THADA regulates the organismal balance between energy storage and heat production

Human susceptibility to obesity is mainly genetic, yet the underlying evolutionary drivers causing variation from person to person are not clear. One theory rationalizes that populations that have adapted to warmer climates have reduced their metabolic rates, thereby increasing their propensity to store energy. We uncover here the function of a gene that supports this theory. THADA is one of the genes most strongly selected during evolution as humans settled in different climates. We report here that THADA knockout flies are obese, hyperphagic, have reduced energy production, and are sensitive to the cold. THADA binds the sarco/ER Ca2+ ATPase (SERCA) and acts on it as an uncoupler. Reducing SERCA activity in THADA mutant flies rescues their obesity, pinpointing SERCA as a key effector of THADA function. In sum, this identifies THADA as a regulator of the balance between energy consumption and energy storage, which was selected during human evolution

The degree of enhancer or promoter activity is reflected by the levels and directionality of eRNA transcription

Gene expression is regulated by promoters, which initiate transcription, and enhancers, which control their temporal and spatial activity. However, the discovery that mammalian enhancers also initiate transcription questions the inherent differences between enhancers and promoters. Here, we investigate the transcriptional properties of enhancers during Drosophila embryogenesis using characterized developmental enhancers. We show that while the timing of enhancer transcription is generally correlated with enhancer activity, the levels and directionality of transcription are highly varied among active enhancers. To assess how this impacts function, we developed a dual transgenic assay to simultaneously measure enhancer and promoter activities from a single element in the same embryo. Extensive transgenic analysis revealed a relationship between the direction of endogenous transcription and the ability to function as an enhancer or promoter in vivo, although enhancer RNA (eRNA) production and activity are not always strictly coupled. Some enhancers (mainly bidirectional) can act as weak promoters, producing overlapping spatio–temporal expression. Conversely, bidirectional promoters often act as strong enhancers, while unidirectional promoters generally cannot. The balance between enhancer and promoter activity is generally reflected in the levels and directionality of eRNA transcription and is likely an inherent sequence property of the elements themselves.Fil: Mikhaylichenko, Olga. European Molecular Biology Laboratory; AlemaniaFil: Bondarenko, Vladyslav. European Molecular Biology Laboratory; AlemaniaFil: Harnett, Dermot. European Molecular Biology Laboratory; AlemaniaFil: Schor, Ignacio Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Males, Matilda. European Molecular Biology Laboratory; AlemaniaFil: Viales, Rebecca R.. European Molecular Biology Laboratory; AlemaniaFil: Furlong, Eileen E. M.. European Molecular Biology Laboratory; Alemani

Depletion of Trypanosome CTR9 Leads to Gene Expression Defects

The Paf complex of Opisthokonts and plants contains at least five subunits: Paf1, Cdc73, Rtf1, Ctr9, and Leo1. Mutations in, or loss of Paf complex subunits have been shown to cause defects in histone modification, mRNA polyadenylation, and transcription by RNA polymerase I and RNA polymerase II. We here investigated trypanosome CTR9, which is essential for trypanosome survival. The results of tandem affinity purification suggested that trypanosome CTR9 associates with homologues of Leo1 and Cdc73; genes encoding homologues of Rtf1 and Paf1 were not found. RNAi targeting CTR9 resulted in at least ten-fold decreases in 131 essential mRNAs: they included several that are required for gene expression and its control, such as those encoding subunits of RNA polymerases, exoribonucleases that target mRNA, RNA helicases and RNA-binding proteins. Simultaneously, some genes from regions subject to chromatin silencing were derepressed, possibly as a secondary effect of the loss of two proteins that are required for silencing, ISWI and NLP1

Upregulation of transcript abundance by <i>CTR9</i> RNAi.

<p>One representative of each of the procyclin locus ORFs is shown, along with the 36 other ORFs showing the greatest increases in RNA after <i>CTR9</i> RNAi. “rpkm” is the reads per kilobase per million reads seen in poly(A)+RNA from normal (wild-type) cells, taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034256#pone.0034256-Manful1" target="_blank">[30]</a>; the median for all ORFs is 30.6. “CTR9/WT” shows the fold change in rpkm after RNAi (2 significant figures); **no signal was seen on a Northern blot using an ORF probe; *no RNA detected by reverse transcription and PCR targeting the ORF. “Features” indicates transcription by polymerase I (pol I), and the distance of the 3′ end of the locus mRNA from a convergent transcription stop area – “0 kb” indicates that this is the last ORF in a transcription unit. “0 kb, rRNA” indicats that the ORF is the last on a pol II transcription unit, next to an rRNA locus read in the opposite direction. “2 kb, ESAG” is a gene upstream of an ESAG. An rpkm of 20 corresponds to 1 mRNA per cell, which is difficult to detect by Northern blotting.</p

Essential genes whose expression is repressed by CTR9 depletion.

<p>Genes were selected by the following criteria: (a) mRNA decreased more than 20-fold by CTR9 RNAi and (b) A significant decrease of at least 5-fold in representation in the RNAi library, using bloodstream forms either 3 or 6 days after tetracycline addition <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034256#pone.0034256-Alsford1" target="_blank">[48]</a>. “rpkm” is the value for wild-type cells from RNASeq: an rpkm value of 20 is equivalent to one mRNA per cell <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034256#pone.0034256-Manful1" target="_blank">[30]</a>. “RNAi” is the rpkm for CTR9 RNAi divided by that for wild-type. “BS3” indicates the representation of this gene in RITSeq analysis in bloodstream forms, at day 3 after RNAi induction, divided by the representation without tetracycline; *not significant <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034256#pone.0034256-Alsford1" target="_blank">[48]</a>. “BS6” is the same for 6 days of tetracycline.</p

Putative homologues of the Paf complex subunits in various organisms.

<p>No homologues were found in either <i>Trichomonas</i> or <i>Giardia</i>. More details are in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034256#pone.0034256.s007" target="_blank">Table S3</a>.</p

Effects of <i>CTR9</i> RNAi on the transcriptome.

<p>A. Effect of <i>CTR9</i> RNAi on RNAseq representation, for poly(A)+ RNA, across two randomly-selected transcription units starting at loci 11.01.5320 and 927.10.4980. Both units have divergent start and convergent stop sites; in the Figure transcription is from left to right. For the values on the Y-axis, the rpkm for each ORF after 24 h RNAi was divided by the rpkm for wild-type cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034256#pone.0034256-Manful1" target="_blank">[30]</a>. Each bar represents an annotated ORF. B. Northern blot confirmation of the RNASeq results for <i>EP</i>, Histone H4 (<i>HISH4</i>), and tubulin (<i>TUB</i>) ORFs, using new RNA preparations. The rRNA control is from a scan of the methylene blue-stained blot. RNAi was induced for 24 h. C. Northern blot confirmation of the RNASeq results for <i>GPEET</i>, <i>PAG2</i>, Tb11.01.4120 and <i>ISWI</i> ORFs, using new RNA preparations. The rRNA control is a scan of the ethidium-bromide stained gel. D. <i>CTR9</i> RNAi effect on <i>EP</i> procyclin mRNA abundance and degradation. The left hand panel shows a Northern blot and the graph includes results from two different experiments, one with time points 0, 10 and 20 min and the other with time points 0, 10 and 30 min. Results were normalised to the 7SL RNA from the signal recognition particle (SRP). The half-life of <i>EP</i> mRNA is less than 8 min, as previously observed.</p

Proteins that co-purified with CTR9-TAP.

<p>Proteins were denatured, separated by SDS-PAGE and the whole gel section containing proteins was analysed by mass spectrometry. The numbers of different peptides detected are indicated. Possible contaminants are judged as such because of their appearance in other, unrelated purifications.</p

Properties of CTR9 complex components.

<p>A. The locations of TPR repeats (SMART SM00028) in trypanosome (Tb), <i>Saccharomyces cerevisiae</i> (Sc), human (Hs) and <i>Arabidopsis thaliana</i> (At) Ctr9 homologues. The maps are taken from the Interpro scan web site. Domains are shown as thickened bars. B. The most conserved portion of the Leo1 domain. The alignment was made using the MegAlign portion of the DNAStar package; for details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034256#pone.0034256.s003" target="_blank">Figure S3</a>. Identical amino acids are underlain in black and functionally conserved amino acids are underlain in grey. Dd: <i>Dictyostelium disoideum</i>; Pi: <i>Phytophthora infestans</i> – Tp: <i>Thalassiosira pseudonana</i>; Tt: <i>Tetrahymena thermophila</i>; Ng: <i>Naegleria gruberi</i>. C. Myc-tagged CTR9 fractionates into both the nucleus and the cytoplasm. Cells expressing CTR9-myc were lysed with NP40, centrifuged, and the extracts subjected to Western blotting using antibodies to myc (Santa Cruz), XRND (nuclear marker) and trypanothione reductase (cytoplasmic marker). T: total; N: nuclear fraction; C: cytoplasmic fraction. D. Effect of RNAi against CTR9. Two separate bloodstream trypanosome lines (represented by different symbols) were incubated with or without 100 ng/ml tetracycline. The starting cell density was 2×10⁵/ml and cultures were diluted to that level daily as required.</p