The control of promoter-proximal pausing and the release of RNA polymerase II (Pol II) is a widely used mechanism for regulating gene expression in metazoans, especially for genes that respond to ...environmental and developmental cues. Here, we identify that Pol-II-associated factor 1 (PAF1) possesses an evolutionarily conserved function in metazoans in the regulation of promoter-proximal pausing. Reduction in PAF1 levels leads to an increased release of paused Pol II into gene bodies at thousands of genes. PAF1 depletion results in increased nascent and mature transcripts and increased levels of phosphorylation of Pol II’s C-terminal domain on serine 2 (Ser2P). These changes can be explained by the recruitment of the Ser2P kinase super elongation complex (SEC) effecting increased release of paused Pol II into productive elongation, thus establishing PAF1 as a regulator of promoter-proximal pausing by Pol II.
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•PAF1 loss results in release of Pol II into gene bodies at thousands of genes•Genes exhibiting high degrees of pausing are more affected by loss of PAF1•Redistribution of Pol II is associated with increased transcription•PAF1 depletion leads to increased recruitment of the super elongation complex
PAF1 plays the role of gatekeeper for Pol II promoter-proximal pausing, with its loss enhancing the transcription of thousands of genes.
Diffuse intrinsic pontine glioma (DIPG) is a highly aggressive pediatric brainstem tumor characterized by rapid and uniform patient demise. A heterozygous point mutation of histone H3 occurs in more ...than 80% of these tumors and results in a lysine-to-methionine substitution (H3K27M). Expression of this histone mutant is accompanied by a reduction in the levels of polycomb repressive complex 2 (PRC2)-mediated H3K27 trimethylation (H3K27me3), and this is hypothesized to be a driving event of DIPG oncogenesis. Despite a major loss of H3K27me3, PRC2 activity is still detected in DIPG cells positive for H3K27M. To investigate the functional roles of H3K27M and PRC2 in DIPG pathogenesis, we profiled the epigenome of H3K27M-mutant DIPG cells and found that H3K27M associates with increased H3K27 acetylation (H3K27ac). In accordance with previous biochemical data, the majority of the heterotypic H3K27M-K27ac nucleosomes colocalize with bromodomain proteins at the loci of actively transcribed genes, whereas PRC2 is excluded from these regions; this suggests that H3K27M does not sequester PRC2 on chromatin. Residual PRC2 activity is required to maintain DIPG proliferative potential, by repressing neuronal differentiation and function. Finally, to examine the therapeutic potential of blocking the recruitment of bromodomain proteins by heterotypic H3K27M-K27ac nucleosomes in DIPG cells, we performed treatments in vivo with BET bromodomain inhibitors and demonstrate that they efficiently inhibit tumor progression, thus identifying this class of compounds as potential therapeutics in DIPG.
Although it is established that some general transcription factors are inactivated at mitosis, many details of mitotic transcription inhibition (MTI) and its underlying mechanisms are largely ...unknown. We have identified mitotic transcriptional activation (MTA) as a key regulatory step to control transcription in mitosis for genes with transcriptionally engaged RNA polymerase II (Pol II) to activate and transcribe until the end of the gene to clear Pol II from mitotic chromatin, followed by global impairment of transcription reinitiation through MTI. Global nascent RNA sequencing and RNA fluorescence in situ hybridization demonstrate the existence of transcriptionally engaged Pol II in early mitosis. Both genetic and chemical inhibition of P-TEFb in mitosis lead to delays in the progression of cell division. Together, our study reveals a mechanism for MTA and MTI whereby transcriptionally engaged Pol II can progress into productive elongation and finish transcription to allow proper cellular division.
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•Mitotic transcription inhibition occurs in early mitosis•P-TEFb is required for mitotic transcriptional activation and release of paused Pol II•Nascent RNA-seq and RNA FISH reveal active transcription at the onset of mitosis•Inhibition of mitotic transcriptional activation delays cell-cycle progression
How transcription is shut down as cells begin to condense chromosomes during mitosis is poorly understood. Liang et al. report the requirement of mitotic transcriptional activation by P-TEFb to release paused Pol II as a prerequisite for this process, and ultimately for proper cell-cycle progression.
The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity ...with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.
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•Discovery of small-molecule inhibitors of SEC and transcription elongation by Pol II•KL-1 and KL-2 disrupt the cyclin T1-AFF4 interaction within SEC•SEC inhibitors attenuate SEC-dependent rapid transcriptional responses•MYC transcriptional programs are inhibited by SEC chemical disruptors KL-1/KL-2
Targeting transcriptional elongation with small-molecule inhibitors of the super elongation complex blocks transcriptional programs driven by the oncogene MYC
Polycomb response elements (PREs) are specific DNA sequences that stably maintain the developmental pattern of gene expression. Drosophila PREs are well characterized, whereas the existence of PREs ...in mammals remains debated. Accumulating evidence supports a model in which CpG islands recruit Polycomb group (PcG) complexes; however, which subset of CGIs is selected to serve as PREs is unclear. Trithorax (Trx) positively regulates gene expression in Drosophila and co-occupies PREs to antagonize Polycomb-dependent silencing. Here we demonstrate that Trx-dependent H3K4 dimethylation (H3K4me2) marks Drosophila PREs and maintains the developmental expression pattern of nearby genes. Similarly, the mammalian Trx homolog, MLL1, deposits H3K4me2 at CpG-dense regions that could serve as PREs. In the absence of MLL1 and H3K4me2, H3K27me3 levels, a mark of Polycomb repressive complex 2 (PRC2), increase at these loci. By inhibiting PRC2-dependent H3K27me3 in the absence of MLL1, we can rescue expression of these loci, demonstrating a functional balance between MLL1 and PRC2 activities at these sites. Thus, our study provides rules for identifying cell-type-specific functional mammalian PREs within the human genome.
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•Trx-dependent H3K4me2 marks the majority of Drosophila Polycomb response elements•This H3K4-dimethylase function is conserved in MLL1, the mammalian Trx homolog•MLL1-dependent H3K4me2 occurs predominantly at CpG islands•In HCT116 cells, ∼300 active genes require MLL1 to block PRC2-dependent repression
The extent to which mammalian CpG islands functionally resemble Drosophila Polycomb response elements (PREs) remains unclear. Rickels et al. describe a conserved role for Trx/MLL1 as an H3K4-dimethylase at PREs and CpG islands, respectively. HCT116 cells express ∼300 genes that require MLL1, not for activation but to block PRC2-dependent repression.
Chromosomal translocations of the mixed-lineage leukemia (MLL) gene with various partner genes result in aggressive leukemia with dismal outcomes. Despite similar expression at the mRNA level from ...the wild-type and chimeric MLL alleles, the chimeric protein is more stable. We report that UBE2O functions in regulating the stability of wild-type MLL in response to interleukin-1 signaling. Targeting wild-type MLL degradation impedes MLL leukemia cell proliferation, and it downregulates a specific group of target genes of the MLL chimeras and their oncogenic cofactor, the super elongation complex. Pharmacologically inhibiting this pathway substantially delays progression, and it improves survival of murine leukemia through stabilizing wild-type MLL protein, which displaces the MLL chimera from some of its target genes and, therefore, relieves the cellular oncogenic addiction to MLL chimeras. Stabilization of MLL provides us with a paradigm in the development of therapies for aggressive MLL leukemia and perhaps for other cancers caused by translocations.
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•UBE2O acts downstream of the interleukin-1 pathway to regulate MLL/COMPASS stability•Stabilizing wild-type MLL protein inhibits MLL leukemia cell proliferation•UBE2O and IRAK inhibition alters a common set of MLL chimera target genes•Targeting the IL-1 pathway is a potential therapeutic strategy for MLL leukemia
Stabilizing wild-type MLL proteins is a potential therapeutic approach for leukemia resulting from MLL translocations.
RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor ...(NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5′ cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.
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•Acute NELF depletion reveals 2-step pausing of RNA Pol II at promoters•The 1st-to-2nd pausing transition is independent of P-TEFb/SEC activity•The heat shock response remains intact in the absence of NELF•NELF recruits the cap-binding complex with modest effects on 5′ cap stability
Metazoan RNA Pol II-transcribed genes exhibit post-initiation regulation called pausing. Aoi et al. find that loss of the protein complex thought to maintain pausing, NELF, does not result in global release of RNA Pol II but instead may regulate other promoter-proximal regulatory steps such as 5′ mRNA cap stability.
Genomic imprinting is a critical developmental process characteristic of parent of origin-specific gene expression. It is well accepted that differentially DNA-methylated regions (DMRs) and enhancers ...are two major classes of cis-elements determining parent of origin-specific gene expression, with each recruiting different sets of transcription factors. Previously, we identified the AF4/FMR2 (AFF) family protein AFF3 within the transcription elongation complex SEC-L3. Here, we report that AFF3 can specifically bind both gametic DMRs (gDMRs) and enhancers within imprinted loci in an allele-specific manner. We identify the molecular regulators involved in the recruitment of AFF3 to gDMRs and provide mechanistic insight into the requirement of AFF3 at an enhancer for the expression of an ∼200-kb polycistronic transcript within the imprinted Dlk1-Dio3 locus. Our data suggest that the heterochromatic environment at the gDMR reinforces silencing of its related enhancer by controlling the binding and activity of AFF3 in an allele-specific manner. In summary, this study provides molecular details about the regulation of dosage-critical imprinted gene expression through the regulated binding of the transcription elongation factor AFF3 between a DMR and an enhancer.
The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with ...plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.
Even though transcription factors (TFs) are central players of gene regulation and have been extensively studied, their regulatory trans‐activation domains (tADs) often remain unknown and a ...systematic functional characterization of tADs is lacking. Here, we present a novel high‐throughput approach tAD‐seq to functionally test thousands of candidate tADs from different TFs in parallel. The tADs we identify by pooled screening validate in individual luciferase assays, whereas neutral regions do not. Interestingly, the tADs are found at arbitrary positions within the TF sequences and can contain amino acid (e.g., glutamine) repeat regions or overlap structured domains, including helix–loop–helix domains that are typically annotated as DNA‐binding. We also identified tADs in the non‐native reading frames, confirming that random sequences can function as tADs, albeit weakly. The identification of tADs as short protein sequences sufficient for transcription activation will enable the systematic study of TF function, which—particularly for TFs of different transcription activating functionalities—is still poorly understood.
Synopsis
The high‐throughput method tAD‐seq allows the functional identification of trans‐activation domains (tADs) in transcription factor sequences. These tADs contain simple sequence signatures such as regions rich in particular amino acids, e.g. glutamine and can overlap structural domains.
tAD‐seq combines reporter expression, FACS and Next‐Generation‐Sequencing to identify tADs directly based on their trans‐activating functions.
tADs occur at arbitrary positions within transcription factors, contain simple sequence signatures (e.g. glutamine rich) and can overlap structured domains.
Sequences found in non‐native reading frames can function as weak tADs.
tADs exist in short and long forms, and flanking sequences can positively or negatively affect tAD function.
tADs exist in short and long forms, and flanking sequences can positively or negatively affect tAD function.
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