nucleosomes

SCIENTIFIC ILLUSTRATION:  Nucleosome
The Mediterranean Institute for Life Sciences
Split, Croatia

High resolution ray-traced model of a nucleosome, isolated on black.

A nucleosome is the basic unit of DNA packaging in eukaryotes, consisting of a segment of DNA wound in sequence around four histone protein cores.  This structure is often compared to thread wrapped around a spool.

Nucleosomes form the fundamental repeating units of eukaryotic chromatin, which is used to pack the large eukaryotic genomes into the nucleus while still ensuring appropriate access to it.  In mammalian cells approximately 2 m of linear DNA have to be packed into a nucleus of roughly 10 µm diameter.  

Nucleosomes are folded through a series of successively higher order structures to eventually form a chromosome; this both compacts DNA and creates an added layer of regulatory control, which ensures correct gene expression.

(Nucleosome - Wikipedia)

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It has already been determined that silencing near tRNA genes requires nucleolar localisation through work with yeast, and now a follow up paper from David Engelke’s lab at the University of Michigan tracks the process ‒ tRNA gene mediated (tgm) silencing ‒ to the nucleosomes.

Elaborating on what makes this form of silencing distinct from other forms of transcriptional silencing or boundary element (cis-acting elements which also serve a structural role) function in yeast, the group discuss models for communication between the tRNA gene transcription complexes and local chromatin.

These RNA polymerase III transcription units are dispersed in high copy throughout nuclear genomes and act counter to RNA polymerase II transcription in their immediate vicinity on a chromosome. 

In some respects tgm silencing may be incompatible with other forms of silencing, since tRNA genes are able to block propagation of silenced chromatin states caused by other silencing mechanisms. It is not clear what the relationship is between tgm silencing and this “insulator” or “boundary element” function.

To test the possible involvement of nucleosomes in an unbiased manner, we screened a comprehensive library of alanine substitution mutations in the four core histones for the ability to release tgm silencing. Over 10% of the substitutions strongly release the silencing, with many residing in the histone H3 and H4 N-terminal tails. In addition, a large group of alleviating mutations defines a surface on the nucleosome disk that has not been implicated in other silencing forms. In light of these observations, we also tested additional mutations in a broad group of chromatin modification and remodeling enzymes, and find that the Rpd3 and Hos1 histone deacetylases, the Glc7 phosphatase, and the RSC nucleosome remodeling activity are also required for silencing.

As can be seen from the images above,

The identified residues map to three general areas of the nucleosome structure: the N-terminal tails of H3 and H4, other residues likely in near contact with the DNA near the nucleosome dyad, and a broad surface on the nucleosome disk face.

The H3 and H4 tails are particularly intensive regions of post-translational modifications that affect transcription and have the densest clusters of mutations that alleviate silencing. They emerge from the nucleosome core nearly opposite each other across the DNA helix and can interact with both the DNA and external proteins.

The left view is from the edge of the disc to optimise visualisation of residues in contact with DNA, while the view alongside it optimises visualisation of residues on the disc surface.

Light blue = DNA, Grey = histones (except residues affecting tgm silencing) : Blue = H2A, Cyan = H2B, Yellow = H3, Red = H4.

An “intriguing possibility" arises from the finding that Maf1, a direct target of TOR signaling which represses RNA pol. III, is required for tgm silencing.

When MAF1 is deleted, tRNA transcription increases, which we originally expected would intensify silencing near tRNA genes. When tgm silencing was instead abolished by maf1Δ , it indicated Maf1 was exerting an influence beyond its physical interaction with pol III and the tRNA gene-bound transcription factors. A link between Maf1 and RSC [chromatin remodelling complex] is thought to be mediated through the Rsc9 subunit, consistent with the involvement of the RSC complex in transcriptional regulation in response to TOR signaling.
Unlike boundary element function, which requires only bound TFIIIC, tgm silencing seems to require the presence of the full RNA polymerase III complex (pol III, TFIIIC, TFIIIB, 1), containing at least 25 polypeptides. Thus, it is not surprising that silencing near the tRNA genes involves interactions with activities not observed in boundary function.

Since both tRNA genes and some tRNA-derived short interspersed elements (SINEs) have been shown to have boundary element properties in larger eukaryotic genomes, it will be interesting to examine whether the other interactions with local chromatin structure are retained as well in these highly reiterated elements.

Good et al., “Silencing near tRNA genes is nucleosome-mediated and distinct from boundary element function”. Gene, vol 526, pp. 7‒15 (2013)

Nucleosome hopping and sliding kinetics determined from dynamics of single chromatin fibers in Xenopus egg extracts (2007)

Chromatin function in vivo is intimately connected with changes in its structure: a prime example is occlusion or exposure of regulatory sequences via repositioning of nucleosomes. Cell extracts used in concert with single-DNA micromanipulation can control and monitor these dynamics under in vivo-like conditions. We analyze a theory of the assembly–disassembly dynamics of chromatin fiber in such experiments, including effects of lateral nucleosome diffusion (“sliding”) and sequence positioning. Experimental data determine the force-dependent on- and off-rates as well as the nucleosome sliding diffusion rate. The resulting theory simply explains the very different nucleosome displacement…

  • Nucleosome displacement in transcription (2007)
  • "It is becoming increasingly clear that the eukaryotic transcriptional machinery is adapted to exploit the presence of nucleosomes in very sophisticated ways."

…kinetics observed in constant-force and constant-pulling velocity experiments. We also show that few-piconewton tensions comparable to those generated by polymerases and helicases drastically affect nucleosome positions in a sequence-dependent manner and that there is a long-lived structural “memory” of force-driven nucleosome rearrangement events.

VolitionRx to Initiate Study with University Hospital Bonn to Confirm NuQ(R) Test Accuracy in Lung Cancer

Confirmatory study will assess VolitionRx’s proprietary Nucleosomics? platform for non-invasive diagnosis of lung cancer as part of larger ongoing, prospective clinical trial

Initiation based on findings from pilot study that demonstrated NuQ? tests able to detect lung cancer in 76% of patients using blood

NAMUR, Belgium, December 17, 2014 /PRNewswire/ — VolitionRx Limited (OTCQB: VNRX), a life sciences company focused on developing diagnostic tests for cancer and other conditions, today announced that the University Hospital Bonn in Germany will initiate a clinical confirmatory study to assess VolitionRx’s proprietary Nucleosomics® platform technology for the diagnosis of lung cancer through a blood test in individuals.

The confirmatory study will be conducted as a separate trial in conjunction with VolitionRx’s larger 4,000-subject prospective study evaluating VolitionRx’s NuQ® assays in the 20 most prevalent cancers with the University Hospital Bonn, in which hospital researchers are currently collecting blood samples from healthy individuals, patients with cancers and patients with other conditions and for which analysis will begin in 2015.

The confirmatory study will include a portion of the samples from the 4,000-subject trial, with both studies being led by Priv-Doz. Dr. Stefan Holdenrieder at the Institute of Clinical Chemistry and Clinical Pharmacology, University Hospital Bonn. In the confirmatory study, approximately 600 blood samples will be analyzed, including approximately 400 samples from patients with lung cancer with different histological subtypes and diverse stages of disease; 100 from patients with benign lung diseases that are relevant for differential diagnosis; as well as 100 samples from healthy subjects tested to determine distribution of nucleosome modifications compared to those modifications in malignant samples.

Priv-Doz. Dr. Stefan Holdenrieder, remarked, “Following the encouraging results reported from VolitionRx’s pilot lung cancer study in both blood and sputum at the BioWin Day 2014 in Belgium last month, I am looking forward to conducting a subsequent study in blood to further validate the accuracy of the Nucleosomics® technology in lung cancer. Early detection of lung cancer remains a high unmet medical need across the globe and the use of promising biomarkers such as modifications of nucleosomes - which play an important role in the development and progression of lung cancer - may offer the potential to enhance the accuracy of early cancer detection.”

Chief Scientific Officer Dr Jake Micallef commented, “The confirmatory study led by Dr. Holdenrieder at University Hospital Bonn serves as further validation of the data we have shown to date demonstrating the accuracy and sensitivity of our NuQ® test not only lung cancer, but also in other prevalent cancers including colorectal and prostate. We look forward to initiating the confirmatory study and hope that the results will confirm the accuracy of the test in detecting lung cancer, this time in a larger number of subjects.”

VolitionRx’s pilot lung cancer study assessed the ability of the NuQ® platform to detect lung cancer in both blood and sputum (airway secretions, or mucus coughed up from the lower respiratory tract). Samples were collected from 46 individuals with either non-small cell lung cancer (NSCLC), chronic obstructive pulmonary disease (COPD) or with no disease (healthy).

Overall, the NuQ® technology was able to detect both early and late stage lung cancer with high sensitivity and specificity in both blood and sputum samples.

In sputum samples, the NuQ® test was able to detect 85% of lung cancer cases, with no false positive results for healthy subjects, and discriminate lung cancer from COPD. The sputum assay data is age and smoking independent.

In blood samples from the same patients adjusted for age and smoking risk, the NuQ® assays were able to detect 76% of patients with cancer, with a single false positive result for a healthy subject, and to also discriminate lung cancer from COPD.

The NuQ® tests utilize the Company’s proprietary Nucleosomics® platform, which identifies and measures circulating nucleosome structures for the presence of epigenetic cancer and signals within the blood, and now within sputum.

In addition to the confirmatory lung cancer study, other clinical trials assessing the effectiveness of VolitionRx’s assays include:

  • A 4,800 patient retrospective symptomatic population study in colorectal cancer at Hvidovre Hospital, University of Copenhagen, Denmark
  • A 14,000 patient prospective screening study in colorectal cancer at Hvidovre Hospital, University of Copenhagen, Denmark
  • A 4,000 patient prospective study that involves patients with the 20 most prevalent cancers at University Hospital in Bonn, Germany
  • A 250 patient prospective study in colorectal cancer at CHU-UCL Mont Godinne Hospital, Belgium
  • A retrospective study with MD Anderson, Texas, to establish the efficacy of VolitionRx’s NuQ® tests to distinguish anaplastic prostate cancer, a particularly aggressive form of the disease, from typical castration resistant prostate cancer (CRPC), the less aggressive form.
  • A prospective study with the University of Oxford, United Kingdom, to assess VolitionRx’s NuQ® tests for the diagnosis of endometriosis.

- End -

About VolitionRx

VolitionRx is a life sciences company focused on developing diagnostic tests for cancer and other conditions. The tests are based on the science of Nucleosomics, which is the practice of identifying and measuring nucleosomes in the bloodstream or other bodily fluid - an indication that disease is present.

VolitionRx’s goal is to make the tests as common and simple to use, for both patients and doctors, as existing diabetic and cholesterol blood tests. VolitionRx’s research and development activities are currently centred in Belgium as the company focuses on bringing its diagnostic products to market first in Europe, then in the US and ultimately, worldwide.

Visit VolitionRx’s website (www.volitionrx.com) or connect with us via Twitter, LinkedIn or Facebook.

Media Contacts

Charlotte Reynolds, VolitionRx
Charlotte.Reynolds@volitionrx.com
Telephone: +44 (0) 795 217 7498

Kirsten Thomas, The Ruth Group
kthomas@theruthgroup.com
Telephone: +1 (646) 536-7014

Investor Contacts

Scott Powell, Investor Relations
S.Powell@volitionrx.com
Telephone: +1 (646) 650-1351

Lee Roth, The Ruth Group
lroth@theruthgroup.com
Telephone: +1 (646) 536-7012

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Statements in this press release may be “forward-looking statements” within the meaning of Section 27A of the Securities Act of 1933, as amended, and Section 21E of the Securities Exchange Act of 1934, as amended, that concern matters that involve risks and uncertainties that could cause actual results to differ materially from those anticipated or projected in the forward-looking statements. Words such as “expects,” “anticipates,” “intends,” “plans,” “believes,” “seeks,” “estimates,” “optimizing,” “potential,” “goal,” “suggests” and similar expressions identify forward-looking statements. These forward-looking statements relate to the effectiveness of the Company’s bodily-fluid-based diagnostic tests as well as the Company’s ability to develop and successfully commercialize such test platforms for early detection of cancer. The Company’s actual results may differ materially from those indicated in these forward-looking statements due to numerous risks and uncertainties. For instance, if we fail to develop and commercialize diagnostic products, we may be unable to execute our plan of operations. Other risks and uncertainties include the Company’s failure to obtain necessary regulatory clearances or approvals to distribute and market future products in the clinical IVD market; a failure by the marketplace to accept the products in the Company’s development pipeline or any other diagnostic products the Company might develop; the Company will face fierce competition and the Company’s intended products may become obsolete due to the highly competitive nature of the diagnostics market and its rapid technological change; and other risks identified in the Company’s most recent Annual Report on Form 10-K and Quarterly Report on Form 10-Q, as well as other documents that the Company files with the Securities and Exchange Commission. These statements are based on current expectations, estimates and projections about the Company’s business based, in part, on assumptions made by management. These statements are not guarantees of future performance and involve risks, uncertainties and assumptions that are difficult to predict. Forward-looking statements are made as of the date of this release, and, except as required by law, the Company does not undertake an obligation to update its forward-looking statements to reflect future events or circumstances.

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Read this news on PR Newswire Asia website: VolitionRx to Initiate Study with University Hospital Bonn to Confirm NuQ(R) Test Accuracy in Lung Cancer
TNF¿ signalling primes chromatin for NF-¿B binding and induces rapid and widespread nucleosome repositioning

Background: The rearrangement of nucleosomes along the DNA fiber profoundly affects gene expression, but little is known about how signalling reshapes the chromatin landscape, in three-dimensional space and over time, to allow establishment of new transcriptional programs. Results: Using micrococcal nuclease treatment and high-throughput sequencing, we map genome-wide changes in nucleosome positioning in primary human endothelial cells stimulated with tumour necrosis factor alpha (TNFα) - a proinflammatory cytokine that signals through nuclear factor kappa-B (NF-κB). Within 10 min, nucleosomes reposition at regions both proximal and distal to NF-κB binding sites, before the transcription factor quantitatively binds thereon. Similarly, in long TNFα-responsive genes, repositioning precedes transcription by pioneering elongating polymerases and appears to nucleate from intragenic enhancer clusters resembling super-enhancers. By 30 min, widespread repositioning throughout megabase pair-long chromosomal segments, with consequential effects on three-dimensional structure (detected using chromosome conformation capture), is seen. Conclusions: Whilst nucleosome repositioning is viewed as a local phenomenon, our results point to effects occurring over multiple scales. Here, we present data in support of a TNFα-induced priming mechanism, mostly independent of NF-κB binding and/or elongating #RNA polymerases, leading to a plastic network of interactions that affects DNA accessibility over large domains. http://bit.ly/1BUJd34 #BMC