiation in Streptomyces fradiae by TylP, a deduced g-butyrolactone receptor activator, adpA (Ohnishi et al., 1999). A variation on this theme is seen in Streptomyces virginiae, in which gbutyrolactones ('butanolides') bind to BarA and derepress virginiamycin biosynthesis with no concurrent effect on sporulation (Nakano et al., 1998). In the latter case, BarA controls the expression of various genes, including barB. Because the tylosin biosynthetic (tyl ) gene cluster of Streptomyces fradiae (Fig. 1) contains obvious orthologues of barA and barB (namely tylP and tylQ respectively; Bate et al., 1999), it seemed likely a priori that one or more g-butyrolactone(s) regulate tylosin production and that the TylP protein (deduced to be a g-butyrolactone receptor) might be a repressor involved in such regulation. The aim of the present work was to test that hypothesis and (if substantiated) to identify targets for TylP. In doing so, we were mindful that g-butyrolactones and their receptors typically operate at relatively 'high' levels in regulatory cascades that control antibiotic production in actinomycetes. That being so and given that the tyl cluster is remarkable in containing at least five regulatory genes (including tylR, tylS and tylT, in addition to tylP and tylQ; Bate et al., 1999;, we were also interested in the possibility of establishing a hierarchical order among the tyl regulators. Accordingly, gene expression analysis (involving reverse transcription-polymerase chain reaction, RT-PCR) was applied to the entire tyl cluster before and after the onset of tylosin production in S. fradiae wild type and, concurrently, in engineered strains in which tylP was constitutively overexpressed or had been specifically disrupted. Such studies were complemented by fermentation analysis and by characterization of tyl promoters transplanted into S. lividans. Results Gene expression analysisWhen transcript analysis was first applied to the tyl gene cluster , it became clear that the TylQ protein is a repressor that controls tylR (a global activator of the tyl cluster; Bate et al., 1999), and that tylQ must be repressed before tylosin production can begin. Thus, in S. fradiae wild type, although tylQ was expressed before the onset of tylosin biosynthesis (i.e. at 18 h; Fig. 2), it was the only silent gene in the cluster after tylosin production had commenced (i.e. at 40 h; Fig. 2). In Molecular Microbiology (2002) 45(3), [735][736][737][738][739][740][741][742][743][744] George Stratigopoulos, Atul R. Gandecha and Eric Cundliffe* Department of Biochemistry, University of Leicester, Leicester LE1 7RH, UK. SummaryDuring promoter-probe analysis carried out in Streptomyces lividans, the TylP protein powerfully inhibited reporter gene expression from the tylP promoter, raising the likelihood that tylP is autoregulated in its native host, Streptomyces fradiae. Also in S. lividans, TylP negatively controlled the tylQ promoter, even though tylQ could still be switched off in ...