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."