Proteomic Analysis of Azacitidine-Induced Degradation Profiles

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Proteomic analysis of Azacitidine-induced degradation profiles identifies multiple chromatin and epigenetic regulators including Uhrf1 and Dnmt1 as sensitive to Azacitidine Emily H. Bowler, Joseph Bell, Nullin Divecha, Paul Skipp, and Rob M. Ewing J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00745 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 25, 2019

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Journal of Proteome Research

Proteomic

analysis

of

Azacitidine-induced

degradation profiles identifies multiple chromatin and epigenetic regulators including Uhrf1 and Dnmt1 as sensitive to Azacitidine AUTHOR NAMES Emily H Bowler1, Joseph Bell1, Nullin Divecha1, Paul Skipp1, Rob M. Ewing1*

AUTHOR ADDRESS

1

School of Biological Sciences, University of Southampton, Southampton, SO17

1BJ, UK

Corresponding Author

ACS Paragon Plus Environment

1

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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* e-mail: [email protected]; Computational and Systems Biology, School of Biological Sciences, University of Southampton, Southampton SO17 1BJ, UK; Tel: +44 (0) 2380 594401


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ABSTRACT DNA methylation is a critical epigenetic modification that is established and maintained across the genome by DNA methyltransferase enzymes (Dnmts). Altered patterns of DNA methylation are a frequent occurrence in many tumour genomes, and inhibitors of Dnmts have become important epigenetic drugs. Azacitidine is a cytidine analog that is incorporated into DNA and induces specific inhibition and proteasomal-mediated degradation of Dnmts. The downstream effects of Azacitidine on CpG methylation and on genetranscription have been widely studied in many systems, but how Azacitidine impacts the proteome is not well understood. In addition, with its specific ability to induce rapid degradation of Dnmts (in particular the primary maintenance DNA methyltransferase, Dnmt1), it may be employed as a specific chemical knockdown for investigating the Dnmt1-associated functional or physical interactome. In this study, we use quantitative proteomics to analyze the degradation profile of proteins in the nuclear proteome of cells treated with Azacitidine. We identify specific proteins as well as multiple pathways and processes that are impacted by

Azacitidine.

The

Dnmt1

interaction

partner,


Uhrf1,

exhibits

significant

3


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Azacitidine-induced degradation, and this Azacitidine-induced degradation is independent of the levels of Dnmt1 protein. We identify multiple other chromatinand

epigenetic-associated

factors,

including

the

Bromodomain-containing

transcriptional regulator, Brd2. We show that Azacitidine induces highly specific perturbations of the Dnmt1-associated proteome, and whilst interaction partners such as Uhrf1 are sensitive to Azacitidine, others such as the Dnmt1 interaction partner and stability regulator, Usp7, are not. In summary, we have conducted the first comprehensive proteomic analysis of the Azacitidine-sensitive nuclear proteome, and we show how 5-azacitdine can be used as a specific probe to explore Dnmt and chromatin related protein networks.

KEYWORDS DNMT1; Azacitidine; UHRF1; chemical knock-down

Introduction


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Patterns of DNA methylation, a key epigenetic mark across the eukaryotic genome, are established and maintained by the DNA methyltransferase proteins (Dnmts) which catalyse the addition of methyl groups to cytosine (at CpG dinucleotides) from S-adenosyl-methionine. CpG methylation is broadly distributed throughout vertebrate genomes, and typically 80% of these residues are methylated. The exception to this are CpG islands, dense clusters of CpG sequences that typically remain unmethylated, and that are associated with the majority of gene promoters 1. All Dnmt proteins consist of a C-terminal catalytic domain which catalyses the transfer of the methyl group, and an N-terminal regulatory region that undergoes diverse protein-protein interactions to regulate Dnmt activity

2.

DNA Methyltransferase I (Dnmt1) is the principal DNA

methyltransferase in eukaryotic cells, preferentially methylating hemi-methylated CpG sites to replicate patterns of methylation after DNA replication 1. Although Dnmt1 is the primary catalyst of DNA methylation, other proteins such as Uhrf1 (Ubiquitin-like with PHD and Ring Finger Domains 1) interact with Dnmt1


3

and

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serve to target Dnmt1 towards CpG methylation sites by binding hemi-methylated sites cooperatively with Dnmt1 4.

Alteration of Dnmt1 activity and patterns of CpG methylation are frequently observed in many different cancers and hyper-methylation of CpG islands may lead to transcriptional repression, a mechanism through which cancer cells silence tumour suppressor genes

5.

Re-activation of silenced genes is an important

avenue for cancer therapy and several cytosine analogs have been identified

6

including Azacitidine, shown to specifically inhibit Dnmt1 inducing demethylation of hyper-methylated regions with concomitant re-expression of silenced genes. Mechanism of action studies have showed that Azacitidine causes Dnmt1 to be trapped in the nucleus

7

. In the presence of Azacitidine, Dnmt1 is not released

following transfer of the methyl group and instead forms a covalent adduct with the associated DNA that results in proteasomal-mediated degradation of Dnmt1 in the nucleus 8.


6

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Although mechanistic studies have shown Dnmt1 is degraded in Azacitidinetreated cells, there has been little attention on the effects of Azacitidine on the wider proteome. Previous proteomic analysis of the effects of Azacitidine have used whole cell proteomic analysis and focused on the cytotoxic effects of Azacitidine

9

or for the identification of pathways to optimize clinical usage of

Azacitidine

10.

Using Azacitidine as a chemical knockdown agent for degradation

of Dnmt1, we aimed to characterize nuclear Dnmt1-associated proteins and protein networks that are sensitive to Azacitidine-mediated degradation. We first measured the effects of Azacitidine dosages on Dnmt1 protein abundance across a timecourse, and then used this to conduct a systematic quantitative proteomic analysis of the nuclear proteome of 5-azactidine treated-cells with the goal of identifying proteins and protein networks that are perturbed in response to Azacitidine treatment and Azacitidine-induced degradation of Dnmt1. We identify proteins, including multiple chromatin and epigenetic regulators such as Uhrf1 that are specifically degraded by Azacitidine. We show that Uhrf1 and associated chromatin-modification

factors

are

sensitive

to


Azacitidine

and

that

their

7


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degradation is not associated with knock-down of Dnmt1 per se (through RNAi) but is a specific effect of Azacitidine degradation.

MATERIALS AND METHODS Cell culture and Azacitidine treatment Colorectal cancer cell lines HCT116 were regularly maintained in McCoy-5A media (Life Technologies, 16600-108, Carlsbad, CA) containing 10% fetal bovine serum (Life Technologies, 10438-026, Carlsbad, CA) and 1% streptomycinpenicillin (Life Technologies, 15140-148, Carlsbad, CA) at 37°C in CO2 incubator (5% CO2, 100% H2O). Cells were washed with warm PBS and then cells were detached using TrypLE express 1X (ThermoFisher). Detached cells were then washed with ice cold PBS, and lysed according to the following protocols. Cells were seeded 24 hours prior to replacement of standard growth media with growth media supplemented with 1M Azacitidine (Sigma 5-Azacytidine, A2385). Treated cells were then grown in Azacitidine supplemented media for the time periods described, at which point media was removed, cells were washed with warm PBS


8

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and then detached using TrypLE express 1X (ThermoFisher). Detached cells were then washed with ice cold PBS, and lysed according to the following protocols. Cell viability was assayed as follows. 5 x 10x3 HCT116 cells were seeded in each well of a 96 well plate and treated at the described time points with 1M Azacitidine, 10M

Azacitidine, or PBS control supplemented growth media.

Following completion of the time course, spent media was removed and assessed for cell cytotoxicity, and replaced with fresh growth media. For quantification a viable cell count standard curve was seeded using a dilution series from 12 x 104 cells. 20l of MTS/PMS solution was added and then assay was returned to cell culture incubator for 3 hours before quantification of absorbance at 490nm using PolarStar Omega (BMG Labtech) and Mars-data analysis software (BMG Labtech).

Protein sample preparation Cell pellets were re-suspended in RIPA buffer (50mM Tris-HCl pH8, 150mM NaCl, 0.1% SDS, 0.5% Deoxycholic acid, 1% NP-40.) supplemented with 1l of


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100x Protease inhibitor cocktail (ThermoFisher Scientific). Lysate was incubated for five minutes at room temperature before centrifugation at 16,000 x g at 4oC for 10 minutes (Eppendorf Centrifuge 5415R), supernatant was then removed and stored at -20oC or at -80oC for long term storage. Nuclear protein sample extract from washed cell pellets was carried out according to the manufactures protocol using cell fraction kit – standard (Abcam, ab109719). Nuclear pellets were resuspended in 100mM ammonium bicarbonate and lysed by sonication at 26 joules. Cell protein lysates were quantified using the manufactures protocol, PierceTM BCA protein assay kit (ThermoFisher Scientific). Colour change was read and protein quantifications calculated using PolarStar Omega (BMG Labtech) and Mars-data analysis software (BMG Labtech).

SDS-PAGE & Immunoblotting 15µg of protein lysate, was incubated in a heating block (Benchmark) at 95oC for five minutes with Tris-glycine sample buffer (252mM Tris-HCl ph 6.8, 40% glycerol, 8% SDS, 0.01% bromophenol blue) and 1M DTT. Samples were then


10

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loaded and run on an 8% acrylamide gel (4ml of 2x gel buffer (160mM Tris-HCl pH7.4, 0.2M serine, 0.2M asparagine, 0.2M glycine),1.6ml of 40% acrylamide, 2.4ml

H2O,

32µl

10%

ammonium

persulfate

(APS),

16µl

of

tetramethylethylenediamine (TEMED)) with 1x Tris-glycine running buffer (25mM Tris, 192mM Glycine, 0.1% SDS). The gel was run for ten minutes at 100 volts (Biorad), then one hour and twenty minutes at 120 volts (Biorad) at 4oC. Proteins were transferred (XCell2 blot module, Invitrogen) to a nitrocellulose membrane (Amersham Protran 0.45m NC) at 80 volts, 0.4 amps (Biorad) for three hours at 4oC in 1x Towbin transfer buffer (0.25M Tris pH8.6, 1.92M glycine, 0.025% SDS) using a XCell2 module (Invitrogen). Nitrocellulose membrane was then washed with 1xTBST twice and blocked for thirty minutes in 5% 1xTBST milk solution at room temperature. Membrane was then washed twice more in 1xTBST and incubated for two hours with primary antibody at 4oC on a rocker. Membrane was then washed twice, for five minutes at room temperature, with 1xTBST, before incubating for one hour at room temperature with secondary LICOR antibody (1xTBST 5% milk solution). Membrane was then washed three


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times for five minutes at room temperature with 1xTBST before imaging on a LICOR Odyssey® CLx and analysing with Image Studio Lite V5.2 software.

Antibody

Product

Company

Host

code

Dilution

species

Primary antibodies Brd2

ab139690

Beta-Actin

8H10D10

β-catenin/

Abcam

Rabbit

1:1000

Cell signalling

Rabbit

1:1000

MA1-301

ThermoFisher

Rabbit

1:1000

Dnmt1

D63A6

Cell signalling

Rabbit

1:1000

Gsk3β

27C10

Cell signalling

Rabbit

1:1000

Lamin A/C

2032S

Cell signalling

Rabbit

1:1000

Alpha-Tubulin

2144S

Cell signalling

Rabbit

1:1000

Uhrf1

PA5-29884

Invitrogen

Rabbit

1:1000

Usp7

Sc-30164

Santa

Rabbit

1:1000

Ctnnb1

Cruz

Biotechnology Secondary antibodies


12

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Rabbit 800CW

925-32211

Li-Cor

-

1:10,00 0

Mouse 680CW

925-68020

Li-Cor

-

1:10,00 0

Table 1 List of all primary and secondary antibodies and sources used in the study

Proteomic sample preparation and mass-spectrometry Nuclear pellets were lysed (0.1M TEAB, 0.1% SDS) with pulsed sonication, samples were centrifuged for 10 minutes, 13,000 x g, and supernatant protein quantified as described previously. Methanol/chloroform extraction was performed on 100g of protein for each lysate and pellet finally re-dissolved in 100ul of 6M Urea (Sigma), 2M thiourea (Sigma), 10mM HEPES buffer (Sigma), ph7.5. Samples were reduced (5mM DTT), alkylated (25mM iodoacetamide) then diluted in 400µl of 20mM ammonium biocarbonate (Sigma) and digested with trypsin (Promega) (1/50 w/w) overnight. Each fraction was acidified to 2

and where the p-value for 48 vs 0 hours

aza < 0.05 were used to create the set of significantly altered 5-azactidine proteins at 48 hours (and similarly for 10 hours). These protein sets were combined to create two sets of proteins: significantly increased by Azacitidine and significantly decreased by Azacitidine. Gene Ontology term enrichment was computed for these sets of proteins using the Pathway Studio database (Elsevier), version 9.0. Gene/Protein identifiers were imported and networks created by selecting all direct edges between the imported nodes (Physical Interactions, Expression Regulation and Protein Modification relations). Pathway Studio


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database network nodes and edges were supplemented by addition of STRING database interactions.

Generation of shRNA-containing lentivirus.

One million Hek293 FT cells were plated per well in a 6 well culture plate (Corning) and transfected 2 hours post-seeding with a 4-2-1 ratio of Plasmid: gagpol:vsvg using polyethyleneimine (3ug:1ug DNA) in 100µl serum-free DMEM (LifeTech). Cells were transfected overnight, and media was replaced the following morning with 2ml DMEM, 10%FCS, and 5% PenStrep. Cells were incubated for a further 24 hours before lentiviral-containing media was removed, filtered through a 0.45µm filter and lentiviral-containing media was stored at -80oC until required.

Transduction of cells with shRNA lentivirus

1 x 105 HCT116 cells were plated per well in a 6 well culture plate (Corning). Once adhered

culture medium was removed and replaced with lentiviral particles


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diluted in media (500µl viral media, 1.5ml DMEM, 10% FCS, 5% PenStrep, 8µg Polybrene (Sigma)). Cells were incubated with virus-containing media overnight and then grown for a further 24 hours in fresh culture medium (DMEM, 10% FCS, 5% PenStep). Stable cell lines were selected with puromycin (2µg/ml) over five day.

RESULTS Azacitidine degradation of Dnmt1 occurs over a period of hours, with a range of time points and dose concentrations used in previously published research, both to investigate the inhibitory effects on DNA methyltransferase activity and to assess downstream changes in CpG methylation

12,13.

Since our goal is to use

Azacitidine to knockdown Dnmt1, we first established appropriate Azacitidine concentrations and time points for use in our model HCT116 colorectal cancer cells. Previous research showed that degradation of Dnmt1 in monolayer culture conditions was evident following treatment with approximately 0.1M in HCT116 cell with a maximal degradation at 1 M

12.


We therefore assessed the

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degradation of Dnmt1 using concentrations of Azacitidine from 0.1 M to 10M as shown in Figure 1A. Dnmt1 degradation was evident at all concentrations, although a clear increase in Dnmt1 degradation was observed after 10hours in cells treated with 1M or higher concentrations of Azacitidine, showing that Azacitidine-induced degradation of Dnmt1 occurs in a dose- and time-dependent manner in HCT116 cells.


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Figure 1 A Western Blot analysis of Dnmt1 protein levels in HCT116 cells treated with Azacitidine. Concentrations of 5-Azactidine between 0.1M and 10 M were used to treat cells over a time course. 15 g of whole cell protein lysates from HCT116 Azacitidine treated and control cells were loaded onto a single phase 8% SDS gel and analyzed by western blot analysis for protein expression of Dnmt1. Tubulin protein expression was used as a loading control. The associated plot shows the relative abundance of Dnmt1 protein levels across the time course following treatment with Azacitidine dose-course. Dnmt1 protein abundance was normalized to loading control and plotted for each concentration of Azacitidine.

Azacitidine treatment of cells has previously been shown to cause cell toxicity and reduced cell viability

14.

A cell viability assay to assess the effect of 1M

and 10 M Azacitidine treatment on HCT116 cells over the proposed time course indicated that following 10 hours of treatment with both 1M and 10 M of


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Azacitidine there was a comparable decrease in cell viability of 8.76% and 9% respectively (Figure 1B). Following longer periods of treatment 10 M of Azacitidine showed a significant decrease in viability compared to cells treated with 1 M of Azacitidine by 6.38%, this was particularly evident following 48 hours of treatment with cells treated with 10 M of Azacitidine showing a 51.66% reduction in cell viability.


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Figure 1B Cell titre assay of viable cells following treatment with Azacitidine. Cells were treated for the specified time points with 1 M and 10 M concentrations of Azacitidine over a period of 48 hours. The number of viable cells was assessed using a CellTitre assay (Promega). * = significant difference between control and 10 M treated cells. ** = significant difference between control and 1 M treated cells (Student’s t-test; p-value

Proteomic Analysis of Azacitidine-Induced Degradation Profiles

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