Amanda J. Guise

The functional interactome landscape of the human histone deacetylase family

Histone deacetylases (HDACs) are a diverse family of essential transcriptional regulatory enzymes, that function through the spatial and temporal recruitment of protein complexes. As the composition and regulation of HDAC complexes are only partially characterized, we built the first global protein interaction network for all 11 human HDACs in T cells. Integrating fluorescence microscopy, immunoaffinity purifications, quantitative mass spectrometry, and bioinformatics, we identified over 200 unreported interactions for both well‐characterized and lesser‐studied HDACs, a subset of which were validated by orthogonal approaches. We establish HDAC11 as a member of the survival of motor neuron complex and pinpoint a functional role in mRNA splicing. We designed a complementary label‐free and metabolic‐labeling mass spectrometry‐based proteomics strategy for profiling interaction stability among different HDAC classes, revealing that HDAC1 interactions within chromatin‐remodeling complexes are largely stable, while transcription factors preferentially exist in rapid equilibrium. Overall, this study represents a valuable resource for investigating HDAC functions in health and disease, encompassing emerging themes of HDAC regulation in cell cycle and RNA processing and a deeper functional understanding of HDAC complex stability.

Nuclear Import of Histone Deacetylase 5 by Requisite Nuclear Localization Signal Phosphorylation

Histone deacetylase 5 (HDAC5), a class IIa deacetylase, is a prominent regulator of cellular and epigenetic processes that underlie the progression of human disease, ranging from cardiac hypertrophy to cancer. Although it is established that phosphorylation mediates 14–3-3 protein binding and provides the essential link between HDAC5 nucleo-cytoplasmic shuttling and transcriptional repression, thus far only four phospho-acceptor sites have been functionally characterized. Here, using a combinatorial proteomics approach and phosphomutant screening, we present the first evidence that HDAC5 has at least 17 in vivo phosphorylation sites within functional domains, including Ser278 and Ser279 within the nuclear localization signal (NLS), Ser1108 within the nuclear export signal, and Ser755 in deacetylase domain. Global and targeted MS/MS analyses of NLS peptides demonstrated the presence of single (Ser278 and Ser279) and double (Ser278/Ser279) phosphorylations. The double S278/279A mutation showed reduced association with HDAC3, slightly decreased deacetylation activity, and significantly increased cytoplasmic localization compared with wild type HDAC5, whereas the S278A and S1108A phosphomutants were not altered. Live cell imaging revealed a deficiency in nuclear import of S278/279A HDAC5. Phosphomutant stable cell lines confirmed the cellular redistribution of NLS mutants and revealed a more pronounced cytoplasmic localization for the single S279A mutant. Proteomic analysis of immunoisolated S278/279A, S279A, and S259/498A mutants linked altered cellular localization to changes in protein interactions. S278/279A and S279A HDAC5 showed reduced association with the NCoR-HDAC3 nuclear corepressor complex as well as protein kinase D enzymes, which were potentiated in the S259/498A mutant. These results provide the first link between phosphorylation outside the known 14–3-3 sites and downstream changes in protein interactions. Together these studies identify Ser279 as a critical phosphorylation within the NLS involved in the nuclear import of HDAC5, providing a regulatory point in nucleo-cytoplasmic shuttling that may be conserved in other class IIa HDACs—HDAC4 and HDAC9.

Aurora B-dependent Regulation of Class IIa Histone Deacetylases by Mitotic Nuclear Localization Signal Phosphorylation

Class IIa histone deacetylases (HDACs 4/5/7/9) are transcriptional regulators with critical roles in cardiac disease and cancer. HDAC inhibitors are promising anticancer agents, and although they are known to disrupt mitotic progression, the underlying mechanisms of mitotic regulation by HDACs are not fully understood. Here we provide the first identification of histone deacetylases as substrates of Aurora B kinase (AurB). Our study identifies class IIa HDACs as a novel family of AurB targets and provides the first evidence that HDACs are temporally and spatially regulated by phosphorylation during the cell cycle. We define the precise site of AurB-mediated phosphorylation as a conserved serine within the nuclear localization signals of HDAC4, HDAC5, and HDAC9 at Ser265, Ser278, and Ser242, respectively. We establish that AurB interacts with these HDACs in vivo, and that this association increases upon disruption of 14-3-3 binding. We observe colocalization of endogenous, phosphorylated HDACs with AurB at the mitotic midzone in late anaphase and the midbody during cytokinesis, complemented by a reduction in HDAC interactions with components of the nuclear corepressor complex. We propose that AurB-dependent phosphorylation of HDACs induces sequestration within a phosphorylation gradient at the midzone, maintaining separation from re-forming nuclei and contributing to transcriptional control.

Histone Deacetylases in Herpesvirus Replication and Virus-Stimulated Host Defense

Emerging evidence highlights a critical role for protein acetylation during herpesvirus infection. As prominent modulators of protein acetylation, histone deacetylases (HDACs) are essential transcriptional and epigenetic regulators. Not surprisingly, viruses have evolved a wide array of mechanisms to subvert HDAC functions. Here, we review the mechanisms underlying HDAC regulation during herpesvirus infection. We next discuss the roles of acetylation in host defense against herpesvirus infection. Finally, we provide a perspective on the contribution of current mass spectrometry-based “omic” technologies to infectious disease research, offering a systems biology view of infection.

Post-translational Modifications Regulate Class IIa Histone Deacetylase (HDAC) Function in Health and Disease

Class IIa histone deacetylases (HDACs4, -5, -7, and -9) modulate the physiology of the human cardiovascular, musculoskeletal, nervous, and immune systems. The regulatory capacity of this family of enzymes stems from their ability to shuttle between nuclear and cytoplasmic compartments in response to signal-driven post-translational modification. Here, we review the current knowledge of modifications that control spatial and temporal histone deacetylase functions by regulating subcellular localization, transcriptional functions, and cell cycle-dependent activity, ultimately impacting on human disease. We discuss the contribution of these modifications to cardiac and vascular hypertrophy, myoblast differentiation, neuronal cell survival, and neurodegenerative disorders.

Proteomics of yeast telomerase identified Cdc48-Npl4-Ufd1 and Ufd4 as regulators of Est1 and telomere length

Almost 400 genes affect yeast telomere length, including Est1, which is critical for recruitment and activation of telomerase. Here we use mass spectrometry to identify novel telomerase regulators by their co-purification with the telomerase holoenzyme. In addition to all known subunits, over 100 proteins are telomerase associated, including all three subunits of the essential Cdc48-Npl4-Ufd1 complex as well as three E3 ubiquitin ligases. The Cdc48 complex is evolutionarily conserved and targets ubiquitinated proteins for degradation. Est1 levels are ∼40-fold higher in cells with reduced Cdc48, yet, paradoxically, telomeres are shorter. Furthermore, Est1 is ubiquitinated and its cell cycle-regulated abundance is lost in Cdc48-deficient cells. Deletion of the telomerase-associated E3 ligase, Ufd4, in cdc48-3 cells further increases Est1 abundance but suppresses the telomere length phenotype of the single mutant. These data argue that, in concert with Ufd4, the Cdc48 complex regulates telomerase by controlling the level and activity of Est1.

Probing phosphorylation‐dependent protein interactions within functional domains of histone deacetylase 5 (HDAC5)

Class IIa histone deacetylases (HDACs) are critical transcriptional regulators, shuttling between nuclear and cytoplasmic cellular compartments. Within the nucleus, these HDACs repress transcription as components of multiprotein complexes, such as the nuclear corepressor and beclin‐6 corepressor (BCoR) complexes. Cytoplasmic relocalization relieves this transcriptional repressive function. Class IIa HDAC shuttling is controlled, in part, by phosphorylations flanking the nuclear localization signal (NLS). Furthermore, we have reported that phosphorylation within the NLS by the kinase Aurora B modulates the localization and function of the class IIa HDAC5 during mitosis. While we identified numerous additional HDAC5 phosphorylations, their regulatory functions remain unknown. Here, we studied phosphorylation sites within functional HDAC5 domains, including the deacetylation domain (DAC, Ser755), nuclear export signal (NES, Ser1108), and an acidic domain (AD, Ser611). We have generated phosphomutant cell lines to investigate how absence of phosphorylation at these sites impacts HDAC5 localization, enzymatic activity, and protein interactions. Combining molecular biology and quantitative MS, we have defined the interactions and HDAC5‐containing complexes mediated by site‐specific phosphorylation and quantified selected changes using parallel reaction monitoring. These results expand the current understanding of HDAC regulation, and the functions of this critical family of proteins within human cells.

Pfh1 Is an Accessory Replicative Helicase that Interacts with the Replisome to Facilitate Fork Progression and Preserve Genome Integrity

Replicative DNA helicases expose the two strands of the double helix to the replication apparatus, but accessory helicases are often needed to help forks move past naturally occurring hard-to-replicate sites, such as tightly bound proteins, RNA/DNA hybrids, and DNA secondary structures. Although the Schizosaccharomyces pombe 5’-to-3’ DNA helicase Pfh1 is known to promote fork progression, its genomic targets, dynamics, and mechanisms of action are largely unknown. Here we address these questions by integrating genome-wide identification of Pfh1 binding sites, comprehensive analysis of the effects of Pfh1 depletion on replication and DNA damage, and proteomic analysis of Pfh1 interaction partners by immunoaffinity purification mass spectrometry. Of the 621 high confidence Pfh1-binding sites in wild type cells, about 40% were sites of fork slowing (as marked by high DNA polymerase occupancy) and/or DNA damage (as marked by high levels of phosphorylated H2A). The replication and integrity of tRNA and 5S rRNA genes, highly transcribed RNA polymerase II genes, and nucleosome depleted regions were particularly Pfh1-dependent. The association of Pfh1 with genomic integrity at highly transcribed genes was S phase dependent, and thus unlikely to be an artifact of high transcription rates. Although Pfh1 affected replication and suppressed DNA damage at discrete sites throughout the genome, Pfh1 and the replicative DNA polymerase bound to similar extents to both Pfh1-dependent and independent sites, suggesting that Pfh1 is proximal to the replication machinery during S phase. Consistent with this interpretation, Pfh1 co-purified with many key replisome components, including the hexameric MCM helicase, replicative DNA polymerases, RPA, and the processivity clamp PCNA in an S phase dependent manner. Thus, we conclude that Pfh1 is an accessory DNA helicase that interacts with the replisome and promotes replication and suppresses DNA damage at hard-to-replicate sites. These data provide insight into mechanisms by which this evolutionarily conserved helicase helps preserve genome integrity.

The Proteomic Profile of Deleted in Breast Cancer 1 (DBC1) Interactions Points to a Multifaceted Regulation of Gene Expression.

Deleted in breast cancer 1 (DBC1) has emerged as an important regulator of multiple cellular processes, ranging from gene expression to cell cycle progression. DBC1 has been linked to tumorigenesis both as an inhibitor of histone deacetylases, HDAC3 and sirtuin 1, and as a transcriptional cofactor for nuclear hormone receptors. However, despite mounting interest in DBC1, relatively little is known about the range of its interacting partners and the scope of its functions. Here, we carried out a functional proteomics-based investigation of DBC1 interactions in two relevant cell types, T cells and kidney cells. Microscopy, molecular biology, biochemistry, and mass spectrometry studies allowed us to assess DBC1 mRNA and protein levels, localization, phosphorylation status, and protein interaction networks. The comparison of DBC1 interactions in these cell types revealed conserved regulatory roles for DBC1 in gene expression, chromatin organization and modification, and cell cycle progression. Interestingly, we observe previously unrecognized DBC1 interactions with proteins encoded by cancer-associated genes. Among these interactions are five components of the SWI/SNF complex, the most frequently mutated chromatin remodeling complex in human cancers. Additionally, we identified a DBC1 interaction with TBL1XR1, a component of the NCoR complex, which we validated by reciprocal isolation. Strikingly, we discovered that DBC1 associates with proteins that regulate the circadian cycle, including DDX5, DHX9, and SFPQ. We validated this interaction by colocalization and reciprocal isolation. Functional assessment of this association demonstrated that DBC1 protein levels are important for regulating CLOCK and BMAL1 protein oscillations in synchronized T cells. Our results suggest that DBC1 is integral to the maintenance of the circadian molecular clock. Furthermore, the identified interactions provide a valuable resource for the exploration of pathways involved in DBC1-associated tumorigenesis.

Determining the Composition and Stability of Protein Complexes Using an Integrated Label-Free and Stable Isotope Labeling Strategy.

In biological systems, proteins catalyze the fundamental reactions that underlie all cellular functions, including metabolic processes and cell survival and death pathways. These biochemical reactions are rarely accomplished alone. Rather, they involve a concerted effect from many proteins that may operate in a directed signaling pathway and/or may physically associate in a complex to achieve a specific enzymatic activity. Therefore, defining the composition and regulation of protein complexes is critical for understanding cellular functions. In this chapter, we describe an approach that uses quantitative mass spectrometry (MS) to assess the specificity and the relative stability of protein interactions. Isolation of protein complexes from mammalian cells is performed by rapid immunoaffinity purification, and followed by in-solution digestion and high-resolution mass spectrometry analysis. We employ complementary quantitative MS workflows to assess the specificity of protein interactions using label-free MS and statistical analysis, and the relative stability of the interactions using a metabolic labeling technique. For each candidate protein interaction, scores from the two workflows can be correlated to minimize nonspecific background and profile protein complex composition and relative stability.