Jesper Svejstrup

Mechanisms underlying transcript elongation by RNA polymerase II

RNA polymerase II (RNAPII) transcribes all protein-encoding genes in eukaryotes and is the endpoint for a plethora of cell regulatory pathways. The overall aim of our research is to understand the basic mechanisms underlying transcription by RNAPII, in particular transcript elongation. We believe that only a detailed insight into this process will make it possible for us to understand the mechanisms underlying multiple human diseases.

Numerous connections between RNAPII transcript elongation and human disease, including cancer, have become evident over the last several years. For example the Cockayne's syndrome group B gene-product (CSB), which is known to be required for transcription-coupled DNA repair, is an elongation factor. Likewise, mutations in the gene encoding IKAP, the largest subunit of the RNAPII-associated Elongator complex discovered in our lab, cause a severe neuro-degenerative disorder called familial dysautonomia (FD).

We employ a combination of biochemical and genetic approaches in yeast and metazoan cells to gain information about RNAPII transcript elongation, and more specifically the fundamental biological mechanisms underlying transcription-coupled DNA repair and RNAPII ubiquitylation, as well as RNAPII transcript elongation through nucleosomes.

We isolated the hyper-phosphorylated, elongating form of RNAPII from yeast chromatin and found it to be a holoenzyme, consisting of core polymerase and a complex, we named Elongator (Wittschieben et al., 1999; Otero et al., 1999). The Elp3 subunit of Elongator is a histone acetyltransferase (HAT), suggesting that the complex helps RNAPII contend with chromatin structure.

The Elongator complex is highly conserved in evolution, with all six Elongator subunits from yeast having a sequence counterpart in the human Elongator complex (Hawkes et al., 2002). Our results indicate that defects in Elongator function result in reduced histone acetylation and transcriptional elongation of several genes, suggesting that FD should be classified as a transcription disease. Indeed, cell motility deficiencies resulting from Elongator depletion may underlie the neuropathology of FD patients (Close et al., 2006).

The second major area of our research relates to our finding that RNAPII is ubiquitylated and degraded in response to DNA damage (Woudstra et al., 2002). We use purified factors to reconstitute RNAPII ubiquitylation in vitro (Somesh et al., 2005). Using this system in combination with yeast genetics, we have shown that RNAPII elongation complexes are the preferred substrates for ubiquitylation. Importantly, not only DNA-damage-, but also DNA damage-independent transcription problems result in RNAPII ubiquitylation in vivo, suggesting that RNAPII ubiquitylation/degradation is generally used to clear genes of RNA polymerases, and not only in response to DNA damage.

In all likelihood, this mechanism represents a 'last resort' enabling cells to remove RNAPII complexes that block genes, for example at DNA lesions that cannot be repaired. Our recent results have outlined the molecular mechanism of RNAPII ubiquitylation and provided compelling evidence for its importance (Somesh et al., 2007; Svejstrup, 2007).

A number of different projects focused on the issues outlined above will be available.

References

Close, et al. Transcription impairment and cell migration defects in elongator-depleted cells: implication for familial dysautonomia. Mol Cell 2006; 22: 521-531.

Hawkes, et al. Purification and characterization of the human elongator complex. J Biol Chem 2002; 277: 3047-3052.

Otero, et al. Elongator, a multisubunit component of a novel RNA polymerase II holoenzyme for transcriptional elongation. Mol Cell 1999; 3: 109-118.

Somesh, et al. Multiple mechanisms confining RNA polymerase II ubiquitylation to polymerases undergoing transcriptional arrest. Cell 2005; 121: 913-923.

Somesh, et al. Communication between distant sites in RNA polymerase II through ubiquitylation factors and the polymerase CTD. Cell 2007; 129: 57-68.

Svejstrup JQ. Contending with transcriptional arrest during RNAPII transcript elongation. Trends Biochem Sci 2007; 32: 165-171.

Wittschieben, et al. A novel histone acetyltransferase is an integral subunit of elongating RNA polymerase II holoenzyme. Mol Cell 1999; 4: 123-128.

Woudstra, et al. A Rad26-Def1 complex coordinates repair and RNA pol II proteolysis in response to DNA damage. Nature 2002; 415: 929-933.