| 2012 |
Gdown1 specifically blocks elongation stimulation by TFIIF in vitro, inhibits the termination activity of TTF2, and stabilizes promoter-proximally paused Pol II in the presence of nuclear extract. P-TEFb can overcome the Gdown1-mediated block, and ChIP-Seq confirms Gdown1 co-localizes with essentially all poised Pol II across the human genome. |
In vitro transcription assays, ChIP-Seq |
Molecular cell |
High |
22244331
|
| 2012 |
Gdown1 competes with TFIIF for binding to the RPB1 and RPB5 subunits of Pol II, inhibiting TFIIF function in preinitiation complex assembly. Mediator can facilitate Pol II(G) binding to promoters and overcome the Gdown1-mediated block, establishing Pol II(G) as a Mediator-dependent form of Pol II. |
In vitro transcription with purified factors, ChIP, RNAi |
Molecular cell |
High |
22244332
|
| 2012 |
Cryo-EM maps of RNAPII(G) at ~19 Å localized Gdown1 primarily to the RPB5 shelf–RPB1 jaw region of Pol II, with Gdown1 binding sites overlapping extensively with TFIIF binding sites. Competition assays by size exclusion chromatography confirmed that Gdown1 sterically excludes TFIIF from RNAPII. |
Single-particle cryo-EM, antibody labeling, size exclusion chromatography competition assays |
The EMBO journal |
High |
22850672
|
| 2014 |
Two functional domains of Gdown1 were identified: the N-terminal domain is responsible for blocking TTF2 (via a conserved LPDKG motif) and the C-terminal domain blocks TFIIF. Both domains are required for tight Pol II association. Gdown1 is phosphorylated at Ser-270 during mitosis, which reduces its affinity for Pol II and consequently reduces its ability to block both TTF2 and TFIIF. An S270A mutation abolishes this phosphorylation and S270E partially mimics phospho-Gdown1. |
In vitro transcription assays, deletion mutagenesis, partial purification of a mitotic kinase from HeLa nuclear extract, mass spectrometry, site-directed mutagenesis (S270A, S270E) |
The Journal of biological chemistry |
High |
24634214
|
| 2014 |
Gdown1 does not functionally associate with Pol II preinitiation complexes (PICs) that contain TFIIF, despite completely displacing TFIIF from free Pol II and elongating Pol II. Gdown1 can associate with Pol II during early transcript elongation in competition with TFIIF; phosphorylation of TFIIF provides a pathway for efficient Gdown1 loading early in elongation. |
In vitro transcription with purified factors, phosphorylation of TFIIF, functional association assays |
The Journal of biological chemistry |
High |
24596085
|
| 2016 |
Gdown1 cannot functionally associate with Pol II in open complexes or during abortive synthesis of very short RNAs. Efficient Gdown1 association occurs only after the upstream portion of the transcription bubble reanneals (early elongation complexes with 5–9 nt RNAs), and Gdown1 displaces TFIIF slowly (~5 min), suggesting Gdown1 locks in the paused state after pausing has already been initiated by other factors (DSIF, NELF). |
In vitro transcription assays with promoter variants, timed competition assays with purified Gdown1 and TFIIF |
PloS one |
Medium |
27716820
|
| 2018 |
Although Gdown1 is intrinsically disordered, its Pol II-interacting domains were mapped and shown to occlude both TFIIF and TFIIB binding sites on Pol II. Robust Pol II binding requires cooperative interactions of a strong binding region and two weaker modulatory regions. In Drosophila embryos, Gdown1 co-localizes with Pol II in transcriptionally silent nuclei but re-localizes to the cytoplasm upon zygotic genome activation, demonstrating a physiological role in transcription repression. |
Integrative structural modeling (cross-linking/MS, SAXS, negative-stain EM), domain mutagenesis, immunofluorescence in Drosophila embryos |
Nature structural & molecular biology |
High |
30190596
|
| 2020 |
Hepatocyte-specific ablation of Gdown1 in mice leads to down-regulation of highly expressed metabolic genes (plasma protein synthesis), reduced Pol II recruitment to these genes, and concomitant cell cycle re-entry with induction of cyclin D1 and p21 via p53 signaling. In the absence of p53, Gdown1-deficient hepatocytes display dysregulated mitosis and premalignant transformation. Gdown1 is associated with elongating (not initiating) Pol II on these highly expressed genes in vivo. |
Hepatocyte-specific conditional knockout mouse, ChIP, gene expression profiling, p53 pathway analysis |
Genes & development |
High |
32381628
|
| 2022 |
Gdown1 localizes predominantly in the cytoplasm of interphase somatic cells, regulated by CRM1-dependent nuclear export signals (NES) and a novel cytoplasmic anchoring signal (CAS) that retains it outside the nuclear pore complex. Mutagenesis of CAS increases nuclear Gdown1 accumulation, causing a drastic reduction in Pol II levels and global transcription. Gdown1 translocates to the nucleus in response to oxidative stress, and Gdown1 ablation weakens cellular stress tolerance. |
Mutagenesis of NES and CAS, live-cell microscopy/immunofluorescence, CRM1 inhibition, global transcription assays, oxidative stress experiments in human cell lines |
eLife |
High |
36476745
|
| 2022 |
Gdown1 resides predominantly in the cytoplasm of interphase cells and enters the nucleus at mitotic onset. Acute depletion of Gdown1 in human cell lines has minimal direct effects on interphase transcription but is associated with partial de-repression of mitotic transcription and aberrant mitoses coupled to p53 pathway activation. In vitro, Gdown1 modulates the combined functions of elongation factors PAF1C, RTF1, SPT6, DSIF, and P-TEFb. |
Genetic depletion (acute and KO), microscopy, multi-omics (ChIP-seq, RNA-seq), in vitro reconstitution with purified elongation factors |
Nucleic acids research |
High |
35048979
|
| 2021 |
A genome-wide CRISPR-Cas9 knockout screen identified POLR2M as a regulatory factor for MIR139 silencing in MLL-AF9 AML, operating downstream of MLL-AF9 and PRC2. POLR2M knockout de-repressed MIR139 expression, placing POLR2M in the epigenetic silencing pathway for this tumor suppressor locus. |
Genome-wide CRISPR-Cas9 knockout screen, genetic validation in human AML cell lines |
Leukemia |
Medium |
34741119
|