Effects of triptolide treatment on Ser5P Pol II, Nipped-B, TBPH and Lark occupancy at active promoters, extragenic enhancers and PREs in BG3 cells.

<p>The log2 ChIP-seq enrichment for Ser5P Pol II, Nipped-B, TBPH and Lark at all individual active promoters, enhancers and PREs in control untreated (Mock) cells is plotted against the enrichment after treatment of cells with 10 μM triptolide for 1, 2 and 4 hours.</p

TBPH and Lark interact with Nipped-B.

<p>(<b>A</b>) The western blots show the binding of Nipped-B and cohesin (Rad21) in BG3 cell nuclear extracts to NTA-Zn²⁺ agarose beads with immobilized His₆-SUMO-TBPH and -Lark fusion proteins probed with both Nipped-B and Rad21 antibodies. The western on the far right (Input) shows the Nipped-B and Rad21 proteins in the BG3 cell nuclear extracts used for the binding experiments. Nuclear extracts were prepared from control (Mock) cells and cells depleted for Rad21 and Nipped-B by RNAi. The results shown for mock nuclear extract are representative of five independent experiments. The results shown for nuclear extracts depleted for Rad21 and Nipped-B are representative of two technical replicates. <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s012" target="_blank">S12 Fig</a> shows that native TBPH and Lark co-immunoprecipitate with Nipped-B from nuclear extracts, that Lark and TBPH do not co-precipitate, and that pre-treatment of BG3 nuclear extract with ribonucleases does not prevent binding of Nipped-B to TBPH and Lark beads. (<b>B</b>) Western blot of Nipped-B binding to the indicated TBPH fragments and full-length TBPH to map the protein domains interacting with Nipped-B. The bottom panel is a longer exposure of the same blot to show low levels of Nipped-B binding to some fragments. The fragments are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s010" target="_blank">S10 Fig</a>. Most of the Nipped-B binding occurs with the RRM1 domain of TBPH. The blots shown are representative of three independent experiments. (<b>C</b>) Western blot of Nipped-B binding to the indicated fragments of Lark and full-length Lark. The fragments are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s010" target="_blank">S10 Fig</a>. The blot shown is representative of three independent experiments.</p

Drosophila TDP-43 RNA-Binding Protein Facilitates Association of Sister Chromatid Cohesion Proteins with Genes, Enhancers and Polycomb Response Elements

<div><p>The cohesin protein complex mediates sister chromatid cohesion and participates in transcriptional control of genes that regulate growth and development. Substantial reduction of cohesin activity alters transcription of many genes without disrupting chromosome segregation. Drosophila Nipped-B protein loads cohesin onto chromosomes, and together Nipped-B and cohesin occupy essentially all active transcriptional enhancers and a large fraction of active genes. It is unknown why some active genes bind high levels of cohesin and some do not. Here we show that the TBPH and Lark RNA-binding proteins influence association of Nipped-B and cohesin with genes and gene regulatory sequences. In vitro, TBPH and Lark proteins specifically bind RNAs produced by genes occupied by Nipped-B and cohesin. By genomic chromatin immunoprecipitation these RNA-binding proteins also bind to chromosomes at cohesin-binding genes, enhancers, and Polycomb response elements (PREs). RNAi depletion reveals that TBPH facilitates association of Nipped-B and cohesin with genes and regulatory sequences. Lark reduces binding of Nipped-B and cohesin at many promoters and aids their association with several large enhancers. Conversely, Nipped-B facilitates TBPH and Lark association with genes and regulatory sequences, and interacts with TBPH and Lark in affinity chromatography and immunoprecipitation experiments. Blocking transcription does not ablate binding of Nipped-B and the RNA-binding proteins to chromosomes, indicating transcription is not required to maintain binding once established. These findings demonstrate that RNA-binding proteins help govern association of sister chromatid cohesion proteins with genes and enhancers.</p></div

Hypothetical models for roles of TBPH and Lark in Nipped-B and cohesin binding to genes and enhancers, enhancer-promoter interactions, and processing of nascent RNA transcripts.

<p>At promoters (top row) we posit that TBPH binds to UG repeats in the first nascent transcripts produced by elongating Pol II (Ser2P / Ser5P) when a gene is initially activated. TBPH then recruits Nipped-B, which interacts with DNA, loads cohesin and recruits the Lark RNA-binding protein. At enhancers (middle row) activator proteins (purple oval) recruit Mediator (large tan circle) and Nipped-B. Nipped-B then loads cohesin and recruits TBPH and Lark, and TBPH stabilizes binding of the complex. The protein complexes at the enhancers and promoters form enhancer-promoter complexes (bottom row) that are stable for hours even in the absence of new transcription initiation. TBPH contributes to their stability, while Lark destabilizes cohesin and Nipped-B binding, particularly to the promoter. P-TEFb in the enhancer-promoter complex phosphorylates paused Pol II and the pausing factors (not depicted), leading to transcriptional elongation (lower right). Some TBPH and Lark present in the enhancer-promoter complex binds to the nascent RNA produced by the elongating polymerase and can facilitate RNA processing. For example, they can regulate intron removal by splicing as depicted in the lower right diagram, in addition to other processes.</p

TBPH and Lark depletion alter Nipped-B and cohesin occupancy at gene regulatory sequences in BG3 cells.

<p>The browser tracks show the log2 enrichment for TBPH, Lark, Nipped-B, SA, and Nipped-B and SA after RNAi depletion of TBPH (iTBPH) and Lark (iLark) for the <i>string</i> gene and its upstream enhancers. The <i>string</i> enhancers that are most resistant to decreases in Nipped-B and SA occupancy upon TBPH depletion are marked with asterisks. The tracks shown are the average of two to five independent biological replicates. The boxplots show the distributions of the average log2 enrichment for all active promoters (gray), extragenic enhancers (yellow) and PREs (orange) in mock-treated (Mock) cells, and cells in which TBPH (iTBPH) or Lark (iLark) have been depleted by RNAi for four to five days. Gene regulatory elements are defined as shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.g002" target="_blank">Fig 2</a>. Asterisks indicate distributions that differ significantly from the mock control using a paired t test. The p values are given in the main text. A paired t test was used because each regulatory sequence is matched with itself in the control and depleted cells and the distributions are close to normal. All comparisons indicated as significant are also significant using the non-parametric Wilcoxon signed rank test. Plots of the values for all individual regulatory sequences in mock control versus the depleted cells are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s006" target="_blank">S6 Fig</a>.</p

TBPH and Lark preferentially bind RNAs from cohesin-binding genes in vitro using known RNA-binding domains.

<p>Diagrams of the TBPH and Lark protein structures with known sequence domains shown at the top: RRM, RNA Recognition Motif; ZnF, Zinc Finger. The smallest fragments tested that bind RNA in vitro are underlined in red. The bar graphs below the protein diagrams show examples of RNA-binding competition experiments with TBPH (left) and Lark (right). Whether or not the RNA is from a cohesin-binding gene or contains UG repeats is indicated (Y = yes, N = no). The sequences of all short RNAs tested from cohesin-binding and non-binding genes are in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s013" target="_blank">S1 Table</a>, along with a summary of their abilities to bind TBPH and Lark derived from multiple experiments. As detailed in the text, soluble His₆-SUMO fusion proteins were immobilized on NTA-Zn²⁺ agarose beads and incubated with equimolar mixtures of the indicated short RNAs. RNAs that were retained after washing were quantified by real-time PCR and binding was defined by enrichment relative to the amount of the CG6310-3 control RNA, which does not bind to either protein. All RNA fragments were tested in two independent binding experiments with freshly made fusion proteins, and each RNA was measured twice in each experiment. Error bars are the standard deviations of all measurements in all experiments. Enrichment of 10-fold or greater is defined as specific binding when recorded in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s013" target="_blank">S1 Table</a>. The bottom bar graphs show example RNA-binding experiments with the indicated fragments of TBPH and Lark to map the domains that bind RNA. SDS-PAGE characterization of the immobilized protein fragments and the residues contained within each fragment are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006331#pgen.1006331.s010" target="_blank">S10 Fig</a>. For truncated proteins, RNA enrichment greater than or equal to half the enrichment obtained with the full-length protein in the same experiment was defined as binding. With TBPH, the RRM1-containing region is necessary and sufficient for RNA-binding. For Lark, the zinc finger (ZnF) is required in addition to the RRM-containing domain. It is unknown if one or both of the Lark RRM domains is required to bind RNA.</p

Inhibition of transcription initiation with triptolide does not ablate binding of Nipped-B, TBPH and Lark to gene regulatory sequences in BG3 cells.

<p>The boxplots show the average ChIP-seq enrichment of Ser5P Pol II, Nipped-B, TBPH and Lark at active promoters, extragenic enhancers, and PREs in control cells, and cells treated with 10 μM triptolide for 1, 2, and 4 hours. The genome browser tracks at the right show the log2 ChIP-seq enrichment for the same proteins at the <i>string</i> gene and enhancers over the same time course of triptolide treatment.</p

Nipped-B facilitates binding of TBPH and Lark to gene regulatory sequences in BG3 cells.

<p>The browser view at the upper left shows log2 ChIP-seq enrichment tracks for Nipped-B in control cells, and for TBPH and Lark in control cells and cells depleted for Nipped-B (iNipped-B) at the <i>string</i> gene and its enhancers. The boxplots at the upper right show the distributions of ChIP-seq enrichment for TBPH and Lark at active promoters (PRO) extragenic enhancers (ENH) and PREs (PRE) in control (Mock) cells and cells depleted for Nipped-B (iNipped-B). Four biological replicates were averaged for the TBPH ChIP-seq and two for Lark ChIP-seq in Nipped-B depleted cells. Distributions that differ significantly from the Mock control by the paired t test are marked with asterisks, and the p values are given in the main text. All marked comparisons are also significant by the Wilcoxon signed rank test. For each regulatory sequence, the log2 enrichment values in Mock control cells (Mock log2 enrichment) are plotted against the enrichment values in cells depleted for Nipped-B (iNipped-B log2 enrichment) in the dot plots.</p

Additional file 2: of The effect of dietary resistant starch type 2 on the microbiota and markers of gut inflammation in rural Malawi children

16S rRNA raw reads analyzed in this study

Additional file 1: Table S1. of The effect of dietary resistant starch type 2 on the microbiota and markers of gut inflammation in rural Malawi children

Number of 16S rRNA and wide genome sequences generated per sample. This table shows the number of raw (unprocessed) reads and the number of reads remaining after analytical processing of 18 samples from children fed with the habitual diet and plus the addition of RS. Table S2 Comparison of the number of reads obtained by V1–V3 and V3–V5 sequencing. Figure S1 Venn diagram showing the degree of overlap for genera captured with the V1–V3 and V3–V5 regions. While the majority of taxa are captured by both sets of primers, the fact that a significant subset were captured by only one primer set made the use of both sets more valuable. Figure S2 LEfse rank plot of differentially abundant genes in gut microbiomes initial samples vs. final samples. LDA scores were given for different abundance of genes before (habitual diet: green) and after the resistant starch was added to the habitual diet (habitual diet + RS: red). Figure S3 Principal component analysis (PCA) plot showing the differential clustering of metabolites in fecal samples collected before (blue-habitual diet) and after (red-habitual diet plus RS) the diet supplementation with RS. Brown circles represent replicate analyses of the pooled quality control (QC) fecal samples. The tight clustering of these QC replicate analyses indicates the high reproducibility and low amount of drift associated with the GC/MS-based fecal metabolomic profiling analyses conducted over several hours while the individual fecal extracts are analyzed at the same time, under identical GC/MS conditions, and on the same GC/MS instrument