Graduate Training at the Interface of Computational and Experimental Biology: An Outcome Report from a Partnership of Volunteers between a University and a National Laboratory.

Leading voices in the biological sciences have called for a transformation in graduate education leading to the PhD degree. One area commonly singled out for growth and innovation is cross-training in computational science. In 1998, the University of Tennessee (UT) founded an intercollegiate graduate program called the UT-ORNL Graduate School of Genome Science and Technology in partnership with the nearby Oak Ridge National Laboratory. Here, we report outcome data that attest to the program's effectiveness in graduating computationally enabled biologists for diverse careers. Among 77 PhD graduates since 2003, the majority came with traditional degrees in the biological sciences, yet two-thirds moved into computational or hybrid (computational-experimental) positions. We describe the curriculum of the program and how it has changed. We also summarize how the program seeks to establish cohesion between computational and experimental biologists. This type of program can respond flexibly and dynamically to unmet training needs. In conclusion, this study from a flagship, state-supported university may serve as a reference point for creating a stable, degree-granting, interdepartmental graduate program in computational biology and allied areas.

The Circadian Clock Modulates Global Daily Cycles of mRNA Ribosome Loading.

Circadian control of gene expression is well characterized at the transcriptional level, but little is known about diel or circadian control of translation. Genome-wide translation state profiling of mRNAs in Arabidopsis thaliana seedlings grown in long day was performed to estimate ribosome loading per mRNA. The experiments revealed extensive translational regulation of key biological processes. Notably, translation of mRNAs for ribosomal proteins and mitochondrial respiration peaked at night. Central clock mRNAs are among those subject to fluctuations in ribosome loading. There was no consistent phase relationship between peak translation states and peak transcript levels. The overlay of distinct transcriptional and translational cycles can be expected to alter the waveform of the protein synthesis rate. Plants that constitutively overexpress the clock gene CCA1 showed phase shifts in peak translation, with a 6-h delay from midnight to dawn or from noon to evening being particularly common. Moreover, cycles of ribosome loading that were detected under continuous light in the wild type collapsed in the CCA1 overexpressor. Finally, at the transcript level, the CCA1-ox strain adopted a global pattern of transcript abundance that was broadly correlated with the light-dark environment. Altogether, these data demonstrate that gene-specific diel cycles of ribosome loading are controlled in part by the circadian clock.

Analysis of mRNA translation states in Arabidopsis over the diurnal cycle by polysome microarray.

Gene regulation at the level of translation occurs in response to environmental perturbation and is increasingly recognized as a factor affecting plant development. Despite extensive knowledge of transcriptional control, very little is known about translational regulation of genes in response to the daily light/dark cycles. Here we describe the experimental layout designed to address how the translation states of genes change at various times during a diurnal cycle in Arabidopsis thaliana seedlings. We have adopted a strategy combining sucrose-gradient profiling of ribosomes and high-throughput microarray analysis of the ribosome-associated mRNA to investigate the translational landscape of the Arabidopsis genome. This is a powerful technique that can be easily extended to study translation regulation in different genetic backgrounds and under various environmental conditions.

Negative elongation factor (NELF) coordinates RNA polymerase II pausing, premature termination, and chromatin remodeling to regulate HIV transcription.

A barrier to eradicating HIV infection is targeting and eliminating latently infected cells. Events that contribute to HIV transcriptional latency include repressive chromatin structure, transcriptional interference, the inability of Tat to recruit positive transcription factor b, and poor processivity of RNA polymerase II (RNAP II). In this study, we investigated mechanisms by which negative elongation factor (NELF) establishes and maintains HIV latency. Negative elongation factor (NELF) induces RNAP II promoter proximal pausing and limits provirus expression in HIV-infected primary CD4(+) T cells. Decreasing NELF expression overcomes RNAP II pausing to enhance HIV transcription elongation in infected primary T cells, demonstrating the importance of pausing in repressing HIV transcription. We also show that RNAP II pausing is coupled to premature transcription termination and chromatin remodeling. NELF interacts with Pcf11, a transcription termination factor, and diminishing Pcf11 in primary CD4(+) T cells induces HIV transcription elongation. In addition, we identify NCoR1-GPS2-HDAC3 as a NELF-interacting corepressor complex that is associated with repressed HIV long terminal repeats. We propose a model in which NELF recruits Pcf11 and NCoR1-GPS2-HDAC3 to paused RNAP II, reinforcing repression of HIV transcription and establishing a critical checkpoint for HIV transcription and latency.

Negative elongation factor accelerates the rate at which heat shock genes are shut off by facilitating dissociation of heat shock factor.

Promoter-proximal pausing of RNA polymerase II (Pol II) occurs on thousands of genes in animal cells. This pausing often correlates with the rapid induction of genes, but direct tests of the relationship between pausing and induction rates are lacking. hsp70 and hsp26 in Drosophila are rapidly induced by heat shock. Contrary to current expectations, depletion of negative elongation factor (NELF), a key factor in setting up paused Pol II, reduced pausing but did not interfere with rapid induction. Instead, depletion of NELF delayed the time taken for these genes to shut off during recovery from heat shock. NELF depletion also delayed the dissociation of HSF from hsp70 and hsp26, and a similar delay was observed when cells were depleted of the histone acetyltransferase CBP. CBP has been reported to associate with Pol II, and acetylation of HSF by CBP has been implicated in inhibiting the DNA-binding activity of HSF. We propose that NELF-mediated pausing allows Pol II to direct CBP-mediated acetylation of HSF, thus causing HSF to dissociate from the gene. Activators are typically viewed as controlling Pol II. Our results reveal a possible reciprocal relationship in which paused Pol II influences the activator.

Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex.

Negative elongation factor (NELF) and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole sensitivity-inducing factor (DSIF) are involved in pausing RNA Polymerase II (Pol II) in the promoter-proximal region of the hsp70 gene in Drosophila, before heat shock induction. Such blocks in elongation are widespread in the Drosophila genome. However, the mechanism by which DSIF and NELF participate in setting up the paused Pol II remains unclear. We analyzed the interactions among DSIF, NELF, and a reconstituted Drosophila Pol II elongation complex to gain insight into the mechanism of pausing. Our results show that DSIF and NELF require a nascent transcript longer than 18 nt to stably associate with the Pol II elongation complex. Protein-RNA cross-linking reveals that Spt5, the largest subunit of DSIF, contacts the nascent RNA as the RNA emerges from the elongation complex. Taken together, these results provide a possible model by which DSIF binds the elongation complex via association with the nascent transcript and subsequently recruits NELF. Although DSIF and NELF were both required for inhibition of transcription, we did not detect a NELF-RNA contact when the nascent transcript was between 22 and 31 nt long, which encompasses the region where promoter-proximal pausing occurs on many genes in Drosophila. This raises the possibility that RNA binding by NELF is not necessary in promoter-proximal pausing.