Mapping the chromatin landscape

One of the most interesting features of all immune cells is their rapid, precise and highly organised response to infection or other assaults on the body. They are therefore set up to dramatically change their gene and protein expression profile rapidly following a signal, which come in many and varied forms - from infections and allergens to signals from within the body that lead to an aberrant autoimmune response.

During this process a plethora of genes that are normally silent are switched on and an equal number that are normally active are turned off. The types of genes that respond are either generic - no matter what the signal is these genes will change their expression - or they are exquisitely specific to the assault; for example, bacterial pathogens will induce one suite of genes and viral attack will produce a different array. There is a very controlled and organised response at the gene level.

Professor Frances Shannon's group at the John Curtin School of Medical Research at ANU is trying to unravel the molecular mechanisms by which these genes in the nucleus see the immune signal and respond appropriately. They are addressing this aim at two levels: by looking at single genes and on a genome-wide scale.

The work has led to important findings in defining the role of architectural proteins and the packaging of DNA in the cell nucleus in controlling immune-related gene expression. Shannon sees this as some of her group's most important findings.

The single genes Shannon's team study encode cytokines, which are secreted by cells to mediate the immune response. One reason to look at this family of genes is that they usually undergo very large changes in transcription upon stimulation.

Shannon originally became interested in inducible gene expression as a postdoc with Dr Julian Wells at Adelaide University, investigating the role of histones in chromatin structure and gene expression. That was long before this area became so popular - chromatin was regarded as somewhat of a "backwater" in the gene transcription world back then, Shannon says.

Shannon then went on to establish her own lab using cells of the immune system and the recently cloned cytokine genes as good models of inducible gene expression. The two cytokine genes that she studies in particular are interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF). IL-2 is a T-cell cytokine important in T cell activation and immune regulation. GM-CSF is also produced by T cells but is more involved in regulating the myeloid response.

For some time, Shannon has researched the transcription factors controlling these genes, and mapping their critical promoter/enhancer regions. "We then became interested in how the packaging of these genes into chromatin changes in response to an immune stimulus," she says. "Also, we asked how the interplay between transcription factors and this packaging works to control gene expression."

Gene promoters

One of the interesting and landmark findings underlying the work Shannon will present at Lorne was published by the group in 2005. Gene promoters are normally covered by nucleosomes, the fundamental packaging unit of chromatin that comprises DNA wrapped around a core of histone proteins.

It was thought until a few years ago that nucleosomes were very stable and that they were rarely removed from the DNA. If so, how then did the transcription machinery assemble on the genes? Shannon says the previous idea of chromatin remodelling at gene promoter was rather vague: "somehow, in a very hand-waving manner, the nucleosomes became more loosely associated with the DNA to allow the transcription complex to assemble," she says.

"Sliding was also invoked as a model and probably applies to some genes. What we found in T cells during immune activation was that the nucleosomes actually fall off the IL-2 gene control region (the promoter) completely, but not off the rest of the gene, and that it happens in a very precise and organised manner.

"Then, the transcriptional machinery assembles in its place - the transcriptional factors, the polymerase enzymes, and all of the other goodies needed to generate a transcriptional complex ... and all of this happens only at the promoter of the gene."

This process is highly dynamic - if the signal is removed, the nucleosome assembles again very rapidly, and it is possible to shuttle between the two states by removing and replacing the signal. Further work also showed that the nucleosome loss was exquisitely dependent on the integrity of the transcriptional complex and if just one transcriptional factor is deleted or the relevant signalling pathway blocked by inhibitors, then the disassembly of the nucleosomes at the promoter will not occur.

"We were very surprised with this result that just removing one transcription factor had such a dramatic effect," she says.

These findings went against prevailing dogma, but actually served to explain some of the group's past results on histone acetylation. At the same time, researchers working on inducible genes in yeast published exactly the same findings, showing that the mechanism Shannon's group observed in immune cells was applicable to other organisms.

"These findings had major implications for how the field views chromatin remodelling events at gene control regions. It also reminded us: never dismiss a crazy result!"

---PB--- Inducible genes

The findings on chromatin packaging and transcription led Shannon to ask whether this was a common phenomenon: do all inducible genes lose nucleosomes at their promoters when you activate them?

The question also arose as to how these inducible genes are packaged in the chromatin in a resting or unstimulated cell compared to non-expressed or constitutive genes. Shannon knew a lot already about inducible genes in T cells from microarray expression studies, including the temporal and quantitative nature of their response to a given stimulus.

She hypothesised that the highly inducible genes are marked in some way to facilitate this rapid and transient swapping of the nucleosome and transcriptional complex. "So the cell in a sense 'knows' that they will need to express these genes at some stage. We are taking two approaches to answer these questions and I will talk about both at Lorne."

The researchers first looked at about 20 genes that are induced at different times and levels. The results thus far are showing that very highly induced genes do lose promoter nucleosomes in response to activation, while those induced only weakly show no detectable loss of histones.

Shannon speculates that the response is simply too small to detect, as it is known from other studies that the strength of response for many genes is dictated by the percentage of cells in the population that respond rather than the level of response in each cell.

Whatever the reason, Shannon's findings are establishing a correlation between both nucleosome density and histone acetylation at gene promoters and the kinetics of gene activation. When the group asked whether inducible genes are tagged in some form in chromatin, another interesting finding has emerged.

By focusing on the primary response genes - the ones that come on very rapidly and don't need protein synthesis for activation - the team has evidence that they already show a very low level of nucleosome occupancy and a relatively high level of histone acetylation at their promoter regions, rendering them 'transcription-ready'. "We also find that a form of polymerase is also sitting on the DNA ready to go when those genes get the activation signal," she says.

On the other hand, the later or secondary response genes more closely resemble those genes that will never be activated by the signal. This result pointed to the early inducible genes being marked or primed in some way in resting cells, and again this activity was only at the gene promoter regions: upstream or downstream, not much was happening.

Protein binding

The most recent work is on a genome-wide scale and Shannon is very excited at the new ability to investigate chromatin in this way. The approach uses ChIP-on-chip (ChIP on microarray) assays to examine the whole genome.

"Essentially you crosslink proteins to DNA in the cell and then use an antibody to pull down any DNA that is binding to that protein. The complexes are then separated and the DNA put onto a promoter or whole-genome microarray for expression analysis. We are looking at histones, polymerases and transcription factors and asking when during the immune activation these proteins are bound to the DNA and what is the level of gene expression.

"ChIP-on-chip is a huge technique at the moment. It is just exploding across the literature in the transcription/chromatin field. We started using this approach only in recent months and are already getting some really interesting results, which I will talk about at the meeting."

So far, this new methodology is yielding promising indications that some of the patterns observed with the small number of genes may hold true on a genome-wide scale. It is also allowing them to map the direct targets of certain transcription factors that control T cell function.

This developing area of whole-genome analysis of chromatin and gene transcription is being referred to as epigenomics, which describes the study of the DNA packaging or the layer of information that is laid down across the genome to control various functions such as gene expression.

This layer includes histones and modifications of the histones, transcription factors and various chromatin-modifying complexes. As Shannon told an international immunology conference recently, a major challenge in the field now is how best to use these new technologies to answer meaningful biological questions in the field of gene expression and genome biology in general.

"My research aims to understand basic molecular mechanisms of gene expression which at this stage is far removed from clinical practice. However, a nice dream is that one day we will be able to control gene expression in a targeted fashion for certain clinical outcomes."