Genome Spot

Gene regulation is a really complicated thing. We have covalent marks to DNA, histones and transcription factors. Chromatin remodeling and long range enhancer interactions. Enhancer elements located in introns of genes hundreds of kilobases away from the gene they're controlling. Transcriptional control from microRNA networks and now there is an emerging model for the function of some of the thousands of long non-coding RNAs which are just now being uncovered with high resolution (directional) transcriptome analysis.

Many of you which studied molecular biology at Uni would (should) remember the model for how X chromosome inactivation is achieved. The mechanism centers around XIST, one of the first non-coding RNA genes identified. Expression of XIST from the inactive X chromosome essentially wraps it up at the same time that repressive epigenetic marks are established through its interaction with the Polycomb Repressive Complex 2 (PRC2). Sounds simple enough, but the model also inv…

We are coming to the end of a big year in the field of genome science, one which was dominated by the swarm of papers from the ENCODE consortium. (The number of ENCODE articles, mostly in Nature, has reached 92! and has no sign of slowing down.) If you haven't had time an read them all, do so here.

Apart from ENCODE, there has been a quite astonishing pace in genomic studies. Here are just a few highlights from me:
The 1000 genomes project published it's main findings in Nature (link).Non-invasive prenatal genome sequencing was described in PNAS (link), triggering a debate about the ethics of genomic studies of the unborn. Single cell sequencing techniques improve with better methods of amplification, even allowing sequencing of 99 individual sperm (link) giving new insights into patterns of recombination, and opening new avenues for IVF testing.Metagenomics analysis takes off. Whether it's environmental samples from the sea, hot springs or soil (JGI website) or even micro…

There have been a number of high profile profile articles in recent times discussing the function of TET proteins, mostly in the conversion of methylated cytosine (5mC) into hydroxymethylated cytosine (5hmC), the 5th base. Hydroxymethylcytosine is much rarer than methylcytosine and is thought to be an intermediate towards demethylation of cytosine, a mechanism which remains incompletely resolved. A paper last year showed that TET proteins also convert 5hmc to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), termed the 6th and 7th bases.


OGT on the other hand is a fairly unique protein because it is the only known known O-GlcNAc transferase in mammals. What is GlcNAc you say? It stands for N-acetylglucosamine, a hexosamine. There has been a series of papers (here, here, here) discussing OGT as a nutrient sensor, transferring GlcNAc during period of surplus nutrient supply. GlcNAc can be transferred to the same amino acids as phosphorylation, so there is a suggested crosstalk betwee…

Its no secret that repeat DNA makes up the majority of the genome in a majority of "higher" eukaryotes. For a long time this repeat DNA has been considered "selfish" or "parasitic" DNA, its only feature was its prolific self-propagation as it hitchhiked a ride throughout evolutionary history at the energetic expense of the host.

Nina Fedoroff argues in a recent review in Science that these transposable elements are not "junk" at all, and in fact they play "a profoundly generative role in genome evolution" where the transposons provide novel mechanisms to generate genetic diversity. Nina explores the relationship between genome size and epigenetic complexity in the comparison of eukaryotes and prokaryotes, postulating that the innovation of epigenetic elaborate control mechanisms in bacteria paved the way for increases in genome size and complexity we see in eukaryotes. So rather than epigenetic mechanisms evolving to keep "parasit…

Nearly every baby born in Australia since 1970 has had a few drops of blood taken and stored on a so-called Guthrie card, and this practise is widely adopted in the developed world. As DNA analysis technologies become ever more sensitive and economical, these cards will become ever more important in diagnosis of genetic disease and also in identifying genetic and epigenetic variations which contribute to complex disease. The paper I showcase today from Beyan et al, describes the development of genome-wide assays for DNA methylation using methylation microarrays and methylcytosine immunoprecipitation followed by Illumina sequencing (MeDIP-Seq). Authors find differential methylation regions which are stable from birth to 3 years of age.

The methodology is fairly novel, but the conclusions are a bit vague and it would have been best to apply Guthrie card analysis for a specific disease. It would be really neat if they analysed material from discordant twins for a complex disease i.e; juv…

This paper was featured in the 12 October issue of Science, so really isn't from this week, nevertheless I thought it would be relevant given the previous post on library preparation.

Sequencing ancient DNA is a hugely challenging task. Not only is it very difficult to get any sort of yield of DNA from bones tens of thousands of years old, but the DNA itself is normally degraded to such an extent that conventional library preparation is highly inefficient. On top of this, there is the challenge to eliminate environmental contamination. To avoid much of this, the team, lead by Matthias Meyer at the Max Planck Institute for in Leipzig came up with a simple but efficient method to generate sequencing libraries from single stranded DNA. The basic steps in the library prep are:

DephosphorylateHeat denatureLigate single stranded biotinylated adaptorsImmobilize on streptavidin beadsGenerate the second strand with DNA polymeraseLigate a second adaptor by blunt end ligation

The main benefit…

3' Polyadenylation is a key mechanism whereby mRNAs are stabilised and made ready for protein translation. One of the mysteries of molecular biology of late is how long non coding RNA (lncRNA) stability is achieved given that these molecules don't have a polyadenylation signal. Wilusz et al published a paper in Genes and Development predicting that MALAT1 is protected from 3' to 5' exonuclease activity by an RNA triple helix structure. The researchers used molecular modeling to resolve that the 3' terminus is neatly tied into a triple helix and thus likely protected from degradation. This was confirmed by mutagenesis, showing that altering bases in these regions led to a reduction in transcript stability.

MALAT1 is transcribed to form a ~6.7 kb lncRNA which is abundant in the nucleus, and also producing a small tRNA like transcript which is processed into a mature 61 nt hairpin localised to the cytosol. Both transcripts are dependant on RNase P, a ribozyme which is …