It’s not all transcription! New insights on how biological rhythms are generated

Sleepy during the day? Hungry at night? You should check your biological clock!

As in every organism, humans are exposed to daily variations of their environment. There is obviously the day/night cycle, but significant variations of temperature and humidity also occur in temperate regions of the globe. To survive to these environmental changes, organisms have evolved so that their biology, biochemistry, physiology and behavior are rhythmically regulated on a 24hr-basis. Humans are no exception, and most (if not all) of our biological functions are set to function optimally at the most appropriate time of the day. For example, the physiology of muscle cells is rhythmic so that their capacity of coping with physical activity is maximal during the day.

A lot of progress has been made over the last two decades to uncover the molecular underpinnings of circadian (for circa, about and dies, day) rhythms. To keep the story short, in all eukaryotes the circadian system relies on transcriptional feedback loops that operate at the level of individual cells (see figure 1). In mammals, these loops are composed of the two transcription factors CLK and BMAL1, which act as a heterodimeric complex to activate the expression of the transcriptional repressors Period (Per1, Per2 and Per3) and Cryptochrome (Cry1 and Cry2). When expressed, these repressor proteins are post-translationally modified (e.g., phosphorylation) and feedback to inhibit the transcriptional activity of CLK:BMAL1. As a result, transcription of Per and Cry genes is shut-off. The progressive degradation of the PER and CRY proteins then leads to a new cycle of CLK:BMAL1-mediated transcription. Importantly, these transcriptional oscillations regulate the rhythmic expression of a large fraction of the transcriptome (up to 10-15% of all mRNAs). These output genes, also called “clock-controlled genes”, are rhythmically regulated in a tissue-specific manner, and are responsible for the daily oscillations of biological functions.

As in other biological systems, it is generally assumed that daily variations of mRNA levels are a direct consequence of transcription regulation. However, there is growing evidence that post-transcriptional events such as mRNA splicing, polyadenylation, nuclear export and half-life also contribute to changes in the amount of mRNA expressed by particular genes. Such post-transcriptional processes are known to have a role in other areas of cell biology but until very recently this had not been studied in detail at a genome-wide level.

This is the question addressed by Jerome Menet, Joseph Rodriguez, Katharine Abruzzi and Michael Rosbash, in a paper recently published at eLife (Menet et al., 2012). The authors directly assayed rhythmic transcription by measuring the amount of nascent RNA being produced at a given time, six times a day, across all the genes in mouse liver cells using a high-throughput sequencing approach called Nascent-Seq (see figure 2). They compared this with the amount of liver mRNA expressed at six time points of the day. Although the authors found that many genes exhibit rhythmic mRNA expression in the mouse liver, about 70% of them did not show comparable transcriptional rhythms. Post-transcriptional regulations have therefore a major role in the circadian system of mice. Interestingly, similar experiments performed by Joe Rodriguez in the Rosbash lab using Drosophila as the model system led to the same conclusions, suggesting that the contribution of post-transcriptional events to the generation of circadian rhythms is common to all animals (Rodriguez et al., in press).

To assess the contribution of the core molecular clock to genome-wide transcriptional rhythms, Menet et al. also examined how rhythmic CLK:BMAL1 DNA binding directly affects the transcription of its target genes. They found that although maximal binding occurs at an apparently uniform phase, the peak transcriptional phases of CLK:BMAL1 target genes are heterogeneous, which indicates a disconnect between CLK:BMAL1 DNA binding and its transcriptional output.

The data taken together reveal novel regulatory features of rhythmic gene expression and illustrate the potential of Nascent-Seq as a genome-wide assay technique for exploring a range of questions related to gene expression and gene regulation.

Menet JS, Rodriguez J, Abruzzi KC, Rosbash M. Nascent-Seq Reveals Novel Features of Mouse Circadian Transcriptional Regulation. elife. 2012;1:e00011. doi: 10.7554/eLife.00011.

Rodriguez J, Tang CHA, Khodor YL, Vodala S, Menet JS, Rosbash M. Post-transcriptional events regulate genome-wide rhythmic gene expression in Drosophila. Proc Natl Acad Sci U S A. (In press).

Finding novel antibiotics in dirt using unculturable bacteria

Sean Brady from The Rockefeller University will be visiting campus to lecture on Culture Independent Approaches for the Discovery of New Bacterial Metabolites as part of the Joint Biology/Biochemistry Colloquium Series, Wednesday, Oct 13, at 4:00 pm in Gestenzang 121.

Sean’s research centers on the discovery, biosynthesis and characterization of new, genetically encoded small molecules from microbial sources, with a special focus on soil bacteria and pathogenic bacteria. One area of particular interest is the development of methods to access new biologically active small molecules from bacteria that cannot be grown in culture. Soil bacteria that can’t yet be cultured outnumber those that have been by orders of magnitude, and provide a huge pool of genetic diversity that can be searched for novel useful natural products.

Sean is a Howard Hughes Medical Institute Early Career Scientist. He was named a Searle Scholar, an Irma T. Hirschl Scholar, an Alexandrine and Alexander L. Sinsheimer Scholar and an Arnold and Mabel Beckman Young Investigator.

Smiths Detection and Novartis Diagnosics sign deal for LATE PCR technology

Smiths Detection and Novartis Diagnostics have entered an collaboration and license agreement for marketing the Bio-Seeq instrument. This uses LATE PCR technology developed in the Wangh lab at Brandeis and licensed by Smiths Detection.

Dilute-’N’-Go sequencing

Prof. Larry Wangh and his lab are interested in detecting changes in mitochondrial genomic sequences that result from aging, disease, or drugs.  To do this, they use LATE-PCR, an advanced form of asymmetric PCR, to detect mutations in the mitochondria by using multiplexes to study many mitochondrial genes at the same time.  LATE-PCR generates single DNA strands that are easily diluted for sequencing.  In the past. they have only been able to sequence one DNA strand from these multiplex reactions.

In a recent publication in Nucleic Acid Research, staff members Yanwei Jia and John Rice, along with Molecular and Cell Biology grad student Adam Osborne, describe the development of a blocking reagent that allows them to sequence both strands of the product DNA, thus allowing for the easy verification of mutations.

The figure at right shows that without a blocker (BLK), one is not able to obtain the excess (XP) strand sequence from a multiplex reaction.  Using a blocker one is able to get not only the limiting (LP) strand, but also the excess strand from the same multiplex

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