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in the news: CpG methylation is not always repressive

10/8/2013

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A recent work published in eLife (Hu et al. DNA methylation presents distinct binding sites for human transcription factors. eLife 2013) challenges the notion of suppressive methylated CpG islands by reporting a significant number of transcription factors binding preferentially to methylated cytosines of CpG islands. In mammals, the methylation of CpG sites—which consist of a cytosine base next to a guanine base—is typically thought to reduce gene expression by preventing proteins called transcription factors from binding to regions of DNA called promoters. This can occur directly if methylation disrupts interactions between the DNA and the transcription factors, or indirectly if other proteins that bind to the methylated DNA compete with the transcription factors for binding sites. However, only a small number of proteins that bind to methylated DNA have so far been identified.

The data used: The authors use protein arrays for 1300 TF and their co-factors in order to assess their binding affinity on unmethylated or methylated DNA. The DNA stretches used were in total 154 sequences selected on the basis of high probability to form part of human promoters, being representative of known TF-binding sites and carrying at least one CpG site. TF-binding intensities were then measured for both the unmethylated and the methylated version of each of the sequences. 

The analysis: Differential TF binding for methylated and unmethylated revealed a significant subset (47 proteins) showed increased binding for the CpG-methylated DNA instead of the unmethylated one. The authors showed that this represents an inherent property of the proteins by showing selective binding of specific TF towards different DNA sequences when they contain methylated cytosines and when not. In this sense, the authors coin mC (methylcytosine) as the "fifth base".

What's next: As the authors note the number of TF identified in this study is probably an under-estimation since only a very limited number of DNA targets was used. High-throughput techniques coupling of high-resolution DNA methylation (RRBS) with ChIPSeq to define regions of TF binding that are effectively methylated are probably the most effective way to probe methylated DNA binding directly.

Read more: A work published a bit earlier (Spruijt et al. Cell 2012) where specific direct binding of hydroxy-methylcytosines is assayed at genome-scale. hmC (hydroxy-methylcytosine) is probably the primary candidate for being coined as the "sixth base".

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in the news: Variability of the human transcriptome

10/8/2013

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A recent advanced online publication in Nature (Lappalainen et al, Transcriptome and genome sequencing uncovers functional variation in humans. Nature. 2013 Sep 15) features the first concise attempt to assess transcriptome variation in the human genome. This forms part of the Geuvadis consortium which brings together scientists from various research centers in Europe and the US under the coordination of X. Estivill and R. Guigo from CRG, Barcelona.

The data used: Primary lymphoblastoid cells of 462 individuals of various ethnic backgrounds were analyzed for total mRNA and miRNA levels by applying high-throughput RNASeq in different platforms and in different scientific institutes. 452 out of those were also genotyped as part of the human 1000 genomes project. To cut the long story short this is a huge amount of data at both genome and transcriptome level.

The analysis: The analysis doesn't involve really complex approach but is made extremely complicated from the sheer amount of data analyzed. Two of the main goals of the Geuvadis consortium are precisely to design efficient manipulation and storage techniques for big data and to test the robustness and replicability of transcriptome analysis. Both objectives were met. On the other hand very few novel or unexpected conclusion were drawn from this study. Yes, there is variation in transcriptome, and yes for the most part it is regulatory meaning that differences in gene expression are not qualitative but quantitative. This is not unexpected. On the other hand one interesting finding, that variation takes place mostly at the relative transcript ratio of the same genes instead of gene expression levels (depicted in the Figure) is not very straight-forward and will probably prove extremely difficult to further validate.

What's next: Let me guess; EVEN more sequencing about to come.

Read more: On the assessment of RNASeq variability and whether this is an efficient method to accurately reflect transcription levels in human (spoiler alert: It is!) (t' Hoen et al., 2013) and on the 1000 genomes project assessing the genomic variation in the human population (Mc Vean et al., 2012)

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in the news: The "get together" of exons and promoters 

10/8/2013

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Last week's Nature Genetics featured a cover story (Mercer et al. (2013) DNase I–hypersensitive exons colocalize with promoters and distal regulatory elements. Nat. Genet 45, 852-859) originating from the labs of John Stamatoyannopoulos at the University of Washington and John Matick that brings a new perspective on the way gene structure and splicing regulation may be entangled with higher level organization of chromatin.

The main idea: Exons have been recently shown to be spliced co-transcriptionally, which means that splicing takes place while the DNA is still being transcribed. The implications of the coupling of these process are various. A very important one is the definition of exons to be spliced-in (thus retained in the initial mRNA transcript) taking place through the interaction of DNA regions in the three dimensional space. The authors examine this possibility through the combination of chromosome interactions in the three dimensional space, DNAseI hypersensitivity assays and RNA sequencing.

The data used: The authors performed a DNAaseI digital footprinting assay to obtain information of chromatin accessibility throughout the genome, ChIA-PET in order to determine distant interactions at chromatin level coupled with RNA sequencing in order to obtain information on the structure of transcripts (effectively which exons are included in mRNAs)

The analysis: After going through all necessary steps for the initial mapping and normalization of sequencing data, the analyses was quite straight-forward. The authors looked for exons that appear to be alternatively spliced in specific tissues (that is non-constitutive exons) to demonstrate that they are positioned in the proximity of promoters and are thus affected by DNAseI. Thus they hypothesize that promoter and enhancer activity can be exerted at the level of splicing by effectively modulating genome proximity. In other words, promoters and enhancers are physically interacting with those exons that will be alternatively spliced in mRNA transcripts and mark them accordingly.

What's next: One of the most interesting aspects of this work is that it extends the notion of "transcription factories" according to which genes that are to be transcribed are organized to concrete entities by approaching each other in physical space. According to Mercer et al., findings this view may be further extended to the level of gene, where exons to be alternatively spliced are co-localized with promoter and enhancer elements and thus gain the necessary "markings" that will eventually define their inclusion or not. Further experimental validation will prove this exciting new feature of gene expression regulation at the level of mRNA transcripts.

Read more: On the DNAaseI digital footprinting method (Neph et al., 2012) and on chromosome conformation capture approaches (Bau et al., 2010) and ChIA-PET (Fullwood et al, 2009). Read more on the transcriptional-factory hypothesis in (Cook, 2010)

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in the news: Splicing regulation of cytokine signaling

10/8/2013

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A recent PNAS paper from the group of David Baltimore at CalTech provides evidence for an interesting link between splicing and the coordinated response to cytokine stimulus. 

Hao and Baltimore (2013). RNA splicing regulates the temporal order of TNF-induced gene expression. PNAS.

The main idea: In many inducible systems the mRNA of different genes being induced appears sequentially. This may be either due delayed access of transcription factors at the genes to appear later but one cannot exclude the possibility for all genes' pre-mRNA being readily produced followed by a subsequent delay in maturation due to splicing. The authors test the latter hypothesis in the context of TNF-induced B-cells.

The data used: Immortalized mouse embryonic fibroblasts treated with TNF and analyzed at the levels of gene expression, transcript stability and splicing speed.

The analysis: The authors have previously defined that upon TNF induction, different sets of genes appear to be upregulated in three distinct "waves". An immediate (within 30 minutes of induction), an intermediate (2h after induction) and a late one (12h or more after induction). In this work they measure primary and mature mRNA levels to show that although the primary (unspliced) mRNA levels appear comparable for genes of all three groups, mature (spliced) mRNA levels seem to follow a certain trend that is related to the gene groups. Thus they argue that the 3 "waves" of induction are regulated at the level of mRNA splicing.

What's next: It remains to be seen whether this splicing-regulated time dependence may be reflected in specific properties of the underlying sequence. It would be interesting to see whether splicing may be guided by other measurable properties of the genes' sequences.

Read more: A previous work of the same team that first proposed the "3 waves" of TNF-inducible genes. Hao and Baltimore (2009). The stability of mRNA influences the temporal order of the induction of genes encoding inflammatory molecules. Nature Immunology 10, (13), 281-288.


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in the news: Bacterial Genome Architecture

10/8/2013

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In a PNAS paper from back in 2010, Yin and colleagues from the University of Georgia propose a very interesting hypothesis according to which the arrangement of operons in the genomes of bacteria is constrained by the degree of their participation in common pathways.The main idea: Operons whose genes are involved in the same pathways tend to be more closely located in the genome so as for their coordinated expression to take place more efficiently.

The data used: The complete genomes of E. coli and B. subtilis, their operon annotation (genomic coordinates and gene content) and the connections between operons and biological (mostly metabolic) pathways. Moreover, genes involved in pathways that are more active belong to operons that are located in smaller distances from each other.

The analysis: The authors devise a simple measure of operon compactness, that is how closely operons involved in the same pathway tend to occur in the linear genome. By comparing the actual average compactness value of E.coli and B. subtilis with one thousand random arrangements of operons (shuffling of the genome) they show that the natural arrangement is much more compact than the one that could have been produced by chance, therefore proposing that the observed compactness is the result of selection.

What's next: In a more recent paper (which we will be discussing soon), this idea is being further elaborated to incorporate data related to the 3-dimensional structure of the genome. It remains to be seen when and how such approaches will be extended to eukaryotic genomes with multiple chromosomes (and without operons)

Read more: Yin et al. (2010) Genomic arrangement of bacterial operons is constrained by biological pathways encoded in the genome. PNAS

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