Structure-Function Analysis of Sds3, Suppressor of Defective Silencing 3, a Key Component of the Histone Deacetylase-Containing Mammalian Sin3L/Rpd3L Corepressor Complex
At the most fundamental level, organisms must have a method to translate information stored in the genetic code into proteins that keep organisms alive. Gene transcription thus plays an important role in the survival of an organism and is predictably tightly regulated. Regulatory machinery exists at the molecular level and is of particular interest to researchers as there is a lack of understanding in how the structure of these complexes contributes to their function. One of these complexes, the Sin3L/Rpd3L HDAC corepressor complex, has been implicated to play a major role in but not limited to cancers and cardiac diseases. Though the role the complex plays in transcription is well studied, the structure and molecular mechanisms through which the Sin3L/Rpd3L complex acts is not well understood.
My studies focused on Sds3, one of the key binding partners and the supposed oligomerization domain that recruits histone deacetylase activity to the site of transcriptional repression. Understanding the molecular mechanism of how Sds3 functions at the molecular level opens up an avenue for the discovery of small molecule therapeutics aimed at blocking the normal functions of histone deacetylase complexes as a treatment for various diseases. The work presented here characterizes the minimal dimerization domain of Sds3 and its behavior in solution, contributing to future work of reconstituting the Sin3L/Rpd3L corepressor complex from its core subunits.
Introduction and Literature Survey
Eukaryotic transcription is a fundamental process that is tightly regulated at multiple levels. One of these levels of regulation occurs through the chemical modification of chromatin. Post-translational modifications such as phosphorylation, methylation and acetylation alter the ability of the transcriptional machinery to physically access the DNA via diverse mechanisms. Among these modifications, histone acetylation is the most prevalent, with two groups of enzymes including histone acetyltransferases (HATs) and histone deacetylases (HDACs) working antagonistically to regulate the steady-state levels. The effect of histone acetylation is well understood and is almost universally correlated with transcriptional activation. HDACs play fundamentally important roles in many biological processes and yet they have been shown to be attractive therapeutic targets for treating diseases such as inflammatory disorders, cancer, neurodegenerative and cardiovascular diseases.
Gene knockouts have implicated HDACs in diseased conditions such as cardiac malformation or embryonic lethality (Haberland, Montgomery, & Olson, 2009). Certain forms of cancer can also result from aberrant gene silencing caused by poor or failed recruitment of HDAC-containing complexes (Farias et al., 2010; Silverstein & Ekwall, 2005). HDAC inhibitors are thought to be a viable and effective treatment for a variety of diseases not only in animal models but also clinically. At least two HDAC inhibitors including SAHA (suberoylanilide hydroxamic) acid and the cyclic peptide Romidepsin have been approved for the treatment of cutaneous T-cell lymphoma (Haberland et al., 2009). However, despite having these drugs in the market, it is still unknown how HDAC inhibitors function at the molecular level. My work seeks to address this issue by clarifying the structure and function of a key protein member of an HDAC-containing complex, Sin3L/Rpd3L.
A) HDAC-containing complexes
The mammalian HDAC superfamily of enzymes is comprised of 18 enzymes divided into four classes by sequence homology (Gregoretti, Lee, & Goodson, 2004; Hayakawa & Nakayama, 2011). Eleven HDACs spanning classes I, II and IV share a common structure and Zn2+-dependent enzyme mechanism. HDACs upon nuclear localization are not known to function on their own; they are commonly found as part of large, multi-protein complexes with several binding partners that target HDAC activity to specific regions of the genome (Hayakawa & Nakayama, 2011). Class I HDACs 1 and 2 are of particular interest as they are exclusively nuclear proteins and are found in at least four distinct multi-protein complexes, including the Sin3L/Rpd3L, Sin3S/Rpd3S, NuRD, and CoREST complexes (Hayakawa & Nakayama, 2011).
B) Sin3L/Rpd3L corepressor complex
Sin3L/Rpd3L is a 1.2-2 megadalton multi-protein corepressor complex conserved in organisms as diverse as yeast, plants and mammals (Figure 1; (Grzenda, Lomberk, Zhang, & Urrutia, 2009; Shi, Seldin, & Garry, 2012)). A key component of the complex is Sin3A (and the paralogous Sin3B), an approximately 150-kilodalton protein that serves as the molecular scaffold for the assembly of the complex. Sin3A/B was originally thought to function as a global regulator of transcription playing an essential role in negatively regulating gene transcription despite lacking any DNA binding activity. The protein is recruited to specific regions of the genome through protein-protein interactions and via its associated HDAC activity plays a role in repression and in the maintenance of native chromatin structure (Ahringer, 2000; Farias et al., 2010; Silverstein & Ekwall, 2005).
Gene knockout studies have demonstrated the essential function Sin3 plays in the growth and viability of early embryonic cells. Sin3 deletions in fibroblasts results in a significant increase in growth defects, as well as an increase in apoptosis in conjunction with the derepression of several target proteins that play a significant role in cell cycle progression, DNA replication and repair, and cell death (McDonel, Demmers, Tan, Watt, & Hendrich, 2012). Other knockout studies have demonstrated that genes involved in both non-homologous end-joining and homologous recombination repair pathways are aberrantly upregulated, suggesting a novel role Sin3 plays in the balance of double stranded break repair mechanisms in the cell (Dannenberg et al., 2005; McDonel et al., 2012). Aside from its role in transcription repression, the Sin3L/Rpd3L complex has been shown to be involved in diverse cancer signaling pathways (David et al., 2006). The Sin3L/Rpd3L complex plays a role in cell growth as the ability of the negative growth regulator p33ING1b and a member of this complex to inhibit cell growth is dependent on its interaction with the complex (Kuzmichev, Zhang, Erdjument-Bromage, Tempst, & Reinberg, 2002).
The novel suppressor of defective silencing 3 gene (Sds3) encodes a 328-residue protein that shows high sequence similarity to its yeast homologue (Alland et al., 2002). Sds3 is an integral component of the Sin3L/Rpd3L complex, discovered in the context of transcriptional silencing in Saccharomyces cervisiae (Vannier, Balderes, & Shore, 1996). The encoded protein is shown to retain critical protein-protein interaction sites crucial to the assembly of the Sin3L/Rpd3L corepressor complex, helping to maintain the physical structure of the complex (Alland et al., 2002; Dorland, Deegenaars, & Stillman, 2000; Vannier et al., 1996). Sds3 deletions in yeast produce similar effects on transcription regulation as does sin3 deletions (Alland et al., 2002; Lechner et al., 2000). Further studies in both yeast and mammals have shown Sds3 to be an integral subunit of the Sin3L/Rpd3L complex, required for efficient recruitment of histone deacetylase enzymatic activity. Cells lacking Sds3 fail to incorporate HDAC into such complexes, while Sds3 knockdowns lead to reduced HDAC recruitment of HDACs (Alland et al., 2002; Lechner et al., 2000). Additionally, Sds3 and Sin3 knockouts in mouse embryonic fibroblasts individually produce cells that result in cell cycle arrest at the G2/M phase, resulting in early embryonic lethality (Dannenberg et al., 2005). The similarity in phenotype of the fibroblasts suggests there is an interaction between Sin3 and Sds3, that without one or the other, cells will fail to develop properly. Biochemical studies have revealed that the histone deacetylase interaction domain (designated HID) of Sin3 is critical for HDAC1/2 recruitment; incidentally, the Sin3 HID domain is also the site of interaction with Sds3 (Alland et al., 2002; Laherty et al., 1997).
Despite the important role that Sds3 plays in the Sin3/HDAC corepressor complex, little is know about its structural features. Studies of an Sds homologue known as the breast cancer metastasis suppressor 1 (BRMS1) have shown that the metastasis suppressor contains an antiparallel coiled-coil motif at the N-terminus (designated CC1) that forms a trimer of dimers (Spínola-Amilibia et al., 2011). BRMS1 exists in the Sin3L/Rpd3L complex but can interact with HDAC1 to form smaller complexes (Meehan et al., 2004). The sites of interaction, however, have not been clearly defined but it has been shown through yeast two-hybrid and co-immunoprecipitation studies that there may be more than one point of contact between BRMS1 and the rest of the complex (Meehan et al., 2004). Additionally, both Sds3 and BRMS1 are predicted to harbor a second coiled-coil motif at the C-terminus (designated CC2) that has potential roles in not only recruiting HDACs but also other subunits of the Sin3L/Rpd3L complex (Meehan et al., 2004).
A) Limited Proteolysis
To determine the minimal coiled-coil region of Sds3, a construct spanning the two putative coiled-coil segments of Sds3 (aa 43-234) was overexpressed in bacteria and purified to homogeneity using chromatographic approaches. The protein was then subjected to limited proteolysis, in combination with electrospray ionization mass spectrometry (ESI-MS). Trypsin and protein were combined in a 1:150 ratio w/w and the reaction was allowed to run for varying times before the was reaction stopped by placing samples in a mixture of isopropanol and dry ice and saved at -80 °C. Samples were collected at different time intervals following addition of trypsin and analyzed by both SDS-PAGE (Figure 2) and ESI-MS.
Two protein products and the intact protein were determined from analysis of the mass spectrum for the 30-second sample following trypsin addition. A 141-residue fragment (Sds343-183) spanning both CC1 and CC2 regions, with and without the N-terminal His6-tag, was found to be the dominant species in the spectrum although a second species containing an intact His6-tag but terminating at R183 was also found. In spite of the presence of at least twenty-six potential trypsin cleavage sites (Figure 2), cleavage is detected only at R43 and R183. These patterns are also confirmed by SDS-PAGE (Figure 2). This suggests that trypsin-sensitive sites within these bounds are relatively inaccessible to the protease, thus defining a minimal structure domain(s) within the protein. Incubating trypsin with the protein sample for periods longer than 120 s indicated completed degradation of the protein, indicative of an all-or-none property for the folded domain.
A) Paircoil2 analysis
The coiled-coil structural motif is a commonly found occurrence in nature, particularly in proteins with biological implications. The coiled-coil motif is most commonly found in rod-like proteins spanning long distances in the cell (Steven, Baumeister, Parry, Fraser, & Squire, 2008). In addition to their role in facilitating the assembly of mechanically rigid structures, coiled-coil motifs play a role in mediating oligomerization in biologically significant systems such as transcription factors involved in cell growth and proliferation, signaling molecules, and molecular motors (Mason & Arndt, 2004; Steven et al., 2008). Not surprisingly then, proteins containing the coiled-coil motif are targets of much investigation due to their potential to be targeted by small therapeutic compounds in treatment of diseases.
Coiled-coil motifs are unique compared to α-helices in that rather than having 3.6 residues per turn, there are 3.5 residues, allowing for one heptad repeat to occur every two turns. This heptad repeat contains residues that form a binding interface not only between two coiled-coils but also between the protein and its environment. Of the seven residues, the first and fourth – designated ‘a’ and ‘d’ – must be hydrophobic residues, stabilizing the protein through hydrophobic interactions (Mason & Arndt, 2004). Further, residues five and seven – designated ‘e’ and ‘g’ – often bear complementary charges to facilitate favorable electrostatic interactions between interacting helices.
With the Paircoil2 coiled-coil prediction algorithm, residues are classified as adopting a coiled-coil or not based on a pre-determined P-score of 0.025. By these criteria, CC2 is not predicted to be a coiled-coil while CC1 is. When the P-score is raised to 0.03, however, CC1 is predicted to span amino acids 60-89 (the same as when the P-score is 0.025) whereas CC2 is predicted to span residues 129-170. On the other hand, when the P-score cutoff is raised to 0.05, CC1 and CC2 are predicted to span residues 59-104 and 127-178, respectively.
B) Sds3 and BRMS1
Guided by the question of whether or not Sds3 resembles its homolog BRMS1, the work presented here suggests that Sds3 behaves in a remarkably different manner. Using the Paircoil2 coiled-coil prediction algorithm, putative coiled-coil domains were established, with Sds3 CC1 ranging from residues 61 to 89. Initially, we surmised that CC1 was the dimerization site for Sds3, as is the case for BRMS1. However, constructs containing CC1 alone did not show any dimerization activity, an indication that Sds3 and BRMS1 have contrasting structures/function. Rather puzzlingly, analysis of BRMS1 CC1 has demonstrated that BRMS151-98 and BRMS151-84 adopt different oligomerization states in the crystal with the former forming a hexamer and the latter a dimer (Spínola-Amilibia et al., 2013). In addition, thermal denaturation of both constructs showed non-sigmoidal behavior, suggesting a mixed population of states for these constructs (Spínola-Amilibia et al., 2013).
C) Sds3 CC1 and CC2
The results presented here strongly suggest that Sds3 exclusively forms dimers and the dimerization activity resides within the CC2 domain and the linker segment N-terminal to it. The role of the CC2 domain and its structure for the BRMS1 protein is currently unknown. Structural studies are required in order to clarify the basis for dimerization, as Sds3 CC2 is not predicted by Paircoil2 to form a coiled-coil, and residues in the linker region between CC1 and CC2 are also not predicted to adopt a coiled-coil motif; portions of these segments were predicted to form a coiled-coil but only with low confidence.
With Sds3 CC1 not being involved in dimerization, the question then presents itself as to the role of this domain in Sds3 function; the answer to this question is presently unknown. Rather intriguingly, both NMR and CD analysis suggest that CC1 adopts a helical conformation with moderate stability. Isolated helices are rare and the significance of the presence of such a segment in Sds3 is presently unknown. Thermal denaturation experiments of Sds343-234 found a thermal melting temperature of ~45°C, consistent with the lack of direct interactions between the CC1 and CC2 domains. Although further studies are needed to clarify the role of this segment in Sds3 function, we speculate that this segment is involved in interactions with another subunit of the Sin3L/Rpd3L complex.
Though it is known that diseases such as cancers can arise from failure of transcriptional regulation, how this occurs is not well understood on a molecular level. The work presented here deepens our understanding of the molecular mechanism of assembly of the Sin3L/Rpd3L complex. My studies show for the first time that Sds3 provides a critical dimerization function that likely increases the potency of the deacetylase activity recruited into the complex. Continued structural and functional studies of core components of the Sds3/Rpd3L complex can open the door to the potential of developing small therapeutics that can target and block protein-protein interactions involved in the assembly of the complex rather than targeting the active sites found on HDAC1/2 leading to a new class of HDAC complex-specific inhibitors.
Yujia Ding (’14) is the recipient of the Irving M. Klotz Basic Prize in Research for her senior thesis (2014) and a Northwestern Undergraduate Research Grant (2013). She is currently an IBiS (Interdisciplinary Biological Sciences) PhD student in Life and Biomedical Sciences at Northwestern University.
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