The KAT module of the SAGA complex maintains the oncogenic gene expression program in MYCN-amplified neuroblastoma.

Pediatric cancers are frequently driven by genomic alterations that result in aberrant transcription factor activity. Here, we used functional genomic screens to identify multiple genes within the transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex as selective dependencies for MYCN-amplified neuroblastoma, a disease of dysregulated development driven by an aberrant oncogenic transcriptional program. We characterized the DNA recruitment sites of the SAGA complex in neuroblastoma and the consequences of loss of SAGA complex lysine acetyltransferase (KAT) activity on histone acetylation and gene expression. We demonstrate that loss of SAGA complex KAT activity is associated with reduced MYCN binding on chromatin, suppression of MYC/MYCN gene expression programs, and impaired cell cycle progression. Further, we showed that the SAGA complex is pharmacologically targetable in vitro and in vivo with a KAT2A/KAT2B proteolysis targeting chimeric. Our findings expand our understanding of the histone-modifying complexes that maintain the oncogenic transcriptional state in this disease and suggest therapeutic potential for inhibitors of SAGA KAT activity in MYCN-amplified neuroblastoma.

The SAGA acetyltransferase module is required for the maintenance of MAF and MYC oncogenic gene expression programs in multiple myeloma.

Despite recent advances in therapeutic treatments, multiple myeloma (MM) remains an incurable malignancy. Epigenetic factors contribute to the initiation, progression, relapse, and clonal heterogeneity in MM, but our knowledge on epigenetic mechanisms underlying MM development is far from complete. The SAGA complex serves as a coactivator in transcription and catalyzes acetylation and deubiquitylation. Analyses of datasets in the Cancer Dependency Map Project revealed many SAGA components are selective dependencies in MM. To define SAGA-specific functions, we focused on ADA2B, the only subunit in the lysine acetyltransferase (KAT) module that specifically functions in SAGA. Integration of RNA-seq, ATAC-seq, and CUT&RUN results identified pathways directly regulated by ADA2B include MTORC1 signaling, MYC, E2F, and MM-specific MAF oncogenic programs. We discovered that ADA2B is recruited to MAF and MYC gene targets, and that MAF shares a majority of its targets with MYC in MM cells. Furthermore, we found the SANT domain of ADA2B is required for interaction with both GCN5 and PCAF acetyltransferases, incorporation into SAGA, and ADA2B protein stability. Our findings uncover previously unknown SAGA KAT module-dependent mechanisms controlling MM cell growth, revealing a vulnerability that might be exploited for future development of MM therapy.

USP22 overexpression fails to augment tumor formation in MMTV-ERBB2 mice but loss of function impacts MMTV promoter activity.

The Ubiquitin Specific Peptidase 22 (USP22), a component of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) histone modifying complex, is overexpressed in multiple human cancers, but how USP22 impacts tumorigenesis is not clear. We reported previously that Usp22 loss in mice impacts execution of several signaling pathways driven by growth factor receptors such as erythroblastic oncogene B b2 (ERBB2). To determine whether changes in USP22 expression affects ERBB2-driven tumorigenesis, we introduced conditional overexpression or deletion alleles of Usp22 into mice bearing the Mouse mammary tumor virus-Neu-Ires-Cre (MMTV-NIC) transgene, which drives both rat ERBB2/NEU expression and Cre recombinase activity from the MMTV promoter resulting in mammary tumor formation. We found that USP22 overexpression in mammary glands did not further enhance primary tumorigenesis in MMTV-NIC female mice, but increased lung metastases were observed. However, deletion of Usp22 significantly decreased tumor burden and increased survival of MMTV-NIC mice. These effects were associated with markedly decreased levels of both Erbb2 mRNA and protein, indicating Usp22 loss impacts MMTV promoter activity. Usp22 loss had no impact on ERBB2 expression in other settings, including MCF10A cells bearing a Cytomegalovirus (CMV)-driven ERBB2 transgene or in human epidermal growth factor receptor 2 (HER2)+ human SKBR3 and HCC1953 cells. Decreased activity of the MMTV promoter in MMTV-NIC mice correlated with decreased expression of known regulatory factors, including the glucocorticoid receptor (GR), the progesterone receptor (PR), and the chromatin remodeling factor Brahma-related gene-1 (BRG1). Together our findings indicate that increased expression of USP22 does not augment the activity of an activated ERBB2/NEU transgene but impacts of Usp22 loss on tumorigenesis cannot be assessed in this model due to unexpected effects on MMTV-driven Erbb2/Neu expression.

Now open: Evolving insights to the roles of lysine acetylation in chromatin organization and function.

Protein acetylation is conserved across phylogeny and has been recognized as one of the most prominent post-translational modifications since its discovery nearly 60 years ago. Histone acetylation is an active mark characteristic of open chromatin, but acetylation on specific lysine residues and histone variants occurs in different biological contexts and can confer various outcomes. The significance of acetylation events is indicated by the associations of lysine acetyltransferases, deacetylases, and acetyl-lysine readers with developmental disorders and pathologies. Recent advances have uncovered new roles of acetylation regulators in chromatin-centric events, which emphasize the complexity of these functional networks. In this review, we discuss mechanisms and dynamics of acetylation in chromatin organization and DNA-templated processes, including gene transcription and DNA repair and replication.

Naked (N) mutant mice carry a nonsense mutation in the homeobox of Hoxc13.

Loss of function mutations in HOXC13 have been associated with Ectodermal Dysplasia-9, Hair/Nail Type (ECTD9) in consanguineous families, characterized by sparse to complete absence of hair and nail dystrophy. Here we characterize the spontaneous mouse mutation Naked (N) as a terminal truncation in the Hoxc13 (homeobox C13) gene. Similar to previous reports for homozygous Hoxc13 knock-out (KO) mice, homozygous N/N mice exhibit generalized alopecia with abnormal nails and a short lifespan. However, in contrast to Hoxc13 heterozygous KO mice, N/+ mice show generalized or partial alopecia, associated with loss of hair fibres, along with normal lifespan and fertility. Our data point to a lack of nonsense-mediated Hoxc13 transcript decay and the presence of the truncated mutant protein in N/N and N/+ hair follicles, thus suggesting a dominant-negative mutation. To our knowledge, this is the first report of a semi-dominant and potentially dominant-negative mutation affecting Hoxc13/HOXC13. Furthermore, recreating the N mutant allele in mice using CRISPR/Cas9-mediated genome editing resulted in the same spectrum of deficiencies as those associated with the spontaneous Naked mutation, thus confirming that N is indeed a Hoxc13 mutant allele. Considering the low viability of the Hoxc13 KO mice, the Naked mutation provides an attractive new model for studying ECTD9 disease mechanisms.

Usp22 Overexpression Leads to Aberrant Signal Transduction of Cancer-Related Pathways but Is Not Sufficient to Drive Tumor Formation in Mice.

Usp22 overexpression is observed in several human cancers and is correlated with poor patient outcomes. The molecular basis underlying this correlation is not clear. Usp22 is the catalytic subunit of the deubiquitylation module in the SAGA histone-modifying complex, which regulates gene transcription. Our previous work demonstrated that the loss of Usp22 in mice leads to decreased expression of several components of receptor tyrosine kinase and TGFß signaling pathways. To determine whether these pathways are upregulated when Usp22 is overexpressed, we created a mouse model that expresses high levels of Usp22 in all tissues. Phenotypic characterization of these mice revealed over-branching of the mammary glands in females. Transcriptomic analyses indicate the upregulation of key pathways involved in mammary gland branching in mammary epithelial cells derived from the Usp22-overexpressing mice, including estrogen receptor, ERK/MAPK, and TGFß signaling. However, Usp22 overexpression did not lead to increased tumorigenesis in any tissue. Our findings indicate that elevated levels of Usp22 are not sufficient to induce tumors, but it may enhance signaling abnormalities associated with oncogenesis.

Conservation and diversity of the eukaryotic SAGA coactivator complex across kingdoms.

The SAGA complex is an evolutionarily conserved transcriptional coactivator that regulates gene expression through its histone acetyltransferase and deubiquitylase activities, recognition of specific histone modifications, and interactions with transcription factors. Multiple lines of evidence indicate the existence of distinct variants of SAGA among organisms as well as within a species, permitting diverse functions to dynamically regulate cellular pathways. Our co-expression analysis of genes encoding human SAGA components showed enrichment in reproductive organs, brain tissues and the skeletal muscle, which corresponds to their established roles in developmental programs, emerging roles in neurodegenerative diseases, and understudied functions in specific cell types. SAGA subunits modulate growth, development and response to various stresses from yeast to plants and metazoans. In metazoans, SAGA further participates in the regulation of differentiation and maturation of both innate and adaptive immune cells, and is associated with initiation and progression of diseases including a broad range of cancers. The evolutionary conservation of SAGA highlights its indispensable role in eukaryotic life, thus deciphering the mechanisms of action of SAGA is key to understanding fundamental biological processes throughout evolution. To illuminate the diversity and conservation of this essential complex, here we discuss variations in composition, essentiality and co-expression of component genes, and its prominent functions across Fungi, Plantae and Animalia kingdoms.

Complex functions of Gcn5 and Pcaf in development and disease.

A wealth of biochemical and cellular data, accumulated over several years by multiple groups, has provided a great degree of insight into the molecular mechanisms of actions of GCN5 and PCAF in gene activation. Studies of these lysine acetyltransferases (KATs) in vitro, in cultured cells, have revealed general mechanisms for their recruitment by sequence-specific binding factors and their molecular functions as transcriptional co-activators. Genetic studies indicate that GCN5 and PCAF are involved in multiple developmental processes in vertebrates, yet our understanding of their molecular functions in these contexts remains somewhat rudimentary. Understanding the functions of GCN5/PCAF in developmental processes provides clues to the roles of these KATs in disease states. Here we will review what is currently known about the developmental roles of GCN5 and PCAF, as well as emerging role of these KATs in oncogenesis.

Transcriptional Activation of MYC-Induced Genes by GCN5 Promotes B-cell Lymphomagenesis.

Overexpression of the MYC oncoprotein is an initiating step in the formation of several cancers. MYC frequently recruits chromatin-modifying complexes to DNA to amplify the expression of cancer-promoting genes, including those regulating cell cycle, proliferation, and metabolism, yet the roles of specific modifiers in different cancer types are not well defined. Here, we show that GCN5 is an essential coactivator of cell-cycle gene expression driven by MYC overexpression and that deletion of Gcn5 delays or abrogates tumorigenesis in the Eµ-Myc mouse model of B-cell lymphoma. Our results demonstrate that Gcn5 loss impacts both expression and downstream functions of Myc. SIGNIFICANCE: Our results provide important proof of principle for Gcn5 functions in formation and progression of Myc-driven cancers, suggesting that GCN5 may be a viable target for development of new cancer therapies.

Targeting the SAGA and ATAC Transcriptional Coactivator Complexes in MYC-Driven Cancers.

Targeting epigenetic regulators, such as histone-modifying enzymes, provides novel strategies for cancer therapy. The GCN5 lysine acetyltransferase (KAT) functions together with MYC both during normal development and in oncogenesis. As transcription factors, MYC family members are difficult to target with small-molecule inhibitors, but the acetyltransferase domain and the bromodomain in GCN5 might provide alternative targets for disruption of MYC-driven functions. GCN5 is part of two distinct multiprotein histone-modifying complexes, SAGA and ATAC. This review summarizes key findings on the roles of SAGA and ATAC in embryo development and in cancer to better understand the functional relationships of these complexes with MYC family members, as well as their future potential as therapeutic targets.

GCN5 HAT inhibition reduces human Burkitt lymphoma cell survival through reduction of MYC target gene expression and impeding BCR signaling pathways.

GCN5, the catalytic subunit in the acetyltransferase modules of SAGA and ATAC, functions as a coactivator of gene transcription. The SAGA complex is recruited to chromatin by transcription factors such as MYC and E2F1 to facilitate acetylation of histones, especially H3 at lysine 9 (H3K9). Burkitt lymphoma is an aggressive subtype of Non-Hodgkin lymphoma driven by the overexpression of MYC. Comparison of GCN5 expression in normal human B cells versus human Burkitt Lymphoma cell lines indicates overexpression of GCN5 in lymphoma. Treatment of Burkitt lymphoma cell lines with a specific inhibitor indicates that decreased GCN5 HAT activity reduces viability and proliferation of these cells. Inhibition of GCN5 HAT activity also induces apoptosis in lymphoma cells. Expression of MYC target genes as well as genes associated with B cell receptor signaling are significantly downregulated upon inhibition of GCN5 enzymatic activity. This downregulation leads to diminished PI3K signaling, a critical pathway in lymphomagenesis. Our data indicate that inhibition of GCN5 HAT activity reduces the tumorigenic properties of human Burkitt lymphoma cells by attenuating BCR signaling and that GCN5 may be a viable target for lymphoma drug therapy.

Complementary Roles of GCN5 and PCAF in Foxp3+ T-Regulatory Cells.

Functions of the GCN5-related N-acetyltransferase (GNAT) family of histone/protein acetyltransferases (HATs) in Foxp3+ T-regulatory (Treg) cells are unexplored, despite the general importance of these enzymes in cell biology. We now show that two prototypical GNAT family members, GCN5 (general control nonrepressed-protein 5, lysine acetyltransferase (KAT)2a) and p300/CBP-associated factor (p300/CBP-associated factor (PCAF), Kat2b) contribute to Treg functions through partially distinct and partially overlapping mechanisms. Deletion of Gcn5 or PCAF did not affect Treg development or suppressive function in vitro, but did affect inducible Treg (iTreg) development, and in vivo, abrogated Treg-dependent allograft survival. Contrasting effects were seen upon targeting of each HAT in all T cells; mice lacking GCN5 showed prolonged allograft survival, suggesting this HAT might be a target for epigenetic therapy in allograft recipients, whereas transplants in mice lacking PCAF underwent acute allograft rejection. PCAF deletion also enhanced anti-tumor immunity in immunocompetent mice. Dual deletion of GCN5 and PCAF led to decreased Treg stability and numbers in peripheral lymphoid tissues, and mice succumbed to severe autoimmunity by 3-4 weeks of life. These data indicate that HATs of the GNAT family have contributions to Treg function that cannot be replaced by the functions of previously characterized Treg HATs (CBP, p300, and Tip60), and may be useful targets in immuno-oncology.

USP22 controls multiple signaling pathways that are essential for vasculature formation in the mouse placenta.

USP22, a component of the SAGA complex, is overexpressed in highly aggressive cancers, but the normal functions of this deubiquitinase are not well defined. We determined that loss of USP22 in mice results in embryonic lethality due to defects in extra-embryonic placental tissues and failure to establish proper vascular interactions with the maternal circulatory system. These phenotypes arise from abnormal gene expression patterns that reflect defective kinase signaling, including TGFß and several receptor tyrosine kinase pathways. USP22 deletion in endothelial cells and pericytes that are induced from embryonic stem cells also hinders these signaling cascades, with detrimental effects on cell survival and differentiation as well as on the ability to form vessels. Our findings provide new insights into the functions of USP22 during development that may offer clues to its role in disease states.

TGFß-Activated USP27X Deubiquitinase Regulates Cell Migration and Chemoresistance via Stabilization of Snail1.

In cancer cells, epithelial-to-mesenchymal transition (EMT) is controlled by Snail1, a transcriptional factor also required for the activation of cancer-associated fibroblasts (CAF). Snail1 is short-lived in normal epithelial cells as a consequence of its coordinated and continuous ubiquitination by several F-box-specific E3 ligases, but its degradation is prevented in cancer cells and in activated fibroblasts. Here, we performed an siRNA screen and identified USP27X as a deubiquitinase that increases Snail1 stability. Expression of USP27X in breast and pancreatic cancer cell lines and tumors positively correlated with Snail1 expression levels. Accordingly, downregulation of USP27X decreased Snail1 protein in several tumor cell lines. USP27X depletion impaired Snail1-dependent cell migration and invasion and metastasis formation and increased cellular sensitivity to cisplatin. USP27X was upregulated by TGFß during EMT and was required for TGFß-induced expression of Snail1 and other mesenchymal markers in epithelial cells and CAF. In agreement with this, depletion of USP27X prevented TGFß-induced EMT and fibroblast activation. Collectively, these results indicate that USP27X is an essential protein controlling Snail1 expression and function and may serve as a target for inhibition of Snail1-dependent tumoral invasion and chemoresistance. SIGNIFICANCE: These findings show that inhibition of USP27X destabilizes Snail1 to impair EMT and renders tumor cells sensitive to chemotherapy, thus opening new strategies for the inhibition of Snail1 expression and its protumoral actions.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/1/33/F1.large.jpg.

Cross-talk between chromatin acetylation and SUMOylation of tripartite motif-containing protein 24 (TRIM24) impacts cell adhesion.

Proteins with domains that recognize and bind post-translational modifications (PTMs) of histones are collectively termed epigenetic readers. Numerous interactions between specific reader protein domains and histone PTMs and their regulatory outcomes have been reported, but little is known about how reader proteins may in turn be modulated by these interactions. Tripartite motif-containing protein 24 (TRIM24) is a histone reader aberrantly expressed in multiple cancers. Here, our investigation revealed functional cross-talk between histone acetylation and TRIM24 SUMOylation. Binding of TRIM24 to chromatin via its tandem PHD-bromodomain, which recognizes unmethylated lysine 4 and acetylated lysine 23 of histone H3 (H3K4me0/K23ac), led to TRIM24 SUMOylation at lysine residues 723 and 741. Inactivation of the bromodomain, either by mutation or with a small-molecule inhibitor, IACS-9571, abolished TRIM24 SUMOylation. Conversely, inhibition of histone deacetylation markedly increased TRIM24's interaction with chromatin and its SUMOylation. Of note, gene expression profiling of MCF7 cells expressing WT versus SUMO-deficient TRIM24 identified cell adhesion as the major pathway regulated by the cross-talk between chromatin acetylation and TRIM24 SUMOylation. In conclusion, our findings establish a new link between histone H3 acetylation and SUMOylation of the reader protein TRIM24, a functional connection that may bear on TRIM24's oncogenic function and may inform future studies of PTM cross-talk between histones and epigenetic regulators.

Histone modification profiling in breast cancer cell lines highlights commonalities and differences among subtypes.

BACKGROUND: Epigenetic regulators are frequently mutated or aberrantly expressed in a variety of cancers, leading to altered transcription states that result in changes in cell identity, behavior, and response to therapy. RESULTS: To define alterations in epigenetic landscapes in breast cancers, we profiled the distributions of 8 key histone modifications by ChIP-Seq, as well as primary (GRO-seq) and steady state (RNA-Seq) transcriptomes, across 13 distinct cell lines that represent 5 molecular subtypes of breast cancer and immortalized human mammary epithelial cells. DISCUSSION: Using combinatorial patterns of distinct histone modification signals, we defined subtype-specific chromatin signatures to nominate potential biomarkers. This approach identified AFAP1-AS1 as a triple negative breast cancer-specific gene associated with cell proliferation and epithelial-mesenchymal-transition. In addition, our chromatin mapping data in basal TNBC cell lines are consistent with gene expression patterns in TCGA that indicate decreased activity of the androgen receptor pathway but increased activity of the vitamin D biosynthesis pathway. CONCLUSIONS: Together, these datasets provide a comprehensive resource for histone modification profiles that define epigenetic landscapes and reveal key chromatin signatures in breast cancer cell line subtypes with potential to identify novel and actionable targets for treatment.

GCN5 Regulates FGF Signaling and Activates Selective MYC Target Genes during Early Embryoid Body Differentiation.

Precise control of gene expression during development is orchestrated by transcription factors and co-regulators including chromatin modifiers. How particular chromatin-modifying enzymes affect specific developmental processes is not well defined. Here, we report that GCN5, a histone acetyltransferase essential for embryonic development, is required for proper expression of multiple genes encoding components of the fibroblast growth factor (FGF) signaling pathway in early embryoid bodies (EBs). Gcn5-/- EBs display deficient activation of ERK and p38, mislocalization of cytoskeletal components, and compromised capacity to differentiate toward mesodermal lineage. Genomic analyses identified seven genes as putative direct targets of GCN5 during early differentiation, four of which are cMYC targets. These findings established a link between GCN5 and the FGF signaling pathway and highlighted specific GCN5-MYC partnerships in gene regulation during early differentiation.

Enhancer transcription reveals subtype-specific gene expression programs controlling breast cancer pathogenesis.

Noncoding transcription is a defining feature of active enhancers, linking transcription factor (TF) binding to the molecular mechanisms controlling gene expression. To determine the relationship between enhancer activity and biological outcomes in breast cancers, we profiled the transcriptomes (using GRO-seq and RNA-seq) and epigenomes (using ChIP-seq) of 11 different human breast cancer cell lines representing five major molecular subtypes of breast cancer, as well as two immortalized ("normal") human breast cell lines. In addition, we developed a robust and unbiased computational pipeline that simultaneously identifies putative subtype-specific enhancers and their cognate TFs by integrating the magnitude of enhancer transcription, TF mRNA expression levels, TF motif P-values, and enrichment of H3K4me1 and H3K27ac. When applied across the 13 different cell lines noted above, the Total Functional Score of Enhancer Elements (TFSEE) identified key breast cancer subtype-specific TFs that act at transcribed enhancers to dictate gene expression patterns determining growth outcomes, including Forkhead TFs, FOSL1, and PLAG1. FOSL1, a Fos family TF, (1) is highly enriched at the enhancers of triple negative breast cancer (TNBC) cells, (2) acts as a key regulator of the proliferation and viability of TNBC cells, but not Luminal A cells, and (3) is associated with a poor prognosis in TNBC breast cancer patients. Taken together, our results validate our enhancer identification pipeline and reveal that enhancers transcribed in breast cancer cells direct critical gene regulatory networks that promote pathogenesis.

YEATS2 links histone acetylation to tumorigenesis of non-small cell lung cancer.

Recognition of modified histones by "reader" proteins constitutes a key mechanism regulating diverse chromatin-associated processes important for normal and neoplastic development. We recently identified the YEATS domain as a novel acetyllysine-binding module; however, the functional importance of YEATS domain-containing proteins in human cancer remains largely unknown. Here, we show that the YEATS2 gene is highly amplified in human non-small cell lung cancer (NSCLC) and is required for cancer cell growth and survival. YEATS2 binds to acetylated histone H3 via its YEATS domain. The YEATS2-containing ATAC complex co-localizes with H3K27 acetylation (H3K27ac) on the promoters of actively transcribed genes. Depletion of YEATS2 or disruption of the interaction between its YEATS domain and acetylated histones reduces the ATAC complex-dependent promoter H3K9ac levels and deactivates the expression of essential genes. Taken together, our study identifies YEATS2 as a histone H3K27ac reader that regulates a transcriptional program essential for NSCLC tumorigenesis.