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.

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.

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.

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.

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.

Human mediator subunit MED26 functions as a docking site for transcription elongation factors.

Promoter-proximal pausing by initiated RNA polymerase II (Pol II) and regulated release of paused polymerase into productive elongation has emerged as a major mechanism of transcription activation. Reactivation of paused Pol II correlates with recruitment of super-elongation complexes (SECs) containing ELL/EAF family members, P-TEFb, and other proteins, but the mechanism of their recruitment is an unanswered question. Here, we present evidence for a role of human Mediator subunit MED26 in this process. We identify in the conserved N-terminal domain of MED26 overlapping docking sites for SEC and a second ELL/EAF-containing complex, as well as general initiation factor TFIID. In addition, we present evidence consistent with the model that MED26 can function as a molecular switch that interacts first with TFIID in the Pol II initiation complex and then exchanges TFIID for complexes containing ELL/EAF and P-TEFb to facilitate transition of Pol II into the elongation stage of transcription.

The language of histone crosstalk.

It has been suggested that a specific pattern of histone posttranslational modifications and their crosstalk may constitute a code that determines transcriptional outcomes. However, recent studies indicate that histone modifications have context-dependent effects, making their interplay more like a language within the chromatin signaling pathway than a code.

Not All H3K4 Methylations Are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification.

The spatiotemporal regulation of gene expression is central for cell-lineage specification during embryonic development and is achieved through the combinatorial action of transcription factors/co-factors and epigenetic states at cis-regulatory elements. Here, we show that in addition to implementing H3K4me3 at promoters of bivalent genes, Mll2 (KMT2B)/COMPASS can also implement H3K4me3 at a subset of non-TSS regulatory elements, a subset of which shares epigenetic signatures of active enhancers. Our mechanistic studies reveal that association of Mll2's CXXC domain with CpG-rich regions plays an instrumental role for chromatin targeting and subsequent implementation of H3K4me3. Although Mll2/COMPASS is required for H3K4me3 implementation on thousands of loci, generation of catalytically mutant MLL2/COMPASS demonstrated that H3K4me3 implemented by this enzyme was essential for expression of a subset of genes, including those functioning in the control of transcriptional programs during embryonic development. Our findings suggest that not all H3K4 trimethylations implemented by MLL2/COMPASS are functionally equivalent.

A ChIP-exo screen of 887 Protein Capture Reagents Program transcription factor antibodies in human cells.

Antibodies offer a powerful means to interrogate specific proteins in a complex milieu. However, antibody availability and reliability can be problematic, whereas epitope tagging can be impractical in many cases. To address these limitations, the Protein Capture Reagents Program (PCRP) generated over a thousand renewable monoclonal antibodies (mAbs) against human presumptive chromatin proteins. However, these reagents have not been widely field-tested. We therefore performed a screen to test their ability to enrich genomic regions via chromatin immunoprecipitation (ChIP) and a variety of orthogonal assays. Eight hundred eighty-seven unique antibodies against 681 unique human transcription factors (TFs) were assayed by ultra-high-resolution ChIP-exo/seq, generating approximately 1200 ChIP-exo data sets, primarily in a single pass in one cell type (K562). Subsets of PCRP mAbs were further tested in ChIP-seq, CUT&RUN, STORM super-resolution microscopy, immunoblots, and protein binding microarray (PBM) experiments. About 5% of the tested antibodies displayed high-confidence target (i.e., cognate antigen) enrichment across at least one assay and are strong candidates for additional validation. An additional 34% produced ChIP-exo data that were distinct from background and thus warrant further testing. The remaining 61% were not substantially different from background, and likely require consideration of a much broader survey of cell types and/or assay optimizations. We show and discuss the metrics and challenges to antibody validation in chromatin-based assays.

Regulation of the imprinted Dlk1-Dio3 locus by allele-specific enhancer activity.

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.

Zic2 is an enhancer-binding factor required for embryonic stem cell specification.

The Zinc-finger protein of the cerebellum 2 (Zic2) is one of the vertebrate homologs of the Drosophila pair-rule gene odd-paired (opa). Our molecular and biochemical studies demonstrate that Zic2 preferentially binds to transcriptional enhancers and is required for the regulation of gene expression in embryonic stem cells. Detailed genome-wide and molecular studies reveal that Zic2 can function with Mbd3/NuRD in regulating the chromatin state and transcriptional output of genes linked to differentiation. Zic2 is required for proper differentiation of embryonic stem cells (ESCs), similar to what has been previously reported for Mbd3/NuRD. Our study identifies Zic2 as a key factor in the execution of transcriptional fine-tuning with Mbd3/NuRD in ESCs through interactions with enhancers. Our study also points to the role of the Zic family of proteins as enhancer-specific binding factors functioning in development.

DOT1L-controlled cell-fate determination and transcription elongation are independent of H3K79 methylation.

Actively transcribed genes in mammals are decorated by H3K79 methylation, which is correlated with transcription levels and is catalyzed by the histone methyltransferase DOT1L. DOT1L is required for mammalian development, and the inhibition of its catalytic activity has been extensively studied for cancer therapy; however, the mechanisms underlying DOT1L's functions in normal development and cancer pathogenesis remain elusive. To dissect the relationship between H3K79 methylation, cellular differentiation, and transcription regulation, we systematically examined the role of DOT1L and its catalytic activity in embryonic stem cells (ESCs). DOT1L is dispensable for ESC self-renewal but is required for establishing the proper expression signature of neural progenitor cells, while catalytic inactivation of DOT1L has a lesser effect. Furthermore, DOT1L loss, rather than its catalytic inactivation, causes defects in glial cell specification. Although DOT1L loss by itself has no major defect in transcription elongation, transcription elongation defects seen with the super elongation complex inhibitor KL-2 are exacerbated in DOT1L knockout cells, but not in catalytically dead DOT1L cells, revealing a role of DOT1L in promoting productive transcription elongation that is independent of H3K79 methylation. Taken together, our study reveals a catalytic-independent role of DOT1L in modulating cell-fate determination and in transcriptional elongation control.

Predicting childhood chronic kidney disease severity in infants with posterior urethral valve: a critical analysis of creatinine values in the first year of life.

BACKGROUND: Posterior urethral valve (PUV) is a leading cause of chronic kidney failure in children. Studies have shown that a creatinine nadir above historical cutoff values of 0.8 or 1.0 mg/dL correlates with worse kidney outcomes. The ability to use nadir creatinine more discriminately as a test of kidney outcomes is otherwise limited. METHODS: We performed a retrospective review of 102 infants treated with primary valve ablation prior to 1 year of age. Patient factors including creatinine at presentation (Cr-P), nadir within 6 weeks after ablation (Cr-6 W), and nadir within 1 year after ablation (Cr-1Y) were assessed as predictors of final chronic kidney disease (CKD) severity. An optimal threshold for four CKD levels was defined in incremental fashion using binary outcome with receiver operating characteristic (ROC). Multivariable logistic regression models compared Cr-P, Cr-6 W, and Cr-1Y while adjusting for patient factors. RESULTS: Boys were ablated at mean age of 36.3 days and followed for 6.6 years (± 3.7). When compared to other demographics, only creatinine remained independently predictive of CKD outcomes on multivariable analysis. ROC analysis demonstrated excellent diagnostic accuracy for Cr-6 W and Cr-1Y (p < 0.001) and acceptable accuracy for Cr-P (p < 0.005). Using the Cr-6 W and Cr-1Y models, high sensitivity and specificity creatinine nadir cutoffs were determined to predict each CKD outcome. CONCLUSIONS: The severity of childhood CKD can be predicted with high accuracy using the creatinine nadir within 6 weeks of ablation. The cutoff values described can be incorporated into a clinical setting for patient counseling and individual risk stratification.

Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation.

The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. Here, we used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. We also performed detailed screens and identified yeast strains lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. Our study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner.

Role of a Preoperative Catheter Regimen in Achieving Early Primary Endoscopic Valve Ablation in Neonates with Posterior Urethral Valves.

PURPOSE: Primary valve ablation is preferred to vesicostomy in the initial management of posterior urethral valves. However, some neonates have a prohibitively small urethra. We describe our experience with a preoperative urethral catheter regimen to enhance the likelihood of neonatal valve ablation. MATERIALS AND METHODS: We performed a retrospective review of 126 neonates with posterior urethral valves treated between 2003 and 2019 with valve ablation prior to 10 weeks of age. The preoperative indwelling catheter either was gradually upsized to an 8Fr (progressive urethral dilation), was not upsized (nondilated) or was initially larger bore (8Fr only). The primary outcome was the ability to perform primary ablation by neonatal resectoscope. The secondary objective was to establish the parameters for considering progressive urethral dilation as well as its associated risks. RESULTS: Overall 97% could be ablated. The progressive urethral dilation group had the lowest mean weight (p <0.001). Only a larger catheter at the time of ablation was significantly associated with feasible ablation (p <0.001) and not urethral dilation, the infant's weight or his gestational age. Progressive urethral dilation was associated with a longer duration of catheterization as well as double the rate of febrile urinary tract infections (8.5%) over the nondilated group (3.6%). CONCLUSIONS: A much higher rate of primary ablation is feasible (97%) than previously reported (82%). More important than the infant's weight is whether a 6Fr to 8Fr catheter is in place at ablation. If an initial 6Fr to 8Fr catheter cannot be placed, urethral dilation to 8Fr should be performed before attempting ablation. This is both a technique and preoperative assessment that is useful for operative planning.

The little elongation complex functions at initiation and elongation phases of snRNA gene transcription.

The small nuclear RNA (snRNA) genes have been widely used as a model system for understanding transcriptional regulation due to the unique aspects of their promoter structure, selectivity for either RNA polymerase (Pol) II or III, and because of their unique mechanism of termination that is tightly linked with the promoter. Recently, we identified the little elongation complex (LEC) in Drosophila that is required for the expression of Pol II-transcribed snRNA genes. Here, using Drosophila and mammalian systems, we provide genetic and molecular evidence that LEC functions in at least two phases of snRNA transcription: an initiation step requiring the ICE1 subunit, and an elongation step requiring ELL.

Transcriptional elongation checkpoint control in development and disease.

Transcriptional elongation control by RNA polymerase II and its associated factors has taken center stage as a process essential for the regulation of gene expression throughout development. In this review, we analyze recent findings on the identification of factors functioning in the regulation of the transcriptional elongation checkpoint control (TECC) stage of gene expression and how the factors' misregulation is associated with disease pathogenesis, including cancer.

The little elongation complex regulates small nuclear RNA transcription.

Eleven-nineteen lysine-rich leukemia (ELL) participates in the super elongation complex (SEC) with the RNA polymerase II (Pol II) CTD kinase P-TEFb. SEC is a key regulator in the expression of HOX genes in mixed lineage leukemia (MLL)-based hematological malignancies, in the control of induced gene expression early in development, and in immediate early gene transcription. Here, we identify an SEC-like complex in Drosophila, as well as a distinct ELL-containing complex that lacks P-TEFb and other components of SEC named the "little elongation complex" (LEC). LEC subunits are highly enriched at RNA Pol II-transcribed small nuclear RNA (snRNA) genes, and the loss of LEC results in decreased snRNA expression in both flies and mammals. The specialization of the SEC and LEC complexes for mRNA and snRNA-containing genes, respectively, suggests the presence of specific classes of elongation factors for each class of genes transcribed by RNA polymerase II.