Neighborhood regulation by lncRNA promoters, transcription, and splicing

Mammalian genomes are pervasively transcribed to produce thousands of spliced long noncoding RNAs (lncRNAs), whose functions remain poorly understood. Because recent evidence has implicated several specific lncRNA loci in the local regulation of gene expression, we sought to determine whether such local regulation is a property of many lncRNA loci. We used genetic manipulations to dissect 12 genomic loci that produce lncRNAs and found that 5 of these loci influence the expression of a neighboring gene in cis. Surprisingly, however, none of these effects required the specific lncRNA transcripts themselves and instead involved general processes associated with their production, including enhancer-like activity of gene promoters, the process of transcription, and the splicing of the transcript. Interestingly, such effects are not limited to lncRNA loci: we found similar effects on local gene expression at 4 of 6 protein-coding loci. These results demonstrate that 'crosstalk' among neighboring genes is a prevalent phenomenon that can involve multiple mechanisms and cis regulatory signals, including a novel role for RNA splicing. These mechanisms may explain the function and evolution of some genomic loci that produce lncRNAs

Changes in suspected adverse drug reaction reporting via the Yellow Card scheme in Wales following the introduction of a National Reporting Indicator

AIMS: This study aimed to assess the impact of a National Reporting Indicator (NRI) on rates of reporting of suspected adverse drug reactions using the Yellow Card scheme following the introduction of the NRI in Wales (UK) in April 2014. METHODS: Yellow Card reporting data for general practitioners and other reporting groups in Wales and England for the financial years 2014–15 (study period 1) and 2015–16 (study period 2) were obtained from the Medicines and Healthcare Products Regulatory Agency and compared with those for 2013–14 (pre‐NRI control period). RESULTS: The numbers of Yellow Cards submitted by general practitioners in Wales were 271, 665 and 870 in the control period, study period 1 and study period 2, respectively. This is equivalent to an increase of 145% in study period 1 and 221% in study period 2 compared with the 12‐month control period (2013–14). Corresponding increases in England were 17% and 37%, respectively (P < .001 chi–squared test). The numbers of Yellow Cards submitted by other groups in Wales were 906, 795 and 947 in each of the study periods. CONCLUSIONS: Introduction of the NRI corresponded with a significant increase in the number of Yellow Cards submitted by general practitioners in Wales. General practitioner reporting rates continued to increase year on year through to 2018–19 with the NRI still in place. No concomitant change was found in reporting rates by other groups in the health boards in Wales

Patterns of chromatin accessibility along the anterior-posterior axis in the early Drosophila embryo.

As the Drosophila embryo transitions from the use of maternal RNAs to zygotic transcription, domains of open chromatin, with relatively low nucleosome density and specific histone marks, are established at promoters and enhancers involved in patterned embryonic transcription. However it remains unclear how regions of activity are established during early embryogenesis, and if they are the product of spatially restricted or ubiquitous processes. To shed light on this question, we probed chromatin accessibility across the anterior-posterior axis (A-P) of early Drosophila melanogaster embryos by applying a transposon based assay for chromatin accessibility (ATAC-seq) to anterior and posterior halves of hand-dissected, cellular blastoderm embryos. We find that genome-wide chromatin accessibility is highly similar between the two halves, with regions that manifest significant accessibility in one half of the embryo almost always accessible in the other half, even for promoters that are active in exclusively one half of the embryo. These data support previous studies that show that chromatin accessibility is not a direct result of activity, and point to a role for ubiquitous factors or processes in establishing chromatin accessibility at promoters in the early embryo. However, in concordance with similar works, we find that at enhancers active exclusively in one half of the embryo, we observe a significant skew towards greater accessibility in the region of their activity, highlighting the role of patterning factors such as Bicoid in this process

Chromatin accessibility differences and similarities at A-P and D-V patterning loci.

<p>Normalized ATAC-seq signal of anterior (orange), posterior (blue), whole embryo (gray) is depicted at <i>even-skipped</i> (A), <i>giant</i> (B), and <i>hunchback</i> (C) loci which contain A-P patterning enhancers and promoters and at <i>decapentaplegic</i> (D), a D-V patterning gene. Chromatin accessibility signal derived from DNaseI data for stage 5 <i>Drosophila</i> embryos is depicted in green [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref035" target="_blank">35</a>]. Colored bars represent peaks called in anterior (orange), posterior (blue), whole (gray), and in DNaseI data (green). Asterisks denote annotated features that show significant changes in accessibility between the anterior and posterior halves. Light gray bars denote the gene annotation while the black bars denote annotated enhancers. Colored annotation bars represent enhancers analyzed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.g003" target="_blank">Fig 3</a>. Dashed lines in the enhancer bars signify overlapping enhancers.</p

A-P patterning gene promoter accessibility does not correlate with activity.

<p>Scatter plots showing normalized ATAC-seq signal in anterior (x axis) and posterior (y axis) halves at (A) anterior (orange) and posterior (blue) and (B) dorsal (purple) and ventral (green) patterning promoters active in Stage 5 embryos and at 1kb adjacent windows tiling the genome (A and B, gray). (C) Box plots showing the difference in mean and variation between overall positional skew scores (methods) for anterior (orange), posterior (blue), dorsal (purple), and ventral (green) promoters of zygotically expressed genes only. Zygotic genes were classified as such from previously published transcriptome data [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref011" target="_blank">11</a>]. Positional skew scores at random genomic regions (excluding genes and enhancers) with the same number and distribution of total ATAC-seq signal as the patterning enhancer and promoter set are in gray (methods). NS (not significant) refers to pairwise t.tests which confirm that the mean positional skew score of anterior and posterior patterning promoters is not significantly different than dorsal and ventral patterning promoters or random regions. (D) Bar graph shows positional skew scores calculated for all anterior (orange) and posterior (blue) patterning promoters in the dataset (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.s013" target="_blank">S1 File</a>). Asterisks denote promoters whose accessibility skew scores show statistical significance over random regions (p < 0.05). (E-J) Normalized ATAC-seq signal across 1kb windows centered around <i>CG13894</i> (E), <i>men</i> (F), <i>mcm2</i> (G), <i>dib</i> (H), <i>atg1</i> (I), and <i>bbg</i> (J) with anterior signal in orange and posterior signal in blue. Gray denotes the location and size of the promoter. Arrow denotes the direction of the gene. Published <i>in situ</i> hybridization images depicting gene expression patterns driven by each promoter are below each graph [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref046" target="_blank">46</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref048" target="_blank">48</a>].</p

ATAC-seq on dissected, frozen, embryo halves.

<p>(A) Stage 5, hand sorted <i>Drosophila</i> embryos were flash frozen over dry ice in a buffer containing 5% glycerol and manually sliced in half with a scalpel. Twenty anterior and posterior halves were collected, homogenized, and the nuclei were isolated. ATAC-seq was then performed as described in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref034" target="_blank">34</a>] with three times Tn5 transposase. (B) Scatter plot of normalized ATAC-seq signal over 1kb adjacent windows that tile the <i>Drosophila</i> genome in posterior (x) and anterior (y) samples shows high degree of correlation between the anterior and posterior halves. The Spearman correlation coefficient (denoted by <i>r</i>_S) is 0.81. The Pearson correlation coefficient (denoted by <i>r</i>_p) is 0.94. X and Y are log transformed. Light blue circles denote point density.</p

A-P patterning enhancers tend to be more accessible where they are active.

<p>Scatter plots showing normalized ATAC-seq signal in anterior (x axis) and posterior (y axis) halves at (A) anterior (orange) and posterior (blue) and (B) dorsal (purple) and ventral (green) patterning enhancers active in Stage 5 embryos and at 1kb adjacent windows tiling the genome (A and B, gray). (C) Box plots showing the difference in mean and variation between average positional skew scores (methods) for anterior (orange), posterior (blue), dorsal (purple), and ventral (green) enhancers. Positional skew scores at random genomic regions (excluding genes and enhancers) that are selected so that their total ATAC-seq signal is of the same magnitude and distribution as the patterning enhancer and promoter set depicted here and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.g004" target="_blank">Fig 4</a> (methods). Pairwise t.tests confirm that the means of anterior patterning enhancers are significantly different than dorsal and ventral patterning enhancers and selected random regions. The mean positional skew score of posterior enhancers is not significantly different than dorsal, ventral, or random regions. (D) Bar graph shows the positional skew scores (methods) calculated for all anterior (orange) and posterior (blue) patterning enhancers in the dataset (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.s013" target="_blank">S1 File</a>). Asterisks denote enhancers whose accessibility skew scores show statistical significance over random regions (p < 0.05). (E-J) Normalized ATAC-seq signal across 1kb windows centered around <i>eve</i> stripe 1 (E), the <i>hunchback</i> anterior activator (F), Kvon region VT47407 (G), <i>hairy</i> stripe 5 (H), <i>giant</i> -3 construct (I), and Kvon region VT42837 (J) with anterior signal in orange and posterior signal in blue. Gray rectangles denote the location and size of the enhancer. Published <i>in situ</i> hybridization images depicting gene expression patterns driven by each enhancer are below each graph [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref039" target="_blank">39</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref086" target="_blank">86</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref088" target="_blank">88</a>].</p

Single nuclei ATAC-seq from cellular blastoderm embryos largely agrees with data from embryo halves.

<p>Recently published single nuclei ATAC-seq data [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref050" target="_blank">50</a>] corresponding to the cellular blastoderm were separated into anterior and posterior groups following designations determined by [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref050" target="_blank">50</a>]. (A) Genome browser trace at the eve locus. Whole embryos, anterior halves, and posterior halves are in grey, orange, and blue respectively. Merged anterior single nuclei and merged posterior single nuclei from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref050" target="_blank">50</a>] are shown in peach and grey blue or cesious. Finally, single anterior and single posterior halves from our study are shown in rusty red and light blue. DnaseI hypersensitivity data is in green. Peaks are depicted in bars below the pooled halves and whole data. Light gray bars denote the gene annotation while the black bars denote annotated enhancers. Colored annotation bars represent enhancers analyzed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.g003" target="_blank">Fig 3</a>. (B) Positional skew scores calculated for single nuclei and halves ATAC-seq data at all A-P and D-V patterning regions (enhancers and promoters) are plotted with halves on X and single nuclei on Y. Dotted line indicates X = Y. (C) Bar graph shows the positional skew scores calculated from single nuclei ATAC-seq data at all anterior (orange) and posterior (blue) patterning enhancers in the dataset. Asterisks denote enhancers whose accessibility skew scores show statistical significance over random regions (p < 0.05). (D) Bar graph shows the positional skew scores calculated from single nuclei ATAC-seq data at all anterior (orange) and posterior (blue) patterning promoters in the dataset. Asterisks denote promoters whose accessibility skew scores show statistical significance over random regions (p < 0.05).</p

A-P patterning transcription factor binding at similarly and differentially accessible A-P patterning enhancers.

<p>A-P patterning enhancers from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.g003" target="_blank">Fig 3</a> are ordered by positional skew score. Positional skew score is indicated by the colored bar above each panel–orange indicates more accessible in the anterior, blue indicates more accessible in the posterior, and white is similarly accessible in both halves. Each panel consists of normalized wig signal in a 3kb window around each enhancer (the actual enhancer region is denoted by a gray rectangle). The first panel shows normalized, merged, ATAC-seq signal in the anterior (orange) and posterior (blue) halves. The second panel shows DNaseI signal from stage 5 embryos [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007367#pgen.1007367.ref035" target="_blank">35</a>] in green. The third through eleventh panels are normalized wig signal from ChIP-seq experiments of the following proteins: Bicoid (red), Hunchback (orange), Kruppel (yellow), Giant (green), Zelda from stage 3,4,and 5 embryos (dark, medium, and light blue), Caudal (purple), and Knirps (brown). The name of the enhancer is above each panel.</p