Final Activity Report Summary - KLW-HMGN-CHEMOKINES (The role of the chromatin protein HMGN3 in cytokine-induced gene expression)
This project concerned the way in which our genetic information is decoded and turned into instructions for the making of cells and organisms. The genes in each of our cells are carried on 23 different chromosomes. The Deoxyribonucleic acid (DNA) in one cell chromosomes would stretch for 1.5 m if it were unravelled and laid end to end, but protein molecules help to fold and compact the DNA in a highly organised manner so that it fits inside the cell nucleus. The compacted DNA and protein structure is called chromatin.
The 'Human Genome' project has allowed scientists to identify the DNA sequence in each chromosome, and the current challenge was to understand how the, over 30 000, genes within the DNA combined to produce a living organism. One of the most exciting areas of research at the moment was how folding of the DNA by proteins restricted access to the genetic information.
This project investigated how the regulation of chromatin folding controlled access to a group of genes called chemokines. Chemokines were important for the development and function of the immune system, as they recruited white blood cells to sites of infection. They also played a variety of roles in cancer development. Some chemokines had anti-tumour activity in mice and were investigated as possible targets for cancer therapy in humans. This project focussed on how the expression of the TCA3 chemokine was regulated. TCA3 was shown to help combat tumour growth in mice via attacking neutrophils and monocytes to the tumour, thus promoting the hosts immune response to the tumour.
We were interested in the roles of chromosomal High mobility group nucleosome-binding (HMGN) proteins in the regulation of gene expression. These proteins unfolded the higher order chromatin structure, thereby improving access to the underlying DNA sequence and facilitating the orderly progression of various DNA-dependent activities in the context of chromatin. Our two major questions were:
1. what were the mechanisms used by HMGN proteins to modulate transcription; and
2. how were HMGN proteins recruited to specific gene loci.
The second question arose from the observations that HMGN proteins regulated only a small subset of genes, yet they did not display DNA sequence specificity when binding to chromatin. It was not clear how their actions were restricted to particular genes.
For this project, we developed cell lines in which the expression of HMGN family members was induced by the drug doxycycline providing a highly controlled system in which to study the role of HMGN3 in vivo. Using this system, we showed that HMGN3 and HMGN1 modulated the induction of the chemokine TCA3 by TNFalpha. By the time of the project completion we were investigating this activity mechanism. We developed robust chromatin immunoprecipitation assays for HMGN proteins and were investigating the way they bound to the TCA3 gene and how they affected the pattern of histone modifications. We also investigated how HMGN proteins influenced signalling by TNFalpha through the NFkappaB and p38 kinase pathways.
This work established a system that would provide valuable insights into how HMGN proteins modulated chromatin structure to influence gene expression in vivo. It was also anticipated to reveal how HMGN proteins were recruited to specific regions of the genome and the extent of redundancy between family members. In addition to answering fundamental questions about the roles of HMGN proteins in gene expression, this system would also provide important information on how cytokines induced gene expression and how chemokine expression was regulated. A deeper knowledge on how the expression of chemokines, and TCA3 in particular, was controlled, could provide insights that would help in the refinement or development of cytokine-based therapies for cancer and other diseases.
The 'Human Genome' project has allowed scientists to identify the DNA sequence in each chromosome, and the current challenge was to understand how the, over 30 000, genes within the DNA combined to produce a living organism. One of the most exciting areas of research at the moment was how folding of the DNA by proteins restricted access to the genetic information.
This project investigated how the regulation of chromatin folding controlled access to a group of genes called chemokines. Chemokines were important for the development and function of the immune system, as they recruited white blood cells to sites of infection. They also played a variety of roles in cancer development. Some chemokines had anti-tumour activity in mice and were investigated as possible targets for cancer therapy in humans. This project focussed on how the expression of the TCA3 chemokine was regulated. TCA3 was shown to help combat tumour growth in mice via attacking neutrophils and monocytes to the tumour, thus promoting the hosts immune response to the tumour.
We were interested in the roles of chromosomal High mobility group nucleosome-binding (HMGN) proteins in the regulation of gene expression. These proteins unfolded the higher order chromatin structure, thereby improving access to the underlying DNA sequence and facilitating the orderly progression of various DNA-dependent activities in the context of chromatin. Our two major questions were:
1. what were the mechanisms used by HMGN proteins to modulate transcription; and
2. how were HMGN proteins recruited to specific gene loci.
The second question arose from the observations that HMGN proteins regulated only a small subset of genes, yet they did not display DNA sequence specificity when binding to chromatin. It was not clear how their actions were restricted to particular genes.
For this project, we developed cell lines in which the expression of HMGN family members was induced by the drug doxycycline providing a highly controlled system in which to study the role of HMGN3 in vivo. Using this system, we showed that HMGN3 and HMGN1 modulated the induction of the chemokine TCA3 by TNFalpha. By the time of the project completion we were investigating this activity mechanism. We developed robust chromatin immunoprecipitation assays for HMGN proteins and were investigating the way they bound to the TCA3 gene and how they affected the pattern of histone modifications. We also investigated how HMGN proteins influenced signalling by TNFalpha through the NFkappaB and p38 kinase pathways.
This work established a system that would provide valuable insights into how HMGN proteins modulated chromatin structure to influence gene expression in vivo. It was also anticipated to reveal how HMGN proteins were recruited to specific regions of the genome and the extent of redundancy between family members. In addition to answering fundamental questions about the roles of HMGN proteins in gene expression, this system would also provide important information on how cytokines induced gene expression and how chemokine expression was regulated. A deeper knowledge on how the expression of chemokines, and TCA3 in particular, was controlled, could provide insights that would help in the refinement or development of cytokine-based therapies for cancer and other diseases.