The roles of Polycomb repressive complexes in mammalian development and cancer.

More than 80 years ago, the first Polycomb-related phenotype was identified in Drosophila melanogaster. Later, a group of diverse genes collectively called Polycomb group (PcG) genes were identified based on common mutant phenotypes. PcG proteins, which are well-conserved in animals, were originally characterized as negative regulators of gene transcription during development and subsequently shown to function in various biological processes; their deregulation is associated with diverse phenotypes in development and in disease, especially cancer. PcG proteins function on chromatin and can form two distinct complexes with different enzymatic activities: Polycomb repressive complex 1 (PRC1) is a histone ubiquitin ligase and PRC2 is a histone methyltransferase. Recent studies have revealed the existence of various mutually exclusive PRC1 and PRC2 variants. In this Review, we discuss new concepts concerning the biochemical and molecular functions of these new PcG complex variants, and how their epigenetic activities are involved in mammalian development and cancer.

Structure and Conformational Dynamics of a COMPASS Histone H3K4 Methyltransferase Complex.

The methylation of histone 3 lysine 4 (H3K4) is carried out by an evolutionarily conserved family of methyltransferases referred to as complex of proteins associated with Set1 (COMPASS). The activity of the catalytic SET domain (su(var)3-9, enhancer-of-zeste, and trithorax) is endowed through forming a complex with a set of core proteins that are widely shared from yeast to humans. We obtained cryo-electron microscopy (cryo-EM) maps of the yeast Set1/COMPASS core complex at overall 4.0- to 4.4-Å resolution, providing insights into its structural organization and conformational dynamics. The Cps50 C-terminal tail weaves within the complex to provide a central scaffold for assembly. The SET domain, snugly positioned at the junction of the Y-shaped complex, is extensively contacted by Cps60 (Bre2), Cps50 (Swd1), and Cps30 (Swd3). The mobile SET-I motif of the SET domain is engaged by Cps30, explaining its key role in COMPASS catalytic activity toward higher H3K4 methylation states.

Targeting Processive Transcription Elongation via SEC Disruption for MYC-Induced Cancer Therapy.

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.

Therapeutic Targeting of MLL Degradation Pathways in MLL-Rearranged Leukemia.

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.

Transcriptional elongation control in developmental gene expression, aging, and disease.

The elongation stage of transcription by RNA polymerase II (RNA Pol II) is central to the regulation of gene expression in response to developmental and environmental cues in metazoan. Dysregulated transcriptional elongation has been associated with developmental defects as well as disease and aging processes. Decades of genetic and biochemical studies have painstakingly identified and characterized an ensemble of factors that regulate RNA Pol II elongation. This review summarizes recent findings taking advantage of genetic engineering techniques that probe functions of elongation factors in vivo. We propose a revised model of elongation control in this accelerating field by reconciling contradictory results from the earlier biochemical evidence and the recent in vivo studies. We discuss how elongation factors regulate promoter-proximal RNA Pol II pause release, transcriptional elongation rate and processivity, RNA Pol II stability and RNA processing, and how perturbation of these processes is associated with developmental disorders, neurodegenerative disease, cancer, and aging.

Distinct layers of BRD4-PTEFb reveal bromodomain-independent function in transcriptional regulation.

The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release. Because its tandem bromodomains interact with acetylated histone lysine residues, it has long been thought that BRD4 requires these bromodomains for its recruitment to chromatin and transcriptional regulatory function. Here, using rapid depletion and genetic complementation with domain deletion mutants, we demonstrate that BRD4 bromodomains are dispensable for Pol II pause release. A minimal, bromodomain-less C-terminal BRD4 fragment containing the PTEFb-interacting C-terminal motif (CTM) is instead both necessary and sufficient to mediate Pol II pause release in the absence of full-length BRD4. Although BRD4-PTEFb can associate with chromatin through acetyl recognition, our results indicate that a distinct, active BRD4-PTEFb population functions to regulate transcription independently of bromodomain-mediated chromatin association. These findings may enable more effective pharmaceutical modulation of BRD4-PTEFb activity.

Born to run: control of transcription elongation by RNA polymerase II.

The dynamic regulation of transcription elongation by RNA polymerase II (Pol II) is an integral part of the implementation of gene expression programmes during development. In most metazoans, the majority of transcribed genes exhibit transient pausing of Pol II at promoter-proximal regions, and the release of Pol II into gene bodies is controlled by many regulatory factors that respond to environmental and developmental cues. Misregulation of the elongation stage of transcription is implicated in cancer and other human diseases, suggesting that mechanistic understanding of transcription elongation control is therapeutically relevant. In this Review, we discuss the features, establishment and maintenance of Pol II pausing, the transition into productive elongation, the control of transcription elongation by enhancers and by factors of other cellular processes, such as topoisomerases and poly(ADP-ribose) polymerases (PARPs), and the potential of therapeutic targeting of the elongation stage of transcription by Pol II.

PAF1, a Molecular Regulator of Promoter-Proximal Pausing by RNA Polymerase II.

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.

It's a DoG-eat-DoG world-altered transcriptional mechanisms drive downstream-of-gene (DoG) transcript production.

The past decade has revolutionized our understanding of regulatory noncoding RNAs (ncRNAs). Among the most recently identified ncRNAs are downstream-of-gene (DoG)-containing transcripts that are produced by widespread transcriptional readthrough. The discovery of DoGs has set the stage for future studies to address many unanswered questions regarding the mechanisms that promote readthrough transcription, RNA processing, and the cellular functions of the unique transcripts. In this review, we summarize current findings regarding the biogenesis, function, and mechanisms regulating this exciting new class of RNA molecules.

PALI1 promotes tumor growth through competitive recruitment of PRC2 to G9A-target chromatin for dual epigenetic silencing.

PALI1 is a newly identified accessory protein of the Polycomb repressive complex 2 (PRC2) that catalyzes H3K27 methylation. However, the roles of PALI1 in cancer are yet to be defined. Here, we report that PALI1 is upregulated in advanced prostate cancer (PCa) and competes with JARID2 for binding to the PRC2 core subunit SUZ12. PALI1 further interacts with the H3K9 methyltransferase G9A, bridging the formation of a unique G9A-PALI1-PRC2 super-complex that occupies a subset of G9A-target genes to mediate dual H3K9/K27 methylation and gene repression. Many of these genes are developmental regulators required for cell differentiation, and their loss in PCa predicts poor prognosis. Accordingly, PALI1 and G9A drive PCa cell proliferation and invasion in vitro and xenograft tumor growth in vivo. Collectively, our study shows that PALI1 harnesses two central epigenetic mechanisms to suppress cellular differentiation and promote tumorigenesis, which can be targeted by dual EZH2 and G9A inhibition.

SPT6 functions in transcriptional pause/release via PAF1C recruitment.

It is unclear how various factors functioning in the transcriptional elongation by RNA polymerase II (RNA Pol II) cooperatively regulate pause/release and productive elongation in living cells. Using an acute protein-depletion approach, we report that SPT6 depletion results in the release of paused RNA Pol II into gene bodies through an impaired recruitment of PAF1C. Short genes demonstrate a release with increased mature transcripts, whereas long genes are released but fail to yield mature transcripts, due to a reduced processivity resulting from both SPT6 and PAF1C loss. Unexpectedly, SPT6 depletion causes an association of NELF with the elongating RNA Pol II on gene bodies, without any observed functional significance on transcriptional elongation pattern, arguing against a role for NELF in keeping RNA Pol II in the paused state. Furthermore, SPT6 depletion impairs heat-shock-induced pausing, pointing to a role for SPT6 in regulating RNA Pol II pause/release through PAF1C recruitment.

SPT5 stabilization of promoter-proximal RNA polymerase II.

Based on in vitro studies, it has been demonstrated that the DSIF complex, composed of SPT4 and SPT5, regulates the elongation stage of transcription catalyzed by RNA polymerase II (RNA Pol II). The precise cellular function of SPT5 is not clear, because conventional gene depletion strategies for SPT5 result in loss of cellular viability. Using an acute inducible protein depletion strategy to circumvent this issue, we report that SPT5 loss triggers the ubiquitination and proteasomal degradation of the core RNA Pol II subunit RPB1, a process that we show to be evolutionarily conserved from yeast to human cells. RPB1 degradation requires the E3 ligase Cullin 3, the unfoldase VCP/p97, and a novel form of CDK9 kinase complex. Our study demonstrates that SPT5 stabilizes RNA Pol II specifically at promoter-proximal regions, permitting RNA Pol II release from promoters into gene bodies and providing mechanistic insight into the cellular function of SPT5 in safeguarding accurate gene expression.

Acute perturbation strategies in interrogating RNA polymerase II elongation factor function in gene expression.

The regulation of gene expression catalyzed by RNA polymerase II (Pol II) requires a host of accessory factors to ensure cell growth, differentiation, and survival under environmental stress. Here, using the auxin-inducible degradation (AID) system to study transcriptional activities of the bromodomain and extraterminal domain (BET) and super elongation complex (SEC) families, we found that the CDK9-containing BRD4 complex is required for the release of Pol II from promoter-proximal pausing for most genes, while the CDK9-containing SEC is required for activated transcription in the heat shock response. By using both the proteolysis targeting chimera (PROTAC) dBET6 and the AID system, we found that dBET6 treatment results in two major effects: increased pausing due to BRD4 loss, and reduced enhancer activity attributable to BRD2 loss. In the heat shock response, while auxin-mediated depletion of the AFF4 subunit of the SEC has a more severe defect than AFF1 depletion, simultaneous depletion of AFF1 and AFF4 leads to a stronger attenuation of the heat shock response, similar to treatment with the SEC inhibitor KL-1, suggesting a possible redundancy among SEC family members. This study highlights the usefulness of orthogonal acute depletion/inhibition strategies to identify distinct and redundant biological functions among Pol II elongation factor paralogs.

A trivalent nucleosome interaction by PHIP/BRWD2 is disrupted in neurodevelopmental disorders and cancer.

Mutations in the PHIP/BRWD2 chromatin regulator cause the human neurodevelopmental disorder Chung-Jansen syndrome, while alterations in PHIP expression are linked to cancer. Precisely how PHIP functions in these contexts is not fully understood. Here we demonstrate that PHIP is a chromatin-associated CRL4 ubiquitin ligase substrate receptor and is required for CRL4 recruitment to chromatin. PHIP binds to chromatin through a trivalent reader domain consisting of a H3K4-methyl binding Tudor domain and two bromodomains (BD1 and BD2). Using semisynthetic nucleosomes with defined histone post-translational modifications, we characterize PHIPs BD1 and BD2 as respective readers of H3K14ac and H4K12ac, and identify human disease-associated mutations in each domain and the intervening linker region that likely disrupt chromatin binding. These findings provide new insight into the biological function of this enigmatic chromatin protein and set the stage for the identification of both upstream chromatin modifiers and downstream targets of PHIP in human disease.

A histone modifier's ill-gotten copy gains.

Little is known about the molecular machinery that contributes to site-specific copy number variations or how CNVs fit into the chronology of tumor progression. Black et al. (2013) now demonstrate that the overexpression of a histone demethylase induces transient copy gain of specific genomic loci known to harbor proto-oncogenes.

The RNA Pol II elongation factor Ell3 marks enhancers in ES cells and primes future gene activation.

Enhancers play a central role in precisely regulating the expression of developmentally regulated genes. However, the machineries required for enhancer-promoter communication have remained largely unknown. We have found that Ell3, a member of the Ell (eleven-nineteen lysine-rich leukemia gene) family of RNA Pol II elongation factors, occupies enhancers in embryonic stem cells. Ell3's association with enhancers is required for setting up proper Pol II occupancy at the promoter-proximal regions of developmentally regulated genes and for the recruitment of the super elongation complex (SEC) to these loci following differentiation signals. Furthermore, Ell3 binding to inactive or poised enhancers is essential for stem cell specification. We have also detected the presence of Pol II and Ell3 in germ cell nuclei. These findings raise the possibility that transcription factors could prime gene expression by marking enhancers in ES cells or even as early as in the germ cell state.

NELF Regulates a Promoter-Proximal Step Distinct from RNA Pol II Pause-Release.

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.

The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis.

The Saccharomyces cerevisiae Set1/COMPASS was the first histone H3 lysine 4 (H3K4) methylase identified over 10 years ago. Since then, it has been demonstrated that Set1/COMPASS and its enzymatic product, H3K4 methylation, is highly conserved across the evolutionary tree. Although there is only one COMPASS in yeast, Drosophila possesses three and humans bear six COMPASS family members, each capable of methylating H3K4 with nonredundant functions. In yeast, the histone H2B monoubiquitinase Rad6/Bre1 is required for proper H3K4 and H3K79 trimethylations. The machineries involved in this process are also highly conserved from yeast to human. In this review, the process of histone H2B monoubiquitination-dependent and -independent histone H3K4 methylation as a mark of active transcription, enhancer signatures, and developmentally poised genes is discussed. The misregulation of histone H2B monoubiquitination and H3K4 methylation result in the pathogenesis of human diseases, including cancer. Recent findings in this regard are also examined.

A small UTX stabilization domain of Trr is conserved within mammalian MLL3-4/COMPASS and is sufficient to rescue loss of viability in null animals.

Catalytic-inactivating mutations within the Drosophila enhancer H3K4 mono-methyltransferase Trr and its mammalian homologs, MLL3/4, cause only minor changes in gene expression compared with whole-gene deletions for these COMPASS members. To identify essential histone methyltransferase-independent functions of Trr, we screened to identify a minimal Trr domain sufficient to rescue Trr-null lethality and demonstrate that this domain binds and stabilizes Utx in vivo. Using the homologous MLL3/MLL4 human sequences, we mapped a short ∼80-amino-acid UTX stabilization domain (USD) that promotes UTX stability in the absence of the rest of MLL3/4. Nuclear UTX stability is enhanced when the USD is fused with the MLL4 HMG-box. Thus, COMPASS-dependent UTX stabilization is an essential noncatalytic function of Trr/MLL3/MLL4, suggesting that stabilizing UTX could be a therapeutic strategy for cancers with MLL3/4 loss-of-function mutations.