Gene Reprogramming cells moves a step forward

The idea of taking a mature cell and removing its identity (nuclear reprogramming), so that it can then become any kind of cell, holds great promise for repairing damaged tissue, or replacing bone marrow after chemotherapy. Hot on the heels of his recent Nobel prize, Dr John B. Gurdon has published research showing that histone H3.3 deposited by the histone-interacting protein HIRA is a key step in reverting nuclei to a pluripotent type, capable of being any one of many cell types. The research was published October 28, 2012 in BioMed Central’s open access journal Epigenetics & Chromatin.

One way to reprogram DNA is to transfer the nucleus of a mature cell into an unfertilised egg. Proteins and other factors inside the egg alter the DNA, switching some genes on and others off until it resembles the DNA of a pluripotent cell. However, there seems to be some difficulties with this method in completely wiping the cell’s ‘memory’.

One of the mechanisms regulating the activation of genes is chromatin and in particular histones. DNA is wrapped around histones; alterations in how the DNA is wound changes which genes are available to the cell.

In order to understand how nuclear reprogramming works Dr Gurdon’s team transplanted a mouse nucleus into a frog oocyte (Xenopus laevis). They added fluorescently tagged histones by microinjection, so that they could see where in the cell and nucleus the these histones collected.

Dr Gurdon said: “Using real-time microscopy it became apparent that from 10 hours onwards H3.3 (the histone involved with active genes) expressed in the oocyte became incorporated into the transplanted nucleus. When we looked in detail at the gene Oct4, which is known to be involved in making cells pluripotent, we found that H3.3 was incorporated into Oct4, and that this coincided with the onset of transcription from the gene.” The team also found that Hira, a protein required to incorporate H3.3 into chromatin, was also required for nuclear reprogramming.

Dr Steven Henikoff, from the Fred Hutchinson Cancer Research Center, commented: “Manipulating the H3.3 pathway may provide a way to completely wipe a cell’s ‘memory’ and produce a truly pluripotent cell. Half a century after showing that cells can be reprogrammed this research provides a link to the work of Shinya Yamanaka (who shared the prize), and suggests that chromatin is a sticking point preventing artificially induced reprogramming being used routinely in the clinic.”

Immune system plays a part in AMD

Changes in how genes in the immune system function may result in age-related macular degeneration (AMD), the leading cause of visual impairment in older adults, based on preliminary research conducted by researchers of the US National Institutes of Health (NIH).

“Our findings are epigenetic in nature, meaning that the underlying DNA is normal but gene expression has been modified, likely by environmental factors, in an adverse way,” said Dr Robert Nussenblatt, chief of the National Eye Institute (NEI) Laboratory of Immunology. Environmental factors associated with AMD include smoking, diet, and ageing. The study identified decreased levels of DNA methylation, a chemical reaction that switches off genes, on the interleukin-17 receptor C gene (IL17RC). The lack of DNA methylation led to increased gene activity and, in turn, increased levels of IL17RC proteins in patients with AMD. IL17RC is a protein that promotes immune responses to infections, such as fungal attacks.

Recent studies have identified several genes with alterations that increase the risk of developing the disease. In addition, environmental risk factors have also been suggested as possible causes of the disease. One explanation may be that environmental exposures influence DNA methylation, which regulates gene expression. Changes in this process may result in the production of too much or too little of a gene’s protein, leading to cellular dysfunction and disease. Changes in DNA methylation have been implicated in cancer, lupus, multiple sclerosis, and many other diseases.

To test whether changes in DNA methylation might play a role in AMD, the investigators evaluated three pairs of twins – one pair identical and two pairs fraternal – where only one of the siblings had AMD. When compared with the unaffected twins, methylation patterns were altered in 231 genes of affected twins. This finding is consistent with the hypothesis that environmental exposures may epigenetically regulate expression of many genes and lead to AMD.

Among the 231 genes, the investigators found that DNA methylation was absent in a region of the IL17RC gene in twins with AMD. The lack of methylation in the IL17RC gene led to increased gene activity and, in turn, increased levels of its protein in circulating blood. Based on these results, the authors propose that chronic increased levels of the IL17RC protein in the retina likely promote inflammation and recruitment of immune cells that damage the retina and lead to AMD.

The investigators next plan to evaluate what environmental factors may be responsible for the regulation of IL17RC and how the epigenetic regulation leading to the chronic inflammation in AMD patients can be reversed by novel therapies. They will also evaluate the role of epigenetics in other eye diseases.

Genetic link found for uterine cancer

Researchers from the US-based National Human Genome Research Institute (NHGRI), have identified several genes that are linked to one of the most lethal forms of uterine cancer, serous endometrial cancer. The researchers describe how three of the genes found in the study are frequently altered in the disease, suggesting that the genes drive the development of tumours.

Cancer of the uterine lining, or endometrium, is the most commonly diagnosed gynaecological malignancy in the United States. Also called endometrial cancer, it is diagnosed in about 47,000 American women and leads to about 8,000 deaths each year.

Each of its three major subtypes – endometrioid, serous and clear-cell – is caused by a different constellation of genetic alterations and has a different prognosis. Endometrioid tumours make up about 80% of diagnosed tumours and is usually diagnosed at an early stage and treated with surgery.

Compared to other subtypes, the 2 to 10% of uterine cancers that comprise the serous subtype do not respond well to therapies. The five-year survival rate for serous endometrial cancer is 45%, compared to 65% for clear-cell and 91% for endometrioid subtypes.

Serous and clear-cell endometrial tumour subtypes are clinically aggressive and quickly advance beyond the uterus.

To determine which genes are altered in serous endometrial cancer, the team, headed up by Dr Daphne W. Bell from the Reproductive Cancer Genetics Section of NHGRI’s Cancer Genetics Branch, undertook a comprehensive genomic study of tumours by sequencing their exomes, the critical 1 to 2% of the genome that codes for proteins.

They began their study by examining serous tumour tissue and matched normal tissue from 13 patients. National Cancer Institute and Massachusetts General Hospital pathologists processed the 26 tissue samples, which subsequently underwent whole-exome sequencing at the NIH Intramural Sequencing Center.

Then the researchers filtered through millions of data points to locate alterations, or mutations. They disqualified from the analysis any mutation found in a tumour and its matched healthy tissue, looking expressly for mutations that occurred exclusively in the tumour cells.

They also eliminated one of the 13 tumours from analysis because its exome had hundreds more unique mutations than any other tumour.

The researchers detected more than 500 somatic mutations within the remaining 12 tumours. They next looked for genes that were mutated in more than one of the tumours.

Dr Bell said: “When you identify a set of mutations, they could either be drivers that have caused the cancer or incidental passengers that are of no consequence; our goal is to identify the drivers. One way to do this is to home in on genes that are mutated in more than one tumour, because we know from experience that frequently mutated genes are often driver genes.”

The team felt confident that alterations in nine genes could be driver genes in serous endometrial cancer. Three of the nine genes had previously been recognised by researchers in the cancer genetics field as a cause of serous endometrial cancer. To get a clearer picture of driver gene status among the other six genes, the researchers sequenced each gene in 40 additional serous endometrial tumours. They discovered that three of the six genes – CHD4, FBXW7 and SPOP – are altered at a statistically high frequency in serous endometrial cancer.

The team also found that this set of three genes is mutated in 40% of the serous endometrial cancer tumours and in 15 to 26% of the other endometrial cancer subtypes.

Researchers identify origin of chronic lymphocytic leukaemia

Researchers of Blueprint consortium have deciphered the first epigenomes of chronic lymphocytic leukaemia. The research, published in Nature Genetics, involved whole-genome DNA methylation analysis of 140 patients and identified the cell of origin of the disease as well as new molecular mechanisms involved in its development. This research, directed by Iñaki Martin-Subero from Universidad de Barcelona and IDIBAPS, represents the first collaborative effort between two European high-impact research initiatives, such as the EU-funded Blueprint Consortium and the CLL Genome Project funded by the Spanish Government. These two initiatives are set within the context of two world-wide Consortia, the International Human Epigenome Consortium and the International Cancer Genome Consortium, respectively.

Elias Campo, co-coordinator of the Spanish CLL Genome Project, said: “In the previous research activities of Spanish CLL Genome Consortium we focused on the analysis of mutated genes involved in the development of this disease. Now, thanks to our work within the Blueprint Consortium, we have analysed the full DNA methylome of CLL. This approach has allowed us to identify the cells of origin of this kind of leukaemia and to discover new mechanisms that contribute to its development.”

Dr Lopez-Otin, co-coordinator of the CLL Project, said: “Our previous genetic studies identified over 1,000 genes mutated in the chronic lymphocytic leukaemia, whereas this epigenomic analysis has allowed us to detect over a million epigenetic changes in this disease. This unexpected finding indicates that the epigenome of the cell suffers a massive shift during leukaemia development.” doi:10.1038/ng.2443   


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