Monthly Archives: January 2012

An introduction to Epigenetics.

Multicellular Eukaryotes are intriguing; from one genome in one cell, they are able to generate a wide range of cells, and consequently tissues, and then organs and organisms. The genes contained in the DNA of these cells are more or less identical, but their expression patterns can be widely divergent. There can be genes that are active in neural tissue, for instance, but not in the tissues of the heart, for instance, and this is found across a whole range of tissues.

These expression profiles, as we know them in the language of molecular biology, are distinctive and unique to tissues and can be used to define the normal physiology of a given tissue type, with aberrations from this being associated with disorders. (See http://www.biomedcentral.com/1471-2164/12/439). This raises questions of how tissue specific expression profiles are developed by cells in a given tissue, and how this is modified during development as an organism takes shape from one single cell at conception.

The ability to regulate gene expression is present in all life, even prokaryote, where transcriptional regulation can be seen in the form of operons, and it is true that gene expression may simply be modulated by transcriptional regulation by various promoters and repressors binding to the promoters of genes in various permutations and combinations (See http://www.ploscompbiol.org/article/info%3Adoi%2F10.1371%2Fjournal.pcbi.0020164 for a paper discussing the complexities of such architecture) but this solely gene-based process isn’t all there is to it. There is something above the genetic layer and below the proteomic layer, and the field that studies this is called epigenetics.

Epigenetics is a function of chromosome structure; for transcription to actually take place, all the transcription factors and enzymes that are required for gene expression must be able to access the DNA that contains the genes that are to be expressed, but chromosome structure will determine if this can be done or not, and can therefore determine if a gene can be expressed or not.

In Eukaryotes, chromsomes aren’t made of naked DNA; DNA is instead associated with complexes of histone octamers, these complexes, called nucleosomes, are then folded upon themselves to form one of several structures; roughly a loosely packed structure called heterochromatin, and a tightly packed structure called euchromatin. Only genes in euchromatin can be transcribed; a heterochromatin configuration at the locus of a gene is a harbinger of silenced expression.

Left, schematic of various levels of chromatin compaction, from extended nucleosome arrays to folding of secondary chromatin structures exemplified by the 30-nm chromatin fiber to poorly characterized higher-order structures. There are ten histone tails protruding from each nucleosome core. Right, detail of nucleosome surface showing histones H2A (yellow), H2B (light red), H3 (blue) and H4 (green). Image Reference - http://www.nature.com/nsmb/journal/v14/n11/fig_tab/nsmb1107-1056_F1.html

I’m now going to introduce you to some of the common epigenetic mechanisms of gene silencing that have been documented.

Promoter methylation

This is one of the best documented mechanisms of epigenetic silencing we’ve uncovered so far; promoters have regions of cytosine nucleotides followed by guanine nucleotides in multiple stretches. We call these CpG islands. If these are methylated, there is a conversion of Cytosine to 5-methylcytosine, and this basically reduces the affinity that the transcriptional apparatus in a cell has for promoters of the genes in question.

The methylation patterns of a cell’s DNA can be inherited by daughter cells as well; enzymes such as DNMT1 (a DNA methyltransferase) can seek out hemimethylated regions (one of the two copies of a gene in a daughter cell is methylated) and methylate the other, completing inheritance of an expression profile. Other proteins such as Methyl CpG binding proteins lock methylation in and keep genes repressed.

a | Differential silencing by CpG island or promoter methylation. b | Regulation by antisense transcripts in conjunction with CpG island or promoter methylation; c | Allele-specific regulation of neighbouring genes by differential methylation of boundary elements within a CpG island. Factors such as CCCTC-binding factor (CTCF) (red disc) bind to the unmethylated allele and block the access of upstream promoters to downstream enhancers (green), leading to transcriptional repression of the upstream gene. d | Differential methylation results in differential binding of silencing factors (red, in this case methylation-sensitive), which repress the promoter in cis. Image Reference - http://www.nature.com/nrg/journal/v2/n1/fig_tab/nrg0101_021a_F5.html

Promoter methylation can also act in combination with transcription factors; it may well be possible to see cases where an activator binding site is methylated while a repressor can bind to its site in the promoter, resulting in synergistic repression of transcription.  Since this is inherited, tissue transcriptomes can be regulated and shaped during development and differentiation, and that is one of the reasons why tissues have unique expression profiles in a state of normalcy.

There are other possible combinations for silencing by methylation, too; antisense transcripts (through RNAseH silencing as well as RNAi and miRNA) can act in conjunction with methylation, upstream promoter element methylation can block access to downstream methylation sites and so on.

Histone Modification.

Histones are proteins as well, and the residues that comprise them are subject to posttranslational modifications by enzymes, and the presence of different modifications can alter the shape of the chromatin at the site of modification. This altered conformation then influences how active a gene is by modulating DNA access by transcriptional machinery.

Chromatin remodelling in this way enables the establishment of activation or repression long term and the continued maintenance thereof. One of the rather well studied modifications is histone acetylation/deacetylation at a lysine residue, especially in Histone H3. However, this is not the only known modification, and phosphorylation, ubiquitylation and so on may be of import as well.

Histone modifications occur at selected residues and some of the patterns shown have been closely linked to a biological event (for example, acetylation and transcription). Emerging evidence suggests that distinct H3 (red) and H4 (black) tail modifications act sequentially or in combination to regulate unique biological outcomes. How this hierarchy of multiple modifications extends (depicted as 'higher-order combinations') or how distinct combinatorial sets are established or maintained in localized regions of the chromatin fibre is not known. Relevant proteins or protein domains that are known to interact or associate with distinct modifications are indicated. The CENP-A tail domain (blue) might also be subjected to mitosis-related marks such as phosphorylation; the yellow bracket depicts a motif in which serines and threonines alternate with proline residues. Image source - http://www.nature.com/nature/journal/v403/n6765/fig_tab/403041a0_F2.html

One thing you could carry away from reading the section above is that the mere presence of histones and enzymes that act on and modify them can regulate differentiation, development and tissue homeostasis, and all of that section is somewhat nicely summarised by the following video.

Why does Epigenetics matter and where are we headed?

If there are disorders that are associated with aberrant epigenetic function in a cell, and there is mounting evidence for this already, it would make sense to continue to understand the mechanisms involved and the effects they have, and a range of techniques, ranging from those that map out global methylation to those that look at individual histones, have been, and are, being developed.

Global dysregulation of methylation patterns is a phenomenon in cancer cells and so are aberrations associated with microRNA dysfunction; in fact, this is significant enough that drugs that seek to hit dysregulated epigenetic processes are in development, such as histone deacytlase inhibitors and global methylation modulators (See http://carcin.oxfordjournals.org/content/31/1/27.full for a well-written summary of the field of cancer epigenetics). Sometimes, more complex chromatin remodeling may serve as potential drug targets, too, JQ1 , a drug that tells undifferentiated cancer cells to differentiate into normal cells,being a case in point…

Understanding epigenetics may also have significant impact on regenerative medicine; some cutting-edge work done recently based on the understanding of how miRNA can alter the expression of chromatin remodelling complexes resulted in researchers being able to turn skin fibroblasts into neurons. The relevant paper is brilliant and can be found here http://www.nature.com/nature/journal/v476/n7359/full/nature10323.html .

There are already large scale projects to study epigenetics being put in place, such as the Human Epigenome Project, which is great news for anybody who is interested in the field.There is a Human Epigenome Browser in case you want to play with it, by the way. This has been accompanied by the development of high-throughput screening methods for epigenetic analysis, such as Medip-Seq, which is a natural progression in the evolution of techniques such as ChIP on Chip and ChIP-Seq.

That is all from me this time round. I will be studying cancer epigenetics in about a month’s time on an academic basis so I suggest you stay tuned for more insights into the field.

Cheers,
Exploreable

A few words on charity in Africa

Terrible day. Which may have influenced my response to the UNICEF man who approached me before I got on the bus home. I heard him out, I did, but there he was telling me that my £3 text would feed a child in east Africa for 4 days.

To summarise, I said to him ‘it won’t help’. Here’s why.

I’ve been to east Africa. £3 might feed a child for 4 days. But what about the 4 days after that? And the 4 after that? No one thinks about those because they’re too busy feeling smug for donating the £3 that wouldn’t even pay for their own lunch here in London. UNICEF man even said to me, ‘if you donate then you can have a great weekend and feel good about yourself for making someone’s life a bit better’. So £3 can really make someone’s life a bit better? Really?

Firstly, (and I’m not trying to be a cynic here) on the practical side of things, if I were to donate, I sadly doubt that said individual child I would apparently be feeding would a) see £3 worth of food, or b) not have to share it with their family or be subject to theft by jealous peers.
Secondly, I do honestly believe that the people doing the charity work are doing it all in good faith, and really do think they are helping, but this type of ‘throw money at a problem’ charity is just a plaster. It not only conceals the problem but it prevents a resolution. They say that for a wound to heal it needs fresh air. What is fresh about foreign aid? Foreign aid is not a new thing to the people of east Africa, and guess what, they still need it. How many times have they done Band Aid now? (Just realising the irony of that name in the context of what I’m saying).

My point is, if something works, you don’t have to keep doing it.

After they donate, people get so wrapped up in the fact that they donated in the first place that they forget about the root causes of this crippling poverty, many of which are the legacy of colonialism, and are perpetuated by the modern western world. Most African countries are in debt to rich western governments, and these are debts that they will never be able to pay off without real change; real change that will never happen whilst they are still in debt, still corruptly governed, and still plagued by malaria, AIDS and the pope (or more widely, religion).

These are just a summary of a few of the problems affecting Africa; there are of course many more, and solving them is obviously complex. But that £3 isn’t going anywhere towards a solution.

 

Sorry for the gloomy post!

Science meets simplicity: A coding independent function of mRNA and pseudogene RNA.

The paper I am going to review is a demonstration of how sound logical deduction based on knowledge of basic molecular biology translated into an biological insight that was experimentally confirmed and evidentially supported.

The paper in question is this one http://www.nature.com/nature/journal/v465/n7301/abs/nature09144.html

Now, I’ve posted about miRNA and RNA interference on this blog before, as well as about the central dogma of molecular biology. The primary function of mRNA is to specify sequences that are later translated to proteins, but given that they, being RNA molecules, can interact with RNA processing/regulating machinery, it made sense to investigate if there was a coding-independent regulatory function. Before we get started, you might want to go through the article on RNA interference https://exploreable.wordpress.com/2011/01/14/right-just-as-promised-here-comes-rna-interference/ and watch a video clip on microRNA function.

Coming to the study in question, the fundamental question was; if there are two transcripts that contain binding sites for a microRNA, can levels of one have an influence on the levels of the other? (Could one transcript compete with another transcript for binding and cleavage by miRNA?). To find out, the researchers chose two transcripts; PTENP1 (a pseudogene, which evolves from the duplicate of a gene but loses the ability to form translatable transcripts) and PTEN (which is a tumour suppressor)

Before they actually tested their hypotheses, they had to verify if they could in fact compete for miRNA binding.They did this by using computational analysis of the 3′ UTR sequences to locate possible miRNAs that could bind to them both; they then settled on miR-19b and miR-20a. They verified the prediction that these microRNAs could reduce expression of both PTEN and PTENP1 by introducing them into cells, and subsequently demonstrated a causal link by showing that inhibiting this microRNAs led to derepression (restoration of high expression).

a, Working hypothesis: PTEN is protected from miRNA binding by PTENP1. miRNAs are indicated by red, blue and green structures. 5′ and 3′ UTRs, open rectangles; open reading frames, filled rectangles. b, PTEN (top) and PTENP1 (bottom) 3′ UTRs contain a highly conserved (dark grey) followed by a poorly conserved (light grey) region. PTEN-targeting miRNA seed matches within the high homology region are conserved between PTEN and PTENP1. c, Binding of PTEN-targeting miRNAs to PTENP1. Seeds and seed matches, bold; canonical pairings, solid lines; non-canonical pairings (G·U), dotted lines. d, PTEN-targeting miR-19b and miR-20a decrease PTEN and PTENP1 mRNA abundance. e, miR-17 and miR-19 family inhibitors (Imix) derepress PTENP1 abundance (left). PTEN is used as positive control (right). IC, miRNA inhibitor negative control. d, e, mean ± s.d., n ≥ 3. *P < 0.05; **P < 0.01; ***P < 0.001.

The work discussed till here managed to establish a regulatory triad; PTEN, PTENP1 and the microRNAs in question were involved in a three way relationship.Potentially, altering the levels of PTEN or PTENP1 could result in altered expression of the other gene, with phenotypic consequences. (PTEN is a tumour suppressor, and a haploinsufficient one at that, i.e, levels determine function).

The researchers then examined if knocking down PTENP1 (which has no coding function) also resulted in increased breakdown of PTEN (which is important). They used siRNA to knock out PTENP1 and investigated if levels of PTEN changed, and they found that those levels went down.

Knocking out PTENP1 using an siRNA targeted specifically against it also results in the knockdown of PTEN expression, since that frees up miRNA that would have otherwise bound to PTENP1 to go and repress PTEN expression, the reciprocal nature of the relationship is confirmed by the siRNA knockdown of PTEN causing a reduction in PTENP1 expression. The last diagram (h) confirms loss of expression by looking for reduced protein expression. The other two look at transcription.

So far, so good, they had managed to demonstrate that PTENP1 could serve to regulate PTEN expression by acting as a decoy for the requisite miRNA. How about taking things further to see if there was pathological significance?

They wanted to investigate if this functional relationship held in human tissues as well, and using microarray expression datasets from prostate cancer samples, they confirmed that the expression of both PTEN and PTENP1 was strongly correlated, implying that the demonstrated regulatory relationship held true.

Confirmation of correlation of PTENP1 and PTEN expression levels in Prostate Cancer Samples.

Tumours evolve, and cells in them undergo a host of genomic changes; frequently picking up amplifications or mutantions of genes that enhance their survival and proliferation (Oncogenes) and frequently losing those that would hinder their growth (tumour suppressors). With this in mind, the researchers examined if PTENP1 was lost by cancer cells (if it acted as a tumour suppressor by preventing miRNA from breaking down PTEN, it would make sense for cancer cells to lose PTENP1, since that would also bring about loss of PTEN activity, which is beneficial to cancer cells) and not very surprisingly, they were able to identify a subset of colon cancers that had evolved losses of genomic regions that contain PTENP1, thus demonstrating that the regulatory relationship described experimentally held, and that PTENP1 could act as a tumour suppressor.

Through the simple invocation of logic, and some cutting edge science, they’d identified a function for pseudogenes as microRNA decoys, and uncovered one method by which cancer cells may evade the action of tumour suppressor genes. This has therapeutic implications, for in cases where miRNAs are overexpressed in cancer cells, and where those lead to loss of tumour suppression, or when pseudogenes such as PTENP1 are lost, one might contemplate putting in decoy molecules to enhance the activity of tumour suppressors.

We know that miRNA is dysregulated in cancer, we know that it is a mechanism that may contribute essentially to tumourigenesis (See http://www.nature.com/nrg/journal/v10/n10/full/nrg2634.html) and we know that the evidence seen above may validate approaches that seek to employ decoys to control the effects that said dysregulation has; the future is exciting.

That is all from me this time round.  You may want to hear the author of the most recent paper I quoted natter on about miRNA dysregulation while I contemplate what to write for the next post.