Transcription, Elongation, Chromatin and Human Disease Grant Hartzog, Departmentof MCD Biology Grant Hartzog's laboratory investigates the roles of proteins that regulate the rate of transcription elongation - i.e. how quickly RNA polymerases travel along and transcribe genes into RNA. Many normal cellular genes are known to be regulated at the level of elongation, and the HIV virus specifically co-opts normal transcription elongation factors to regulate its own replication. Thus, understanding how transcription elongation is controlled is important for an understanding of both normal and abnormal gene expression. (Keywords: chromosome biology, genetics, molecular genetics)

Transcription, the process of copying the DNA of genes into RNA, has several distinct phases. In the initiation phase, proteins bind to the promoter, the DNA sequences at the beginning of the gene,  and recruit RNA polymerase. In the elongation phase, RNA polymerase leaves the promoter and copies the DNA sequences of the gene into an RNA transcript. Finally, when RNA polymerase reaches the end of the gene, it terminates transcription, releasing the RNA transcript for export from the nucleus to the cytoplasm, allowing itself to be recycled for a new round of transcription.

The rate and the efficiency of the elongation phase of transcription can be manipulated to regulate the levels of specific RNA transcripts in cells. One of the most important examples of such regulation is found in HIV, the virus that causes AIDS. When a cell is initially infected with HIV, transcription elongation of the HIV DNA is very inefficient. However, when an HIV protein called Tat accumulates, transcription elongation occurs very efficiently and large amounts of viral RNA are produced.

The Tat protein appears to function by recruiting proteins from the infected host to elongating RNA polymerase. These proteins, in turn, stimulate elongation by the polymerase. Interestingly, many host proteins required for Tat function have been identified and, in uninfected cells, appear to regulate transcription elongation of normal cellular genes.

Yeast DNA Transcription is Analogous to Humans

To thoroughly understand how the Tat protein hijacks normal cellular proteins to regulate transcription elongation of HIV DNA, we must understand the functions of the transcription elongation regulators that work with Tat. Hartzog and his colleagues use bakers yeast - Saccharomyces cerevisiae - to study the normal functions of these cellular transcription elongation regulators. Yeast provides an ideal system for studying transcription. The proteins that regulate transcription in yeast are very similar to those of humans. In addition, yeast grow very quickly and can be readily used for both genetic and biochemical analyses of transcription.

Spt5 assembles into distinct protein complexes: Hartzog has used immunoaffinity chromatography to purify Spt5 and proteins that associate with it.  A large number of proteins were found associated with Spt5. Using co-immunoprecipitation studies of these Spt5-associated proteins, Hartzog's lab has found that Spt5 must be assembled into at least  three different complexes. Furthermore, several of these Spt5-associated proteins are known to associate with RNA polymerase at distinct points in the transcription cycle. These data suggest that Spt5 remains associated with RNA polymerase throughout most or all of the process of transcription as part of a dynamic series of protein complexes.

Spt4 and Spt5 Are Host Transcription Elongation Factors Required by HIV Tat

Hartzog's studies focus on the role of Spt4 and Spt5, two proteins required for normal transcription elongation. Spt4 and Spt5 form a complex that regulates transcription elongation of normal cellular genes, and in humans, Spt4 and Spt5 are also required for the function of HIV Tat.

Hartzog has proposed that the Spt4-Spt5 help RNA polymerase to transcribe DNA that is assembled into nucleosomes.

Nucleosomes are complexes of histones and DNA, which enable DNA to be stored compactly within the small space of a cell's nucleus. Using a combination of biochemical and genetic approaches, Hartzog seeks answers to the following questions:

• How does the Spt4-Spt5 complex affect transcription elongation?
• What proteins do Spt4 and Spt5 interact with?
• How is Spt4-Spt5 function regulated?

Identifying Other Proteins Required for Transcription

Hartzog's laboratory has used complementary genetic and biochemical approaches to identify many proteins that associate with Spt5 and RNA polymerase. Many of these proteins are already implicated in transcription elongation and elongation through chromatin. It is likely that Spt5 associates with many of these proteins in a dynamic fashion. Hartzog's group is currently using affinity purification techniques to dissect different Spt5-RNA polymerase complexes. Initial results indicate that these complexes represent snapshots of transcribing RNA polymerase with its associated proteins at different points in the transcription cycle. Other proteins that copurify with Spt5 function in RNA processing events-i.e. pre-mRNA capping, splicing and polyadenylation.

Following up on these findings, the Hartzog group has found that Spt5 interacts genetically and biochemically  with the capping enzyme. In collaboration with the Ares group, they have used whole genome splicing sensitive DNA microarrays to show that Spt4, Spt5 and many other transcription elongation factors make important contributions to splicing in vivo.

Current studies in the Hartzog lab are aimed at understanding how the elongation phase of transcription is linked to these processing events, and to understanding the functional significance of these linkages.