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.

ATXN7L3 and ENY2 Coordinate Activity of Multiple H2B Deubiquitinases Important for Cellular Proliferation and Tumor Growth.

Histone H2B monoubiquitination (H2Bub1) is centrally involved in gene regulation. The deubiquitination module (DUBm) of the SAGA complex is a major regulator of global H2Bub1 levels, and components of this DUBm are linked to both neurodegenerative diseases and cancer. Unexpectedly, we find that ablation of USP22, the enzymatic center of the DUBm, leads to a reduction, rather than an increase, in global H2bub1 levels. In contrast, depletion of non-enzymatic components, ATXN7L3 or ENY2, results in increased H2Bub1. These observations led us to discover two H2Bub1 DUBs, USP27X and USP51, which function independently of SAGA and compete with USP22 for ATXN7L3 and ENY2 for activity. Like USP22, USP51 and USP27X are required for normal cell proliferation, and their depletion suppresses tumor growth. Our results reveal that ATXN7L3 and ENY2 orchestrate activities of multiple deubiquitinating enzymes and that imbalances in these activities likely potentiate human diseases including cancer.

Histone H3K4 methylation regulates deactivation of the spindle assembly checkpoint through direct binding of Mad2.

Histone H3 methylation on Lys4 (H3K4me) is associated with active gene transcription in all eukaryotes. In Saccharomyces cerevisiae, Set1 is the sole lysine methyltransferase required for mono-, di-, and trimethylation of this site. Although H3K4me3 is linked to gene expression, whether H3K4 methylation regulates other cellular processes, such as mitosis, is less clear. Here we show that both Set1 and H3K4 mutants display a benomyl resistance phenotype that requires components of the spindle assembly checkpoint (SAC), including Bub3 and Mad2. These proteins inhibit Cdc20, an activator of the anaphase-promoting complex/cyclosome (APC/C). Mutations in Cdc20 that block Mad2 interactions suppress the benomyl resistance of both set1 and H3K4 mutant cells. Furthermore, the HORMA domain in Mad2 directly binds H3, identifying a new histone H3 "reader" motif. Mad2 undergoes a conformational change important for execution of the SAC. We found that the closed (active) conformation of both yeast and human Mad2 is capable of binding methylated H3K4, but, in contrast, the open (inactive) Mad2 conformation limits interaction with methylated H3. Collectively, our data indicate that interactions between Mad2 and H3K4 regulate resolution of the SAC by limiting closed Mad2 availability for Cdc20 inhibition.

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.

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.

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.

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.

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.

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.

Cloning, chromosomal organization and expression analysis of Neurl, the mouse homolog of Drosophila melanogaster neuralized gene.

The Drosophila neuralized (neur) gene belongs to the neurogenic group of genes involved in regulating cell-cell interactions required for neural precursor development. neur mutant phenotypes include strong overcommitment to neural fates at the expense of epidermal fates. The human neuralized homolog (NEURL) has been recently determined and found to map to chromosome 10q25.1 within the region frequently deleted in malignant astrocytomas. Because of its potential importance in developmental processes, we analyzed the structure of the mouse homolog, Neurl, and its expression pattern in embryonic tissues. Neurl activity is detected from early developmental stages in several tissues and organs including neural tissues, limbs, the skeletal system, sense organs and internal organs undergoing epithelial-mesenchymal interactions. Neurl encodes a polypeptide associated with the plasma membrane but also detected in the cytoplasm. Similarly to the Drosophila gene, mammalian neuralized may code for an important regulatory factor.

Poly(Q) Expansions in ATXN7 Affect Solubility but Not Activity of the SAGA Deubiquitinating Module.

Spinocerebellar ataxia type 7 (SCA7) is a debilitating neurodegenerative disease caused by expansion of a polyglutamine [poly(Q)] tract in ATXN7, a subunit of the deubiquitinase (DUB) module (DUBm) in the SAGA complex. The effects of ATXN7-poly(Q) on DUB activity are not known. To address this important question, we reconstituted the DUBm in vitro with either wild-type ATXN7 or a pathogenic form, ATXN7-92Q NT, with 92 Q residues at the N terminus (NT). We found that both forms of ATXN7 greatly enhance DUB activity but that ATXN7-92Q NT is largely insoluble unless it is incorporated into the DUBm. Cooverexpression of DUBm components in human astrocytes also promoted the solubility of ATXN7-92Q, inhibiting its aggregation into nuclear inclusions that sequester DUBm components, leading to global increases in ubiquitinated H2B (H2Bub) levels. Global H2Bub levels were also increased in the cerebellums of mice in a SCA7 mouse model. Our findings indicate that although ATXN7 poly(Q) expansions do not change the enzymatic activity of the DUBm, they likely contribute to SCA7 by initiating aggregates that sequester the DUBm away from its substrates.

Histone-modifying enzymes: regulators of developmental decisions and drivers of human disease.

Precise transcriptional networks drive the orchestration and execution of complex developmental processes. Transcription factors possessing sequence-specific DNA binding properties activate or repress target genes in a step-wise manner to control most cell lineage decisions. This regulation often requires the interaction between transcription factors and subunits of massive protein complexes that bear enzymatic activities towards histones. The functional coupling of transcription proteins and histone modifiers underscores the importance of transcriptional regulation through chromatin modification in developmental cell fate decisions and in disease pathogenesis.

The role of deubiquitinating enzymes in chromatin regulation.

Post-translational modifications of the histones are centrally involved in the regulation of all DNA-templated processes, including gene transcription, DNA replication, recombination, and repair. These modifications are often dynamic, and their removal is just as important as their addition in proper regulation of cellular functions. Although histone acetylation/deacetylation and histone methylation/demethylation are highly studied, the functions and regulation of histone ubiquitination and deubiquitination are less well understood. This review highlights our current understanding of how histone ubiquitination impacts gene transcription, DNA repair, and cell cycle progression, and stresses the importance of deubiquitinases to normal cellular functions as well as to disease states such as cancer.

Ubp8 and SAGA regulate Snf1 AMP kinase activity.

Posttranslational modifications of histone proteins play important roles in the modulation of gene expression. The Saccharomyces cerevisiae (yeast) 2-MDa SAGA (Spt-Ada-Gcn5) complex, a well-studied multisubunit histone modifier, regulates gene expression through Gcn5-mediated histone acetylation and Ubp8-mediated histone deubiquitination. Using a proteomics approach, we determined that the SAGA complex also deubiquitinates nonhistone proteins, including Snf1, an AMP-activated kinase. Ubp8-mediated deubiquitination of Snf1 affects the stability and phosphorylation state of Snf1, thereby affecting Snf1 kinase activity. Others have reported that Gal83 is phosphorylated by Snf1, and we found that deletion of UBP8 causes decreased phosphorylation of Gal83, which is consistent with the effects of Ubp8 loss on Snf1 kinase functions. Overall, our data indicate that SAGA modulates the posttranslational modifications of Snf1 in order to fine-tune gene expression levels.

Multiple faces of the SAGA complex.

The SAGA complex provides a paradigm for multisubunit histone modifying complexes. Although first characterized as a histone acetyltransferase, because of the Gcn5 subunit, SAGA is now known to contain a second activity, a histone deubiquitinase, as well as subunits important for interactions with transcriptional activators and the general transcription machinery. The functions of SAGA in transcriptional activation are well-established in Saccharomyces cerevisiae. Recent studies in S. pombe, Drosophila, and mammalian systems reveal that SAGA also has important roles in transcript elongation, the regulation of protein stability, and telomere maintenance. These functions are essential for normal embryo development in flies and mice, and mutations or altered expression of SAGA subunits correlate with neurological disease and aggressive cancers in humans.

Neuralized-like 1 (Neurl1) targeted to the plasma membrane by N-myristoylation regulates the Notch ligand Jagged1.

Notch signaling constitutes an evolutionarily conserved mechanism that mediates cell-cell interactions in various developmental processes. Numerous regulatory proteins interact with the Notch receptor and its ligands and control signaling at multiple levels. Ubiquitination and endocytosis followed by endosomal sorting of both the receptor and its ligands is essential for Notch-mediated signaling. The E3 ubiquitin ligases, Neuralized (Neur) and Mind Bomb (Mib1), are crucial for regulating the activity and stability of Notch ligands in Drosophila; however, biochemical evidence that the Notch ligands are directly targeted for ubiquitination by Neur and/or Mib1 has been lacking. In this report, we explore the function of Neurl1, a mouse ortholog of Drosophila Neur. We show that Neurl1 can function as an E3 ubiquitin ligase to activate monoubiquitination in vitro of Jagged1, but not other mammalian Notch ligands. Neurl1 expression decreases Jagged1 levels in cells and blocks signaling from Jagged1-expressing cells to neighboring Notch-expressing cells. We demonstrate that Neurl1 is myristoylated at its N terminus, and that myristoylation of Neurl1 targets it to the plasma membrane. Point mutations abolishing either Neurl1 myristoylation and plasma membrane localization or Neurl1 ubiquitin ligase activity impair its ability to down-regulate Jagged1 expression and to block signaling. Taken together, our results argue that Neurl1 at the plasma membrane can affect the signaling activity of Jagged1 by directly enhancing its ubiquitination and subsequent turnover.