Neurodegenerative Disease Proteinopathies Are Connected to

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Neurodegenerative Disease Proteinopathies are Connected to Distinct Histone Post-Translational Modification Landscapes Karen Chen, Seth A Bennett, Navin Rana, Huda Yousuf, Mohamed Said, Sadiqa Taaseen, Natalie Mendo, Steven Marc Meltser, and Mariana Torrente ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.7b00297 • Publication Date (Web): 15 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

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ACS Chemical Neuroscience

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Neurodegenerative Disease Proteinopathies are Connected to Distinct Histone Post-Translational Modification Landscapes

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Karen Chen1, Seth A. Bennett1,2, Navin Rana1, Huda Yousuf1, Mohamed Said1, Sadiqa Taaseen1, Natalie Mendo1, Steven M. Meltser1, and Mariana P. Torrente1,3*

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Chemistry Department of Brooklyn College, Brooklyn, NY 11210; 2Graduate Program

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in Biochemistry, The Graduate Center of the City University of New York, New York, NY

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10016; 3 Ph.D. Programs in Chemistry, Biochemistry, and Biology, The Graduate Center

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of the City University of New York, New York, NY 10016.

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*Correspondence to: Mariana P. Torrente; Email: [email protected]

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Keywords: ALS, Parkinson’s Disease, FUS, TDP-43, alpha-synuclein, Epigenetics,

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Histone Modifications

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ACS Paragon Plus Environment

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ABSTRACT

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Amyotrophic Lateral Sclerosis (ALS) and Parkinson’s disease (PD) are devastating

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neurodegenerative diseases involving the progressive degeneration of neurons. No cure

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is available for patients diagnosed with these diseases. A prominent feature for both ALS

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and PD is the accumulation of protein inclusions in the cytoplasm of degenerating

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neurons; however, the particular protein comprising these inclusions varies. The RNA-

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binding proteins TDP-43 and FUS are most notable in ALS, while α-synuclein

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aggregates into Lewy bodies in PD. In both diseases, genetic causes fail to explain the

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occurrence of a large proportion of cases and, thus, both are considered mostly

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sporadic. Despite mounting evidence for a possible role for epigenetics in the

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occurrence and progression of ALS and PD, epigenetic mechanisms in the context of

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these diseases remain mostly unexplored. Here, we comprehensively delineate histone

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post-translational modification (PTM) profiles in ALS and PD yeast proteinopathy

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models. Remarkably, we find distinct changes in histone modification profiles for each.

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We detect the most striking changes in the context of FUS aggregation: changes in

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several histone marks support a global decrease in gene transcription. We also detect

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more modest changes in histone modifications in cells overexpressing TDP-43 or α-

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synuclein. Our results highlight a great need for the inclusion of epigenetic mechanisms

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in the study of neurodegeneration. We hope our work will pave the way for discovery of

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more effective therapies to treat patients suffering from ALS, PD, and other

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neurodegenerative diseases.

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KEYWORDS. ALS, Parkison’s Disease, FUS, TDP-43, α-synuclein, Epigenetics, Histone

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Modifications

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INTRODUCTION

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Neurodegenerative diseases are devastating illnesses with few treatment

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options. Among these, amyotrophic lateral sclerosis (ALS) and Parkinson’s disease (PD)

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are characterized by the occurrence of protein inclusions leading to the premature

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demise of neurons. There is no definitively established cause or cure for either disease,

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and available treatments ultimately fail to stop their progression.1-4 For both ALS and PD,

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most cases occur in people with no known family history of the disease and have not

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been linked to a genetic cause; thus, these ailments are considered mostly sporadic.

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Only a relative small proportion of ALS and PD cases are associated with genetic

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mutations and within these cases a multitude of genes have been implicated.5,6 In ALS,

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mutations in TDP-43 (TAR DNA binding protein 43) and FUS (Fused in

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sarcoma/translocated in liposarcoma) are known to lead to protein aggregation and

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formation of cytoplasmic inclusions in motor neurons, while α-synuclein aggregates to

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form Lewy bodies in dopaminergic neurons in PD.3, 7-15 Remarkably, protein aggregation

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occurs in both familial and sporadic forms of ALS as 97 % of patients display TDP-43

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inclusions regardless of whether or not they present mutations in this protein.16 It is thus

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far clear that genetic causes alone fail to explain the development of ALS and PD.5, 6

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Eukaryotic DNA is tightly packed around a complex network of proteins referred

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to as chromatin.17 The organization of chromatin controls gene expression: euchromatin

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is loosely packed and easily transcribed, while heterochromatin is tightly packed and

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transcriptionally silent.18 Changes in the architecture of chromatin and the consequent

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changes in gene expression are sufficient to cause heritable phenotypic alterations.

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These alterations – occurring without changes in DNA sequence – are termed

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epigenetic. Epigenetic mechanisms involve mainly the methylation of DNA and the post-

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translational modification of histone proteins.19


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The basic unit of chromatin comprises approximately 146 base pairs of DNA

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wrapped around a protein core made up of an octamer of histone proteins comprising

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two H2A-H2B dimers and one H3 and H4 tetramer.20 Each histone protein has a long,

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protruding N-terminal tail that can be post translationally modified (PTM) with chemical

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moieties including phosphorylation, acetylation, methylation, ubiquitination and

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sumoylation. These modifications are installed on specific residues.18 The way in which

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these modifications are “read” by other proteins has led to the ‘Histone Code

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Hypothesis,’ which states that specific modifications can be read by ‘reader’ proteins and

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recruit transcription or silencing factors, ultimately leading to repression or activation of

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transcription.21, 22 Histone PTMs are dynamically added and removed; for instance,

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acetyl groups are added by histone acetyltransferases and removed by histone

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deacetylases (HDACs).17 Specific modifications can also affect how other amino acids in

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the same or another histone molecule are modified (reviewed in 23). For example,

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phosphosphorylation of histone H3 at serine 10 (H3S10ph) blocks methylation of histone

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H3 at lysine 9.24 Conversely, H3S10ph increases acetylation of histone H4 on lysine 16

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and creates a nucleosome platform that increases transcription.25

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Is there a role for epigenetics in neurogenerative disease? Recent evidence has

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linked ALS and PD to epigenetic mechanisms (reviewed in6, 26, 27). Thus far, DNA

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methylation has been the dominant subject of research on epigenetics of

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neurodegenerative disease. Nevertheless, there is evidence for the importance of

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histone modification in this context. For instance, overexpression of wild type FUS

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induces hypoacetylation of histone H3 on a specific gene promoter.28 Moreover, the

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levels of HDAC1, HDAC2 and Sirtuins (a family of class III histone deacetylases) are

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impaired in postmortem ALS tissues.29, 30 Indeed, FUS itself has been shown to interact

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with HDAC1 to recruit the latter to sites of DNA damage.31 More recently, redistribution

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of wild-type or mutant FUS to the cytoplasm has been associated with a loss-of-function


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in the arginine methyltransferase PRMT1 resulting in alterations in histone PTMs.32 PD

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models have also provided evidence that epigenetics and histone PTMs may play a role

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in that disease. For instance, SIRT2 inhibitors alleviate α-synuclein toxicity in vitro and in

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a drosophila model.33 Mitochondrial dysfunction has also been observed in PD and is

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related to hypoacetylation of histone H3.34 Furthermore, α-synuclein itself binds to the

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promoter region of certain genes, blocking histone acetylation and leading to reduced

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transcription.35 Despite all the evidence for an epigenetic component in ALS and PD, a

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comprehensive study of the histone modifications involved in these diseases has not

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been carried out to date.

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Yeast is a useful model for studying neurodegenerative diseases because it

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preserves many neuronal cellular pathways and upon TDP-43, FUS, or α-synuclein

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overexpression it recapitulates proteinopathies and cytoplasmic foci seen in ALS and

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PD.36-40 In contrast to other ALS and PD model systems, yeast are inexpensive,

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expeditious and easy to work with. In fact, yeast models have enabled identification of

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ALS genetic risk factors.41 Interestingly, deletion of Set3—a member of a histone

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deacetylase complex, homolog to the human protein ASH1—suppresses the toxicity of

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TDP-43 inclusions in a yeast model.42 Furthermore, yeast models for other ALS-related

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proteinopathies have also identified chromatin proteins as modifiers of protein

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aggregation cytotoxicity.37 Here, we exploit FUS, TDP-43 and α-synuclein yeast

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proteinopathy models to identify perturbations in histone PTMs patterns by way of

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western blotting using modification-specific antibodies. To the best of our knowledge,

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this is the first comprehensive characterization of histone PTMs in any ALS or PD

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models. Remarkably, we find distinct changes in histone modification profiles for each

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proteinopathy model. We show FUS aggregation is associated with the most

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pronounced changes in several histone marks including phosphorylation of histone H2B,


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phosphorylation and acetylation on histone H3, as well as histone H4 arginine

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dimethylation. Furthermore, we observe a global decrease in gene transcription in the

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context of FUS proteinopathy. We also identify more moderate histone PTMs

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disturbances in cells overexpressing TDP-43 and α-synuclein. Small increases in the

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levels of histone acetylation on lysine 12 and 16 on histone H4 as well as a small

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decrease in the level of trimethylation of lysine 36 on histone H3 are apparent in the

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context of TDP-43 overexpression, while small changes on the levels of dimethylation on

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histone H3 on lysine 36 and a significant change on the phosphorylation of H2B

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accompany α-synuclein aggregation. Overall, our results highlight a great need for the

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inclusion of epigenetic mechanisms in the study of neurodegenerative disease and the

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search for more effective treatments for these disorders.

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RESULTS AND DISCUSSION

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TDP-43, FUS and α-synuclein Proteinopathies Elicit Distinct Perturbations in the

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Histone PTMs Landscape

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Using modification-specific antibodies, we have delineated the global epigenetic

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make up of TDP- 43, FUS, and α-synuclein proteinopathies. To do this, we exploited

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well-established yeast models for ALS and PD.38, 40, 43, 46 Previous investigations have

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found these show cytoplasmic foci in 90% of cells in TDP-43 and FUS models and in

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60% of cells for the α-synuclein model.47 Yeast were transformed with either a control

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vector or vectors expressing TDP-43, FUS or α-synuclein. Spotting assays on inducing

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selective media corroborated growth of control cells and cytotoxicity of TDP-43, FUS,

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and α-synuclein. In our experiments, FUS overexpression was the most toxic, followed

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by TDP-43 and α-synuclein overexpression respectively (Supplementary Figure 1a-b).


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Galactose-induced protein expression was verified by western blotting for all three

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constructs (Supplementary Figure 1c-e). In liquid culture, cytotoxic phenotypes are

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much less severe (Supplementary Figure 1f). After galactose induction, we harvested

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and lysed both control yeast and yeast overexpressing TDP-43, FUS, or α-synuclein.

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The timeframe of cell harvest for histone modification analysis precedes the point of

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toxicity in liquid culture (Supplementary Figure 1f). Cell lysates were subjected to SDS-

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PAGE to separate histone proteins from all other proteins. We then probed for individual

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histone modifications using commercially available modification-specific antibodies. We

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selected immunoblotting methods because of their high sensitivity of detection for low-

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abundance proteins and modifications.48 In this work, we focused primarily on histones

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H3 and H4, as these histones are most pervasively modified.49 Furthermore, we included

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histone modifications involved in both transcriptional activation and repression.

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Altogether, we incorporated 24 histone modifications in our experiments. Individual

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histone modifications were quantitated by blot image analysis, normalized to the loading

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control and compared to the control sample to obtain fold change measurements.

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Remarkably, we identified distinct histone PTM panoramas for each proteinopathy

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(Figure 1). P-values were used to assess the statistical significance of observed

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changes. Due to large variations observed in some of the histone modifications, the p-

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values provide a better representation of the observed PTM changes than the averages

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and were thus used to construct the heat map in Figure 1. Overall, we found TDP-43

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overexpression is associated with increases in several histone PTMs, while FUS

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overexpression is associated with highly significant decreases in histone modification

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levels. Aligning with its mild cytotoxicity, α-synuclein overexpression resulted in the least

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amount of changes (Figure 1). As a positive control illustrating the potential range of

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changes on histone modification levels, we treated wild type cells with the DNA-damage


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agent bleomycin, which induces both single-stranded and double-stranded DNA

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damage.50 We explored the levels of phosphorylation on histone H2A on serine 129,

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which is homologous to phosphorylation on S139 in humans and a notable histone

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modification occurring in response to a DNA double strand break.51, 52 Bleomycin

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induces a ~2.5-fold increase in the phosphorylation of serine 129 in histone H2A

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(Supplementary Figure 2). In general, it is important to note that statistical significance

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does not correlate with magnitude of the change in histone PTMs levels. For instance,

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in the context of TDP-43 overexpression, changes on histone H3K36me3 levels are

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highly reproducible and thus significant statistically (Figure 1), but very modest in

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magnitude (Figure 2c). Altogether, our results suggest the observed epigenetic changes

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are specific to each individual proteinopathy and not associated with general protein

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aggregation toxicity pathways. Furthermore, we found that ALS and PD proteinopathies

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are connected to widespread changes in histone modifications throughout the genome

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that are detectable without the need for enrichment of any particular genomic regions.

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TDP-43 and α-synuclein Overexpression is linked to Modest Decreases in Histone

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H3 Methylation on Lysine 36

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To our surprise, many histone H3 methylation sites display similar modification

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levels in both control cells and proteinopathy models. In particular, all degrees of

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methylation (me1, me2, and me3) on lysine 4 and lysine 79 showed no changes

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(Supplementary Figures 3 and 4b-d and 4f-h). We did not study methylation on lysine

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9 because this modification is not present in budding yeast.53 We note no genome-level

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disturbances in the levels of H3K79me3, which along with H3K9me3, H3K27me3 and

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H4K20me3, has been implicated in particular genetic loci in other proteinopathies related

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to ALS.54 However, we found an exception in changes on the levels of di- and tri-

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methylation of histone H3 at lysine 36. In yeast, H3K36 is methylated by the


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methyltransferase Set2, which can install up to three methyl groups on the lysine side

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chain.55 Interestingly, the human counterpart of Set2, SETD2, interacts with huntingtin

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protein pointing to a potential role for this modification in neurodegenerative disease.56

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H3K36 methylation is involved in transcriptional elongation, suppressing histone

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exchange and hyperacetylation as well as promoting maintenance of chromatin structure

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over coding regions in order to prevent aberrant transcriptional initiation within coding

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sequences.57 While the levels of H3K36me2 do not change in FUS and TDP-43 models

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(Figure 2a), the levels of this modification are slightly, but consistently decreased in the

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context of α-synuclein overexpression (Figure 2b). Conversely, the levels of H3K36me3

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were modestly decreased when TDP-43 is overexpressed (Figure 2c), but remained

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unchanged in the FUS and α-synuclein models (Figure 2c-d). Levels of

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monomethylation of H3K36 remained unchanged in all of the proteinopathy models

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(Supplementary Figure 4a and 4e).

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FUS Overexpression is Associated with Important Changes in the

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Phosphorylation and Acetylation of Histone H3

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The most sizeable changes in histone H3 PTMs levels occured in the context of

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FUS overexpression. In particular, we detected a 50% decrease in the levels of

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phosphorylation of H3 on serine 10 (Figure 3a). This change in H3S10ph is unmatched

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in either TDP-43 or α-synuclein overexpression models (Figure 3a and 3d). H3S10ph is

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linked to both transcriptional activation and repression. Ipl1, the yeast homolog of the

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mammalian Aurora B Kinase, is responsible for its installation.58 Highlighting its

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importance, this modification is remarkably conserved throughout the eukaryotic

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kingdom.59 The striking alteration in the levels of H3S10ph might point to an important

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role for Aurora B Kinase in ALS etiology. Indeed, Aurora B Kinase is able to


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phosphorylate Ataxin-10, a key protein in Spinocerebellar ataxia type 10, another

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neurodegenerative disorder.60 Unfortunately, we are unable to pinpoint if decreases in

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H3S10ph arise from alterations in the levels of Ipl1 as antibodies for this protein are

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unavailable. To clarify whether the observed alterations in histone modifications are

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related to protein toxicity and/or expression levels, we lowered toxic protein expression

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levels by growing cells on a combination of galactose and sucrose. Sucrose gets slowly

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metabolized to glucose, and glucose in turn represses the galactose promoter.61 First,

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we verified the sucrose/galactose setup reduced FUS expression (Supplementary

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Figure 5c). Unsurprisingly, decreased expression of FUS lessened the toxic phenotypes

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observed on solid and liquid media (Supplementary Figure 5a and 5b, respectively).

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Indeed, we find lowered levels of FUS resulted in decreased magnitude of change in the

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level of phosphorylation of Histone H3 on Serine 10 (Supplementary Figure 5d and

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5e).

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FUS overexpression also resulted in hypoacetylation of H3 on lysine 14 (Figure

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3b). We did not observe parallel acetylation changes in the TDP-43 or α-synuclein

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overexpression models (Figure 3b and 3e). Furthermore, we detected no alternations in

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the acetylation levels of histone H3 on lysine 9, lysine 18 or lysine 27 in any of our

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proteinopathy models (Supplementary Figure 6). Overexpression of wild type FUS has

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been linked to hypoacetylation on both K9/K14 in the cyclin D1 gene promoter in human

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cells.28 However, as this investigation used an antibody that recognized K9/K14

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acetylation in tandem, it is hard to compare our findings. Generally, hypoacetylation is

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correlated to a tighter association between histones and DNA and the establishment of

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transcriptionally inactive heterochromatin.18, 62, 63 However, it is striking that we did not

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observe a general reduction in acetylation for all sites, but rather FUS proteinopathy is

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associated with loss of acetylation at specific histone H3 sites. H3K14ac is involved in

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DNA damage checkpoint activation and DNA repair,64, 65 and thus lowered levels


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suggest DNA repair is impaired in the FUS model. Furthering this hypothesis, H3K56ac

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– an acetylation site in the core domain of H3- is also significantly lowered in the FUS

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proteinopathy model (Figure 3c). This modification is required to reinstate chromatin

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structure over repaired DNA, and is critical for signaling completion of repair.66 Akin to

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H3K14ac, we did not detect statistically significant changes in the levels of H3K56ac in

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the TDP-43 or α-synuclein overexpression models (Figure 3c and 3f).

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Dimethylation of Histone H4 is Decreased in the Context of FUS Overexpression In conjunction to the changes on H3 modification outlined above, we also

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identified a decrease on the levels of dimethylation on arginine 3 on histone H4 in the

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FUS model (Figure 4a). This agrees with the observed hypo-acetylation on H3K14, as

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H4R3me2 is associated with H3 acetylation at both lysine 9 and lysine 14.67 In yeast,

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Hmt1, the yeast homolog of the protein arginine methyltransferase 1 (PRMT1), catalyzes

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the formation of H4R3me2.68 PRMT1 is known to interact with and methylate FUS,

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affecting its cellular localization.69-73 Consistent with our findings, expression and

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mislocalization of wild type or mutant human FUS in cultured murine motor neurons

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leads to nuclear depletion of PRMT1 and a consequent reduction in the levels of

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H4R3me2.32 It is possible that FUS is sequestering Hmt1 and localizing to the cytoplasm

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resulting in hypo-methylation at this site. It is also conceivable mislocalized protein

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sequesters other histone modifiers resulting in the various changes on histone

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modification levels we detect. Moreover, H4R3me2 is associated with transcriptional

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activation.74 Together with lowered acetylation on H3K14 and H3K56, decreased

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H4R3me2 would suggest a decrease in gene transcription. We found no statistically

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significant changes in the level of H4R3me2 in TDP-43 or α-synuclein models (Figure

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4a-b). Furthermore, we observed no alterations in any of the other histone H4


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modifications studied in the FUS overexpression background, including symmetric

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dimethylation on R3 (Figure 4b and 4c; Supplementary Figure 7a-d).

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TDP-43 Overexpression is Associated with Modest Increases in Histone H4

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Acetylation

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In contrast to the FUS model, we encountered modest hyper-acetylation of

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histone H4 on lysine 12 and lysine 16 in the TDP-43 proteinopathy background (Figure

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4b-c). Histone acetylation has been long correlated with transcriptional activation.75 In

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yeast, the specific removal of H4K16ac enables the formation of a heritably silent state,76

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and thus increases in this modification would suggest enhanced gene transcription in the

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TDP-43 background. Interestingly, increased levels of H4K12ac have been found in

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Alzheimer’s disease mouse models during early development of the disease.77 These

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changes in H4 acetylation are unique to TDP-43 overexpression and were not present in

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FUS or α-synuclein models (Figure 4b-c and 4e-f)

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Phosphorylation of Histone H2B is Decreased in Both FUS and α-synuclein

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Proteinopathy Models

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In both FUS and α-synuclein proteinopathy models, we discovered considerable

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hypo-phosphorylation of histone H2B on threonine 129 (Figure 5). Loss of

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phosphorylation at this site is associated with decreased gene expression as well as

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DNA damage.78, 79 While this modification is not conserved in humans, we included it in

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our analysis as we anticipated it could yield important information on the biological

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pathways linked to ALS and PD pathology. Cells overexpressing FUS show a 50%

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decrease in the level of H2BT129ph (Figure 5a). Changes in this modification fit

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together with changes in H3K14ac and H3K56ac (Figure 3b-c) and stress a role for


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DNA damage and repair in FUS proteinopathy. Furthermore, activation of poly (ADP-

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ribose) polymerase 1 (PARP-1) in response to DNA damage inhibits Aurora B Kinase,80

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and thus changes in H3S10ph levels in the FUS model would also correspond with

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triggering of the DNA damage signaling. Puzzlingly, we found no significant differences

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in the levels of phosphorylation on histone H2A on serine 129 in any of the proteinopathy

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models (Supplementary Figure 8). This raises the possibility that the combination of

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modifications observed in the FUS model is not occurring in association to DNA

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damage, but are instead related to other processes. It is plausible that the histone

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modifications we observe arise in association to unidentified defense mechanisms

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triggered by the protein aggregates.

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Unexpectedly, we also find profound differences in the levels of H2BT129ph in

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the α-synuclein overexpression background (Figure 5b). There is approximately a 40%

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decrease in the levels of this modification in the context of α-synuclein overexpression.

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By far, this is the most severe histone PTM alteration we detect for this model. This

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modification is associated with DNA damage in yeast.79 As other DNA damage and

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repair histone modifications disturbances (i.e. H2AS129ph, Supplementary Figure 2)

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are absent in the α-synuclein proteinopathy background, this suggests yet-undiscovered

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functions for H2BT129ph are likely. Such unknown functions could point to pathways

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important in the occurrence and progression of PD.

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FUS Overexpression is Associated with a Decrease in Total RNA Levels Collectively, many of the histone PTM alterations observed in the FUS model

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point to decreased gene transcription. To further validate this hypothesis, we chose a

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straightforward approach in which we extracted and quantitated total RNA in each

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proteinopathy model and compared them to control. As we expected, total RNA levels


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were lower in the FUS overexpression background compared to control (Figure 6a).

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Together with our histone modification data, we interpret this result to indicate a global

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decrease in gene expression occurs in the FUS model. This finding agrees with previous

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investigations, which found transcriptional activity is decreased in neurons presenting

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FUS mislocalization.32 Notably, we observed this effect despite protein overexpression.

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As FUS is a RNA-binding protein, it is possible that it sequesters RNA in protein

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aggregates. However, we did not observe a similar effect in the context of TDP-43

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overexpression. TDP-43 is also a RNA- binding protein, and thus if the change in RNA

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levels resulted from sequestration then this effect should be present in the TDP-43

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samples as well. We observed no significant differences in total RNA levels in cells

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overexpressing TDP-43 (Figure 6a) or cells overexpressing α-synuclein (Figure 6b).

350

While the histone modification changes observed in the TDP-43 model (increases in

351

histone H4 acetylation as well as H3K36me3) support transcriptional activation, it is

352

possible that the increased gene transcription corresponds to specific genomic loci and

353

thus it would not be detected by changes in total RNA levels.

354 355

Distinct, Non-Overlapping Histone PTMs Landscapes for Each Proteinopathy

356

Model Emphasize Histone Cross-Talk

357

We present a summary of all observed histone PTMs changes in Figure 7.

358

Extraordinarily, we found very little overlap between the histone PTM alterations elicited

359

in each proteinopathy model. Indeed, only H2BT129ph is altered in both FUS and α-

360

synuclein models. Overall, the most intense modification changes are associated with

361

FUS proteinopathy, followed by TDP-43 and α-synuclein proteinopathies. Unsurprisingly,

362

the number and magnitude of changes appear to correlate with cytotoxicity of protein

363

expression. Remarkably, overexpression of TDP-43 is the only one to correlate to


14

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364

increases in any modifications. Altogether, our data indicates important decreases in

365

histone phosphorylation and acetylation are associated with cytotoxic phenotypes.

366

Our data also underscores histone crosstalk between modifications and provides

367

hints regarding the mechanisms of PTM interaction. For instance, we observed

368

decreases in both H3S10ph and H3K14ac in the FUS proteinopathy background (Figure

369

7). In yeast, H3S10ph promotes acetylation of H3K14 at specific gene promoters.81 Thus,

370

it is not surprising to observe that these two modifications decrease in tandem. In the

371

context of FUS overexpression, we also observe decreases in H2BT129ph (Figure 7).

372

Ipl1 –the yeast homolog of Aurora B Kinase- is also able to phosphorylate H2B.82

373

Therefore, decreases in the phosphorylation of these two sites might be pointing at Ipl1

374

impairment in FUS proteinopathy. Lastly, we observed evidence for inter-histone

375

crosstalk, as we identify decreases in both H4R3me2 and H3K14ac (Figure 7).

376

H4R3me2 has been previously linked to histone H3 acetylation at both lysine 9 and

377

lysine 14.67 Other correlations present in our data could arise from yet undiscovered

378

crosstalk processes.

379

It is important to note that our data is limited to yeast models of ALS and PD, and

380

thus verification in other model systems is still lacking. Furthermore, it is difficult to

381

establish causal relationships from our experiments: do changes in histone modifications

382

lead to disease processes or do disease processes lead to changes in histone

383

modifications? Issues of causality are a struggle for the field of histone modifications at

384

large.83 Nevertheless, we establish a clear association between strong decreases in

385

histone phosphorylation and acetylation with most severe cytotoxic phenotypes.

386

Furthermore, changes in histone PTMs occur as early as five hours into protein

387

overexpression. Regardless of causation, histone modification pathways are highly

388

accessible pharmacologically and thus they hold great promise for disease treatment.


15


389

Page 16 of 35

Recent work has targeted several histone ‘writers’ and ‘erasers’ to ameliorate

390

ALS and PD phenotypes. Notably, HDAC inhibitors have been used with success in

391

mice ALS models.84 In ALS mice models, treatment with an HDAC inhibitor led to

392

neuroprotection and improved motor function,85 while treatment with phenylbutyrate (a

393

general HDAC inhibitor) significantly extended survival and improved clinical and

394

neuropathological phenotypes.86 Our findings highlight further histone modifiers as

395

potential targets for pharmaceutical intervention. In addition to reversing decreases in

396

histone acetylation, novel chemical intervention strategies aimed at reversing decreases

397

in histone phosphorylation and methylation could potentially improve cell survival in the

398

context of neurodegenerative disease. Based on our results, inhibitors for Protein

399

Phosphatase 1 and 2, which remove H3S10ph,59, 87 as well as inhibitors of JMJD6, a

400

Jumonji domain-containing protein reported to demethylate H4R3me2,88 could become

401

useful therapeutic avenues for ALS.

402 403

CONCLUSIONS

404

We have carried out the first comprehensive characterization of histone

405

posttranslational modifications in any ALS or PD models. Remarkably, we discover that

406

distinct changes in histone modification profiles for each proteinopathy model evidencing

407

that histone PTM changes are not associated with general protein aggregation toxicity

408

pathways. Furthermore, we find widespread changes detectable in whole genome

409

samples. We show FUS aggregation is associated with many pronounced changes in

410

several histone marks including phosphorylation of histone H2B, lysine phosphorylation

411

and acetylation on histone H3, as well as histone H4 arginine methylation. We also

412

observe a global decrease in gene transcription in the context of FUS proteinopathy.

413

Furthermore, we identify moderate disturbances in histone modification patterns in cells

414

overexpressing TDP-43 and α-synuclein. Our data corroborates previous findings in


16

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415

different model systems, while significantly broadening our knowledge of the epigenetic

416

landscape of neurodegenerative disease. The expanded set of modifications we present

417

here could uncover novel histone ‘writer’ and ‘eraser’ targets for pharmacological

418

intervention. Overall, our results underscore the potential importance of epigenetic

419

mechanisms in the occurrence and progression of ALS, PD, and other

420

neurodegenerative diseases and lead to novel, more effective therapies for these

421

ailments.

422 423

METHODS

424

Materials. All chemicals were purchased from Sigma Aldrich (St. Louis, MO) unless

425

otherwise specified.

426 427

Yeast Strains, Media, and Plasmids. All yeast were W303a (MATa, can1-100, his3-

428

11,15, leu2-3,112, trp1-1, ura3-1, ade2-1).39 Yeast were grown in synthetic dropout

429

media (Clontech Laboratories, Inc., Mountain View, CA). Media was supplemented with

430

2% glucose, raffinose, or galactose. Vectors encoding TDP-43, FUS, and α-synuclein

431

(pAG303GAL-TDP-43, pAG303GAL-FUS, and pAG303GAL-α-synuclein-GFP,

432

respectively) were a gift from A. Gitler. 36, 40, 43 A pAG303GAL-ccdB vector was used as a

433

control. pAG303GAL-ccdB was a gift from Susan Lindquist (Addgene plasmid # 14133).

434 435

Yeast Transformation, Spotting Assays, and Liquid Culture Growth Assay. Yeast

436

were transformed according to standard protocols using polyethylene glycol and lithium

437

acetate.44 For spotting assays, yeast were grown to saturation overnight in raffinose-

438

supplemented dropout media at 30ºC. Cultures were diluted 2-fold, serially diluted 2-fold,

439

and then spotted in duplicate onto synthetic dropout media containing glucose, sucrose

440

galactose or mixtures of sucrose and galactose. Plates were analyzed after growth for 2-


17


Page 18 of 35

441

3 days at 30ºC. For liquid culture growth assays, yeast were grown in galactose-

442

supplemented or sucrose/galactose-supplemented media for 48 hours at 30ºC. Optical

443

density measurements were taken every 24 hours.

444 445

Bleomycin Treatment. Wild-type W303a yeast were grown to saturation overnight at

446

30ºC on YPD media (Dot Scientific, Burton, MI, Cat #DSY20092-1000). Yeast were

447

standardized to an optical density of 0.3 and grown for 4 hours at 30ºC. Cultures were

448

then checked to verify log phase growth and treated with 20 µg/ml bleomycin (EMD

449

Millipore Corp., Billerica, MA, Cat # 203401-10MG) for one hour. After treatment,

450

cultures were normalized to an optical density of 0.9; 10 mL of cells were then harvested

451

and frozen at -80ºC.

452 453

Protein Overexpression. Protein overexpression in yeast was induced in galactose- or

454

sucrose/galactose-containing medium for 5 hours (TDP-43 and FUS) or 8 hours (α-

455

synuclein-GFP) at 30ºC. After induction, cultures were normalized to an optical density

456

of 0.9; 10 mL of cells were then harvested and frozen at -80ºC.

457 458

Western Blotting. Frozen yeast cell pellets were thawed and treated with 0.2M NaOH

459

for 10 minutes on ice, pelleted again and subsequently resuspended in 100 µL 1×SDS

460

sample buffer and boiled for 10 minutes. Cell lysates were separated using SDS-PAGE

461

(15%) and then transferred to a PVDF membrane (EMD Millipore, Billerica, MA).

462

Membranes were blocked using LI-COR blocking buffer (LI-COR Biosciences, Lincoln,

463

NE) for 1 hour at room temperature. Primary antibody incubations were performed at

464

4°C overnight. Antibodies used were: rabbit anti-GFP polyclonal (Sigma Aldrich,

465

Cat#G1544), rabbit anti-TDP-43 polyclonal (Proteintech, Chicago, IL, Cat #10782-2-AP),

466

rabbit anti-FUS polyclonal (Bethyl Laboratories, Montgomery, TX, Cat#A300-302A), and


18

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467

mouse anti-3-phosphoglycerate kinase (PGK) monoclonal (Novex, Frederick, MD,

468

Cat#459250). For histone modifications, all antibodies were purchased from Abcam

469

(Cambridge, MA) unless otherwise specified: H3 (cat #ab24834), H3K4me1 (cat

470

#ab8895), H3K4me2 (cat #ab7766), H3K4me3 (cat #ab8580), H3K9ac (cat #ab10812),

471

H3K14ac (Millipore, cat #07-353), H3K18ac (cat #ab1191), H3K27ac (cat #ab45173),

472

H3K36me1 (cat #ab9048), H3K36me2 (cat #ab9049), H3K36me3 (cat #ab9050),

473

H3K56ac (ActiveMotif, Carlsbad, CA, cat #39281), H3K79me1 (Millipore, cat #ABE213),

474

H3K79me2 (cat #ab3594), H3K79me3 (cat #ab2621), H3S10ph (cat #ab5176), H4 (cat

475

#ab10158), H4K8ac (cat #ab15823), H4K12ac (cat #ab46983), H4K16ac (cat

476

#ab109463), H4K20me1 (cat #ab9051), H4K20me3 (cat #ab9053), symmetric H4R3me2

477

(cat #ab5823), H4R3me2 (cat #ab194696), H2AS129ph (cat #ab15083), and

478

H2BT129ph (cat #ab188292). Blots were processed using goat anti-mouse and anti-

479

rabbit secondary antibodies from LI-COR Biosciences (Lincoln, NE) and imaged using

480

an Odyssey Fc Imaging system (LI-COR Biosciences, Lincoln, NE). All immunoblotting

481

experiments were repeated a minimum of three times with independent cell samples. As

482

blots for TDP-43 and FUS were performed separately from blots for α-synuclein, these

483

are shown separately throughout our results.

484 485

RNA Purification. Yeast were grown and induced in galactose-containing medium for 5

486

hours (TDP-43 and FUS) or 8 hours (α-synuclein-GFP). 2 x 107 cells were harvested for

487

each sample and treated with 100 U Zymolyase-20T (Nacalai USA, San Diego, CA, cat

488

# 07663-91) for 30 minutes at 30°C. RNA was isolated using the RNeasy Mini Kit from

489

Qiagen (Germantown, MD) according to the manufacturer’s instructions. Total RNA

490

levels were measured using a NanoDrop Lite spectrophotometer (Thermo Fisher,

491

Waltham, MA). All experiments were repeated a minimum of three times with

492

independent cell samples.


19


Page 20 of 35

493 494

Data Analysis. Densitometric analysis of Western blots was performed using Image

495

Studio (LI-COR). The signals obtained for histone modifications were normalized to their

496

respective total histone H3 signals (modification/total H3). These values were then

497

compared against the control sample values to obtain fold change values

498

(sample/control), which were used for statistical analysis. A similar procedure was used

499

to analyze Western blot data from sucrose experiments; the normalized signals were

500

compared to the sucrose samples' signals. RNA concentrations were normalized against

501

controls to obtain fold change values used for further analysis.

502 503

Statistical Analysis. Statistical analysis of data was performed in R 3.3.1 using the

504

built-in stats package (R-Project, Vienna, Austria). Significant differences between

505

sample groups (Control vs. TDP-43, Control vs. FUS, and Control vs. α-synuclein) was

506

determined using Welch's t-test with p = 0.05 as the cut-off for significance. Error bars

507

on the graphs represent the standard error of the mean calculated from values obtained

508

in the data analysis step described above. Figure 1 was constructed from these p-

509

values, which were transformed according to a previously described method using the

510

equation, − logሺ‫ݔ‬ሻ ∗ ‫ݕ‬, where ‫ ݔ‬represents the p-value and ‫ ݕ‬represents the sign of the

511

test statistic.45 Western blot data from sucrose experiments were analyzed using one-

512

way ANOVA, followed by Tukey's test for pairwise comparison of the group means.

513 514

ACKNOWLEDGEMENTS AND FUNDING INFORMATION

515

We thank Prof. James Shorter and Prof. Aaron Gitler for kindly sharing reagents.

516

We also thank Marcella Meykler, Farrah Benoit, Farzanna Mohamed and Royena Tanaz

517

for technical help. Brooklyn College and the Advanced Science Research Center (CUNY)


20

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518

as well as an NINDS Advanced Postdoctoral Career Transition Award (K22NS09131401)

519

from the NIH support MPT.

520 521 522

AUTHOR CONTRIBUTIONS Conceived and designed the experiments: KC, SAB, MPT. Performed the

523

experiments: KC, SAB, NR, MS, HY, ST, NM, SMM. Analyzed the data: KC, SAB, NR,

524

MS, HY, ST, NM, SMM, MPT. Contributed reagents/materials/analysis tools: KC, SAB,

525

MPT. Wrote the paper: KC, SAB, MPT. All authors reviewed the manuscript.

526 527 528

SUPPORTING INFORMATION Growth assays, overexpression verification, positive controls, expression tuning

529

experiments as well as representative immunoblots for unchanging histone modifications

530

and their quantitation are presented in Supplementary Figures 1-8.

531 532

REFERENCES

533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548 549 550 551 552

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Figure 1. Histone Modifications are Altered in the Context of Yeast Overexpressing TDP-43, FUS, and αsynuclein (α-Syn). p- values for the ratio of histone H2A, H2B, H3 and H4 PTMs abundances in yeast strains expressing TDP-43, FUS, and α-synuclein (α-Syn) relative to control cells are shown. The scale is based on p-values derived from statistical analysis of Western blotting experiments. p- values were calculated using a two- tailed t-test with Welch's modification Green indicates more statistically significant decreased modification levels, while red indicates more statistically significant increased modification levels. 84x121mm (300 x 300 DPI)


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Figure 2. α-synuclein and TDP-43 and Proteinopathy Models Display Decreases in Histone H3 Di- and Trimethylation on Lysine 36, respectively. Representative immunoblots showing the levels of (a,c) H3K36me2, (b,d) H3K36me3 for TDP-43 and FUS or α-synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside blots. All graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate the +SEM. n = 3-7 for each modification. **p < 0.01, ***p < 0.001. 177x134mm (300 x 300 DPI)



Figure 3. FUS Overexpression Correlates with Changes in the Phosphorylation of Serine 10 and Acetylation of Lysine 14 and Lysine 56 on Histone H3. Representative immunoblots showing the levels of (a,d) H3S10ph, (b,e) H3K14ac, and (c,f) H3K56ac for TDP-43 and FUS or α-synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside blots. All graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate the +SEM. n = 3-6 for each modification. **p < 0.01, ***p < 0.001. 177x93mm (300 x 300 DPI)


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Figure 4. FUS and TDP-43 Overexpression Correlate with Changes in Arginine Di-methylation and Lysine Acetylation on Histone H4. Representative immunoblots showing the levels of (a,d) H4R3me2, (b,e) H4K12ac, and (c,f) H4K16ac for TDP-43 and FUS or α-synuclein yeast proteinopathy models, respectively. Quantitation histograms compiling multiple biological replicates are presented alongside blots. All graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate the +SEM. n = 3-6 for each modification, *p < 0.05. 177x135mm (300 x 300 DPI)



Figure 5. FUS and α-synuclein Proteinopathies are associated with Changes in Histone H2B Phosphorylation Levels. Representative immunoblots showing the levels of H2BT129ph for (a) TDP-43 and FUS or (b) αsynuclein yeast proteinopathy models. Quantitation histograms compiling multiple biological replicates are presented alongside blots. All graphs display the mean fold change in modification levels for each group based on densitometric analysis of Western blots. Error bars indicate the +SEM. n ≥ 3 for each modification. *p < 0.05, **p < 0.01. 177x51mm (300 x 300 DPI)


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Figure 6. RNA Levels Are Decreased in a FUS Overexpression ALS Model (a) Total RNA levels in yeast control cells as well as cells overexpressing TDP-43 and FUS. (b) Total RNA levels in yeast control cells as well as cells overexpressing α-synuclein. All graphs display the mean fold change in total RNA levels for each group. Error bars indicate the SEM. n = 3 for each group. *p < 0.05. 84x54mm (300 x 300 DPI)



Figure 7. Yeast Models of Neurodegenerative Disease Proteinopathies are connected to Distinct Histone PostTranslational Modification Landscapes. Green bars indicate significant decreases, while red bars indicate significant increases relative to control samples. Error bars indicate the +SEM. n ≥ 3 for each modification. 84x154mm (300 x 300 DPI)


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Graphical Abstract- For Table of Contents Only Chen et al " Neurodegenerative Disease Proteinopathies are Connected to Distinct Histone PostTranslational Modification Landscape" 80x39mm (300 x 300 DPI)


Neurodegenerative Disease Proteinopathies Are Connected to

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