Supplementary data for the manuscript Das et al.

, Characterization of nucleolin K88 acetylation defines a new pool of nucleolin colocalizing with pre-mRNA splicing factors

Supplementary Material and Methods Preparation of Nuclear S2 extract from HeLa Cells Nuclei purification was adapted from previously published protocols [1]. For one experiment, 3 108 cells were seeded onto 245245-mm Petri dishes in Eagles minimum essential medium (MEM alpha, Gibco) containing 10% fetal bovine serum (Gibco). Cells were incubated at 37C under a 5% CO2 containing atmosphere. At 80% confluence, cells were washed with cold phosphate-buffered saline, pH 7.4, and detached from the plate by scraping on ice. Cells were collected by centrifugation at 500 g for 5 min and resuspended in 15 volumes of nuclei buffer (10 mM Tris-HCl, pH 7.4, 250 mM sucrose, 2.5 mM MgCl2 and 0.1 mM CaCl2). Cell lysis was performed by sequential addition of a final concentration of 0.3% Nonidet P-40 (Roche Applied Science, Mannheim, Germany), 0.8 ml of collagenase (10mg/ml) and homogenization was performed using an Ultra-Turrax IKA-Werke (Germany). Nuclei were collected by centrifugation at 3500 g for 5 min and resuspended in 10 volumes of nuclei buffer for further washing. Nuclei were then purified by centrifugation at 3500 g for 5 min in 1mM EDTA (pH 8) solution. This supernatant is called nuclear S2. Identification of acetylation sites by mass spectrometry Identification of acetylated residues by mass spectrometry analysis - Mass-acetylated baculovirus expressed nucleolin was resolved by a 10% SDSPAGE, then the band was excised out and subjected to in-gel reduction, carbamidomethylation and tryptic digestion as previously described [2]). Peptide sequences were determined by mass spectrometry performed using a Q-STAR XL instrument (Qq TOF) equipped with a nanospray source (Applied Biosystems) and coupled to an online nanoLC system (Ultimate Famos Switchos; Dionex). A MS survey scan was acquired over the m/z range 400-1600 by data dependent MS/MS scans over the m/z range 65-2000 for the three most intense ions with a charge of 2 to 4. The spectra were recorded using dynamic exclusion of previously analyzed ions for 0.5 min with 50 millimass units (mmu) of mass tolerance. The collision energy was automatically set by the software (Analyst 1.1) and was related to the charge of the precursor ion. The peptide separation was obtained on a C18 PepMap micro-precolumn (5 m; 100 ; 300 m x 5 mm; Dionex) and a C18 PepMap nanocolumn (3 m; 100 ; 75 m x 150 mm; Dionex) using a linear 60-min gradient from 0 to 60% B, where solvent was 0.1% HCOOH in H2O/CH3CN (95/5) and solvent B was 0.1% HCOOH in H2O/CH3CN (20/80) at 300 nL/min flow Rate. Proteins identification and screening for acetylated peptides were performed with the Paragon Algorithm from the ProteinPilot software v. 2.0 (Applied Biosystems) against the SwissProt database (release 56.6) limited to the human species. Acetylation of peptides was confirmed by manual inspection of the corresponding MS/MS spectra. Cell culture and Immunofluorescence experiments Peripheral Blood Mononuclear Cells (PBMC) were grown at 37C with 5% CO2 in RPMI supplemented with 10% fetal bovine serum, glutamax and penicillin/streptomycin. They were isolated from healthy donors using Leucosep (Greiner bio-one) and Ficoll-Paque PLUS (GE Healthcare) according to manufacturers instructions. PBMC stimulation was performed by supplementing the culture medium with 1.5% Phytohemagglutinin (PHA M Form, Life 1

Technology) for 68 h. A million stimulated PBMC were fixed with cold ethanol and stained with propidium iodide (20g/mL, 30min 37C) for cell cycle analysis (LSRII, FACSDivaTM, BD Biosciences). HeLa cells were grown on coverslip and stimulated PBMC were grown on polylysine-coated coverslips for 24 h in DMEM medium supplemented with 10% fetal bovine serum. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 in PBS for 25 min. After two washes in PBS, non-specific binding of antibodies was blocked with blocking buffer containing 10% FCS, 3% BSA, 0.1% Triton X-100. Subsequently, coverslips were incubated in primary antibodies at 37C for 30 min. After two washes in PBS with 0.1% Triton X-100, cover slips were incubated with secondary antibodies coupled with Alexa dyes (A488, A555 or A647) for 30 min. After two more washes in PBS with 0.1% Triton X-100, the coverslips were washed in PBS, rinsed in ddH2O and briefly dipped in 100% ethanol. After a quick dry, cover slips were mounted with Fluoromount G containing 400 ng/ml DAPI. Images were captured with an Axio-Imager microscope. The images for comparative studies were captured at identical microscope settings. For subcellular colocalization analysis images were processed using Image J software and colocalization analysis were performed plotting the cytofluorogram using JACoP plugin [3]. The JACoP plugin was also used to calculate the amount of colocalization using Manders coefficients (M) since they are good indicators of the proportion of the green channel coincident with a signal in the red channel over its total intensity. Chromatin immunoprecipitation For NCL-K88ac ChIP-seq analysis, approximately 100 ng of input DNA and DNA precipitated by AcNcl1 antibody were sequenced with the Illumina/Solexa 1G technique (GATC, Germany). The input DNA sequences (27615364 reads) were used for normalisation of the NCL-K88ac ChIP-seq data (13779360 reads). Unique reads from the NCL-K88ac ChIP data set (9772860 reads) were mapped onto human rDNA reference sequence (NCBI accession number: HSU13369) using BWA [4,5]. We optimised the quality threshold (equal to 30) and mismatch penalty (-M) as 7 and d as 12 to obtain the mapping of unique reads. The mapped NCL-K88ac data and the control input data were then processed and filtered using tools and software algorithms from BioCOS Life Sciences, which uses data map quality-based filtering to eliminate repeated and poorly mapped reads. Furthermore, the mapped and filtered NCL-K88ac data is normalised by the mapped and filtered input control data and plotted as shown on Supplementary Figure 5. References [1] [2] [3] [4] [5] Ochs, R.L. (1998). Methods used to study structure and function of the nucleolus. Methods Cell Biol 53, 303-21. Pavat, C. et al. (2012). The shell matrix of the pulmonate land snail Helix aspersa maxima. Comp Biochem Physiol B Biochem Mol Biol 161, 303-14. Bolte, S. and Cordelieres, F.P. (2006). A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224, 213-32. Li, H. and Durbin, R. (2009). Fast and accurate short read alignment with BurrowsWheeler transform. Bioinformatics 25, 1754-60. Li, H. et al. (2009). The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078-9.

Filter Binding Assay

B
Gel Assay
HeLa core histones + [3H]-Acetyl CoA p300 PAT + p300

Counts per minute

14000 12000 10000 8000 6000 4000 2000 0

+ -

+ + p300

+ + CBP

+ + PCAF

+ + Gcn5

+ + Tip60

+ + Moz

Autoradiogram

Coomassie

Supplementary Figure 1. Normalization of the activity of the different protein acetyltransferases (PAT). (A) Filter-binding assay was performed to normalize the activities of different histone acetyltransferases (HATs) with core histones (purified from HeLa cells) as substrates. (B) An in vitro HAT assay was performed with 2g of HeLa core histones as a substrate with different acetyltransferases (p300, CBP, PCAF, Gcn5, and Tip60 and Moz). The amount of HAT was adjusted in order to get the same level of acetylase activity for each of them.

79 88 AcNcl1: NH2-CA(AcK)KAAVTPGK(AcK)A-COOH 102 110 AcNcl2: NH2-A(AcK)AVTTPGK(AcK)GC-COOH 109 116 AcNcl3: NH2-G(AcK)KGATPG(AcK)ALVATPGKKGC-COOH 9 15 AcNcl4: NH2-AG(AcK)NQGDP(AcK)KMAPC-COOH

3 2,5 2 Non-acetylated peptide Acetylated peptide

C
3 2,5 450nm OD 2 AcNcl1 AcNcl2 AcNcl3 AcNcl4

450nm OD

1,5 1 0,5 0 0 5000 10000 15000

1,5 1

0,5 0 20000 0 10000 20000 1/Dilution 30000 40000

1/Dilution

WB:AcNcl1 WB:H3 acetyl Lysine 14

Supplementary Figure 2. Characterization of AcNcl1 antibody. (A) Peptide sequences that were used for generating different polyclonal antibodies against these acetylated peptides. In red are shown the positions of the acetylated lysines. (B) ELISA test using Acncl1 serum and acetylated and non-acetylated AcNcl1 peptides. (C) ELISA test to check the cross reactivity of AcNcl1 serum with different acetylated peptides (AcNcl2, AcNcl3 and AcNcl4). (D) Western blot analysis of purified HeLa core histones with AcNcl1 and H3 acetyl Lysine 14 antibodies.

HeLa Core Histones

A
AcNcl1_Ctrl: NH2-CAKKAAVTPGKKA-COOH 79 88 AcNcl1: NH2-CA(AcK)KAAVTPGK(AcK)A-COOH 88 AcNcl1_Pep1: NH2-CAKKAAVTPGK(AcK)A-COOH 79 AcNcl1_Pep2: NH2-CA(AcK)KAAVTPGKKA-COOH

3,000 control peptide 2,500 2,000 450nm OD 1,500 1,000 0,500 0,000 0 20000 40000 1/Dilution 60000 80000 AcNcl1 AcNcl peptide 1 AcNcl peptide 2

Supplementary Figure 3. Specificity of the AcNcl1 serum for nucleolin K88. (A) Peptide sequences that were used for ELISA tests. In red are shown the positions of the acetylated lysines. (B) ELISA test with AcNcl1 serum and with the different peptides shown in (A).

Control siRNA

B
Nucleolin siRNA 0,25 Acetylated nucleoin level 0,2 0,15 0,1 0,05 0 1 2 Control siRNA Nucleolin siRNA

WB: AcNcl1 WB: Nucleolin WB: Beta-actin

mNCL

AcNcl1

mNCL+AcNcl1

Merge

DAPI

Control

Nucleolin siRNA

Supplementary Figure 4. Reactivity of AcNcl1 serum in nucleolin depleted cells. (A) HeLa cells were transfected with control (lane 1) or nucleolin siRNA (Lane 2) and cell extracts were analyzed by Western blot with AcNcl1, nucleolin and b-actin antibodies. (B) Quantification of the western blots. (C) Nucleolin silencing in HeLa cells using siRNA was performed as previously described (13). Immunofluorescence was performed with nucleolin monoclonal antibody (mNCL, red) and with AcNcl1 antibody (green). DNA was stained with DAPI (blue). Scale bar, 5m.

A
+1

18S

28S

Nucleolin ChIP-seq

* C
Nucleolin K88ac ChIP-seq

Supplementary Figure 5. Comparison of the nucleolin ChIP-seq data (Rong et al., Nucleic Acids Research, 40 (19):9441-54, 2012) with ChIP-seq for NCL-K88ac. (A) Schematic representation of a human rRNA repeat. (B) ChIP-seq mapping of nucleolin binding sites throughout the rRNA locus in HeLa cells. Reads that map to multiple locations were masked during the bioinformatic analysis, therefore regions rich in repetitive sequences can leave gaps as shown in the 18S region (star). See Rong et al, Nucleic Acids Research, 40 (19):9441-54, 2012 for details.(C) ChIP-seq mapping of NCL-K88ac binding sites throughout the rDNA locus. Data were analysed as explained in the Materials and Methods. Note the absence of nucleolin binding peaks in the promoter region and in the coding region of rDNA. These data were validated by Q-PCR. The genome wide distribution of NCL-K88ac will be published elsewhere.

Input

No Ab

WB : AcNcl1

Supplementary Figure 6. Immunoprecipitation with anti SC35 antibody (Abcam, ab11826). Immunoprecipitation (IP) assay was performed with HeLa cells extracts and with SC35 antibody. Following IP, Western Blot was done with AcNcl1 antibody. IP with No antibody (No ab) or with control IgG (pre-immune serum) were used as control.

IgG

SC35 Ab