Evangelia Koutelou

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.

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.

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.

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

ABSTRACT 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.

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

ABSTRACT 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.

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

Summary 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.

Abstract SY24-01: A SAGA of GCN5 and USP22 in stem cells and cancer

Histone modifying enzymes are important creating and maintaining epigenetic programs that regulate cell identity and growth. Our lab uses genetic approaches to define the full spectrum of functions for these enzymes. For example, we have created a series of mutations in the mouse Gcn5 (KAT2A) gene in order to define the functions of this histone acetyltransferase (AT) in a mammalian system. Gcn5 is the catalytic subunit of the SAGA and ATAC complexes. Deletion of Gcn5 led to early embryonic death and to telomere dysfunction. We are now defining the role of Gcn5 and SAGA in maintenance of pluripotency in mouse ES cells and ES cell differentiation. Our data indicate that Gcn5 is an important cofactor for both Myc and E2F family transcription factors, predicting a role for Gcn5 not only in self renewal of ES cells but also in Myc-driven cancers. Our latest studies are defining the functions of the SAGA deubiquitinase module, and these are revealing new information about SAGA composition and function during mouse development and in human diseases. Citation Format: Sharon Y. R. Dent, Boyko Atanassov, Calley Hirsch, Evangelia Koutelou, Zeynep Coban, Xian Jiang Lan, Li Wang. A SAGA of GCN5 and USP22 in stem cells and cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr SY24-01. doi:10.1158/1538-7445.AM2014-SY24-01

Abstract IA07: New functions for histone modifying enzymes

Histone modifying enzymes are important creating and maintaining epigenetic programs that regulate cell identity and growth. Mutations or abnormal expression of these proteins are associated with multiple cancers. Our lab uses genetic approaches to define the full spectrum of functions for these enzymes. For example, we have created a series of mutations in the mouse Gcn5 gene in order to define the functions of this histone acetyltransferase (HAT) in a mammalian system. Deletion of Gcn5 led to early embryonic death and to telomere dysfunction. Biochemical studies revealed that depletion of Gcn5 or ubiquitin specific protease 22 (Usp22), which is another bona fide component of the Gcn5-containing SAGA-type complex, increases the turnover of two shelterin proteins, TRF1 and POT1a (Atanassov, 2009). Our studies provided the first indication that Gcn5 and mammalian SAGA influence telomere maintenance and the first demonstration that SAGA affects protein stability. We are now defining the role of Gcn5 and SAGA in maintenance of pluripotency in ES cells and ES cell differentiation. Our data indicate that Gcn5 is an important cofactor for both Myc and E2F family transcription factors. We are also identifying additional USP22 protein substrates and are defining USP22 functions during mouse development. We have also discovered unexpected functions for the Set1 lysine methyltransferase, which is highly homologous with the MLL protein, during mitosis. We found that Set1 methylates kinetochore proteins, such as Dam1, in yeast, and we have determined that H2Bub is required in trans for methylation of this protein. This work provides the first example of cross talk between a histone modification and a modification in a non-histone protein, defining a new mode of “chromatin signaling.” Zhang K, Lin W, Latham JA, Riefler GM, Schumacher JM, Chan C, Tatchell K, Hawke DH, Kobayashi R, Dent SY. The Set1 methyltransferase opposes Ipl1 aurora kinase functions in chromosome segregation. Cell 122(5):723-34, 9/2005. PMCID: PMC1794220 Atanassov BS, Evrard YA, Multani AS, Zhang Z, Tora L, Devys D, Chang S, Dent SY. Gcn5 and SAGA Regulate Shelterin Protein Turnover and Telomere Maintenance. Mol Cell 35(3):352-364, 8/2009. PMCID: PMC2749492. Atanassov BS, Dent SY. USP22 regulates cell proliferation by deubiquitinating the transcriptional regulator FBP1. EMBO Rep 12(9):924-30, 9/2011. e-Pub 9/2011. PMCID: PMC3166460. Latham JA, Chosed RJ, Wang S, Dent SY. Chromatin signaling to kinetochores: Trans-regulation of Dam1 methylation by histone H2B ubiquitination. Cell 146(5):709-19, 9/2011. PMCID: PMC3168986. Citation Format: Sharon Y.R. Dent, Boyko Atanassov, Calley Hirsch, Evangelia Koutelou, John Latham. New functions for histone modifying enzymes. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA07.