Epigenetic and non-epigenetic functions of the RYBP protein in development and disease

Accepted Manuscript Title: Epigenetic and non-epigenetic functions of the RYBP protein in development and disease Authors: Carolina J. Simoes da Silva, Roc´ıo Sim´on, Ana Busturia PII: DOI: Reference:

S0047-6374(17)30238-5 https://doi.org/10.1016/j.mad.2018.03.011 MAD 11042

To appear in:

Mechanisms of Ageing and Development

Received date: Revised date: Accepted date:

28-9-2017 22-3-2018 26-3-2018

Please cite this article as: da Silva CJS, Sim´on R, Busturia A, Epigenetic and nonepigenetic functions of the RYBP protein in development and disease, Mechanisms of Ageing and Development (2010), https://doi.org/10.1016/j.mad.2018.03.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Epigenetic and non-epigenetic functions of the RYBP protein in development and disease

Carolina J. Simoes da Silva1*, Rocío Simón1* and Ana Busturia1**

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Key words: RYBP/dRYBP; development; ubiquitin binding protein; Polycomb/trithorax; epigenetic; cancer.

**corresponding

author: [email protected]

office: +34 911 964 689

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1Centro

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Fax: +34 911 964 420

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laboratory:+ 34 911 964 690

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contribution

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Short title: The RYBP/dRYBP protein

de Biología Molecular “Severo Ochoa” CSIC-UAM.

Nicolás Cabrera 1

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28049 Madrid, Spain

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Abstract Over the last decades significant advances have been made in our understanding of the molecular mechanisms controlling organismal development. Among these mechanisms the knowledge gained on the roles played by epigenetic regulation of gene expression is extensive. Epigenetic control of transcription requires the function of protein complexes whose specific biochemical activities, such as histone mono-ubiquitylation, affect chromatin compaction and, consequently activation or repression of gene expression.

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Complexes composed of Polycomb Group (PcG) proteins promote transcriptional

silencing while those containing trithorax group (trxG) proteins promote transcriptional

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activation. However, other epigenetic protein factors, such as RYBP, have the ability to interact with both PcG and trxG and thus putatively participate in the reversibility of chromatin compaction, essential to respond to developmental cues and stress signals. This review discusses the developmental and mechanistic functions of RYBP, a

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ubiquitin binding protein, in epigenetic control mediated by the PcG/trxG proteins to

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control transcription. Recent experimental evidence indicates that proteins regulating chromatin compaction also participate in other molecular mechanisms controlling

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development, such as cell death. This review also discusses the role of RYBP in

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apoptosis through non-epigenetic mechanisms as well as recent investigations linking

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the role of RYBP to apoptosis and cancer.

Abbreviations: RYBP (Ring1 and Ying Yang 1 binding protein), dRYBP (drosophila Ring1 and Ying Yang 1 binding protein), PcG (Polycomb Group), trxG (trithorax

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Group), PRC1/2 (Polycomb Repressive Complex 1/2), PHO/YY1 (Polyhomeotic/Ying Yang 1), PRE (Polycomb Response Element), TRE (Trithorax Response Element), ETP

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(Enhancer of Trithorax and Polycomb), Ad-RYBP (Adenoviral-RYBP), HR (homologous recombination), H2Aub (Histone2A mono-ubiquitylation), H2Bub

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(Histone2B mono-ubiquitylation),

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Contents 1-

Introduction.

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Polycomb and Trithorax proteins.

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The RYBP/dRYBP protein.

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Function of RYBP/dRYBP during development.

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The role of dRYBP/RYBP in epigenetic mechanisms: interaction with PcG and

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trxG proteins and their function in development.

The role of dRYBP/RYBP in non-epigenetic mechanisms: interaction with cell death related proteins and their function in apoptosis.

Correlation between variations in RYBP levels and disease development.

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Conclusions and perspectives.

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Conflict of interest

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Acknowledgements

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References

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1. Introduction Epigenetic regulation of gene expression has been a frequently reviewed theme over the last decade. This is due to the tremendous increase in publications in the field roughly 50,000 in the last 10 years! This great output illustrates its biological importance and relevance in normal and pathological development, including the opportunity to develop “Epi-drugs” to target epigenetic factors in the treatment of many diseases including cancer.

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In this review, we present and discuss the function of the RYBP (Ring1 and YY1 Binding Protein) protein, a phylogenetically conserved epigenetic factor with ubiquitin

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binding activity, which is crucial for development. Most of the research on RYBP’s

function has been performed in mice and Drosophila (where the protein is known as dRYBP), as developmental model systems. Until recently, it was thought that the epigenetically implemented “silenced” or “active” transcriptional states were fixed and

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permanently inherited. However, it now appears that these transcriptional states can be

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dynamically changed thus indicating reversible epigenetic regulation. Here, we discuss studies showing that the RYBP/dRYBP protein is essential for proper development and

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functions in epigenetic regulation as well as studies in flies showing that it has the dependent mechanisms.

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capacity to modulate the “silenced” or “active” transcriptional states by chromatin

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The biological pathways controlling programed cell death/developmental apoptosis as well as those controlling stress induced P53 mediated apoptosis are conserved. In this review, we additionally discuss evidence supporting the roles of the RYBP/dRYBP protein in apoptosis by chromatin- and transcription- independent

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mediated mechanisms. Furthermore, work in flies and mice support the importance of maintaining, perhaps through microRNAs regulation, homeostatic levels of

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RYBP/dRYBP to achieve normal development. Investigations proposing a role for microRNAs in the control of RYBP/dRYBP levels as well as results showing its role in

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cancer are discussed. 2. Polycomb and Trithorax proteins Epigenetic factors i.e., factors that regulate heritable changes in gene function through mechanisms that do not involve changes in DNA sequence (Hendrich &Willard, 1995), include silencing proteins such as those of the Polycomb Group (PcG) and activating factors such as those of the trithorax group (trxG). These proteins were 4

initially discovered in Drosophila based on their function in the regulation of homeotic genes expression. Later it was shown that both the sequence and the function of these proteins were phylogenetically conserved (recently reviewed in (Kassis et al., 2017) and (Schuettengruber et al., 2017)) and it is now widely accepted the fundamental importance of PcG/trxG in the regulation of embryogenesis, growth and adult life homeostasis. PcG and TrxG proteins function in multi-protein complexes by altering chromatin

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structure and compaction through biochemical activity that post-translationally modify

histones. Two main canonical PcG complexes that contribute to chromatin compaction,

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required for transcriptional repression, have been described in flies and vertebrates:

Polycomb Repressive Complex 1 (PRC1, composed of the core components PH/PHC13, PC/CBX2 (4, 6-8), PSC/PCGF1-6, SCE/RING1A-B; names indicated as

fly/vertebrate counterparts) and 2 (PRC2, composed of the core components ESC/EED,

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EZH2/EZH1-2, SUZ12/SUZ12). PRC2 complex is responsible for di- and tri-

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methylation of the Lysine (K) 27 of histone H3 (H3K27me2/3) via its enzymatic subunit EZH2. The PRC1 complex mono-ubiquitylates Lysine 118 in flies and 119 in

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vertebrates of histone H2A (H2AK119ub) via the E3-ubiquitin ligase SCE/RING1A

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subunit. Several trxG complexes have been isolated (recently reviewed in (Kassis et al., 2017)). Among those, the COMPASS family complexes functions to mono-and di-

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methylate Lysine 4 of histone H3 (H3K4me1/2), a prerequisite for the monoubiquitylation of histone H2B by the E3-Ubiquitin ligase Bre1 complex (Mohan et al., 2011), reviewed in (Shilatifard, 2012)) that leads to the expansion of chromatin and the activation of transcription. These protein complexes containing PcG and trxG interact

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with PREs (Polycomb Response Elements) and TREs (Trithorax Response Elements), DNA cis-regulatory elements found at target genes that serve as DNA platforms for

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protein complexes recruitment and subsequent chromatin modification. Moreover, it has been shown in flies that targeting of the respective protein complexes to DNA is

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mediated by PcG/trxG proteins with DNA binding activity, for example PHO (the fly ortholog of the vertebrate YY1 gene) (Brock & Fisher, 2005; Brown et al., 1998; Busturia et al., 2001; Busturia et al., 1997; Fritsch et al., 1999). PREs and TREs in the fly genome have been thoroughly characterized experimentally (reviewed in (Kassis et al., 2017)). Extensive efforts to characterize equivalent elements in vertebrate genomes (Farcas et al., 2012; Herzog et al., 2014; Li et al., 2017b; Woo et al., 2010) indicate that

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CpG islands may function as PREs and TREs (for recent reviews see (Bauer et al., 2016; Blackledge et al., 2015). A less extensively studied group of proteins first discovered in Drosophila (Bejarano & Busturia, 2004; Busturia et al., 2001; Faucheux et al., 2003, reviewed in (Kassis et al., 2017)) exists whose protein members are referred to as Enhancers of Trithorax and Polycomb, or ETP’s. In Drosophila, inactivation of ETP function results in PcG inactivation-related phenotypes and trxG inactivation-related phenotypes.

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Importantly, mutations in ETP genes are able to modulate and counteract either the PcG or the trxG inactivation related phenotypes. Thus, ETPs seem to play an important role

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in the reversibility of the epigenetic regulation of transcription. The ETP protein RYBP/dRYBP is the main subject of this review. 3. The RYBP/dRYBP protein

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The RYBP (Ring1 and YY1 binding protein) protein, also known as DEDAF

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(Zheng et al., 2001) and YEAF1 (Sawa et al., 2002) was first identified in a screen designed to search for proteins interacting with RING1, a E3-ubiquitin ligase (Garcia et

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al., 1999) and a core component of the PRC1 complex (Cao et al., 2005; Gutierrez et al.,

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2012; Wang et al., 2004). The high degree of sequence similarity with YAF2 (YY1 Associated Factor 2), a human protein previously isolated in a search for YY1

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interacting proteins (Kalenik et al., 1997), prompted Vidal´s group to study RYBP’s interaction with YY1. They found that indeed RYBP not only interacted with RING1 but also with YY1, thus forming the RYBP/YAF2 family. RYBP/dRYBP is phylogenetically conserved (Adams et al., 2000; Bejarano et al., 2005) and the fly

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dRYBP was shown to interact with SCE (the RING1 fly homolog) and with PHO (the YY1 fly homolog) (Fereres et al., 2014; Gonzalez et al., 2008).

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RYBP/dRYBP is a nuclear protein that is expressed at very early stages of mouse

(Eid & Torres-Padilla, 2016; Garcia et al., 1999) and fly development (Gonzalez et al.,

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2008) and its expression can be detected in different tissues and levels throughout development (Eid &Torres-Padilla, 2016; Garcia et al., 1999; Gonzalez et al., 2008; Pirity et al., 2005). RYBP/dRYBP is a relatively small protein (mammalian protein 227aa (Garcia et al., 1999); Drosophila protein 150aa (Bejarano et al., 2005)) whose Nterminal domain contains a conserved nucleoporin type C2-C2 Zn finger domain, a motif associated with protein-protein interactions (Meyer et al., 2000). A portion of this domain contains a specific motif surrounding the Zinc coordination site which shows 6

ubiquitin binding activity (Alam et al., 2004). Indeed, it has been shown that RYBP/dRYBP is itself ubiquitylated and binds Ubiquitin (Arrigoni et al., 2006; Fereres et al., 2014). Due to its interaction with PcG proteins, the study of RYBP/dRYBP function was initially focused on its possible function in the epigenetic regulation of gene expression. Moreover, its interaction with YY1/PHO prompted the investigations of RYBP as a

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putative recruiter of PcG complexes to chromatin. 4. Function of RYBP/dRYBP during development

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RYBP knockdown mice display a variety of phenotypes. RYBP has been reported to be essential for early embryonic development as it is required for survival of the embryo, for the establishment of functional extra-embryonic tissues, for neural tube and

neocortex development and for the completion of decidualization, a process required for

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embryo implantation (Bian et al., 2016; Pirity et al., 2005). Moreover, analysis of

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RYBP function in the mouse eye indicates that it is required for the retinal closure and lens development (Pirity et al., 2007). Work from several laboratories indicates a role

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for RYBP in the differentiation of embryonic stem cells (ESCs) - analyses of RYBP null

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ESCs demonstrated that RYBP is required for cardiomyocytes (CMCs) differentiation (Ujhelly et al., 2015) as well as for neural differentiation (Kovacs et al., 2016).

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Consistent with these studies, Hisada et al. demonstrated that RYBP is required for neither maintenance of ESCs nor the pluripotent state of ESCs, but is required for ESCs differentiation (Hisada et al., 2012). In skeletal myogenesis, RYBP has been found to be required for repression of the myoblast myogenic differentiation, thus having a role in

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skeletal muscle cell differentiation (Zhou et al., 2012). Extensive functional analysis has also been performed in Drosophila. Lack of

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dRYBP shows a range of phenotypes, which are variable both in penetrance and expressivity. Homozygous null mutants die progressively during development and the

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phenotypes observed in the survivors include blocked oogenesis, embryonic cuticle pattern defects, wing size reduction and leg malformations (Gonzalez et al., 2008). Moreover, clonal analysis of dRYBP mutant wing cells indicates that dRYBP is also required for their differentiation (Gonzalez et al., 2008). Additionally, inactivation of dRYBP produced apoptosis only in few cells indicating only a weak activation of cell death (Fereres et al., 2013). Curiously, dRYBP null mutants do not present homeoticlike phenotypes, an expected trait due to its interaction with PcG and TrxG proteins. 7

However, when combined with mutations in either PcG or trxG genes, their corresponding homeotic-like phenotypes such as increased/decreased number of sex combs (specialized hairs of the male legs) or the male abdominal segments pigmentation (Figure 1) are affected. This was the first indication that dRYBP could interact with both epigenetic silencers and activators (Gonzalez et al., 2008). Furthermore, mutations in the dRYBP gene have been found to counteract the phenotypes associated with both PcG and trxG mutants (Figure 1A and B) (Fereres et

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al., 2014). Thus, the analysis of dRYBP mutants suggested that dRYBP does not behave as classically defined PcG/trxG genes, i.e., dRYBP mutants do not have an obvious

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impact on the expression of homeotic genes unless the genetic analysis is performed in combination with PcG/trxG mutations (Figure 1). Even though inactivation of dRYBP by RNAi does not produce homeotic-like phenotypes, high levels of dRYBP show strong effects on the homeotic gene expression. Importantly this effect is PcG/trxG

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dependent (Bejarano et al., 2005; Gonzalez et al., 2008).

5. The role of RYBP/dRYBP in epigenetic mechanisms: interactions with

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PcG/trxG proteins and their function in development

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The structure of RYBP/dRYBP led to the suggestion that it is involved in protein ubiquitylation. This, combined with the importance of this post-translational

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modification in the control of epigenetic regulation of transcription and the reported interaction with RING1/SCE and YY1/PHO, first focused the study of RYBP/dRYBP function on its possible role in transcriptional silencing and the recruitment of the PcG complexes to chromatin. Experiments in which RYBP (Garcia et al., 1999) or dRYBP

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(Bejarano et al., 2005) were tethered to DNA indicated that each functions as a PcGdependent transcriptional repressor that, importantly, influences the silencing effect

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produced by PREs (Bejarano et al., 2005). Moreover, RYBP was found to bind to one of the two “PRE-like” sequences found in the vertebrate homeotic HOXD2 gene (Woo

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et al., 2010). However, RYBP has not yet been shown to recruit PcG/trxG to DNA (Basu et al., 2014; Wilkinson et al., 2010). The dRYBP/RYBP interaction with SCE/RING1 strongly suggested that

dRYBP/RYBP is a member of the PRC1 complex. Biochemical analysis focusing on RYBP-containing complexes indicated the existence, both in vertebrates and flies, of several PRC1 complex variants depending on the presence or absence of RYBP/dRYBP (Bauer et al., 2016; Blackledge et al., 2015; Fereres et al., 2014; Gao et al., 2012; 8

Lagarou et al., 2008; Lee et al., 2015; Morey et al., 2013; Schwartz & Pirrotta, 2013; Tavares et al., 2012). In vertebrates, PRC1 complexes are classified as either “canonical” or “non-canonical” depending on the presence of either the chromobox containing CBX subunit (PC in the fly) or RYBP subunit (Bauer et al., 2016; Blackledge et al., 2015; Gao et al., 2012; Lee et al., 2015; Morey et al., 2013; Schwartz & Pirrotta, 2013; Tavares et al., 2012). As proposed by Hisada et al. (Hisada et al., 2012) RYBP and its paralog YAF2 do not form part of the canonical PRC1 complex

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perhaps because of the mutually exclusive association of either RYBP or PRC1

chromobox subunits with RING1 proteins (Wang et al., 2010). Further, the presence of

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RYBP in the “non-canonical” complexes promotes and strongly stimulates

H2AK119ub1 and, thereby, transcriptional repression. Moreover, these non-canonical PRC1-RYBP complexes have been recently found to serve as a “communication bridge” with PRC2 complexes promoting the chromatin compaction required for gene

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silencing. It remains to be determined the distinct functions of the non-canonical PRC1-

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RYBP complexes. Perhaps, RYBP association with specific group of complexes suggests cellular context specificity, precise temporal requirements or particular

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characteristic of target genes (Gao et al., 2012; Morey et al., 2013; Tavares et al., 2012).

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Proteomic and subsequent biochemical analyses in Drosophila have identified a large number of proteins interacting with dRYBP including SCE, dKDM2 and dBre1

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(Fereres et al., 2014) (Table 1). dKDM2 is a member of the dRAF complex (Lagarou et al., 2008) that strongly stimulates H2A mono-ubiquitylation. dBre1 is an E3-ubiquitin ligase required for mono-ubiquitylation of H2B, a histone mark that promotes transcriptional activation (Fereres et al., 2014; Henry et al., 2003; Wood et al., 2003). It

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has been demonstrated that dRYBP is able to modulate repression and activation of transcription by directly modifying levels of H2Aub and H2Bub and promoting their

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mono-ubiquitylation (Fereres et al., 2014). This supported the finding of genetic interactions between dRYBP and PcG and trxG genes (Figure 1) (Bejarano et al., 2005;

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Fereres et al., 2014; Gonzalez et al., 2008; Gonzalez & Busturia, 2009) reinforcing its classification as an ETP protein and suggesting its role in the reversibility of epigenetic regulation (Figure 2A). 6. The role of RYBP/dRYBP in non-epigenetic mechanisms: interaction with cell death related proteins and their function in apoptosis.

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dRYBP/RYBP has been found to interact with proteins other than those of the PcG and trxG (Table 1). Importantly, and the focus of this section, characterization of those interactions is contributing to the understanding of the role dRYBP/RYBP plays not only in the epigenetic mechanisms of gene silencing but also in other biological processes independently of epigenetic mechanisms mediated by PcG/trxG. A role of RYBP/dRYBP in apoptosis is observed when analysing the effect of the modulation of its levels both in vertebrates and flies. Knockout RYBP mice embryos do

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not complete decidualization, a developmental process for which apoptosis is required

(Bian et al., 2016; Pirity et al., 2005). High levels of RYBP in mammalian cells induce

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apoptosis, only in tumor cells (Danen-van Oorschot et al., 2004; Ma et al., 2016; Novak & Phillips, 2008; Stanton et al., 2007). This suggests that in vertebrates RYBP is a proapoptotic protein (Chen et al., 2009; Danen-van Oorschot et al., 2004; Ma et al., 2016; Stanton et al., 2007; Zheng et al., 2001) and its specific tumor killing activity is of

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special interest for cancer therapy. In flies, both low levels of dRYBP and high levels of

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dRYBP induce apoptosis (Fereres et al., 2013; Gonzalez & Busturia, 2009) suggesting that dRYBP functions both as a inhibitor of apoptosis and also as a pro-apoptotic

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protein (see below). Thus, the analyses of mice, flies and cells suggest that

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dRYBP/RYBP, including its YAF2 paralog, (Stanton et al., 2006) have an apoptotic function.

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How does RYBP/dRYBP control cell death? A significant number of RYBP/dRYBP interacting proteins are known to function in cell death (Table 1) and many of these have been shown to be important also for the assembly and activation of inflammatory complexes (Park et al., 2007). RYBP has been found to interact with

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several DED-containing proteins (Valmiki & Ramos, 2009) such as FADD, pro-caspase 8 and 10 and DEDD (Zheng et al., 2001). Furthermore, it has been shown to enhance

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both Death Receptor (CD95-mediated) and DED-mediated apoptosis (Zheng et al., 2001). Similarly, apoptosis induced by high levels of dRYBP in Drosophila is

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dependent of the DED-domain containing proteins dFADD (FADD homologue) and DREDD (Caspase-8 homologue) (Gonzalez & Busturia, 2009). These interactions suggest that RYBP/dRYBP might be regulating apoptosis via the tumor necrosis factor alpha (TNFalpha) receptor family (Chan et al., 2015). On the other hand, the observed interaction of dRYBP with the SCF complex (Fereres et al., 2013), one of the most important super-families of E3-ubiquitin ligases (Cardozo & Pagano, 2004) and the analysis of this interaction in the control of cell 10

death has led to the proposal that upon an apoptotic stimuli -either developmental or stress-related, such as irradiation- dRYBP acts as an ubiquitin adaptor protein. Therefore, the dRYBP/SCF complex facilitates the interaction and/or assembly of SCF complex members (Figure 2B), leading to the ubiquitylation of the pro-apoptotic Reaper protein and its consequently proteasomal degradation, thus inhibiting cell death (Fereres et al., 2013). Curiously, the interaction of dRYBP with the SCF complex members led to the discovery of a role for dRYBP in the inhibition of the innate immune response

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(Aparicio et al., 2013): it has been proposed that dRYBP contributes to the negative

regulation of the IMD signaling pathway by acting, together with SKPA, as an ubiquitin

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adaptor protein to promote SCF-dependent proteasomal degradation of Relish,

inhibiting its translocation to the nucleus and thus impeding Relish transcriptional activation of the IMD pathway effectors.

Several studies have linked dRYBP/RYBP protein and their interaction partners

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with tumor associated apoptosis: interactions with FANK1 (Ma et al., 2016), the viral

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protein Apoptin (Danen-van Oorschot et al., 2004), the E3-ubiquitin ligase MDM2 (Chen et al., 2009). This function is consistent with the reported variations of RYBP

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levels in both hematopoietic and human solid tumor tissues (see below).

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Of special interest to the field of oncology research is the discovery that RYBP, similarly to Apoptin, has a tumor-preferential cell-killing activity, raising the possibility

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that this could be exploited in a gene therapy approach to treat cancer (Danen-van Oorschot et al., 2004). It has been shown that adenoviral-mediated RYBP expression (Ad-RYBP) promotes tumor cell-specific apoptosis supporting the Ad-RYBP approach as a treatment of cancer (Novak & Phillips, 2008). However, the mechanisms as to how

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RYBP is able to induce apoptosis preferentially in tumor cells are poorly understood. Recent results indicate that RYBP negatively regulates the homologous recombination

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(HR) repair pathway (Ali et al., 2018) and that this regulation is independent of transcription. These authors proposed that high levels of RYBP inhibit tumor growth by

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inhibiting DNA repair in malignant cells, an interesting mechanism that may be exploited in cancer therapy. Perhaps, these are similar to the mechanisms, also poorly understood, by which Apoptin induces tumor-cell-specific killing (Danen-van Oorschot et al., 2004). The viral Apoptin protein does not have a cellular counterpart and in flies high levels of dRYBP have a pro-apoptotic effect in non-tumoral cells (Gonzalez & Busturia, 2009). Thus, Drosophila might be a useful model system to analyse the

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mechanisms with which to understand how high levels of RYBP, and Apoptin, induce tumor cells specific apoptosis (Los et al., 2009). Also of special interest in the field of cancer research is the discovery of RYBP/dRYBP interactions with the proteins involved in the process of p53 protein stabilization (Chen et al., 2009). In vertebrates it has been reported that RYBP interacts with the E3-ubiquitin ligase MDM2 to inhibit the MDM2-mediated p53 ubiquitylation, therefore stabilizing p53 and increasing p53 activity (Chen et al., 2009). Chen et al.

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proposed that the interaction between RYBP and MDM2 induces conformational

changes in the MDM2 protein that interfere with the p53 ubiquitylation and subsequent

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proteasomal degradation (Figure 2C). This identifies RYBP as a new regulator of the MDM2-p53 loop and provides evidence that it functions as a tumor suppressor. In Drosophila it has been shown that inactivation of dRYBP inhibits Dp53 induced apoptosis through its interaction with SCE, a E3-ubiquitin ligase. The mechanisms

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involved in this process are not completely clear. One proposal is that dRYBP, together

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with SCE influences Dp53 levels by controlling the stabilization of Dp53 protein by promoting its ubiquitylation and targeting for proteasomal degradation (Figure 2C)

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(Simoes da Silva et al., 2017).

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In summary, the described interactions of RYBP with proteins associated with the regulation of apoptotic pathways and the analysis of its function in this process suggests

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that RYBP/dRYBP controls cell death through non-epigenetic mechanisms. Considering that, 1) RYBP/dRYBP is a ubiquitin binding protein (Arrigoni et al., 2006; Fereres et al., 2014) that may be involved in the assembly of protein complexes (Alam et al., 2004; Gonzalez & Busturia, 2009) and that 2) protein ubiquitylation plays a

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crucial role in apoptosis, immune response and regulation of p53 levels (Bergmann, 2010; Karin & Ben-Neriah, 2000; Shembade et al., 2009; Thompson et al., 2008), it is

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reasonable to propose that RYBP/dRYBP controls cell death/immune response by regulating protein ubiquitylation for proteasomal degradation using a non-epigenetic

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mechanism.

7. Correlation between variations in RYBP levels and disease development There are many published studies linking variations in RYBP levels with the development of specific diseases. Examples outside of the field of cancer include the correlation of low levels of RYBP with Chronic Rhinosinusitis (Zhang et al., 2012) and

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type 2 diabetes (Lee et al., 2017). However, the majority of studies of RYBP and disease have focused on its role in cancer development. Decreases in RYBP levels have been associated with the development of a number of different cancers (Table 2) including hepatocellular carcinoma (Wang et al., 2014; Zhao et al., 2017; Zhu et al., 2017a; Zhu et al., 2017b), lung cancer (Dinglin et al., 2017; Jiang et al., 2016; Voruganti et al., 2015), prostate cancer (Krohn et al., 2013; Taylor et al., 2010; Ulz et al., 2016), breast cancer (Buas et al., 2015; Kenny et al.,

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2017; Zhou et al., 2016), cervical cancer (Lando et al., 2009; Lando et al., 2013),

glioblastoma (Li et al., 2013; Minchenko et al., 2014), as well as in ileal carcinoid

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(Andersson et al., 2009) reviewed in (Nilsson, 2013). Moreover, the genomic region containing the RYBP gene (3p13-14) has been shown to be deleted in a range of

different tumor types (Andersson et al., 2009; Krohn et al., 2013; Lando et al., 2009; Lando et al., 2013; Taylor et al., 2010; Ulz et al., 2016). Importantly, these studies were

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able to show a correlation between loss of RYBP and a worse prognosis in comparison

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with cancer patients without this chromosomal deletion. Additional studies using patient tumor samples, cell lines and mouse xenografts have shown correlations between

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decreased levels of RYBP and increased disease severity and shorter survival times in

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hepatocellular cancer (Wang et al., 2014; Zhao et al., 2017; Zhu et al., 2017b); in glioblastoma (Li et al., 2013); cervical cancer (Lando et al., 2009); in lung cancer

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(Dinglin et al., 2017; Voruganti et al., 2015) and in breast cancer (Kenny et al., 2017; Zhou et al., 2016).

Depending on the cancer type, increased levels of RYBP have been shown to be associated with both beneficial and detrimental effects. Increased levels of RYBP have

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been found in lymphomas and leukemias (Sanchez-Beato et al., 2004; Sasaki et al., 2011; Seitz et al., 2011) and associated with more aggressive cancer forms and poor

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prognosis. However, other studies have shown that high levels of RYBP are harmful for cancer cells: it has been reported that increased levels of RYBP inhibit tumoral growth

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by induction of apoptosis (Andersson et al., 2009; Voruganti et al., 2015; Wang et al., 2014) or by decreasing cell proliferation (Dinglin et al., 2017; Voruganti et al., 2015; Zhou et al., 2016). Also, increased expression of RYBP is associated with increased glioma patient survival times (Li et al., 2013). Finally, hepatocellular and lung cancer tumor cells containing high levels of RYBP show increased sensitivity to chemotherapy (Voruganti et al., 2015; Wang et al., 2014). As stated above, recent results (Ali et al., 2018) indicate that RYBP expression may negatively regulate the homologous 13

recombination (HR) repair pathway and sensitize cancer cells to DNA damage. These authors propose (Ali et al., 2018) that the observed cancer protective function of RYBP (Diglin et al., 2017; Zhou et al., 2016; Wang et al., 2014; Voruganti et al., 2015; Krohn et al., 2013, Lando et al., 2009) may be related to a reduced DNA repair capacity in malignant cells expressing high levels of RYBP. Thus, one can conclude from these studies that, depending on the tumor type, RYBP can have either a tumor repressing or tumor promoting activity thus indicating that it can function as either a tumor

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suppressor gene or as an oncogene.

The question then arises as to how RYBP levels are controlled and how RYBP is

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implicated in cancer initiation and progression. A thorough analysis of the RYBP

promoter region has not yet been performed. However, studies in hepatocellular cancer have identified, in the RYBP promoter functional, SP1 binding sites that act as

activators and functional KLF4 binding sites that act as repressors (Zhao et al., 2017).

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Thus, alteration of the levels of transcription factors that regulate RYBP transcription

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may account for variations of RYBP levels in pathological conditions. Recent studies have shown that RYBP levels are controlled by microRNAs.

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Bioinformatic analyses (www.microRNA.org) show that the 3’-UTRs of both RYBP

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and dRYBP contain a number of microRNA binding sites suggesting that microRNAs contribute to the control RYBP/dRYBP levels. It has been experimentally shown that

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miR-9 (Zhao et al., 2015), miR-27a and miR-27b (Scott et al., 2006), miR-29, miR-1, miR-206 (Zhou et al., 2012), miR-125a (Zhao et al., 2016) and miR-125b (Li et al., 2017a) bind and down-regulate RYBP expression. Interestingly, a microRNA regulatory loop has been proposed that includes YY1, miR-9, miR-29 and miR-125a

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and RYBP. In this proposal, YY1 represses the expression of miR-9, miR-29 and miR125a which down-regulate RYBP expression. Depending on the cellular YY1 levels, the

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regulatory microRNAs will modulate RYBP levels and thus modulate RYBP actions with its binding partner YY1 and/or other interacting proteins (Zhao et al., 2015; Zhao

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et al., 2016; Zhou et al., 2012). The mechanisms by which RYBP influences cancer initiation and/or progression

are far from well understood. On one hand, variations in the levels of RYBP could influence global chromatin compaction and thus global genome transcription. This RYBP action will be executed together with its interacting epigenetic factors whose levels have also been observed to be deregulated in a number of cancers (Mills, 2010; Oktaba et al., 2008; Probst et al., 2009). On the other hand, RYBP has been shown to 14

induce tumor specific apoptosis (Chen et al., 2009; Danen-van Oorschot et al., 2004; Ma et al., 2016; Novak & Phillips, 2008; Stanton et al., 2007) through a mechanism that may involve the reported interaction between RYBP with MDM2 in vertebrates and, in Drosophila, the corresponding SCE protein (Simoes da Silva et al., 2017). This implies a function for RYBP/dRYBP in the process of p53 stabilization (see above). In vertebrates high levels of RYBP recruit MDM2, thus blocking p53 poly-ubiquitylation and proteasomal degradation. This block of P53 degradation then would promote p53

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mediated apoptosis and improve prognosis.

Thus, studies to date demonstrate the importance of maintaining homeostatic

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RYBP levels and raise the possibility of using these levels as a clinical diagnosis

marker. Furthermore, the studies linking increased RYBP levels and tumor cell death suggest the use of RYBP over-expression as a possible cancer treatment (Andersson et al., 2009; Dinglin et al., 2017; Krohn et al., 2013; Voruganti et al., 2015; Wang et al.,

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2014; Zhou et al., 2016). 8. Conclusions and perspectives

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It has been almost 20 years since the discovery of the RYBP protein and the

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research done to analyze its function has mainly focused on its role in development through the epigenetic regulation of transcription. The results from these investigations

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clearly establish RYBP as a potent stimulator of mono-ubiquitylation of Histone2A (H2Aub) and, thus, as a repressor of transcription. Although less extensively analyzed, dRYBP has also been found to stimulate mono-ubiquitylation of (H2Bub) suggesting a role as an activator of transcription. Importantly, dRYBP was shown to modulate both

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repressive and activated transcriptional states (Fereres et al., 2014) (Figure 2A) and control the interplay of repression and activation of homeotic genes during Drosophila

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development. More work is needed to analyze the developmental and/or tissue clues that instruct RYBP to act either as a chromatin dependent repressor/activator. This

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should lead to a deeper understanding of the reversibility of epigenetic regulatory system with an impact on our understanding of disease biology. Proteomic analyses (Fereres et al., 2014;Tavares et al., 2012) as well as analyses

of the function of the described RYBP interactions have revealed that RYBP/dRYBP is involved, most likely through chromatin independent mechanisms, in other processes such as the control of both developmental and stress induced apoptosis (Figure 2B), such as irradiation. Importantly, RYBP has been found to behave as a specific killer of 15

tumor cells and its function in apoptosis may be through the process of p53 stabilization (Figure 2C). Also relevant to its role in cancer, it has been recently found that RYBP inhibits HR through a transcription independent mechanism (Ali et al., 2018). This, along with many other reports describing RYBP function in cancer, establishes RYBP as a candidate to be considered for disease therapy by perhaps modulating its levels. Analysis of the mechanisms of RYBP in apoptosis and as a tumor suppressor will also shed light onto the mechanisms controlling homeostatic RYBP levels including

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transcription initiation through the effect of promoter binding factors and posttranscriptional mechanisms involving microRNAs.

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Finally, understanding the function of RYBP as an ubiquitin binding domaincontaining protein is critical for a mechanistic description of the processes that are under the RYBP’s control. RYBP/dRYBP has been found to self-ubiquitylate, to

interact with ubiquitylated proteins and also to mono-ubiquitylate proteins (Arrigoni et

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al., 2006; Fereres et al., 2014). Ubiquitin binding domains are involved in many

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regulatory mechanisms from protein degradation to vesicular trafficking and epigenetic regulation (Hurley et al., 2006). Protein mono-ubiquitylation has an important impact

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on the regulation of normal and disease development. However, in comparison with the

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study of protein poly-ubiquitylation whose function in most cases is to mark a protein for degradation, the understanding of the function of mono-ubiquitylation is relatively

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incomplete. Analysis of RYBP activity as well as analysis of its targets for monoubiquitylation will help to shed light on this process and its functions. The non-epigenetic functions of RYBP may be relevant for other biological processes such as its function in B-cell development recently shown to be independent

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of PRC1 components (Cales et al., 2015). Also, non-epigenetic functions may also be relevant for the biology of the PcG/trxG proteins. This is the case for both PSC and

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RNF2/SCE, both E3-ubiquitin ligases. PSC has been found to control Cyclin B proteasomal degradation (Mohd-Sarip et al., 2012) and RNF2/SCE has been proposed

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to function in p53 stabilization process (Simoes da Silva et al., 2017; Su et al., 2013; Wen et al., 2014). In summary, the continued study of RYBP/dRYBP and the networks in which it functions will surely prove beneficial to our understanding of the development and control of normal as well as pathological states. Conflict of interest Authors declare that they have no conflict of interest. 16

Aknowledgements We thank Keith Harshman for suggestions and critically reading the manuscript. This work was supported by grants from the Dirección General de Investigación (BFU2008-01154) to A.B., the Consolider Ingenio 2010 Program of the Ministerio de Ciencia e Innovación (CSD 2007-00008) to A.B., and by an institutional grant to the Centro de Biología Molecular Severo Ochoa from the Fundación Ramón

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Areces.

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Figure and Table legends.

Figure 1. dRYBP controls developmental morphogenesis by counteracting both transcriptional repression and activation. The diagrams in A and B represent the segmented dorsal abdomen of a Drosophila male, which is composed of six segments

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(A1-A6, only A4 to A6 are shown). In a wild type male fly, segments A1-A4 are

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weakly pigmented. In contrast segments A5-A6 are strongly pigmented due to the action of the homeotic Abdominal-B (Abd-B) gene that is expressed in the A5 and A6

A

segments and controls their identity. The PcG maintain the repression of the Abd-B gene

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in the A1-A4 segments while the trxG maintain the activation of the Abd-B gene in the A5 and A6 segments. A) In a PcG mutant fly (PcG- ) repression of the Abd-B gene in

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the A4 segment is lost and, as a result, the A4 segment is pigmented indicating its transformation towards the A5 segment (above, red arrow). This phenotype is reverted to wt condition (below, red arrow) when dRYBP is additionally mutated (PcG -, dRYBP ) indicating that dRYBP counteracts the effect of the PcG. B) In a trxG mutant fly (trxG activation of the Abd-B gene in the A5 segment is lost and, as a result, segment A5 is

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-)

de-pigmented indicating its transformation towards the A4 segment (above red arrow).

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This phenotype is reverted to wt condition (below, red arrow) when dRYBP is additionally mutated (trxG -, dRYBP-), indicating that dRYBP counteracts the effect of

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the trxG (model based on data from Fereres et al. 2014). C) Photographs of male abdomens showing A4, A5 and A6 segments. The left panel shows a wt abdomen. Note the strong pigmentation of segments A5 and A6 segments and the weak pigmentation of A4. The middle panel shows a Polycomb-Group mutant (PcG-) abdomen. Note that segment A4 is pigmented, thus resembling A5 and indicating its transformation towards this segment. The right panel shows a trithorax-Group mutant (trxG-) abdomen. Note

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that segment A5 is de-pigmented (arrowhead), thus resembling A4 and indicating its

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transformation towards this segment.

Figure 2. Function of RYBP/dRYBP in epigenetic regulation of gene expression

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and in cell death.

A) RYBP/dRYBP interacts with PcG/trxG proteins to epigenetically control

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transcription. RYBP/dRYBP interacts with PcG proteins and stimulates the monoubiquitylation of H2A (H2Aub) to promote transcriptional repression. Furthermore, it has been shown in Drosophila that dRYBP interacts with trxG to stimulate the mono-

A

ubiquitylation of H2B (H2Bub) and promote transcriptional activation. It is proposed that this dual activating/repressing function is responsible for RYBP´s role in reversibility of epigenetic regulation, being able to modulate both active and/or repressed transcriptional states. B) Role of RYBP in developmental apoptosis. Both in vertebrates and flies, high levels of RYBP/dRYBP interact with pro-apoptotic factors (Table 1) and induce cell death. During normal Drosophila development, dRYBP

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interacts with the SCF complex to inhibit apoptosis. C) Role of RYBP/dRYBP in p53 stabilization. In Drosophila, it has been proposed that the interaction of dRYBP with SCE promotes Dp53 ubiquitylation to maintain homeostatic Dp53 levels while in vertebrates high levels of RYBP recruit MDM2 thus inhibiting MDM2-mediated p53

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ubiquitylation resulting in the stabilization of p53 and increasing p53 apoptotic activity.

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Seitz V, Thomas PE, Zimmermann K, Paul U, Ehlers A, Joosten M, Dimitrova L, Lenze D, Sommerfeld A, Oker E, Leser U, Stein H, Hummel M (2011) Classical Hodgkin's lymphoma shows epigenetic features of abortive plasma cell differentiation. Haematologica 96: 863-70

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Shembade N, Parvatiyar K, Harhaj NS, Harhaj EW (2009) The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-kappaB signalling. EMBO J 28: 51322 Shilatifard A (2012) The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis. Annu Rev Biochem 81: 65-95 Simoes da Silva CJ, Fereres S, Simon R, Busturia A (2017) Drosophila SCE/dRING E3-ligase inhibits apoptosis in a Dp53 dependent manner. Dev Biol 429: 81-91

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SC R

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Taylor BS, Schultz N, Hieronymus H, Gopalan A, Xiao Y, Carver BS, Arora VK, Kaushik P, Cerami E, Reva B, Antipin Y, Mitsiades N, Landers T, Dolgalev I, Major JE, Wilson M, Socci ND, Lash AE, Heguy A, Eastham JA et al. (2010) Integrative genomic profiling of human prostate cancer. Cancer Cell 18: 11-22

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Table 1. RYBP/dRYBP interacting proteins. The first column shows the reported RYBP/dRYBP interacting proteins; the second column shows the protein domains as well as the function of the RYBP interacting protein; the third column shows the described function of the interaction in a variety of biological processes. Asterisks (*) denote when the functional characterization of the interaction has been performed in Drosophila. The absence of an asterisk indicates the characterization has been

A

CC

EP

TE D

M

A

N

U

SC R

IP T

performed in vertebrates.

28

I N U SC R

Table 1. RYBP/dRYBP interacting proteins. Protein

Domain/Function

Zinc finger Ring-type E3 ubiquitin-protein ligase Histone H2A monoubiquitination

YY1/PHO

  

Zinc finger C2H2-type DNA binding protein Transcription factor

HIPPI

  

DED domain DNA binding protein Pro-apoptotic protein

M

ED

PT   

Zinc finger C2H2-type DNA binding protein Transcription factor



Fibronectin type III and ankyrin repeat domains protein Apoptosis regulator

CC E

REX-1/Zfp42

A

RING1/SCE

  

A

FANK1

Apoptin

BCOR FADD

   

Chicken anemia virus protein (without cellular homolog) Transcription regulator Pro-apoptotic protein

   

POZ/Zinc finger BCL6 co-repressor Transcription factor DED domain

Function of the interaction  Epigenetic regulation: stimulation of H2Aub  Apoptosis  Inhibition of P53-induced apoptosis*

References (Garcia et al., 1999) (Gonzalez et al., 2008) (Fereres et al., 2013) (Simoes da Silva et al., 2017)

 

Epigenetic regulation DNA-binding: recruiting PRC1 complex to DNA*

(Garcia et al., 1999) (Gonzalez et al., 2008) (Wilkinson et al., 2010)



Neural developmental apoptosis

(Stanton et al., 2007) (Gdynia et al., 2008)

 

Stem cells self-renewal Epigenetic regulation of REX1 targets

(Garcia-Tunon et al., 2011)



Tumor cell-specific apoptosis

(Ma et al., 2016)



Tumor cell-specific apoptosis

(Danen-van Oorschot et al., 2004) (Novak & Phillips, 2008)



Epigenetic regulation of BCOR targets

(Gearhart et al., 2006)



Apoptosis: DISC complex

(Zheng et al., 2001) 29

I N U SC R



Apoptotic mediator

 

DED-domain Initiator caspase

Caspase-10

 

DED-domain Initiator caspase

DEDD

 

DED-containing DNA binding protein Apoptotic regulator

MDM2

  

Zinc finger Ring-type E3 ubiquitin-protein ligase P53 binding protein

SKP1/SKPA

   

(Zheng et al., 2001)



Apoptosis: DISC complex formation

(Zheng et al., 2001)

 

Apoptosis DEDD subnuclear localization control

(Schickling et al., 2001) (Zheng et al., 2001)

 

Apoptosis. Inhibition of p53 proteaosomal degradation

(Chen et al., 2009)

SKP1/BTB/POZ domain E3 ubiquitin-protein ligase SCF ubiquitin ligase complex

 

SCF-Cul1 scaffold protein



Inhibition of Apoptosis* Inhibition of Immune response Inhibition of Apoptosis*

ED

M

A

Apoptosis: DISC complex formation

CC E

Cullin-1



PT

Caspase-8

A

dBRE-1

dKDM2

formation

  

Zinc finger Ring-type E3-ubiquitin-protein ligase Histone H2B monoubiquitination



JmjC domain-containing histone demethylation protein Zinc finger CXXC-type F-box SCF complex E3-ubiquitin-protein ligase Histone H2A monoubiquitination H3K36 demethylation

    

(Fereres et al., 2013) (Aparicio et al., 2013) (Fereres et al., 2013)



Epigenetic regulation: stimulation of H2Bub*

(Fereres et al., 2014)



Epigenetic regulation: stimulation of H2Aub and H3K36me2 demethylation

(Fereres et al., 2014)

30

I N U SC R

E2F transcription factor DNA binding protein Cell cycle regulator

PCGF1-6

 

Polycomb group ring finger proteins Histone H2A monoubiquitination

Oct-4

 

POU domain class 5 Transcription factor



Epigenetic regulation of E2F6 targets

(Trimarchi et al., 2001)



“Non-canonical” PRC1 complexes Epigenetic regulation in ESC differentiation

(Gao et al., 2012) (Morey et al., 2013) (Tavares et al., 2012) (Si et al., 2016)

Pluripotency IPSCs

(Li et al., 2017)

 

A

CC E

PT

ED

M

A

E2F6

  

31

I N U SC R

Table 2. RYBP levels in different cancer types. RYBP levels

Cancer type

Breast cancer (BC)

M

A

Cervical cancer (CC)

A

CC E

PT

ED

Low

Glioblastoma

Hepatocellular carcinoma (HCC) Ileal carcinoid Lung cancer (LC)

Prostate cancer (PC)

Adult T cell lymphoma/leukemia (ATL)

References (Buas et al., 2015) (Zhou et al., 2016) (Kenny et al., 2017) (Lando et al., 2009) (Lando et al., 2013) (Li et al., 2013) (Minchenko et al., 2014) (Wang et al., 2014) (Zhu et al., 2017a) (Zhu et al., 2017b) (Zhao et al., 2017) (Andersson et al., 2009) (Voruganti et al., 2015) (Jiang et al., 2016) (Dinglin et al., 2017) (Taylor et al., 2010) (Feik et al., 2013) (Krohn et al., 2013) (Cho et al., 2015) (Ulz et al., 2016) (Sasaki et al., 2011)

High Hodgkin lymphoma

(Sanchez-Beato et al., 2004) (Seitz et al., 2011)

32