The “Paranoid Organism”: N. crassa, Telomere Silencing, and DNA Methylation

Introduction

Walk Me Through the Title: Vocabulary & Background

This study focuses on a few basic genetic principles: telomeres, heterochromatin, gene silencing, and DNA methylation.  A clear understanding of these concepts is important for understanding how the researchers advanced the current knowledge about genes, DNA, and genetics. Background in biology? Skip ahead (or look back, no one’s judging).

Two facts about telomeres explain their purpose: first, telomeres are found at the ends of a chromosome and are by definition repetitive DNA sequences, and second, a cell’s replication machinery cannot replicate the chromosome all the way to the end because of its mandatory 5′ to 3′ moving direction.  In humans and Neurospora crassa this repeated sequence is TTAGGG.

Telomeres. Dennis, Lyle. Chronic Endurance Training Linked to Longer Telomeres in Older Adults. 2013. Photograph. Extreme LongevityWeb. 28 Apr 2013. <http://extremelongevity.net/2013/01/10/chronic-endurance-training-linked-to-longer-telomeres-in-older-adults/>.

Their existence has persisted for one important reason: they protect the DNA found at ends of chromosomes from being lost by deterioration which accompanies cell division until the cells reach a “critical level” of genetic information loss.  It is at this critical level that cells stop dividing and the process of aging is first seen at a cellular level.  Without telomeres protecting DNA from damage and mutation, cancer can also become prevalent.  Telomeres are a lot like plastic shoe lace tips that prevent the lace from fraying.

Heterochromatin is a way of describing chromatin: it is tightly coiled, and the opposite is euchromatin.  Heterochromatin is usually found towards the “sides” of a cell, in the periphery.  Examples of heterochromatin include the Barr body of the female X chromosome, centromeres, and more importantly to this study, telomeres.  It is thought that the functionality of heterochromatin is to protect DNA from damage by not allowing it to be exposed to DNA machinery.

When a researcher states that genes were silenced, he/she is saying that the gene was purposefully incorporated into already silent heterochromatin.  The chromosomal DNA that is silenced is not operational and will not be translated or transcribed.  Telomeric silencing is largely not understood in plants and animals because telomeres have such an important role in human genetics, and as such it has previously proven difficult to study the process.

DNA methylation is a modification of DNA; it is the addition of a methyl group to the cytosine or adenine ends of a chromosome.  This modification generally happens during cell division.  This is a cell’s way of safely modifying expression of a gene, and also serves to “put a cap” on viral genes that may have been incorporated into the cell’s genome over its lifetime.  DNA methylation also plays a significant role in the development of cancer. Adding methyl groups to DNA to suppress their expression can be used by researchers who want to eliminate or focus on the role of a specific gene.

For more background on the genetics of the experiment, visit:

Chromatin, Histones and Modifications

The Guest of Honor – Neurospora crassa

Neurospora crassa. Dalke, Kate. Mighty Mold Is Sequenced. 2003. Photograph. Genomes News NetworkWeb. 28 Apr 2013. <http://www.genomenewsnetwork.org/articles/05_03/mold.shtml>.

This fungus is a bread mold reigning from most tropical regions who started its life of fame in the mid-1800s as a contaminant in French bakeries.  It is donned the “nerve spore”, a nick-name earned by its striated appearance. N. crassa is easy to grow and has a haploid life cycle; these two characteristics and its small genome of 7 chromosomes of 43 megabases long make it a suitable model organism for genetic studies (watch out, fruit fly!).   N. crassa has earned its claim to fame by being in the spot light with Edward Tatum and George Wells Beadle on the Nobel Prize stage in 1958. Its genome was completely sequenced in 2003.

Neurospora crassa. Raju, N. BASIC CELL STRUCTURE. 2004. Photograph. n.p. Web. 28 Apr 2013. <http://www.fgsc.net/neurospora/sectionb3.htm>.

N. crassa is an interesting organism because it has been called a “paranoid organism” because it insistently defends its genome with gene silencing strategies.  The subtelomeric regions of the choromosomes of N. crassa do not have a certain class of repeat elements at their telomeres.  Instead, they have short stretches of A-T rich DNA that separate the first telomere repeat unit from the most telomere-proximal gene.  How does this happen?  The organism’s genetic machinery detects duplicated sequences and changes every C:G pair to a T:A basepair, and this T:A pair serves as a target for DNA methylation.  The by-products of this naturally occurring repeat-induced point mutation, (RIP) are found in subtelomeric regions of the genome.  When reviewing the evolutionary history of N. crassa, one can see where they may have showed homology between species, but because of this RIP one wouldn’t be able to tell when comparing two organisms.

Never heard of it. Why read on?

The authors of this study used telomeric silencing to explore & better understand the function of heterochromatin in organisms.  The novelty and importance of this study lies in the fact that all prior research on the structure and sequence of telomeres and subtelomeric regions has been drawn from studies involving organisms who do not have DNA methylation in their arsenal of genetic machinery.  Without this distinction, previous research leaves doubt in the minds of many as to how our information on telomeres can relate to humans, who do have DNA methylation as a resource.

Accurate and reliable knowledge about telomeres and telomeric silencing holds great potential for human health.  How can telomeric silencing be reversed in organisms? Which enzymes, genes, and proteins are important in the process of telomeric silencing? Understanding the process and characteristics of heterochromatin and telomere silencing is extremely important for human health, as you may have gleaned from the aforementioned background.  Knowing how to reverse the process of telomeric silencing may lead to reversing the process of aging and cancer development.  The ultimate goal from this study was to learn as much as possible about telomere silencing from a model organism that has telomere functioning with more in common with humans than previously used organisms.  

Methods & Results

“It’s All Greek to Me!” – Vocabulary List & a Little Background

  • HDACs: histone deacetylases remove acetyl groups on a histone to allow DNA to be wrapped tightly
  • hygromycin: an antibiotic that prevents protein synthesis
  • Basta: an herbicide with glufosinate as the active ingredient
  • Repeat-induced point mutation (RIP): genetic process where G:C basepairs are changed to A:T basepairs

The Concise Version – for the Reader “Short on Time”

This study focuses on and answers 5 main questions:

  1. Does N. crassa show telomeric silencing? Yes.
  2. Is more than one Neurospora sir trans (nst) gene required for telomeric silencing? No.
  3. Does telomeric silencing occur on other chromosomes and with other genes? Yes.
  4. True or False: Classes of HDACs have overlapping functions and drugs can inhibit silencing. True.
  5. Do nst genes behave like HDACs? Yes.

The Nitty Gritty – For the Devoted Reader

(For the organized reader, the paragraphs correspond to the numbers above)

First, researchers inserted a genetic marker near telomeres of N. crassa to test its expression.  Because Sir2p is a gene involved in telomeric silencing in other organisms, it was chosen to be the scape goat for determining if N. crassa shows telomeric silencing.  After making mutants and observing expression levels of their marker, researchers found 7 genes in subtelomeric regions of the VR chromosome known to code NAD+ deacetylase (an important component in DNA methylation), and they named these genes “Neurospora sir two” genes (nst-1, nst-2, nst-3, and so on).  They used repeat induced point-mutation (RIP) to make nonsense mutations in nst-1 genes to create stop codons above the gene (null alleles were also created to serve as a control).  They then crossed their mutants and designed a selectable marker to observe expression levels and determine the activity of telomeric silencing.  The marker was ‘hph’ and coded for hygromycin resistance.  When this resistance was lost in their progeny with the nst-1 wildtype allele, the researchers concluded that N. crassa exhibits telomeric silencing.

Great start! Researchers found seven genes that contribute to telomeric silencing in N. crassa and creatively named them nst- 1-7.  So, does each one have a special role and contribution towards silencing?  To answer this question, the authors combined nst mutants and tagged them with their hph marker.  They found that nst-2 and nst-5 actually relieve silencing, and nst-3 “increases” silencing (the marker had extreme depressed expression).  They combined nst-1, 5, and 3 mutants and compared the expression levels to the nst-3 mutant and saw that the expression levels were extremely similar and therefore concluded that the nst genes are redundant.

But is this an isolated event that happens on the VR chromosome, or will it happen outside this starting place?  Researchers switched to using the marker Bar and monitored Basta resistance in their organisms as they crossed transformants.  As if they crossed their fingers, bar expression was lost and the experiment mirrored the results of the experiment with the hph marker and hygromycin resistance on chromosome VR.  Interestingly, nst-2 actually contributed to loss-of-function on this chromosome (for reasons that the researchers did not attempt to explain).  Telomeric silencing occurs on other chromosomes than VR.

Derepression of the subtelomeric transgenes by inhibitors of histone acetyltransferases and DNA methyltransferases. A) Nicotinamide (NAM) relieved silencing of telomeric hph (N2285) but not hph flanked by amRIP (N2015) at a non-telomeric site. All plates contained 0.6 mg/ml Hygromycin. Plates with NAM contained 0.1 mg/ml (middle plate) or 1 mg/ml (right plate) of the drug. Each plate received approximately 100,000 conidia. B) Approximately 1000 conidia from an nst+ strain (N3120) carrying the telVR hph allele were plated in the presence or absence of 0.5 mg/ml Hygromycin to assay the effect of the drugs 5-azacytidine (5AC), Trichostatin A (TSA) and NAM on telomeric silencing. C) Same as B except strain N3441 carrying the tel VIIL bar allele was plated in the presence and absence of 4 mg/ml Basta. Control plates lacking Basta show a lawn of growth. Smith et al. Epigenetics & Chromatin 2008 1:5 doi:10.1186/1756-8935-1-5

How powerful are the effects of DNA methylation inhibitors and HDACs on the expression of markers on the VR and VIIL chromosomes?  Nicotinamide and 5-azacythindine were two drugs chosen to represent these two groups, and their effects (loss of silencing) were the greatest when they were used together.  This proves that classes of HDACS have overlapping functions and DNA methylation is not universally required for silencing of telomeres.

So, knowing this about HDACs, do nst genes behave like them? With the triple mutant mentioned before (nst-1,3, and 5) significant hyperacetylation was the result, showing that nst proteins are in fact HDACs.

Hug-Slap-Hug Sandwich: The Pros and Cons

Pro: The authors explored a variety of characteristics about telomeric silencing, including the genes, proteins, and drugs involved.

Con: The researchers relied on correlational conclusions to make the basis of their claims (Are the markers they used dependent on other markers? They openly admitted they didn’t research this).

Pro: The study utilizes the unique genetic quirks of N. crassa to make links between past research, current knowledge of human genetics, and future developments for genetic research with telomere silencing.


References:

Dalke, Kate. Mighty Mold Is Sequenced. 2003. Photograph. Genomes News NetworkWeb. 28 Apr 2013. <http://www.genomenewsnetwork.org/articles/05_03/mold.shtml>.

Dennis, Lyle. Chronic Endurance Training Linked to Longer Telomeres in Older Adults. 2013. Photograph. Extreme LongevityWeb. 28 Apr 2013. <http://extremelongevity.net/2013/01/10/chronic-endurance-training-linked-to-longer-telomeres-in-older-adults/>.

Raju, N. BASIC CELL STRUCTURE. 2004. Photograph. n.p. Web. 28 Apr 2013. <http://www.fgsc.net/neurospora/sectionb3.htm>.

Smith, Kristina, et al. “The fungus Neurospora crassa displays telomeric silencing mediated by multiple sirtuins and by methylation of histone H3 lysine 9.”Epigenics & Chromatin. 1.5 (2008): n. page. Web. 2 Apr. 2013. <http://www.epigeneticsandchromatin.com/content/1/1/5>.

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