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Supplementary Table 1

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Supplementary Figs. 1-17 with legends

Purpose:T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes.Experimental Design:To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models.Results:We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo.Conclusions:These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.

Purpose: The epigenetic mechanisms involved in transcriptional regulation leading to malignant phenotype in gliomas remains poorly understood. Topoisomerase IIB (TOP2B), an enzyme that decoils and releases torsional forces in DNA, is overexpressed in a subset of gliomas. Therefore, we investigated its role in epigenetic regulation in these tumors. Experimental Design: To investigate the role of TOP2B in epigenetic regulation in gliomas, we performed paired chromatin immunoprecipitation sequencing for TOP2B and RNA-sequencing analysis of glioma cell lines with and without TOP2B inhibition and in human glioma specimens. These experiments were complemented with assay for transposase-accessible chromatin using sequencing, gene silencing, and mouse xenograft experiments to investigate the function of TOP2B and its role in glioma phenotypes. Results: We discovered that TOP2B modulates transcription of multiple oncogenes in human gliomas. TOP2B regulated transcription only at sites where it was enzymatically active, but not at all native binding sites. In particular, TOP2B activity localized in enhancers, promoters, and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFRA and MYC expression across glioma specimens, which was not seen in nontumoral human brain tissue. In vivo, TOP2B knockdown of human glioma intracranial implants prolonged survival and downregulated PDGFRA. Conclusions: Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas promoting a proliferative phenotype.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

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.

The COMPASS family members Set1A, Set1B, and Mll2 differentially regulate H3K4me3 peak breadth in embryonic stem cells., Set1A and Set1B, two members of the COMPASS family of methyltransferases that methylate the histone H3 lysine 4 (H3K4) residue, have been accredited as primary depositors of global H3K4 trimethylation (H3K4me3) in mammalian cells. Our previous studies in mouse embryonic stem cells (ESCs) demonstrated that deleting the enzymatic SET domain of Set1A does not perturb bulk H3K4me3, indicating possible compensatory roles played by other COMPASS methyltransferases. Here, we generated a series of ESC lines harboring compounding mutations of COMPASS methyltransferases. We find that Set1B is functionally redundant to Set1A in implementing H3K4me3 at highly expressed genes, while Mll2 deposits H3K4me3 at less transcriptionally active promoters. While Set1A-B/COMPASS is responsible for broad H3K4me3 peaks, Mll2/COMPASS establishes H3K4me3 with narrow breadth. Additionally, Mll2 helps preserve global H3K4me3 levels and peak breadth in the absence of Set1A-B activity. Our results illustrate the biological flexibility of such enzymes in regulating transcription in a context-dependent manner to maintain stem cell identity.

Summary 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.

Mutations and translocations within the COMPASS (complex of proteins associated with Set1) family of histone lysine methyltransferases are associated with a large number of human diseases, including cancer. Here we report that SET1B/COMPASS, which is essential for cell survival, surprisingly has a cytoplasmic variant. SET1B, but not its SET domain, is critical for maintaining cell viability, indicating a novel catalytic-independent role of SET1B/COMPASS. Loss of SET1B or its unique cytoplasmic-interacting protein, BOD1, leads to up-regulation of expression of numerous genes modulating fatty acid metabolism, including ADIPOR1 (adiponectin receptor 1), COX7C, SDC4, and COQ7. Our detailed molecular studies identify ADIPOR1 signaling, which is inactivated in both obesity and human cancers, as a key target of SET1B/COMPASS. Collectively, our study reveals a cytoplasmic function for a member of the COMPASS family, which could be harnessed for therapeutic regulation of signaling in human diseases, including cancer.

Histone H3 lysine 4 monomethylation (H3K4me1) is an evolutionarily conserved feature of enhancer chromatin catalyzed by the COMPASS-like methyltransferase family, which includes Trr in Drosophila melanogaster and MLL3 (encoded by KMT2C) and MLL4 (encoded by KMT2D) in mammals. Here we demonstrate that Drosophila embryos expressing catalytically deficient Trr eclose and develop to productive adulthood. Parallel experiments with a trr allele that augments enzyme product specificity show that conversion of H3K4me1 at enhancers to H3K4me2 and H3K4me3 is also compatible with life and results in minimal changes in gene expression. Similarly, loss of the catalytic SET domains of MLL3 and MLL4 in mouse embryonic stem cells (mESCs) does not disrupt self-renewal. Drosophila embryos with trr alleles encoding catalytic mutants manifest subtle developmental abnormalities when subjected to temperature stress or altered cohesin levels. Collectively, our findings suggest that animal development can occur in the context of Trr or mammalian COMPASS-like proteins deficient in H3K4 monomethylation activity and point to a possible role for H3K4me1 on cis-regulatory elements in specific settings to fine-tune transcriptional regulation in response to environmental stress.

Histone H3 Lys4 (H3K4) methylation is a chromatin feature enriched at gene cis-regulatory sequences such as promoters and enhancers. Here we identify an evolutionarily conserved factor, BRWD2/PHIP, which colocalizes with histone H3K4 methylation genome-wide in human cells, mouse embryonic stem cells, and Drosophila. Biochemical analysis of BRWD2 demonstrated an association with the Cullin-4–RING ubiquitin E3 ligase-4 (CRL4) complex, nucleosomes, and chromatin remodelers. BRWD2/PHIP binds directly to H3K4 methylation through a previously unidentified chromatin-binding module related to Royal Family Tudor domains, which we named the CryptoTudor domain. Using CRISPR–Cas9 genetic knockouts, we demonstrate that COMPASS H3K4 methyltransferase family members differentially regulate BRWD2/PHIP chromatin occupancy. Finally, we demonstrate that depletion of the single Drosophila homolog dBRWD3 results in altered gene expression and aberrant patterns of histone H3 Lys27 acetylation at enhancers and promoters, suggesting a cross-talk between these chromatin modifications and transcription through the BRWD protein family.

Zic family proteins regulate GLUT1 gene expression in a β-catenin/Tcf7l2–dependent manner in colorectal cancer cells., The zinc finger of the cerebellum (ZIC) proteins has been implicated to function in normal tissue development. Recent studies have described the critical functions of Zic proteins in cancers and the potential tumor-suppressive functions in colon cancer development and progression. To elucidate the functional roles of Zic proteins in colorectal cancer, we knocked out the Zic5 gene and analyzed the chromatin localization pattern and transcriptional regulation of target gene expression. We found that Zic5 regulates glucose metabolism, and Zic5 knockout is accompanied by an increased glycolytic state and tolerance to a low-glucose condition. Furthermore, loss of β-catenin or TCF7l2 diminishes the chromatin binding of Zic5 globally. Our studies suggest that the Wnt/β-catenin signaling pathway has a strong influence on the function of Zic proteins and glucose metabolism in colorectal cancers through GLUT1. Interfering Wnt/-catenin–Zic5 axis–regulated aerobic glycolysis represents a potentially effective strategy to selectively target colon cancer cells.

The COMPASS protein family catalyzes histone H3 lysine 4 (H3K4) methylation and its members are essential for regulating gene expression. MLL2/COMPASS methylates H3K4 on many developmental genes and bivalent clusters. To understand MLL2-dependent transcriptional regulation, we performed a CRISPR-based screen with an MLL2-dependent gene as a reporter in mouse embryonic stem cells (mESCs) and found that MLL2 functions in gene expression by protecting developmental genes from repression via repelling PRC2 and DNA methylation machineries. Accordingly, repression in the absence of MLL2 is relieved by inhibition of PRC2 and DNA methyltransferases. Furthermore, recruitment of DNA demethylation machineries on such loci leads to reactivation of MLL2-dependent genes not only by removing DNA methylation but also by opening up previously CpG methylated regions for PRC2 recruitment, diluting PRC2 at Polycomb-repressed genes. These findings reveal how the context and function of these three epigenetic modifiers of chromatin can orchestrate transcriptional decisions and demonstrate that prevention of active repression by the context of the enzyme and not H3K4me3 underlies transcriptional regulation on MLL2/COMPASS targets.

Oncogene expression varies across gliomas. We discovered that TOP2B modulates transcription and splicing of multiple oncogenes in Drosophila brain tumors and human gliomas. TOP2B regulated transcription at sites where it was active, but not where found natively binding. TOP2B activity localized in enhancers, promoters and introns of PDGFRA and MYC, facilitating their expression. TOP2B levels and genomic localization was associated with PDGFR and MYC expression across gliomas specimens. Susceptibility to MST-16, a TOP2 inhibitor drug, correlated with baseline PDGRA expression across glioma cell lines. Our results indicate that TOP2B activity exerts a pleiotropic role in transcriptional regulation of oncogenes in a subset of gliomas. Modulation of oncogenes by TOP2-targeting drugs is the basis for a personalized approach for cancer therapy.

Delineating how chromosomes fold at length scales beyond one megabase remains obscure relative to smaller-scale folding into TADs, loops, and nucleosomes. We find that rather than simply unfolding chromatin, histone hyperacetylation results in interactions between distant genomic loci separated by tens to hundreds of megabases, even in the absence of transcription. These hyperacetylated "megadomains" are formed by the BRD4-NUT fusion oncoprotein, interact both within and between chromosomes, and form a specific nuclear subcompartment that has elevated gene activity with respect to other subcompartments. Pharmacological degradation of BRD4-NUT results in collapse of megadomains and attenuation of the interactions between them. In contrast, these interactions persist and contacts between newly acetylated regions are formed after inhibiting RNA polymerase II initiation. Our structure-function approach thus reveals that broad chromatin domains of identical biochemical composition, independent of transcription, form nuclear subcompartments, and also indicates the potential of altering chromosome structure for treating human disease.

An epigenetic axis directs a transcriptional program via enhancer activation by MLL3 COMPASS and TET2-dependent DNA demethylation., The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.

Purpose: T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Chemotherapy treatments show high response rates but have debilitating effects and carry risk of relapse. Previous work implicated NOTCH1 and other oncogenes. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Our goal in this work has been to identify enzymes active in T-ALL whose activity could be targeted for therapeutic purposes. Experimental Design: To identify and characterize new NOTCH1 druggable partners in T-ALL, we coupled studies of the NOTCH1 interactome to expression analysis and a series of functional analyses in cell lines, patient samples, and xenograft models. Results: We demonstrate that ubiquitin-specific protease 7 (USP7) interacts with NOTCH1 and controls leukemia growth by stabilizing the levels of NOTCH1 and JMJD3 histone demethylase. USP7 is highly expressed in T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 levels through deubiquitination. USP7 binds oncogenic targets and controls gene expression through stabilization of NOTCH1 and JMJD3 and ultimately H3K27me3 changes. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and inhibition of USP7 leads to a decrease of the transcriptional levels of NOTCH1 targets and significantly blocks T-ALL cell growth in vitro and in vivo. Conclusions: These results provide a new model for USP7 deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. Our studies also show that targeting USP7 inhibition could be a therapeutic strategy in aggressive leukemia.

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) has the highest mortality of all pediatric solid tumors. Histone H3 mutation (H3K27M) occurs in 80%, altering chromatin structure and function. To characterize the effects of H3K27M as an oncogenic driver, we analyzed the transcriptome and epigenome in DIPG cell lines. METHODS: Transcriptomes (RNA-Seq) and genomic enrichment of Histone proteins (ChIP-Seq) were characterized in H3K27M DIPG (n=7), wild-type pediatric high-grade glioma (n=3), neural stem cells (n=1) and astrocyte cell lines (n=1). Reads were aligned (UCSC hg19, Tophat), normalized and quantified (HTSeq), and formatted \ for peak calling (SICER) and genomic enrichment mapping (Ensembl). Differential expression and enrichment patterns were identified (edgeR) then analyzed for biological relevance (TopGO, IPA). Epigenetic therapeutic targets were studied in vitro and in vivo. RESULTS: Unsupervised clustering revealed two DIPG subgroups, distinct from control and H3 wild-type lines. Increased EZH2 (p

The lysine methyltransferase KMT2C (also known as MLL3), a subunit of the COMPASS complex, implements monomethylation of Lys4 on histone H3 (H3K4) at gene enhancers. KMT2C (hereafter referred to as MLL3) frequently incurs point mutations across a range of human tumor types, but precisely how these lesions alter MLL3 function and contribute to oncogenesis is unclear. Here we report a cancer mutational hotspot in MLL3 within the region encoding its plant homeodomain (PHD) repeats and demonstrate that this domain mediates association of MLL3 with the histone H2A deubiquitinase and tumor suppressor BAP1. Cancer-associated mutations in the sequence encoding the MLL3 PHD repeats disrupt the interaction between MLL3 and BAP1 and correlate with poor patient survival. Cancer cells that had PHD-associated MLL3 mutations or lacked BAP1 showed reduced recruitment of MLL3 and the H3K27 demethylase KDM6A (also known as UTX) to gene enhancers. As a result, inhibition of the H3K27 methyltransferase activity of the Polycomb repressive complex 2 (PRC2) in tumor cells harboring BAP1 or MLL3 mutations restored normal gene expression patterns and impaired cell proliferation in vivo. This study provides mechanistic insight into the oncogenic effects of PHD-associated mutations in MLL3 and suggests that restoration of a balanced state of Polycomb-COMPASS activity may have therapeutic efficacy in tumors that bear mutations in the genes encoding these epigenetic factors.

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.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease, affecting children and adults. Treatments1-6 show high response rates but have debilitating effects and carry risk of relapse5,7,8. Previous work implicated NOTCH1 and other oncogenes1,2,9-20. However, direct inhibition of these pathways affects healthy tissues and cancer alike. Here, we demonstrate that ubiquitin-specific protease 7 (USP7)21-32 controls leukemia growth by stabilizing the levels of the NOTCH1 and JMJD3 demethylase. USP7 is overexpressed T-ALL and is transcriptionally regulated by NOTCH1. In turn, USP7 controls NOTCH1 through deubiquitination. USP7 is bound to oncogenic targets and controls gene expression through H2B ubiquitination and H3K27me3 changes via stabilization of NOTCH1 and JMJD3. We also show that USP7 and NOTCH1 bind T-ALL superenhancers, and USP7 inhibition alters associated gene activity. These results provide a new model for deubiquitinase activity through recruitment to oncogenic chromatin loci and regulation of both oncogenic transcription factors and chromatin marks to promote leukemia. USP7 inhibition33 significantly blocked T-ALL cell growth in vitro and in vivo. Our studies also show that USP7 is upregulated in the aggressive high-risk cases of T-ALL and suggest that USP7 expression might be a prognostic marker in ALL and its inhibition could be a therapeutic tool against aggressive leukemia.

The counterbalance between coactivator and co-repressor complexes at enhancers instructs stem cell pluripotency transition., Chromatin regulators control cellular differentiation by orchestrating dynamic developmental gene expression programs, and hence, malfunctions in the regulation of chromatin state contribute to both developmental disorders and disease state. Mll4 (Kmt2d), a member of the COMPASS (COMplex of Proteins ASsociated with Set1) protein family that implements histone H3 lysine 4 monomethylation (H3K4me1) at enhancers, is essential for embryonic development and functions as a pancancer tumor suppressor. We define the roles of Mll4/COMPASS and its catalytic activity in the maintenance and exit of ground-state pluripotency in murine embryonic stem cells (ESCs). Mll4 is required for ESC to exit the naive pluripotent state; however, its intrinsic catalytic activity is dispensable for this process. The depletion of the H3K4 demethylase Lsd1 (Kdm1a) restores the ability of Mll4 null ESCs to transition from naive to primed pluripotency. Thus, we define an opposing regulatory axis, wherein Lsd1 and associated co-repressors directly repress Mll4-activated gene targets. This finding has broad reaching implications for human developmental syndromes and the treatment of tumors carrying Mll4 mutations.

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.

A complete understanding of transcription and co-transcriptional RNA processing events by polymerase requires precise and robust approaches to visualize polymerase progress and quantify nascent transcripts on a genome-wide scale. Here, we present a transcriptome-wide method to measure the level of nascent transcribing RNA in a fast and unbiased manner.

A complete understanding of transcription and co-transcriptional RNA processing events by polymerase requires precise and robust approaches to visualize polymerase progress and quantify nascent transcripts on a genome-wide scale. Here, we present a transcriptome-wide method to measure the level of nascent transcribing RNA in a fast and unbiased manner.

Recent discovery of somatic histone gene mutations, resulting in replacement of lysine 27 by methionine (K27M) in the encoded histone H3.3 proteins, in diffuse intrinsic pontine glioma (DIPG) has dramatically improved our understanding of disease pathogenesis, and stimulated the development of novel therapeutic approaches targeting epigenetic regulators for disease treatment. K27M mutant DIPG shows a dramatic reduction in global methylation at K27 residues on all 16 H3 proteins. This reduction in H3K27 methylation is believed to modify cellular gene expression in a way that favors tumor development. We have shown that inhibition of the H3K27 demethylase JMJD3 acts to restore K27 methylation in DIPG cells, while demonstrating potent anti-tumor activity, in vitro and in vivo. In addition to H3K27 methylation, H3K27 can also be acetylated (K27ac), which requires bromo- and extra-terminal domain (BET) protein activity. We have recently identified H3K27M-K27ac nucleosomes co-localize with BET bromodomain proteins at actively transcribed genes, whereas a polycomb repressive complex 2 (PRC2) is excluded from these regions, demonstrating that H3K27M and PRC2 occupy distinct chromatin regions in DIPG cells. Despite a major loss of H3K27 methylation, PRC2 activity is still detected in H3K27M DIPG cells, and the residual PRC2 activity is required to maintain DIPG proliferative potential by repressing neuronal differentiation and function. Small molecule inhibitor of EZH2 inhibited cell growth through the upregulation of gene that are normally repressed by PRC2 in DIPG. We also tested the anti-tumor activity of BET bromodomain containing protein 4 (BRD4) inhibitor, JQ1, and EZH2 inhibitor, GSK126, in our human DIPG xenograft model, and JQ1 and GSK126 treatment results in a significant delay of tumor progression and prolonged animal survival. Our findings suggest EZH2 inhibition could be a potential combinatorial strategy with BRD4 inhibition in treating K27M DIPG, that could counteract the development of resistance to single-agent epigenetic therapy.

Multitalented enhancers Productive transcription from DNA demands initiation, elongation, and termination. Enhancers are DNA sequences that loop with promoters to initiate transcription. Chen et al. show that enhancers also regulate gene expression by modulating transcription elongation. PAF1, a RNA polymerase II–associated factor, sits on enhancers. This prevents the full activation of the enhancer required for the release of paused polymerase at promoters to achieve successful transcription elongation. Science , this issue p. 1294

Misharin et al. elucidate the fate and function of monocyte-derived alveolar macrophages during the course of pulmonary fibrosis. These cells persisted throughout the life span, were enriched for the expression of profibrotic genes, and their genetic ablation ameliorated development of pulmonary fibrosis., Little is known about the relative importance of monocyte and tissue-resident macrophages in the development of lung fibrosis. We show that specific genetic deletion of monocyte-derived alveolar macrophages after their recruitment to the lung ameliorated lung fibrosis, whereas tissue-resident alveolar macrophages did not contribute to fibrosis. Using transcriptomic profiling of flow-sorted cells, we found that monocyte to alveolar macrophage differentiation unfolds continuously over the course of fibrosis and its resolution. During the fibrotic phase, monocyte-derived alveolar macrophages differ significantly from tissue-resident alveolar macrophages in their expression of profibrotic genes. A population of monocyte-derived alveolar macrophages persisted in the lung for one year after the resolution of fibrosis, where they became increasingly similar to tissue-resident alveolar macrophages. Human homologues of profibrotic genes expressed by mouse monocyte-derived alveolar macrophages during fibrosis were up-regulated in human alveolar macrophages from fibrotic compared with normal lungs. Our findings suggest that selectively targeting alveolar macrophage differentiation within the lung may ameliorate fibrosis without the adverse consequences associated with global monocyte or tissue-resident alveolar macrophage depletion.

The homeotic (Hox) genes are highly conserved in metazoans, where they are required for various processes in development, and misregulation of their expression is associated with human cancer. In the developing embryo, Hox genes are activated sequentially in time and space according to their genomic position within Hox gene clusters. Accumulating evidence implicates both enhancer elements and noncoding RNAs in controlling this spatiotemporal expression of Hox genes, but disentangling their relative contributions is challenging. Here, we identify two cis-regulatory elements (E1 and E2) functioning as shadow enhancers to regulate the early expression of the HoxA genes. Simultaneous deletion of these shadow enhancers in embryonic stem cells leads to impaired activation of HoxA genes upon differentiation, while knockdown of a long noncoding RNA overlapping E1 has no detectable effect on their expression. Although MLL/COMPASS (complex of proteins associated with Set1) family of histone methyltransferases is known to activate transcription of Hox genes in other contexts, we found that individual inactivation of the MLL1-4/COMPASS family members has little effect on early Hox gene activation. Instead, we demonstrate that SET1A/COMPASS is required for full transcriptional activation of multiple Hox genes but functions independently of the E1 and E2 cis-regulatory elements. Our results reveal multiple regulatory layers for Hox genes to fine-tune transcriptional programs essential for development.

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.

CHAF1B is the p60 subunit of the chromatin assembly factor (CAF1) complex, which is responsible for assembly of histones H3.1/H4 heterodimers at the replication fork during S phase. Here we report that CHAF1B is required for normal hematopoiesis while its overexpression promotes leukemia. CHAF1B has a pro-leukemia effect by binding chromatin at discrete sites and interfering with occupancy of transcription factors that promote myeloid differentiation, such as CEBPA. Reducing Chaf1b activity by either heterozygous deletion or overexpression of a CAF1 dominant negative allele is sufficient to suppress leukemogenesis in vivo without impairing normal hematopoiesis.

INTRODUCTION: Pediatric high-grade glioma (HGG), including H3K27M diffuse midline glioma, has the highest mortality of pediatric solid tumors. Recurrent Histone H3 mutations result in methionine for lysine substitution (H3K27M) in 80% of diffuse midline gliomas, or valine for glycine/arginine substitution (G34V/R) in 50% of hemispheric HGGs. These mutations alter chromatin structure and are associated with distinct patterns of gene expression and poorer response to therapy. To elucidate the mechanism by which these mutations affect chromatin function, we characterized genomic deposition of Histone H3 proteoforms in a large cohort of rare pediatric glioma cell lines. METHODS: H3K27M DIPG (n=6), H3G34V (n=1) and wild-type pediatric high-grade glioma cells (n=2), neural stem cells (n=1) and astrocytes (n=1) were analyzed for genomic deposition patterns of H3.3, H3K27M, H3G34V, H3K27me3, H3K27Ac, H4Kme1, and H4Kme3. Extracted chromatins were sonicated to produce DNA fragments for ChIP. RNA was extracted for whole transcriptome profiling. DNA/RNA libraries were prepared using the KAPA HTP Library Preparation Kit and sequenced (ChIP-and RNA-Seq, Illumina NextSeq 500). Genomic enrichments and gene expressions were determined, quantified, and analyzed for biological relevance. RESULTS: Distinct genomic enrichment of H3 proteoforms was observed between mutant and wild type cell lines, corresponding with respective gene expression levels. Differential co-localization of H3 post-translational marks with mutant vs. wild type H3 protein was also observed, consistent with our prior description of mutant heterotypic nucleosomes. Location of specific marks (promoter, gene body) suggests altered transcription factor recruitment and function may result in observed patterns of gene expression. CONCLUSION: We present the largest known epigenetic analysis of pediatric glioma cell lines to date, indicating distinct patterns of Histone H3 enrichment and gene expression in H3K27M and H3G34V/R mutant lines. These data provide insight into the mechanisms by which H3 mutations impact pediatric glioma biology, which may inform novel therapeutic approaches.

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 Pol II-associated Factor 1 (PAF1) to possess 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 a novel function for PAF1 as a regulator of promoter-proximal pausing by Pol II.

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.

Acute lymphoblastic leukemia (ALL) is a highly aggressive blood cancer affecting children and adults. Certain high-risk disease subsets have poor outcomes and often debilitating therapy-related toxicities stemming from direct inhibition of the oncogenes. We hypothesize that the process of oncogenic transformation is driven by aberrant activity of oncogene-associated chromatin modifying partners. These changes create a chromatin environment unique to the malignant state and, therefore, disruption of critical oncogenic chromatin signatures would not likely affect healthy tissues. We have generated strong evidence for the intertwined roles between the NOTCH1 oncogenic pathway and deubiquitinase enzymes in T cell leukemia, members of the ubiquitin-specific proteases (USP) family in particular. Members of the USP family physically interact with NOTCH1 and the lysine 27 on histone H3 (H3K27) demethylase JMJD3 and this methylation-ubiquitination biology-related axis coordinates regulation of transcriptional initiation and elongation, vital for the survival of leukemia cells. Interestingly transcription of USP genes is positively controlled by NOTCH1 creating a feedback loop in leukemia. We have further characterized this oncogenic axis using a combination of small molecule inhibitors and genetic engineering of USPs in ALL cell lines, primary patient samples and primagraft models of disease. We are able to show that a) USP activity is important for certain oncogenic pathways (such as NOTCH1) in leukemia; b) Oncogenes and USP enzymes co-bind certain areas in the leukemia genome; c) Ubiquitination of histone H2B acts in a combinatorial fashion with H3K27me, is a major epigenetic change affected by the USP activity in leukemia and controls d) transcriptional elongation. Finally we demonstrate that f) chemical inhibition or down-regulation of USPs affect leukemia growth in vitro and in vivo. Ongoing and future studies include manipulation of USP levels in mouse models of leukemia as well as combinatorial use of USP inhibitors with chemotherapeutic regiments in vitro using matched diagnosis-relapsed primary samples and in xenograft studies. Information gained from these studies will lend rationale towards the use of small molecule inhibitors against USP proteins in clinical trials for the treatment of aggressive and relapsed ALL. Disclosures Kumar: Progenra Inc.: Employment. Wang:Progenra Inc.: Employment. Wu:Progenra Inc.: Employment.

The elongation stage of transcription is highly regulated in metazoans. We previously purified the AFF1- and AFF4-containing super elongation complex (SEC) as a major regulator of development and cancer pathogenesis. Here, we report the biochemical isolation of SEC-like 2 (SEC-L2) and SEC-like 3 (SEC-L3) containing AFF2 and AFF3 in association with P-TEFb, ENL/MLLT1, and AF9/MLLT3. The SEC family members demonstrate high levels of polymerase II (Pol II) C-terminal domain kinase activity; however, only SEC is required for the proper induction of the HSP70 gene upon stress. Genome-wide mRNA-Seq analyses demonstrated that SEC-L2 and SEC-L3 control the expression of different subsets of genes, while AFF4/SEC plays a more dominant role in rapid transcriptional induction in cells. MYC is one of the direct targets of AFF4/SEC, and SEC recruitment to the MYC gene regulates its expression in different cancer cells, including those in acute myeloid or lymphoid leukemia. These findings suggest that AFF4/SEC could be a potential therapeutic target for the treatment of leukemia or other cancers associated with MYC overexpression.