Lineage-specific dynamic and pre-established enhancer–promoter contacts cooperate in terminal differentiation

Chromosome conformation is an important feature of metazoan gene regulation; however, enhancer–promoter contact remodeling during cellular differentiation remains poorly understood. To address this, genome-wide promoter capture Hi-C (CHi-C) was performed during epidermal differentiation. Two classes of enhancer–promoter contacts associated with differentiation-induced genes were identified. The first class ('gained') increased in contact strength during differentiation in concert with enhancer acquisition of the H3K27ac activation mark. The second class ('stable') were pre-established in undifferentiated cells, with enhancers constitutively marked by H3K27ac. The stable class was associated with the canonical conformation regulator cohesin, whereas the gained class was not, implying distinct mechanisms of contact formation and regulation. Analysis of stable enhancers identified a new, essential role for a constitutively expressed, lineage-restricted ETS-family transcription factor, EHF, in epidermal differentiation. Furthermore, neither class of contacts was observed in pluripotent cells, suggesting that lineage-specific chromatin structure is established in tissue progenitor cells and is further remodeled in terminal differentiation

Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE

icSHAPE (in vivo click selective 2-hydroxyl acylation and profiling experiment) captures RNA secondary structure at a transcriptome-wide level by measuring nucleotide flexibility at base resolution. Living cells are treated with the icSHAPE chemical NAI-N3 followed by selective chemical enrichment of NAI-N3-modified RNA, which provides an improved signal-to-noise ratio compared with similar methods leveraging deep sequencing. Purified RNA is then reverse-transcribed to produce cDNA, with SHAPE-modified bases leading to truncated cDNA. After deep sequencing of cDNA, computational analysis yields flexibility scores for every base across the starting RNA population. The entire experimental procedure can be completed in ∼5 d, and the sequencing and bioinformatics data analysis take an additional 4-5 d with no extensive computational skills required. Comparing in vivo and in vitro icSHAPE measurements can reveal in vivo RNA-binding protein imprints or facilitate the dissection of RNA post-transcriptional modifications. icSHAPE reactivities can additionally be used to constrain and improve RNA secondary structure prediction models

Sanger-based verification of two novel methylated positions in <i>S. solfataricus</i> rRNA identified using RNA-seq.

<p>Bisulfite-converted sequences were amplified using amplicon-specific primers and sequenced. (A) Position C1369 in the <i>S. solfataricus</i> 16S rRNA. (B) Position C2643 in the <i>S. solfataricus</i> 23S rRNA.</p

Modified m⁵C bases in mRNAs.

<p>Modified m⁵C bases in mRNAs.</p

Flow of the bisulfite-treatment/RNA-seq experiment.

<p>(A) Shown is a schematic representation of RNA molecules; colored boxes represent single bases. Red, blue, green and black represent C, U/T, A, and G residues, respectively. m⁵C modified cytosine bases are marked by a black circle. Total RNA is bisulfite-treated, leading to deamination of ‘C’ residues into ‘U’, except for methylated residues. Bisulfite-treated RNA is reverse transcribed into cDNA, which is then sequenced via an Illumina HiSeq system. This yields short sequence reads, representing random fragments of the sequenced RNAs. Resulting reads are mapped to the genome using an algorithm that allows mapping of ‘T’ residues in the sequenced cDNAs onto genomic ‘C's. Residues in which ‘C's are found to be consistently non-modified are declared as m⁵C (red arrow). (B) Flowchart of data analysis and artifact filtering (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003602#s4" target="_blank">Methods</a>).</p

Sequencing reads covering protein coding genes in the studied organisms.

a<p>Conversion rates were calculated out of the raw data prior to filtering.</p>b<p>Relates to mean coverage of all coding genes with mean coverage >5 in experiment #2.</p>c<p>Data relates to two sequenced lanes combined.</p