Research highlight of our noise paper

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Marathon runners represent the dynamics of the transcription process, influenced by DNA sequence elements. In this issue, single-cell time-lapse microscopy experiments were used to shed light on the ways in which two types of promoter elements—nucleosome disfavoring sequences and transcription factor binding sites—influence transcription dynamics. Promoter dynamics dictate the transcriptional noise across a cell population, as illustrated by the variability between the runners.

Schubert et al. found that RNAs were involved in maintaining the accessibility of chromatin using an assay in which chromatin is digested by a nuclease (DNase). This digestion creates a ladder of DNA of different sizes on a gel, ranging from single nucleosomal fragments to far larger pieces. The extent of digestion is dependent on the level of chromatin condensation; the more compacted the nucleosomal structure, the more refractory it will be to DNase digestion. The researchers found that incubation of chromatin with an RNase prior to DNase treatment resulted in more compacted chromatin; DNase digestion was less efficient. Using different RNases and inhibitors they discovered that the RNA population involved was single-stranded and synthesised by RNA polymerase II.

Similar results were found when the authors used density-gradient centrifugation to isolate chromatin. They found a fraction of RNA associated with chromatin (caRNA) that when digested, resulted in chromatin becoming more compacted and shifting to higher density fractions. Interestingly, this RNA-dependent chromatin condensation effect is reversible; when the compacted chromatin was extracted and incubated with fresh cellular extracts, it reopens, again migrating in lighter fractions, re-associated with RNA. This caRNA-dependent chromatin accessibility is also dependent on chromatin-associated proteins, as it could not be rescued under denaturing conditions.

Using mass spectrometry, the authors identified 59 proteins that had lower affinity for chromatin after removal of RNA. One of these was the highly abundant chromatin decondensation factor 31 (Df31). A study that I have long intended to write about (Filion et al. 2010) used the binding of Df31 and 52 other chromatin associated proteins to determine that rather than dividing Drosophila chromatin into two types: transcriptionally repressed heterochromatin and active euchromatin, we should instead think in terms of five different classes which they colour coded. Green, blue and black chromatin are broadly transcriptionally repressed, whilst red and yellow are euchromatic and more transcriptionally active. Df31 is found bound to these red and yellow types of more open chromatin. Schubert et al found that Df31 chromatin binding is stabilised by caRNAs. Df31 binds histone H3 in the absence of RNA, but its affinity is substantially enhanced by the addition of RNA. RNAi knockdown of Df31 causes a fraction of genomic DNA to be more compacted.

Deep sequencing of the pool of caRNAs revealed that they were enriched for non-coding RNAs, especially a class termed small nucleolar RNAs (snoRNAs). snoRNAs are known to guide the modification of bases in ribosomal, transfer and messenger RNAs. They have also been implicated in RNA editing and splicing. Schubert et al. found that 30 of the 186 snoRNAs expressed in Drosophila embryos were found associated with chromatin. Using fluorescent in situ hybridisation, they showed that two of the most highly enriched snoRNAs could be visualised binding to the interbands of Drosophila polytene chromosomes (ie. euchromatin). These two snoRNAs were able to ‘rescue’ compacted chromatin in the density-gradient experiments, and were shown to directly interact with Df31.

The authors also found that extracts of human cells could rescue RNase treated compacted chromatin, and that snoRNAs are found associated with chromatin in human cells.

Schubert et al have therefore characterised a novel conserved role for snoRNAs mediating the accessibility of higher-order chromatin structures. As none of the protein components known to complex with snoRNAs in snoRNPs were identified in the mass spectrometry experiments, it appears that snoRNAs form distinct ribonucleoprotein complexes to mediate this chromatin associated role. Df31 is one such important interactor, linking chromatin-associated snoRNAs and histone proteins within nucleosomes to maintain accessibility of red and yellow chromatin. However, the knockdown of Df31 resulted in far more limited and localised chromatin compaction than RNase treatment, suggesting that the role of caRNAs goes beyond the maintenance of open euchromatin, but also to regulating heterochromatic accessibility. Seeing as 58 other chromatin proteins, as well as many other snoRNAs and other caRNAs, were implicated in RNA-associated chromatin accessibility functions, this initial study has just revealed the tip of an iceberg. Just how this RNA-chromatin network functions to maintain accessibility requires a lot of work. As with many of the best studies, the light shone serves to partially illuminate the scale of our ignorance.

Schubert T, Pusch MC, Diermeier S, Benes V, Kremmer E, Imhof A, & Längst G (2012). Df31 Protein and snoRNAs Maintain Accessible Higher-Order Structures of Chromatin. Molecular cell PMID: 23022379

Filion GJ, van Bemmel JG, Braunschweig U, Talhout W, Kind J, Ward LD, Brugman W, de Castro IJ, Kerkhoven RM, Bussemaker HJ, & van Steensel B (2010). Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell, 143 (2), 212-24 PMID: 20888037

A team of scientists from the University of Virginia and University of North Carolina in the US have discovered a previously unidentified type of small circular DNA molecule occurring outside the chromosomes in mouse and human cells. The circular DNA is 200-400 base pairs in length and consists of non-repeating sequences. The new type of extra-chromosomal circular DNA (eccDNA) has been dubbed microDNA. Unlike other forms of eccDNA, in microDNA the sequences of base pairs are non-repetitive and are usually found associated with particular genes. This suggests they may be produced by micro-deletions of small sections of the chromosomal DNA.

This result suggests that the DNA found in tissue cells may exhibit more variation than previously thought, and the implication of this is that sequencing of the DNA in blood cells (which are the cells usually used for sequencing) may give misleading results if micro-deletions have occurred in the DNA of other tissues but not in blood cells. Examples in which this might be important are in genetic sequencing for autism or schizophrenia, which could be caused by incorrect functioning of certain genes in brain tissue. Many cancers are also caused by incorrect functioning of genes; in this case tumor suppressor genes, and sequencing of blood cell DNA could also give misleading results.

Excerpts from an article via PhysOrg

Eukaryotic chromosomal DNA is condensed by being wound around octamers of histone proteins to form nucleosomes. Post-translational modifications (PTMs) of histones can modulate chromatin structure, altering its biological activity (for example it’s transcription status). Different combinations of histone proteins and their PTMs are found through the genome and between different cell types. Deciphering this ‘histone code’ is crucial to our understanding of cellular regulation and differentiation, and is therefore the focus of huge amounts of current biological research.

Prior to this new paper at least twelve different types of histone PTM, at over sixty different amino acid residues had been reported. These include the most commonly discussed such as methylation and acetylation, as well as esoterica like citrullination. By performing a highly comprehensive survey of histone PTMs based on mass spectrometry, Tan et al have identified two new types of modification and 67 new histone marks.

The structure of the nucleosome. The four core histones are in different colours. Their N terminal tails are protruding from the nucleosome.

Nucleosomal cores consist of histone octamers containing two molecules each of histones H2A, H2B, H3, and H4. Interactions between histone proteins and between histones and DNA are generally mediated within the globular core domains of the histone proteins, whilst their N-terminal tails protrude from the nucleosome and have been considered the primary sites for post-translational modifications. However, this new study identified many histone PTMs within the globular cores, suggesting that previous methods of PTM identification have been biased against their discovery.

Tan et al also report further characterisation of one of the new types of histone PTM: lysine crotonylation (KCr). Crotonylation was found at 28 different lysine residues from all four core histones and the linker histone H1. KCr was detected in histones isolated from yeast, nematodes and fruit flies, as well as mice and humans.

Using an antibody that recognised all lysine crotonylation, chromatin immunoprecipitation followed by sequencing (ChIP-seq) showed that histone KCr was associated with active chromatin and was particularly enriched at promoter and enhancer regions.

Tan et al went on to find that during mouse spermatogenesis histone KCr is highly enriched in post-meiotic spermatids, coinciding with a general transcriptional shutdown. By using ChIP-seq in combination with transcriptomic data, they showed that KCr was marking a group of genes on the sex chromosomes that are transcriptionally active, whilst the rest of the sex chromosome is inactivated.

Lysine crotonylation appears to be an important new PTM adding even more complexity to an already complex field of study. The comprehensiveness of the technique employed for PTM identification used in this study, however, suggests that there may not be too many more histone marks to add to the list. The next questions to ask will be whether crotonylation of different lysine residues correlates with different biological events? What enzymes are responsible for the addition and removal of crotonyl modification? And what effects does the disruption of their activity have? What proteins interact with KCr? As can be ascertained from this taster, deciphering the histone code is going to keep a lot of people busy for a long time.

Tan, M., Luo, H., Lee, S., Jin, F., Yang, J., Montellier, E., Buchou, T., Cheng, Z., Rousseaux, S., Rajagopal, N., Lu, Z., Ye, Z., Zhu, Q., Wysocka, J., Ye, Y., Khochbin, S., Ren, B., & Zhao, Y. (2011). Identification of 67 Histone Marks and Histone Lysine Crotonylation as a New Type of Histone Modification Cell, 146 (6), 1016-1028 DOI: 10.1016/j.cell.2011.08.008

The nematode worm C. elegans has an invariant cell lineage, meaning that any particular cell is generated from a specific series of mother and grandmother cells. Differences between daughter cells are determined either by non-cell autonomous mechanisms such as signalling by neighbouring cells, or by cell autonomous mechanisms such as the asymmetric inheritance of cell fate determinants, or by both.

The MI motor neuron is a left-right unpaired neuron located in the pharynx. The great-great-grandmother cell of MI gives rise to left and right paired lineages of cells, symmetrical, except for one left-right asymmetry: the MI motor neuron and the e3D pharyngeal epithelial cell. The researchers had previously shown that the MI-e3D asymmetry was dependent on a cascade of transcription factors asymmetrically expressed in the grandmother and mother cells of MI: CEH-36 (an Otx homeodomain protein) promoted the expression of the bHLH containing proneural proteins NGN-1 and HLH-2. When any of these proteins are inactivated, the MI neuron is transformed into an e3D-like cell.

In a genetic screen to find other factors involved in the MI-e3D asymmetry, Nakano et al identified a gain of function allele in the gene his-9 as causing MI-e3D transformation. his-9 encodes one of 14 identical replication-dependent histone H3 proteins in C. elegans.

In eukaryotes, chromosomal DNA is condensed by being wound around octamers of various histone proteins to form nucleosomes. Alterations to nucleosome structure or density can determine the accessibility of the DNA to the transcriptional apparatus, and hence the transcription state of that piece of chromatin. These variable chromatin states are said to be ‘epigenetically’ determined, as they can be maintained through mitoses by the inheritance of the modification status of histones (and other non-DNA sequence chromosomal features).

The nucleosome core contains a tetramer composed of two histone H3/ H4 dimers. This dimerisation occurs due to interactions between the two H3 molecules. It was these H3-H3 interactions that were compromised in the original mutant allele. The addition of similarly mutated versions of other replication dependent histone H3 genes into wild type worms also had the ability to transform the fate of the MI and yet showed no other gross abnormalisties. This showed that MI cell fate specification is very sensitive to gain of function mutations in histone H3 genes.

By generating worms that carried mutant his-9 transgenes on an extrachromasomal array that is mitotically unstable (hence creating mosaic worms), Nakano et al showed that the histone H3 gain of function activity acts cell autonomously within the MI mother cell.

Histone H3-H4 dimers are deposited into the nucleosome by a histone chaperone complex called CAF-1. Compromising the activity of any of the CAF-1 subunits in C. elegans also caused MI transformation. Therefore, replication dependent nucleosome formation mediated by CAF-1 is necessary to generate MI-e3D asymmetry.

To integrate their earlier findings with their new data, Nakano et al suggest that the NGN-1/HLH-2 complex recruits histone modifying enzymes that act on CAF-1 assembled nucleosomal arrays to generate an epigenetically marked MI-neuronal state. They combine this with the idea that CAF-1 can generate differences in the densities of nucleosomes between sister chromatids that upon mitotic segregation would generate a difference between sister cells. MI neuronal fate determination would require NGN-1/HLH-2 mediated histone modifications to be found at a specific (CAF-1 mediated) density.

To my knowledge, the idea that epigenetic marks, asymmetrically inherited, can act as cell fate determinants is novel and potentially a very important mechanism of development. In this case it is only a model that will require a lot more experimentation, however the authors go on to suggest that it could be a conserved mechanism generating bilateral asymmetries in the nervous systems of mammals as well. Mutations in a microtubule-based motor protein called left-right dynein (LRD) randomize visceral left-right asymmetry in the mouse due to defective cilia causing a left-right determining flow in the node to fail. LRD has also been implicated in biased chromatid segregation and interestingly rather than randomized asymmetry in the brain, LRD mutant mouse hippocampuses exhibit a loss of bilateral asymmetry that the authors suggest could be caused by parallel mechanisms as MI-e3D asymmetry. This is probably a leap too far, but fun anyway.

Nakano, S., Stillman, B., & Horvitz, H. (2011). Replication-Coupled Chromatin Assembly Generates a Neuronal Bilateral Asymmetry in C. elegans Cell, 147 (7), 1525-1536 DOI: 10.1016/j.cell.2011.11.053

The GAL1 and GAL10 genes are separated by a 680 bp bidirectional promoter, which contains a region called the GAL upstream activating sequence or UASg, with four GAL4 binding sites in it. Before Galactose induction, Gal4 binds to the UASg but is inhibited by Gal80. Addition of Galactose frees Gal4, and this activator quickly and strongly induces transcription. Addtion of Glucose, a preffered carbon source, to cells growing in Galactose inhibits expression of GAL4 and GAL2, which encodes the Galactose permease.

Nucleosome occupancy is usually measured by Micrococcal Nuclease (MNase) digestion. In a typical modern version of such an experiment, cells are fixed with formaldehyde, and isolated chromatin is lightly digested with a single dose of MNase for a fixed time. Crosslinking is then reversed, and mononucleosomal-sized DNA fragments of about 150 bp are isolated. These recovered DNA fragments can be analysed by qPCR, microarray, and DNA sequencing. Unfortunately, DNA sequence itself influences digestion efficiency of MNase, a bias that can create false apparent protection of ‘naked’ genomic DNA.

In this paper, the authors developed a quantitative MNase protection assay that normalises against such bias. They digest either crosslinked chromatin or un-crosslinked chromatin (so called ‘naked’ genomic DNA) with a wide range of MNase concentration, comparing the digestion rate of the same DNA sequence under two condition (crosslinked and naked) to assay the protection.

As expected, they found that naked genomic DNA exhibits a first-order decay function with monophasic digestion pattern. The digestion rates of the naked segments varied as much as 10-fold, suggesting differences in the intrinsic sensitivities of various DNA sequences. Most crosslinked DNA possess a biphasic digestion pattern: a rapidly-digested portion and a slowly-digested portion, indicating the existence of two states of DNA (unprotected and protected) in the cell population.

Using this method, they found that the UASg is 100% protected both before and after Galactose addition. Moreover, this UASg segment is protected by unknown factors rather than Gal4 or nucleosome, because: 1) the protection remains unchanged upon deletion of GAL4; 2) ChIP experiments show that this region is free of H2B; 3) the digestion rate of the protected UASg is significantly faster than that predicted is the protecting factor a nucleosome; 4) the size of protected UASg is 130 bp, some 30 bp smaller than a typical nucleosome.

This paper is really interesting.

DNA Packaging

The findings concluded that the repeats affect the activity of neighboring genes by way of how tightly the downstream DNA is wrapped around a complex of proteins collectively called a nucleosomes. A nucleosome is one of the half dozen packaging features of the eukaryote genome which allows a genome that is 3 billion base pairs long or 6 feet in length to be squeezed into a tiny little nucleus. About 167 basepairs wrap around one nucelosome. DNA that is more wrapped around a nucleosome is harder to be activated, and thus otherwise non-coding/junk tandem repeats of sequences determine how tightly the local DNA is wrapped around these protein complexes.

The extra cool finding about this paper is that the tandem repeats are very unstable, as you possibly could tell from the title. The authors found out that the number of repeats changes a lot during DNA replication, as if the DNA pol III exonucleases don’t bother proof-reading these areas! These changes affect the local DNA packaging, which in turn alters gene activity. In this way, unstable junk DNA is one of the faster acting mechanisms in altering gene activity with each cellular division.

As an extra step, the researchers conducted a experiment investigating the impact of these tandem repeats on yeast cells. They found out that when a repeat is present near a gene, it is possible to select yeast mutants that show vastly increased activity of this gene. But, when the repeat sequences were removed, this fast evolution was impossible.

So what does this all mean for human evolution? Well, unstable pieces tandem repeats of ‘junk’ non-coding DNA are one of the many ways of regulating gene expression and honing on when a gene’s activity can enable organisms like humans to quickly adapt to changes in their environments.

Vinces, M., Legendre, M., Caldara, M., Hagihara, M., & Verstrepen, K. (2009). Unstable Tandem Repeats in Promoters Confer Transcriptional Evolvability Science, 324 (5931), 1213-1216 DOI: 10.1126/science.1170097