Biology subjects

Furger, A.

Publications and source records attributed to Furger, A..

3 recordsLinked to original sources

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

CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation.

AbstractCDK9 is a kinase critical for the productive transcription of protein-coding genes by RNA polymerase II (pol II). As part of P-TEFb, CDK9 phosphorylates the carboxyl-terminal domain (CTD) of pol II and elongation factors, which allows pol II to elongate past the early elongation checkpoint (EEC) encountered soon after initiation. We show that, in addition to halting pol II at the EEC, loss of CDK9 activity causes premature termination of transcription across the last exon, loss of polyadenylation factors from chromatin, and loss of polyadenylation of nascent transcripts. Inhibition of the phosphatase PP2A abrogates the premature termination and loss of polyadenylation caused by CDK9 inhibition, indicating that this kinase/phosphatase pair regulates transcription elongation and RNA processing at the end of protein-coding genes. We also confirm the splicing factor SF3B1 as a target of CDK9 and show that SF3B1 in complex with polyadenylation factors is lost from chromatin after CDK9 inhibition. These results emphasize the important roles that CDK9 plays in coupling transcription elongation and termination to RNA maturation downstream of the EEC.

molecular biology↗

bioRxiv2020

Cold induced chromatin compaction and nuclear retention of clock mRNAs resets the circadian rhythm

Cooling patients to sub-physiological temperatures is an integral part of modern medicine. We show that cold exposure induces temperature-specific changes to the higher-order chromatin and gene expression profiles of human cells. These changes are particularly dramatic at 18{degrees}C, a temperature synonymous with that experienced by patients undergoing controlled deep-hypothermia during surgery. Cells exposed to 18{degrees}C exhibit largely nuclear-restricted transcriptome changes. These include the nuclear accumulation of core circadian clock suppressor gene transcripts, most notably REV-ERB. This response is accompanied by compaction of higher-order chromatin and hindrance of mRNPs from engaging nuclear pores. Rewarming reverses chromatin compaction and releases the transcripts into the cytoplasm, triggering a pulse of suppressor gene proteins that resets the circadian clock. We show that cold-induced upregulation of REV-ERB alone is sufficient to trigger this resetting. Our findings uncover principles of the cellular cold-response that must be considered for current and future applications involving therapeutic deep-hypothermia.

molecular biology↗