The positive transcriptional elongation factor (P-TEFb) is required for neural crest specification

Regulation of gene expression at the level of transcriptional elongation has been shown to be important in stem cells and tumour cells, but its role in the whole animal is only now being fully explored. Neural crest cells (NCCs) are a multipotent population of cells that migrate during early development from the dorsal neural tube throughout the embryo where they differentiate into a variety of cell types including pigment cells, cranio-facial skeleton and sensory neurons. Specification of NCCs is both spatially and temporally regulated during embryonic development. Here we show that components of the transcriptional elongation regulatory machinery, CDK9 and CYCLINT1 of the P-TEFb complex, are required to regulate neural crest specification. In particular, we show that expression of the proto-oncogene c-Myc and c-Myc responsive genes are affected. Our data suggest that P-TEFb is crucial to drive expression of c-Myc, which acts as a ‘gate-keeper’ for the correct temporal and spatial development of the neural crest

Genome evolution in the allotetraploid frog Xenopus laevis

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of ???fossil??? transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.ope

Decoding the chromatin proteome of a single genomic locus by DNA sequencing

<div><p>Transcription, replication, and repair involve interactions of specific genomic loci with many different proteins. How these interactions are orchestrated at any given location and under changing cellular conditions is largely unknown because systematically measuring protein–DNA interactions at a specific locus in the genome is challenging. To address this problem, we developed Epi-Decoder, a Tag-chromatin immunoprecipitation-Barcode-Sequencing (TAG-ChIP-Barcode-Seq) technology in budding yeast. Epi-Decoder is orthogonal to proteomics approaches because it does not rely on mass spectrometry (MS) but instead takes advantage of DNA sequencing. Analysis of the proteome of a transcribed locus proximal to an origin of replication revealed more than 400 interacting proteins. Moreover, replication stress induced changes in local chromatin proteome composition prior to local origin firing, affecting replication proteins as well as transcription proteins. Finally, we show that native genomic loci can be decoded by efficient construction of barcode libraries assisted by clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9). Thus, Epi-Decoder is an effective strategy to identify and quantify in an unbiased and systematic manner the proteome of an individual genomic locus by DNA sequencing.</p></div

Capturing the chromatin interactome with Epi-Decoder.

<p>(A) Scatter plot of ChIP/input values, which can be interpreted as binding scores, of BC_UP versus BC_DN. For some factors, only the BC_UP or BC_DN value was available. In these cases, the missing value was set to −1 in order to still visualize the remaining value. Factors are coloured based on known functions or complexes. Triangles represent histone proteins. The average values of 6 replicates are shown. (B) Enrichment plot for 3 categories: initiation (TFIID-E and GRFs), termination (Cleavage and Polyadenylation and THO complex), and replication (ORC and MCM) factors. Binders were ranked based on the BC_UP/BC_DN ratio. The top part shows the running sum enrichment for each category. Initiation factors were significantly enriched at BC_UP (<i>p</i> = 3.18⁻³) and termination and replication at BC_DN (<i>p</i> = 2.03⁻⁷ and <i>p</i> = 1.06⁻⁵). The bottom lines indicate the factors represented in the categories. (C) Illustration of the different protein complexes that Epi-Decoder identified at the barcoded <i>KanMX</i> gene at the <i>HO</i> locus. BC_DN, downstream barcode; BC_UP, upstream barcode; ChIP, chromatin immunoprecipitation; GRF, general regulatory factor; IP, immunoprecipitation; MCM, minichromosome maintenance; ORC, origin recognition complex.</p

Application of Epi-Decoder to a native genomic locus.

<p>(A) Procedure to generate a custom Epi-Decoder collection. A selection marker (<i>NatMX</i>) was inserted in proximity to the locus of interest, downstream of the to-be-barcoded <i>ADE2</i> gene. Subsequently, a Cas9- and gRNA-expressing vector was introduced in this strain together with a repair template that is composed of a 15 bp random barcode-sequence (BC) flanked by 40 bp arms with homology to the gRNA target site, here at the 5’ end of the <i>ADE2</i> coding sequence (details see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005542#pbio.2005542.s005" target="_blank">S5A Fig</a>). Transformants, the vast majority of which represent barcoded clones, were transferred to an arrayed format. Rows and columns were pooled for identifying barcodes and their location on the array by high-throughput sequencing. The barcoder library was mated and crossed with the TAP-tagged protein library (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005542#pbio.2005542.g001" target="_blank">Fig 1</a>) to generate heterozygous diploids and then haploids, after which Epi-Decoder screens could be performed. For diploid screens, the insertion of a selectable marker genetically linked to the barcode can be omitted. (B) Epi-Decoder results of BC_5’-ADE2 (<i>n</i> = 2, average) in diploid cells were compared to BC_UP in haploid cells (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005542#pbio.2005542.g002" target="_blank">Fig 2</a>). Binding scores were visualized in a scatterplot with histones shown as triangles and classes of proteins coloured: TFIIH, RNAPII, DSIF. BC, barcode; BC_5’-ADE2, barcode in 5’ region of <i>ADE2</i>; BC_UP, upstream barcode at HO locus; Cas9, CRISPR-associated protein 9; gRNA, guide RNA; IP, immunoprecipitation; TAP, Tandem Affinity Purification.</p

Chromatin rewiring upon HU treatment.

<p>(A) DNA regions replicated in the HU arrest were determined by DNA copy number assessment. The DNA coverage in bins of 200 bp is plotted across the genome for untreated and HU-treated samples. The lower panel shows a zoom-in of chromosome IV, which contains the barcoded locus on the left arm (indicated by the arrow). Replication timing in the absence of HU was obtained from Alvino and colleagues [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005542#pbio.2005542.ref036" target="_blank">36</a>]. The percentage of replicated DNA is plotted for each bin at 10, 12.5, 15, 17.5, 25, and 40 minutes after release from a G1 arrest, as indicated by the shades of grey. (B) RT-qPCR shows relative mRNA expression of <i>KanMX/HphMX</i> in untreated and HU-treated samples. The untreated samples were set to 1. The average of 3 biological replicates is shown; error bars indicate SD. (C) Volcano plots showing the ratio of binding in HU-treated/untreated for factors that were significantly enriched in either one or both of the conditions. The coloured dots are factors with significantly different binding scores (FDR < 0.05). BC_DN, downstream barcode; BC_UP, upstream barcode; FDR, false discovery rate; HU, hydroxyurea; ORC, origin recognition complex; RT-qPCR, quantitative reverse transcription PCR.</p

Identifying chromatin binders with DNA barcode counting.

<p>(A) Volcano plots showing the average log2 ChIP/input versus significance (<i>p</i>-value) calculated for 3,994 factors for BC_UP and 3,995 factors for BC_DN across 6 independent biological experiments. The red colour indicates factors with a Benjamini-Hochberg–adjusted <i>p</i>-value < 0.01. Green triangles represent the barcodes associated with the TAP-tagged histone proteins in the library. (B) Venn diagram indicating the number of factors that are significant (FC > 0; FDR < 0.01) at BC_UP, BC_DN, or both. (C) Bar plot showing the fraction of factors in 4 different categories that were determined by using GO annotations (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005542#sec009" target="_blank">Methods</a>). The first 2 bars show the fractions in the group of binders (at either BC_UP or BC_DN). The right bar shows the fraction in the background set (the entire library). (D) Density plot of GFP abundance using data from the Cyclops database (<a href="http://cyclops.ccbr.utoronto.ca/" target="_blank">http://cyclops.ccbr.utoronto.ca/</a>). The binders were defined as before (FC > 0; FDR < 0.01), and the categories are similar to those in panel C. The ribosome category was defined based on the CYC2008 database (<a href="http://wodaklab.org/cyc2008/" target="_blank">http://wodaklab.org/cyc2008/</a>). Histones are shown separately; they bind at the DNA and are highly abundant. The numbers indicate the size of each group. (E) ChIP-qPCR of selected TAP-tagged strains from the barcoded KanMX Epi-Decoder library, with specific primers in proximity to BC_UP and BC_DN. To compare the barcode counts with ChIP-qPCR signal, the samples were normalized to the Bar1-TAP signal (Bar1 is not expressed in these cells) before calculating ChIP/input. The average of 3 biological replicates is shown; the error bars indicate the SD. Rpl31A—a highly expressed ribosomal subunit frequently used in anchor away studies to deplete proteins from the nucleus—was used as a negative control and as a reference to determine binding specificity in the qPCR analysis. Rpo21 is the largest subunit of RNA polymerase II, Smc3 is a cohesin subunit, Ssa1 and Ssa2 are HSP70 chaperones, Top2 is topoisomerase II, and Cdc19/Pyk1 is pyruvate kinase. The <i>p</i>-values from a two-sided <i>t</i> test compared to Rpl13A are indicated by asterisks. Underlying data for Fig 2C and Fig 2E in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.2005542#pbio.2005542.s013" target="_blank">S1 Data</a>. BC_DN, downstream barcode; BC_UP, upstream barcode; ChIP, chromatin immunoprecipitation; FC, fold change; FDR, false discovery rate; GFP, green fluorescent protein; GO, gene ontology; IP, immunoprecipitation; qPCR, quantitative PCR; TAP, Tandem Affinity Purification.</p