Epigenetics: The sins of the father

In the recent paper in Nature (2014, vol 507, p. 22-24), Virginia Hughes reports the experiments carried out by Dr Dias and Dr Ressler from the University of Atlanta in recent years. They have studied the involvement in inheritance imprints in mice as a result of a fear-based reaction associated to acetophenone. As a result, they found a larger than normal expression of M71 glomeruli receptors in their offspring’s noses. These receptors are encoded by a single gene, known as Olfr151.

This elegant, but still inconclusive cause-effect mechanism approach, brings a possible explanation to a controversial observation back to the 19th century when French biologist Jean-Baptiste Lamark pointed out the pass of acquired traits to future generations. Since then, scientists have observed this phenomenon in plants, animals and even humans.

Although some scientists are still sceptical about the transmitance method, nobody denies the phenomenon. Finding an explanation to this complex problem would involve a deeper study on reproductive biology and to study both mother and father lines over few generations.

The strong suggestion that this heriditary transmission of environmental factors is due to epigenetics, a concept introduced in the 2000’s, where there are some changes in the way that DNA is packed and expressed without altering its sequence, is one of the strong lines of thought, where chemical tags (methylation) on DNA can turn genes on and off.

But even if epigenetics is directly involved in the inheritance, through marks on the material contained in the sperm, the first question to be addressed would be to understand how the effects of environmental/ health legacy get embedded into the animal’s germ cells.

Epigenetics is still unable to explain how this observed phenomenon gets passed down through multiple generations, surviving several rounds of genetic re-programing. Other suggested agents might involve histones (proteins which has been observed that they can be passed down through generations) or short RNA molecules which role would be to latch on DNA and affect further into gene expression.

Scientists are optimistic about finding a cause-effect relationship in the years to come for a phenomenon which has proved elusive for researchers in the past hundred years.

Cancer Genome Landscapes

Vogelstein et al have published a very informative review on the genomic landscapes of cancer.

As the cost of genome sequencing has fallen 100 fold in the last ten years it is becoming commonplace to sequence the exomes of sets of 100+ tumours, which is allowing us to study the genomic make up of tumours.

The number of mutations a tumour has is dependent on its type:  solid adult tumours have between 33-66, where as childhood cancers far fewer.

FP1Exceptions to this are lung cancers in smokers and melanomas that have far more mutations due to the mutagenic impact of the carcinogen (smoke/sunlight) that initiates them. Tumours with defects in DNA Damage Response also accrue disproportionally large number of mutations.

Interesting, the number of mutations observed in solid adult tumours in self-renewing tissues (e.g. colon) are proportional to the age of the patient implying that these mutations may be present at the pre-neoplastic stage.

It is estimated that between two to eight sequential alterations that develop over the course of 20 to 30 years are actually causative of the cancer.  These are termed  “driver mutations” and occur in driver genes.  Each alteration causes a selective growth advantage to the cell in which it resides.  The other mutations occur because the cancer is genetically unstable, are termed passenger mutations, and confer no selective advantage to the cell in which they reside,

Driver genes are genes that contain driver mutations and there are two types:

Tumour suppressors that confer a selective advantage to the cell when the are “broken”

Oncogenes who confer a selective advantage to the cell, if they are “activated”.

Vogelstein et al. estimate how many driver genes exist using the 20:20 rule. In tumour suppressors at least 20% of the mutations cause truncation of the gene product, where in oncogenes at least 20% of the missense mutations occur in a single position along the polypeptide chain. (see figure 2)


PIK3CA and IDH1 are oncogenes, where as RB1 and VHL are tumour suppressors.

Using the 20:20 rule Vogelstein et al.identify ~140 genes whose intragenic mutations contribute to cancer (so-called Mut-driver genes).

Interesting this is far fewer than the ~500 genes identified in the Cancer Gene Census as being causative of cancer.  They suggest that other genes (Epi-driver genes) that are altered by epigenetic mechanisms and cause a selective growth advantage, but the definitive identification of these genes has been challenging.

Although every individual tumor, even of the same histopathologic subtype as another tumor, is distinct with respect to its genetic alterations, but the pathways affected in different tumors are similar.  These driver genes function through a dozen signaling pathways that regulate three core cellular processes: cell fate determination, cell survival, and genome maintenance.


They also briefly discuss that currently most anti-cancer drugs available inhibit the activity of an enzyme. However, of ~140 driver genes identified, only 31 could be targeted in this manner.

Indeed the majority of Mut-driver genes encode tumor suppressors, not oncogenes. Drugs generally interfere in the function of a protein – conceptually very difficult to produce a drug that will restore the function of a protein.

They conclude that in the future, the most appropriate management plan for a patient with cancer will be informed by an assessment of the components of the patient’s germline genome and the genome of his or her tumor.  That the inherent heterogeneity of tumours and their metastases makes resistance to targeted therapies ‘inevitable’ and that it is important to research the efficacy of combination therapies. They also suggest that the information from cancer genome studies should be exploited to improve methods for prevention and early detection of cancer, which will be essential to reduce cancer morbidity and mortality.

Identification of a small molecule inhibitor of BLM helicase

BLM helicase is a member of the RecQ helicase family of ATP-dependent proteins that separate the two strands of duplex DNA, allowing DNA replication and repair processes to proceed. Mutations in this protein result in Bloom’s syndrome (BS), characterised by genomic instability, particularly an increased frequency of sister chromatid exchanges (SCEs), and a predisposition to cancer development, amongst other clinical features. Patients often develop leukaemia’s and lymphoma’s although most types of cancer have been observed in this group. BLM has also been demonstrated to be involved in a process known as Alternative Lengthening of the Telomeres (ALT). This is a homologous recombination-mediated mechanism by which new telomeric DNA is synthesised from an existing DNA template. The finer details of this pathway are unknown but it is known that depletion of BLM by siRNA in ALT+ cells results in telomere shortening.

Telomeres are ‘caps’ at the ends of the chromosomes and consist of repetitive DNA sequence and proteins. They function to protect the chromosome end from degradation or association with other chromosomes. The length of the telomere shortens with each cell division, resulting in replicative  senescence when a critical telomere length is reached. Telomeres are maintained in cancer cells either by up regulation of Telomerase, an enzyme that adds telomeric repeats, or via the use of the ALT pathway, enabling cells to proliferate aberrantly. Approximately 10% of all tumours rely on the ALT pathway for their continued growth and 66% of Osteosarcoma tumours are ALT+. Repression of ALT in ALT+ immortal cell lines results in senescence and cell death, making inhibition of this pathway an attractive therapeutic target.

Nguyen et al., recently described the identification of a novel small molecule BLM inhibitor. The group developed a high-throughput screening assay using a labelled partly duplex DNA substrate. Quenching of the substrate was lost upon separation of the two strands, resulting in increased fluorescence with increasing BLM helicase activity. Hits from this primary screen were then confirmed in a helicase gel assay. Medicinal chemistry optimisation of a hit compound resulted in a lead molecule termed ML216 which inhibited BLM with an IC50 value of 3µM. They went on to show that ML216 affects the DNA binding activity of BLM . Further, the group demonstrated that ML216 inhibited the proliferation of cells transfected with BLM, but not the isogenic BLM negative control cells. Treatment with ML216 also sensitised the BLM+ cells to the replication inhibitor, aphidicolin but with no sensitising effect on the BLM negative controls.  A further important observation was that treatment of cells with ML216 increased the frequency of SCE’s observed, but only in the BLM expressing cells. The group also observed that ML216 could inhibit WRN, a related DNA helicase, in in vitro gel assays. However, they found no inhibitory effects on WRN in the cell-based assays conducted, indicating that the primary target of this compound in human cells is BLM. There is currently no available crystal structure of BLM for further analysis of ML216 binding.sw3sw4


The identification of this novel small molecule could now enable studies of the effects of BLM helicase inhibition in ALT+ tumour cells and potentially uncover a new treatment strategy for ALT+ tumours.



ENCODE: A new tool for drug discovery?

Only a small proportion (<2%) of the total genome codes for proteins and the remainder had up to now been termed non-coding or ‘junk DNA’. The aim of the ENCODE (Encyclopaedia of DNA elements) project was to attempt to characterize these undefined regions.  The consortium has recently published 30 papers detailing, amongst much data, regions of transcription and regulatory areas that were previously unreported.

One of these papers by Maurano et al., used a technique to map sites of regulatory elements within the DNA and compare these with noncoding variant polymorphisms associated with common diseases that have been identified through genome-wide association studies (GWAS).

The group examined many different cell types including primary cells, immortalized, malignancy derived or pluripotent cell lines, hematopoietic cells, progenitor cells as well as some fetal tissue samples. They used Deoxyribonuclease 1 (DNase1) hypersensitive sites (DHSs) of increased chromatin accessibility as a marker for binding sites of regulatory elements such as transcription factors and thus mapped the regulatory regions in this material. In total, they identified DHS positions spanning 42.2% of the genome, a higher density of regulatory regions than previously appreciated. They then examined the position of single nucleotide polymorphisms (SNPs) identified by GWAS and found a 40% enrichment of these SNPs in DHSs. This analysis shows that the common genetic variants associated with disease are often located at recognition sequences of transcription factors. The authors also demonstrated that these regulatory regions may control the expression of genes that are distant (>250kb) rather than solely the expression of the nearest gene.

Further interesting data from the consortium was obtained through the study of cancer lines. Over 40 cancer lines of different origin were examined and data obtained showing that cancer lines possess regulatory DNA regions that are not present in normal cells (Stamatoyannopoulous, J. A., 2012).

The new information provided by ENCODE is not yet readily applicable to drug discovery, however, this data could provide a map of transcriptional and regulatory regions that could help to identify novel therapeutic targets. In a recent article in Nature Drug Discovery, Michael Snyder one of the principal investigators of the ENCODE consortium explains that changes in gene expression through a change in regulatory sequence could enable identification of proteins that could make useful drug targets.

Applications that could be useful in drug discovery settings include the use of knockdown technologies to screen for biological effects, or zinc finger nuclease technology that can introduce mutations to regulatory elements to determine if changes in these regulatory regions are causal of disease.

Systematic localization of common disease-associated variation in regulatory DNA.

Maurano MT, Humbert R, Rynes E, Thurman RE, Haugen E, Wang H, Reynolds AP, Sandstrom R, Qu H, Brody J, Shafer A, Neri F, Lee K, Kutyavin T, Stehling-Sun S, Johnson AK, Canfield TK, Giste E, Diegel M, Bates D, Hansen RS, Neph S, Sabo PJ, Heimfeld S, Raubitschek A, Ziegler S, Cotsapas C, Sotoodehnia N, Glass I, Sunyaev SR, Kaul R, Stamatoyannopoulos JA.

Science. 2012 Sep 7;337(6099):1190-5. doi: 10.1126/science.1222794. Epub 2012 Sep 5.

What does our genome encode?, Stamatoyannopoulous, J. A. 2012, Genome Research 22: 1602-1611

An audience with Michael Snyder, Nature reviews Drug Discovery Oct 2012. 11: 744

The ENCODE papers are available online at go.nature.com/iN6Ezx.