Biology subjects

Fischl, H. J.

Publications and source records attributed to Fischl, H. J..

3 recordsLinked to original sources

bioRxiv2021

Global and Gene-specific Transcriptional Responses to Acute Stress

Nucleosomes may regulate transcription by controlling access to promoters by transcription factors and RNA polymerase II (Pol2). Potentially active genes display nucleosome depleted regions flanked by positioned -1 and +1 nucleosomes. On yeast genes, the transcription start site (TSS) is on the upstream face of the +1 nucleosome, but whether precise +1 nucleosome positioning controls Pol2 access to the TSS remains unclear. Here, using acute nutrient starvation to rapidly reprogramme the genome, we show highly dynamic upstream or downstream shifts in the position of +1 nucleosomes, coincident with levels of transcriptionally engaged Pol2 at 58% of genes. Transcript level changes broadly reflect Pol2 occupancy changes with a delay but can be further influenced by Pub1 or Puf3 dependent changes in transcript degradation rates. The response to acute stress has a second component as we also observed genome-wide changes in Pol2 distribution on genes, independent of changes in Pol2 occupancy, with Pol2 accumulating upstream of a +170 nt stalling site. Mathematical modelling supports a global increase in promoter-proximal early transcription termination as a major component of the global stress response. Thus, we uncover a two-component transcriptional response to stress, one focused on the +1 nucleosome, the second on Pol2 itself. O_LIA two-component responses to acute stress involving a gene-specific response and a global response C_LIO_LIDynamic shifting of +1 nucleosome position with transcriptional activation or repression. C_LIO_LIGlobal targeting of Pol2 leading to early transcription termination on acute stress C_LI

molecular biology↗

bioRxiv2021

Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

Genomes are pervasively transcribed, leading to stable and unstable transcripts that influence 3-dimensional genome organisation and gene regulation. High sensitivity and nucleotide resolution are required to resolve mammalian nascent transcriptomes. Here, we exploit the sensitivity of 4-thio-uridine (4sU) metabolic pulse-labelling to develop two nucleotide-resolution methods: Single-Nucleotide resolution 4sU sequencing (SNU-Seq) and size-fractionated 4sU-Seq (sf4sU-Seq). sf4sU-Seq involves gel isolation of abundant 4sU-labelled promoter proximal nascent transcripts, enabling nucleotide resolution mapping of transcription start sites and promoter proximal pauses (PPPs) on the same transcript. SNU-Seq exploits 3 end RNA-Seq, using bacterial poly(A) polymerase (bPAP) to polyadenylate the 3 ends of nascent transcripts and create oligo(dT)-primed libraries. The artificial poly(A) tail marks the precise position of polymerase on a transcription unit. SNU-Seq read levels are similar at pre-mRNAs and enhancers genome-wide and read spikes in pre-mRNA outputs map pauses, PPPs and polyadenylation sites. SNU-Seq enables discovery of thousands of unannotated regions of divergent transcription and helps define hundreds of the more than 10,000 regions of primed non-transcribed acetylated open chromatin that induce divergent nascent transcripts within 0.5h of IFN-{gamma} treatment in Hep3B cells. Thus, combining chromatin analysis with SNU-Seq reveals the transcriptional responsiveness of an epigenetically primed human genome. HIGHLIGHTSO_LISNU-Seq maps nascent transcripts with bp resolution, high sensitivity and low cost C_LIO_LIsf4sU-Seq resolves TSS and PPP at the same gene, complementing SNU-Seq C_LIO_LI1000s of divergently transcribed enhancers resolved by SNU-Seq C_LIO_LIRapid IFN{gamma} dependent transcriptional induction from primed Hep3B epigenome C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=165 SRC="FIGDIR/small/452379v2_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@da6239org.highwire.dtl.DTLVardef@16692c6org.highwire.dtl.DTLVardef@12e30f7org.highwire.dtl.DTLVardef@190c98f_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

Spt4 Facilitates the Movement of RNA Polymerase II through the +2 Nucleosomal Barrier

Spt4 is a transcription elongation factor, with homologues in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, yet the in vivo function of Spt4 is unclear. Here we assessed the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

genomics↗