mRNA expression phase and correlations with CRBSs.

<p>Analysis of temporal expression profiles of 5708 expressed genes in two microarray replicates identified 118 common genes with circadian expression rhythms. Data from array A is shown. (<b>A</b>) The amplitudes of expressed genes (n = 5708) were plotted versus the circadian phase. The 118 rhythmic genes are indicated by black symbols. The shade of blue corresponds to the rhythmicity of a gene (light blue = low 24 h-rhythmicity and/or phase undefined; dark blue = high 24 h-rhythmicity and highly reliable phase). (<b>B</b>) The amplitudes of the 118 rhythmic genes are plotted against the phase. Genes with CRBSs are shown with black diamonds, the other rhythmic genes are displayed with gray diamonds. Core circadian clock genes are indicated. (<b>C</b>) Phase distribution of rhythmic genes with and without CRBSs.</p

Heat map view of 24 h cycling genes.

<p>Rhythmic genes with CRBSs (<b>A</b>) and without CRBSs (<b>B</b>) were ordered by phase of expression. Temperature entrained U2OS cells were released into constant conditions (37°C) and RNA expression levels were analyzed in 3 h intervals over a period of two days.</p

Relative expression of selected genes in hand mutant animals, compared to wild type.

<p>Expression levels were assessed by Northern blot. Statistically relevant deviations are evident with respect to all genes tested. While <i>akirin</i> displays an expression that is reduced by 40.5% in the <i>hand</i> mutant (<i>hand</i>^<i>173</i>), the expression of <i>kugelei</i> is lowered by 57.5%, that of <i>msp-300</i> by 65% and that of <i>multiplexin</i> by 28.5%, respectively. With regard to <i>zasp52</i>, two major transcripts are detected by the applied riboprobes with the larger one (arrow) being downregulated by 44.8% in the <i>hand</i> mutant and the smaller one (arrowhead) being upregulated by 34.9% in the same line. Bars represent mean values <u>+</u> SD of at least three independent experiments. Asterisks indicate statistical significance (Student´s <i>t</i>-test <i>P</i><0.05). Quantification of the relative band intensities in relation to the loading controls (ribosomal RNA, rRNA) was done by densitometric analysis.</p

Functional classification of genes exhibiting deviant expression levels in <i>hand</i>^<i>173</i> animals.

<p><i>Drosophila</i> Hand is apparently involved in regulating the expression of 545 genes with 385 genes being downregulated in <i>hand</i> mutants (<i>hand</i>^<i>173</i>) and 160 genes displaying an increased expression in the same line, compared to wild type. Functional classification of the corresponding protein products was done manually utilizing data from NCBI (<a href="http://www.ncbi.nlm.nih.gov/" target="_blank">http://www.ncbi.nlm.nih.gov/</a>) and Flybase (<a href="http://flybase.org/" target="_blank">http://flybase.org/</a>). Factors with yet unknown physiological functions are allocated to the category “assorted other”.</p

Rhythmic transcription of genes with CRBSs in their promoter is masked by high baseline expression.

<p>Robust low amplitude transcription rhythms revealed by long-term luminescence measurements of stable U2OS cell lines expressing the destabilized luciferase2P (<i>luc2P</i>) under control of the promoters of <i>ATG3</i>, <i>EIF5A2</i>, and <i>SCN5A</i>. <i>BMAL1</i>-<i>luc</i>, <i>PER2-luc2,</i> and <i>GAPDH-luc2P</i> cell lines are shown for control. Luminescence of synchronized cultures was measured at 30 min intervals. Raw expression data of the indicated reporter genes for day 1 to day 5 after synchronization are shown in the left panels. The middle panels show the 24 h moving average of the luciferase activity to estimate the non-rhythmic contribution and the right panels show the de-trended data to estimate the rhythmic contribution. The black and grey traces show expression profiles in the absence and presence of 200 nM rapamycin, respectively.</p

Temporal expression profiles of rhythmic and non-rhythmic genes.

<p>RNA levels of U2OS cells were analyzed in 3(0–45 h) relative to the average were calculated and plotted on a log2 scale versus the circadian time (CT). For each gene up to 10 probes were spotted on the arrays. Light blue lines correspond to expression profiles based on individual probes. The black triangles and the fitted dark blue sine curve correspond to the median of the data. Light and dark areas in the background indicate subjective day and night, respectively. Examples are shown for genes that fall in various categories. (<b>A</b>) Clock genes with CRBSs. (<b>B</b>) Clock-genes without CRBSs. (<b>C</b>) Rhythmic genes with CRBSs. (<b>D</b>) Rhythmic genes without CRBSs. (<b>E</b>) Genes not expressed in a rhythmic fashion. <i>AGAP11</i> harbors a highly enriched binding site for BMAL1, CLOCK, and CRY1, <i>CHP</i> has a CRY1 binding site. In both genes the CRBSs were close to the TSS. <i>CSNK2B</i> and <i>KDELR1</i> do not have a CRBS in a window of ±20 kb.</p

Sequences of primers and probes for q-RT-PCR.

<p>Sequences of primers and probes for q-RT-PCR.</p

Expression analysis of genes with CRBSs in their promoters.

<p>(<b>A</b>) UCSC browser views of BMAL1 (top), CLOCK (middle) and CRY1 (bottom) occupancy at the promoters of <i>ATG3</i>, <i>EIF5A2</i>, and <i>SCN5A</i>. Binding sites of the individual transcription regulators are indicated by colored bars. Black bars indicate common binding sites of the three regulators. (<b>B</b>) Temporal expression profiles (microarray analysis) of <i>ATG3</i>, <i>EIF5A2</i>, and <i>SCN5A</i>, which have strong CRBSs in their promoters. (<b>C, D</b>) Around the clock qRT-PCR analysis of preRNA (<b>C</b>) and mRNA (<b>D</b>) levels of <i>ATG3</i>, <i>EIF5A2</i>, and <i>SCN5A</i>. qRT-PCR analysis was carried out with intron- and exon-specific probes, respectively, of <i>ATG3</i>, <i>EIF5A2,</i> and <i>SCN5A</i> using RNA of time courses A and B. Expression levels of <i>HPRT1</i> were used for normalization.</p

The bHLH Transcription Factor Hand Regulates the Expression of Genes Critical to Heart and Muscle Function in <i>Drosophila melanogaster</i>

<div><p>Hand proteins belong to the highly conserved family of basic Helix-Loop-Helix transcription factors and are critical to distinct developmental processes, including cardiogenesis and neurogenesis in vertebrates. In <i>Drosophila melanogaster</i> a single orthologous <i>hand</i> gene is expressed with absence of the respective protein causing semilethality during early larval instars. Surviving adult animals suffer from shortened lifespan associated with a disorganized myofibrillar structure being apparent in the dorsal vessel, the wing hearts and in midgut tissue. Based on these data, the major biological significance of Hand seems to be related to muscle development, maintenance or function; however, up to now the physiological basis for Hand functionality remains elusive. Thus, the identification of genes whose expression is, directly or indirectly, regulated by Hand has considerable relevance with respect to understanding its biological functionality in flies and vertebrates. Beneficially, <i>hand</i> mutants are viable and exhibit affected tissues, which renders <i>Drosophila</i> an ideal model to investigate up- or downregulated target genes by a comparative microarray approach focusing on the respective tissues from mutant specimens. Our present work reveals for the first time that <i>Drosophila</i> Hand regulates the expression of numerous genes of diverse physiological relevancy, including distinct factors required for proper muscle development and function such as Zasp52 or Msp-300. These results relate Hand activity to muscle integrity and functionality and may thus be highly beneficial to the evaluation of corresponding <i>hand</i> phenotypes.</p></div

Relative expression of <i>Drosophila</i> homologs of selected vertebrate Hand target genes in <i>hand</i> mutant <i>Drosophila</i>, compared to wild type.

<p>Expression levels were assessed by Northern blot. Statistically relevant deviations are evident with respect to all genes tested. While <i>mef2</i> and <i>hedgehog</i> display an increased expression of 40.6% and 19.8% in <i>hand</i>^<i>173</i> animals, respectively, the expression of <i>cubitus interruptus</i> is decreased by 19.8% in the same line. Bars represent mean values ± SD of at least three independent experiments. Asterisks indicate statistical significance (Student´s <i>t</i>-test <i>P</i><0.05). Quantification of the relative band intensities in relation to the loading controls (ribosomal RNA, rRNA) was done by densitometric analysis.</p