Feature: Epigenetic mischief makers

This issue appeared in the November/December 2011 edition of Australian Life Scientist. To subscribe, click here.

As a PhD student in Madrid, Dr Fatima Valdés-Mora explored the role of the Rho GTPase family of signalling molecules in epigenetic gene silencing in colorectal cancer, and became intrigued by the epigenetic mechanisms involved in wholesale dysregulation of genes in cancerous cells.

Her interest in the epigenetics brought her to the other side of the world to Sydney and Professor Susan Clark’s Epigenetics Laboratory at the Garvan Research Institute, where she undertook a postdoctoral project to resolve an enigma involving a histone protein variant, H2A.Z.

Over the past decade a new understanding of the basic mechanisms involved in cancer has emerged from studies of the way DNA is packed into chromosomes as chromatin, and the various epigenetic mechanisms that keep chromatin condensed and quiescent, or unpack it to make gene promoters accessible to the cell’s transcription machinery.

The genome’s basic packaging unit for chromosomal DNA is the nucleosome, a DNA-protein complex comprising some 146 base pairs of DNA wound around a barrel-shaped core of eight protein molecules.

Chromatin’s octamer core usually consists of dimers of four different histones: H2A, H2B, H3 and H4. At particular times, a variant of histone H2A, called H2A.Z, substitutes for H2A in nucleosomes and modifies their activity.

H2A.Z has only about 60 per cent sequence homology with H2A at the peptide level, indicating that it diverged from archetypal H2A long ago. It is highly conserved in multicellular species, suggesting some unique aspect of its structure modulates structural changes in chromatin that are literally vital to normal gene regulation, with gene-silencing experiments showing that metazoan life forms are unviable without H2A.Z.

Previous research suggested molecular geneticists were dealing with a molecular version of the Roman god Janus: one entity with two different faces. Depending on the organism, or the cell type, some earlier studies identified H2A.Z as a gene activator, while others linked it to gene repression.

H2A.Z is known to participate in a variety of nuclear processes, including nucleosome turnover, DNA-repair, heterochromatin silencing, chromosome segregation, progression through the cell cycle, suppression of anti-sense RNAs, and methylation-mediated gene suppression. It is also a key player in the differentiation of embryonic stem cells.

That was the state of knowledge when Valdés-Mora began her study of H2A.Z’s apparently opposing functions, and the histone’s role in remodelling tumour-cell genomes.

The study involved several colleagues in Professor Clark’s Garvan Institute research group, the Bioinformatics Division of the Walter and Eliza Hall Medical Research Institute in Melbourne and the Australian National University’s John Curtin School of Medical Research.

Valdés-Mora was lead author on a paper published in Genome Research from the Cold Spring Harbour Laboratory Press in July, announcing the identification of a new epigenetic mechanism where H2A.Z and its acetylated form have a key role in gene deregulation in prostate cancer cells. She believes their results will apply to all cancers including leukaemias and lymphomas.

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Epigenome map

She chose prostate cancer as a model because Professor Clark’s team had previously built the first integrated prostate cancer epigenome map, combining gene expression changes with post-translational histone modifications and DNA methylation alterations.

With the funding support of the Prostate Cancer Foundation of Australia, she set out to investigate whether H2A.Z was also an important player on the prostate cancer epigenome map.

Valdés-Mora says the great advantage of the Garvan’s prostate cells lines for her epigenetic studies is that both cancerous and normal prostate epithelial cells replicate readily in vitro, and are easily maintained and studied. There are no similarly tractable cell lines for colorectal cancer.

“We’re also studying breast cancer, as it is one of the most common types of cancer in Australian women. Epigenetic changes are strikingly important during breast carcinogenesis and, again, we have a good model cell line,” Valdés-Mora says.

H2A.Z’s seemingly contradictory effects in different cell lines turn out to involve a familiar biochemical mechanism: histone acetylation. Earlier studies reported that H2A.Z acetylation occurs at peptide sequences with three adjacent lysine residues.

By combining new chromatin immunoprecipitation and tiling microarray chip technologies (ChIP-on-chip) or next generation sequencing (ChIP-seq), researchers can now perform high-precision, whole- genome surveys to identify all sites where regulatory proteins such as histones interact with DNA.

Valdés-Mora and her colleagues determined that H2A.Z and its acetylated form (acH2A.Z) is enriched at many gene promoters in humans and other species, and that there is a dynamic process in cancer where deacetylated H2A.Z is incorporated at the 5’ ends of silenced tumour-suppressor genes and acetylated H2A.Z is decreased at promoters of up-regulated oncogenes.

“One other paper had previously reported that H2A.Z is decreased at promoters in B-cell lymphoma, but they didn’t look for acetylated H2A.Z. But the decrease indicates that H2A.Z is probably important in leukaemias and lymphomas, as well as in solid tumours,” she says.

“H2A.Z occupancy is highly correlated with patterns of gene expression, both in our normal and cancerous prostate cell lines. We found it around the transcriptional start site of active genes, and poised genes.”

Poised genes are found in regions of open chromatin; they are primed for rapid activation, with most of the components of their transcription complexes in place, but special repressor proteins prevent them being expressed.

“In our prostate cell lines , we observed that in suppressed genes, H2A.Z is present along the entire promoter, but at relatively low levels,” she says. “As enrichment increases and narrows to only the transcriptional star site, gene expression increases. We found the same patterns of H2A.Z occupancy in several other human cell lines. Another published study had previously reported similar patterns in human T-cells.

“H2A.Z has a bimodal distribution on active gene promoters around the transcriptional start site, but the pattern of enrichment is different in inactive genes, where H2A.Z spreads out to the entire promoter at relatively lower levels.”

In its acetylated form, acH2A.Z, is restricted to transcription start sites (TSSs) of highly expressed genes in both normal and cancerous prostate cells, and is absent from the promoters of poised, lowly expressed and inactive genes.

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Tuning the balance

Valdés-Mora says their comparisons of H2A.Z and H2A occupancy in normal and cancerous prostate cells showed that increased occupation of gene promoters by H2A.Z, and a corresponding reduction in H2A, correlates with increasing gene expression. The balance between “normal” H2A and H2A.Z appears to tune gene expression.

Valdés-Mora said they found a relocalisation of H2A.Z occupancy and also an alteration the acetylation state in their prostate cancer cell lines: up-regulation of oncogenes correlated with sparse occupation at promoters, while H2A.Z was enriched at the promoters of down-regulated tumour-suppressor genes. Their observations strongly implicate H2A.Z as a major player in tumorigenic patterns of gene expression.

“When we selected genes that decreased in expression in tumorigenic cell lines, we found that the down-regulation of tumour-suppressor genes involved the loss of acetylated H2A.Z at the promoters, and a gain of non-acetylated H2A.Z,” Valdés-Mora says. “In contrast, activated oncogenes in cancerous cells gained acetylated H2A.Z, although total levels of the histone at the promoter decreased.”

At the same time, H2A appears to migrate to sites downstream of a point about 4000 base pairs from the transcription start site in cancerous prostate cells. In cancerous cells, down-regulated genes exhibited a loss of acetylated H2A.Z and combined acH2A.Z/H2A.Z along their entire promoters, indicating that deacetylation of H2A.Z – or a failure to reacetylate – results in gene inactivation.

The study concluded that gene repression in cancerous cells is not due to complete loss of H2A.Z, but specifically, to loss of acetylated H2A.Z. Conversely, in up-regulated genes, increased activity is not due to increased promoter occupation by H2A.Z, but to the increasing presence of acetylated H2A.Z.

Next, they checked three well-known oncogenes that are activated in LNCaP prostate cancer cell lines, and found that their activation was correlated with loss of acH2A.Z/H2A.Z at their promoters.

“That’s the most important finding in our paper: the changing patterns of gene activity in our cancerous cell lines were strongly correlated with changes in H2A.Z enrichment at the promoter, and in its acetylation state.”

Valdés-Mora says when they looked for other epigenetic marks in their cancerous and normal prostate cell lines, to see if they were correlated with loss of H2A.Z and acetylated H2A.Z, they found that as genes were down-regulated by hypermethylation at CpG residues in their DNA, acetylated H2A.Z disappeared from their promoters.

More strong support for H2A.Z’s involvement in methylation-induced gene silencing in prostate cancer cells emerged from a comparison of gene activity in healthy and cancerous cells. They identified 903 genes were active in normal cells, but which were significantly down-regulated by hypermethylation in cancerous cells, along with 75 genes that were hypomethylated and active in cancerous but not in normal cells.

Again, they found that acetylated H2A.Z, but not total H2A.Z, was absent from the promoters of the aberrantly hypermethylated genes in the cancerous cells. The presence of another very important gene-repressing epigenetic mark in cancer, polycomb marks, also correlated with loss of acetylated H2Z from gene promoters.

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Partners

“So H2A.Z is not working alone in epigenetic remodelling of gene expression patterns in cancer. But we have identified a previously unreported histone modification as an important player in cancer.”

Summarising their resolution of the H2A.Z enigma, Valdés-Mora et al. write: “We found that the landscape of H2A.Z occupancy and its acetylation state directly correlates with the level of gene expression, arguing that acetylation of H2A.Z plays a key role in gene regulation in normal cells, and gene deregulation in cancer.

We have shown for the first time that it is acetylation of H2A.Z-occupied nucleosomes that potentially drives active gene expression in normal cells, and deacetylation of H2A.Z that drives gene repression in cancer cells, rather than a change in H2A.Z occupancy.”

Over the past decade, oncologists have taken a growing interest in a novel class of cancer therapeutics, histone deacetylation inhibitors (HDAC inhibitors), that are able to reactivate repressed genes in cancerous cells, including tumor-suppressors, restoring the capacity of cancer tells to undergo terminal differentiation and apoptosis.

When Valdés-Mora and her colleagues treated prostate cancer cell lines with the HDAC inhibitor Trichostatin A, they found it increased levels of acH2A.Z at the promoter of a particular tumour suppressor genes, switching on its activity. “This is a promising result, because it suggests that the effect of HDAC inhibitors on acH2A.Z is one mechanism by which this class of drugs inhibit the progress of cancer,” she says.

“The problem is that HDAC inhibitors are non-specific in their activity – they can promote the acetylation of proteins other than histones and also could activate undesired genes, so we need to devise ways of targeting inhibitors to specific proteins we know are important.”

Valdés-Mora says the next step is to identify and target upstream proteins that coordinate remodelling of the cancer cell genome through the gain or loss of acH2A.Z from promoters, and devise ways to selectively suppress them with inhibitors. “RNA-interference therapies look promising, but we need to find better ways of delivering them into cancerous cells.”

She says the Alliance for the Human Epigenome and Disease (AHEAD) is working to identify all of the components of the epigenomic code that are important in tumorigenesis. “Our finding is important because if we find patterns of gene deregulation involving acH2A.Z/H2A.Z that are common to different types of cancer, it would be an important diagnostic as well as prognostic tool.”