Inventory of Supplemental Information presented in...

1

Inventory of Supplemental Information presented in relation to each of the main figures in the

manuscript

1. Figure S1. Related to Figure 1.







8. Table S1. Related to Figures 1 and S1.

9. Table S2. Related to Figures 3, 4, 6 and 7.

10. Table S3. Related to Figure 3.

Supplemental Experimental Procedures

1. Patients and samples

2. Cell culture

3. Long term culture-initiating cells (LTC-IC) assay

4. Differentiation of CD34+ cord blood cells

5. Bi-phasic erythroid differentiation assay

6. Clonal assay

7. Mutation and sequencing analysis

8. Bisulphite sequencing and quantitative pyrosequencing

9. Human SNP genotyping

10. Strand-specific cDNA synthesis and PCR

11. Chromatin Immuno-precipitation (ChIP)

12. Immunoprecipitation and western blot

13. Colony genotyping

14. shRNA generation and viral infection

15. Retrovirus generation and transduction

16. Study approval

17. Statistical Methods

18. Primers list

Supplementary References (19)

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10

Figure S1. Additional families showing SGK2 and GDAP1L1 are paternally expressed. Family

studies demonstrate that SGK2 and GDAP1L1 are expressed from the paternally derived allele in T

cells and erythroblasts respectively. These cell types were chosen because they express readily

detectable levels of SGK2 and GDAP1L1 respectively. Family 1 can be found in Figure 1. The

expressed allele is indicated by an arrow.

Figure S2. The L3MBTL1 gene cluster shows biallelic expression in mouse and wallaby but

predominantly monoallelic expression in the macaque. A Expression analysis in mouse tissue.

Sequences are shown for SNPs in exon 6 of L3MBTL1 (rs27347041), exon 6 of SGK2 (rs27331555)

and exon 5 of GDAP1L1 (rs28243099), each experiment was performed twice, representative traces

are shown. BM, bone marrow; PBMNC, peripheral blood mononuclear cells. B Expression analysis in

the Wallaby. Head and yolk sac membrane cDNA were analysed in four animals, a representative

animal is shown. C L3MBTL1 and SGK2 show polymorphic tissue-specific monoallelic expression in

the macaque (Macaca fascicularis). Two representative animals are shown. M1-male 1, F4/5 –

females 4 (for the kidney and liver samples) and 5 (for the blood sample), at least three SNPs were

tested for each gene per tissue from each animal; a representative trace is shown per tissue. Arrows

indicate the position of the SNP; WBC, white blood cells. D Phylogenetic tree demonstrating the

evolutionary relationships between species studied, MYA, million years ago.

Figure S3. Characterization of L3MBTL1 antisense transcript and microsatellite quantitation of

20q deletion. A Diagram of the genomic region between human L3MBTL1 and SGK2 demonstrating

genomic arrangement, putative promoters and alternatively spliced isoforms of L3MBTL1 around

exons 11-13, with corresponding encoded mRNAs and proteins annotated with known domains.

MBT, malignant brain tumour repeat; C2HC, C2HC-type Zinc Finger domain; SAM, Sterile Alpha

Motif. Arrows indicate locations for primers A, B and C for different mRNA transcripts, and P3 and

11

P5 between which lies the start point of the L3MBTL1 antisense RNA. B Characterization of

L3MBTL1 antisense RNA. Strand specific cDNAs for the sense and antisense strands were analysed

by RT-PCR. Controls are a primer control lacking template, indicating no template independent

amplification, and RT- control (reverse transcriptase minus), indicating no DNA contamination of the

RNA. C Stand specific RT-PCR and sequencing of sense and antisense transcript from K562 and a

normal informative individual. L3MBTL1 exon 24 expression is detected with a SNP in K562

(rs6124571) and normal PB granulocytes (rs6030948). D Strand specific quantitative RT-PCR for

antisense RNA on two normal individuals and two patient samples, one 20q normal cell line (K562)

and one 20q del cell line (HNT-34) using primer set for L3MBTL1 antisense RNA in exon 18 region

and cDNA synthesis using primer L3MBTL1-AS-P2 and ABL cDNA exon 4 primer. E Microsatellite

PCR analysis of Patient 6, MDS (RAEB 2) with 20q deletion in bone marrow (BM) sample compared

to peripheral blood mononuclear (PBMNC) cells control (Con). At least 3 microsatellites were

analysed per patient, representative traces shown. The percentages of cells carrying the deletion were

calculated as described in the materials and methods. Arrows indicate the allele lost.

Figure S4. Microsatellite quantitation and expression analysis to characterise hematopoietic

colonies carrying the 20q deletion. A Patient 11, MPN (ET) with 20q deletion in granulocytes

(Gran), compared to peripheral blood T cells control, and representative traces for each marker from

BFU-E colonies with deletion (n=9). The percentages of granulocytic cells carrying the deletion were

calculated as described in the materials and methods. Arrows indicate the allele lost. Note the

complete loss of the deleted allele in the colony-derived samples. Expression of imprinted genes in

hematopoietic colonies from B, normal individuals and C-E, from patients 8, 9, 12, 13 and 14. E,

BFU-E colonies; GM, CFU-GM colonies. Each point represents the mean of two PCR technical

replicates for a single colony. C Patient 8, CFU-GM colonies; D Patient 9, BFU-E colonies; E Patient

12, BFU-E colonies; F Patient 13, BFU-E colonies; G Patient 14, CFU-GM colonies; H Patient 14,

BFE-U colonies. See also figure 4.

12

Figure S5. Cooperative and compensatory effects of L3MBTL1 and SGK2 on erythroid and

megakaryocytic differentiation. A L3MBTL1 and SGK2 expression kinetics during in vitro CD34+

cord blood erythroid ( ) and megakaryocytic differentiation ( ). B Quantitative RT PCR for

expression of L3MBTL1 and SGK2 following infection of CD34+ cord blood cells with shRNAs

against L3MBTL1 (L3-shRNA1 and L3-shRNA2) and SGK2 (S2-shRNA1 and S2-shRNA2) at day 4

of differentiation. Expression levels relative to ABL were normalized to results obtained using

scrambled control shRNA. C Differentiation of cord blood CD34+ cells. Effect on erythroid

(CD71+GPA+) or megakaryocytic (CD41+CD61+) cell number of shRNA-mediated knockdown of

L3MBTL1 (L3-shRNA2 ), SGK2 (S2-shRNA2 ) or scrambled control (Scr-shRNA ). Each

data point is the mean of three independent cord blood differentiation experiments each performed in

triplicate. Error bars indicate standard error of the mean (SEM), **, p<0.01 relative to scramble

control. D Quantitative RT-PCR for expression of and globins (HBA and HBB) in day 4 infected

cells (GFP+) cells in erythroid differentiation conditions. E Quantitative RT-PCR for glycoprotein IX

(gpIX) expression in day 4 infected cells (GFP+) in megakaryocytic differentiation conditions. See

also figure 5.

Figure S6: Cooperative effect of knocking down L3MBTL1 and SGK2 on erythropoiesis. Bi-

phasic erythroid culture of CD34+ cells (see legend to Figure 4D). A Effect on percentage of CD71+ or

GPA+ cells of shRNA-mediated knockdown of L3MBTL1 (L3-shRNA1 ), SGK2 (S2-shRNA1 ),

L3MBTL1 and SGK2 (L3+S2-shRNA ) or scrambled control (Scr-shRNA ). B Lentiviral

infected erythroid progenitor cells (GFP+CD71+ or GFP+CFP+CD71+) were analysed for proliferation

at day 8 of phase I differentiation. Representative FACS profiles showing BrdU/7AAD analysis of

infected erythroid progenitor cells (GFP+CD71+ or GFP+CFP+CD71+). Percentage of cells in S-phase;

presented here as the histogram summarising results from three independent experiments each

performed in duplicate, *, p<0.05; **, p<0.01 relative to scramble control. Error bars indicate

13

standard error of the mean (SEM). C Apoptosis analysis at day 8 of phase I differentiation.

Representative FACS profiles showing Annexin V/7AAD analysis of infected erythroid progenitor

cells (GFP+CD71+ or GFP+CFP+CD71+) together with a histogram summarising results from three

independent experiments each performed in duplicate. Error bars indicate standard error of the mean

(SEM), *: p<0.05 relative to scramble control. D Quantitative RT-PCR analysis of retrovirally

expressed L3MBTL1 or SGK2 transcript levels in HNT-34 cells compared to endogenous transcript

levels in K562 and HEL cells. E FACS analysis shows that hemin-induced GPA expression is

inhibited by introduction of L3MBTL1 and/or SGK2. HNT-34 cells were infected with retroviruses

expressing L3MBTL1, or SGK2 or both before induction of erythroid differentiation by hemin.

Analysis was performed five days after induction. F. Quantitative RT-PCR analysis showing that

hemin-induced -globin (HBA) and -globin (HBB) expression is inhibited by introduction of

L3MBTL1 and/or SGK2. Error bars indicate standard error of mean. G Expression of imprinted genes

relative to ABL after 5-azacytidine induction at differing concentrations in 20q deleted HNT-34 cell

line. H Hemin induced expression of GPA with or without 5-azacytidine induction. Analysis was

performed 3 days after induction. I LTC-IC assay as in figure 6. Left panel shows the number of GM

colonies, middle panel - erythroid colonies (BFU-E) and right panel - mix colonies (CFU-GEMM).

Histograms represent mean of three independent experiments. J Lineage negative bone marrow cells

from normal C57B6 mice were infected with lentivirus expressing shRNA against L3mbtl1 and/or

Sgk2 or scrambled shRNA control. Transcript levels of L3mbtl1 and Sgk2 were analysed by

quantitative RT-PCR 3 days post infection. K CFU-S assay. Lineage negative bone marrow cells were

infected with lentivirus expressing shRNA against L3mbtl1 (mL3-shRNA), Sgk2 (mS2-shRNA), both

(mL3+mS2) or scramble control shRNA (Scr-ShRNA), sorted and injected into lethally irradiated

mice (5 per genotype) CFU-S were counted at day 10. L Assessment of knock-down efficiency in

recipient mice 16 weeks after competitive bone marrow transplantation. Quantitative RT-PCR was

performed on CD45.2+ lineage negative bone marrow cells. M Assessment of chimerism in recipients

of the competitive bone marrow transplant. Box plots represent the ratio of donor (CD45.2+) to

competitor (F1: CD45.1+CD45.2+) and host (CD45.1+) in the progenitor compartments (LSK, CMP,

14

GMP MEP) of five mice for each shRNA experiment. N Same as in panel K but analysis of mature

cells positive for B220, CD71, Mac-1, or CD3 in bone marrow, spleen and thymus.

Figure S7: L3MBTL1 and SGK2 collaborate to regulate MYC expression. A-E Analysis of cord

blood derived erythroid cells 8 days after lentiviral shRNA infection in phase I of biphasic erythroid

differentiation assay. One experiment from each independent cord blood sample is shown per panel,

the first experiment is shown in figure 7B-D. A Quantitative RT-PCR of MYC transcript levels

relative to ABL. ChIP-PCR analysis of B H3K4me3 on the MYC promoter, C phospho-S2 PolII

binding on the MYC transcriptional start site, D L3MBTL1 and E BRG1 binding to the MYC

promoter. F Analysis of HNT-34 cells, ChIP-PCR analysis of BRG1 binding to the MYC promoter

following retroviral expression of SGK2. Fold enrch. Rel. to IgG – QPCR quantitation Mean value

normalised to IgG control, with IgG value normalised to 1.

15

Gene PB Gr

PB T

CD34+ BM

mesenchymal cells from BM

GPA+ cells K562 293T

L3MBTL1 M M M M NI M M

SGK2 M M LE ND NI NI NI

GDAP1L1 M LE LE ND M NI NI

Supplementary table S1: Summary of cell types tested for monoallelic expression. All primary

cell types were derived from hematologically normal individuals. PB – peripheral blood, Gr –

granulocytes, T – CD2 positive T-lymphocytes, CD34+ - CD34 expressing hematopoietic

stem/progenitor cells, BM – bone marrow, mesenchymal cells – adherent CD34 negative bone

marrow cells cultured for 3 weeks. GPA+ cells – isolated GPA expressing erythroblasts. M-

monoallelic expression, NI – gene expressed, but no informative SNPs in samples tested, LE – low

expression precludes allelic expression analysis, ND – not done.

16

ID Dx

Age Dx

Dis dur (yrs)

WBC at Dx

Hb at Dx

Plt at Dx BM Cytogenetics JAK2

1 PV 59 21 8.5 20.7 467 46,XX del(20q) [10/10] V617F 2 PV 75 >6 12.8* 13.4* 315* 46,XX del(20q) [7/12] V617F 3 PV 79 NA** 5.7* 11.4* 90* 46,XY del(20q) V617F

4 PMF 77 8 22.1 11.5 196 46,XY del(20q)(q11.2q13.3) [20/20] V617F

5 CMML 75 2 14.7 14.9 35 46,XY del(20q)(q11,q13) WT

6 RAEB 65 0 2.1 9.1 38 46,XY del(20q)(q12) [x/20] , 45,X-Y del(20q)(q12) [2/20] WT

7 RCMD 57 2 5.2 12.8 58

47,XY,+8,del(20q) [12/12]; nuc ish 20q11.2(D20S108x1) [129/200] WT

8 MDS 67 2 3.1 10.4 108

46, XY, del(20q)(q11,q13) [22], 45, X-Y, idem [2], 46, X-Y [3], 46,XY [4] WT

9 RCMD 41 9 5.5 9.4 80 46,XY del(20q) 100% ND

10 CML 52 8 13.4 26.8 167 FISH: BCR/ABL 66%, del(20q) D20S108 77% WT

11 ET 69 10 8.1 14.2 991 46,XY del(20q) [7/10] V617F

12 PV/AML 43 26 12*** 1.3*** 81*** 46,XY del(20q)(q11,q13) [9], 46,XY [5]

N542-E543del

13 ET 55 5 8.3 12.4 >2000 46/XX del(20q). nuc ish 20q11.2(D20S108x1) 19% WT

14 ET 45 14 14.1 12.7 1080 Karyotyping failed. nuc ish 20q11.2(D20S108x1) 38% WT

*At time of sample collection

**Not Available

***At transformation to AML

Supplementary Table S2. Summary of patient disease characteristics. Patients 1 to 5 were studied

for expression and mutation screening in peripheral blood granulocytes, patient 6 was studied for

expression and mutation screening in bone marrow, and patients 7-14 were studied for expression in

hematopoietic colonies. See materials and methods for diagnosis abbreviations used.

17

Gene name (genbank id)

Exon Coding region

SNP amino-acid

Proportion with SNP

SNP id

GDAP1L1 (NM_024034)

4 Y 669c/t S204S 3/42 Rs2425632

6 N 1373a/g na 10/42 Rs12479863

6 N 1386c/g na 1/42 Na

6 N 2196g/c na 3/42 Rs3810511

SGK2 (NM_016276)

2 N c/g na 7/42 Rs3827067

3 N 44g/a na 1/42 Na

3 N 66a/g na 10/42 Rs6065627

3 Y 253t/a S12T 3/42 Rs33969356

8 Y 816c/t Y199Y 1/42 Na

11 Y 1092g/a L291L 1/42 Na

Supplementary Table S3. Exonic SNPs identified by mutation screening analysis of GDAP1L1

and SGK2. GenBank identifiers for the mRNA and Rs numbers are given where previously published

for each gene, and novel SNPs were confirmed by sequencing of constitutional samples where

available. All patients were previously screened by either microsatellite analysis or FISH in the

common deleted region of chromosome 20q12, results for L3MBTL1 have been published by Bench

et al (1).

18

SUPPLEMENTARY METHODS

Patients and samples

50ml of peripheral blood was collected from 12 patients with informed consent into an ethically

approved research study at Addenbrooke’s Hospital, Cambridge, UK; Belfast City Hospital, Belfast,

UK; King’s College Hospital, London, UK; Derriford Hosptial, Plymouth, UK; Hôpital Avicenne and

Hôpital St Louis, Paris, France and Karolinska University Hosptial, Stockholm, Sweden. Diagnoses of

myeloproliferative neoplasm (MPN), myelodysplastic syndrome (MDS), acute myeloid leukemia

(AML) and their subtypes were made according to World Health Organization (WHO) criteria (2).

Cell fractions were obtained with Ficoll separation followed by magnetic bead isolation from

mononuclear cells of T cells using CD2 Dynabeads (Invitrogen, Paisley, UK) and CD34+ cells using

MACS CD34 selection kit (Miltenyi Biotech); and granulocytes. Mature cell population purities were

95% or higher and were confirmed by cytospin or counting of cells attached to beads in a

hemocytometer.

For granulocyte or bone marrow gene expression analysis, RNA samples were available from 6

individuals. Peripheral blood granulocyte samples were obtained from three patients with

polycythemia vera (PV, patients 1-3), one with primary myelofibrosis (PMF, patient 4) and one with

chronic myelomonocytic leukemia (CMML, patient 5). A bone marrow sample with 20q deletion in

all cells was from patient 6 who had refractory anemia with excess of blasts (RAEB). All were shown

to have >98% 20q deletion in these samples by microsatellite PCR for at least three different markers

per individual. Samples were obtained from 8 additional patients with less than 95% deletion in

granulocytes, for colony analysis. The diagnoses of these patients were: refractory cytopenias with

multi-lineage dysplasia (RCMD, patients 7 and 9), unclassified MDS (patient 8), chronic myeloid

leukemia (CML, patient 10), essential thrombocythemia (ET, patients 11, 13 and 14), and PV

19

transformed to AML (patient 12). For mutation screening, granulocyte DNA was available from 15

samples with MPN (8 PV, 4 PMF and 3 unclassified MPN), and 8 with MDS (5 Refractory anemia

(RA), 1 RAEB, 2 unclassified MDS), with 20q deletion demonstrated by microsatellite PCR present

in at least 90% of cells (3); (4); (5). An additional 19 bone marrow DNA samples from PV patients

without 20q abnormality were available as described before (1).

Fluorescence In situ Hybridization (FISH)

FISH was performed on cell lines as described previously (6), using the probes 20ptel (Abbott Vysis)

and RP11-1108D11 which contains the JPH2 and GDAP1L1 genes (GeneService Ltd, Cambridge,

UK). 20 metaphases were scored for each cell line, and copy number of the CDR (in figure 3A) as

indicated by the number of RP11-1108D11 signals was recorded as the primary 20q karyotype in the

majority of metaphases analysed. In cell lines HEL, SET-2 and 293T, two signals were scored on the

same chromosome in each metaphase analysed, In the cell line K562, 20ptel and CDR signals were

seen on the same chromosome once, and separated onto different chromosomes, suggesting a

translocation event centromeric of the CDR. In the cell lines UKE-1 and HNT-34, FISH showed one

chromosome 20 with signals for both probes, but the other chromosome 20 had just the 20ptel signal,

with no other CDR signals seen on other chromosomes. In the cell line CMK, the karyotype was

complex, with four signals for both the 20ptel and the CDR probe on different chromosomes, with the

20ptel probe always at the end of small chromosomes, and the CDR probe always close to the

centromere on small chromosomes, suggesting translocation events have occurred followed by

duplication of the translocated chromosomes.

Bi-phasic erythroid differentiation assay

A bi-phasic erythroid differentiation method was modified from van den Akker et al (7). Briefly,

100,000 CD34+ cells/condition were cultured for 14 days in StemSpan® (Stem Cell Technologies)

20

medium supplemented with SCF (100ng/ml, Peprotech), Erythropoietin (2U/ml NeoRecormon®),

Dexamethasone (1µM, Sigma-Aldrich), Insulin-like growth factor-1 (40ng/ml, R&D Systems), and

Cholesterol rich lipids (40µg/ml Sigma-Aldrich). After 14 days in culture, cells were flow-sorted for

GFP+ and/or CFP+ and CD71+ expression and transferred to the second phase culture medium

comprising of StemSpan medium supplemented with 3% human AB serum (Sigma-Aldrich),

Erythropoietin (10U/ml, Insulin (10µg/ml, R&D), Insulin-like growth factor-1 (40ng/ml), and iron

saturated transferrin (0.5mg/ml, Sigma-Aldrich). In both cultures, cells were counted and

supplementary fresh medium was added every second day with FACS analysis for infection and

differentiation.

Long term culture-initiating cells (LTC-IC) assay

LTC-IC assays were performed as described previously (8) with slight modification. Briefly, cord

blood CD34+ cells infected with lentivirus and co-cultured for 5 weeks with feeder cells producing

human IL-3, SCF, IL-6 and Flt-3l, with media change every week. After 5 weeks, infected cells flow

sorted and used for CFC assay performed using MethoCult H4435 (StemCell Technologies) number

and types of colonies formed were scored at day 14.

Single Cell Clonality Assay

Single cell assays were performed as described before (9). Briefly, 50ml peripheral blood was

obtained from consented and informed patients. Mononuclear cells were isolated by density

centrifugation of 1:1 PBS diluted blood on Lymphoprep® (Axis-Shield Biotech). Mononuclear cells

washed with PBS were used for MACS purification of CD34+ cells using CD34-positive selection kit

(Human CD34-EasySep Positive selection Kit, StemCell Technology). Isolated CD34+ cells cultured

as with Serum Free Expansion Medium (SFEM, StemCell Technology) and with 1X StemSpan

CC110 (StemCell Technology) and 1X Pen/Strep on a 24-well Fibronactin coated plate (BD BioCoat)

21

pre-coated with concentrated retrovirus (retroviral preparation described below). Cells were spin

infected for 30 minutes with retrovirus at 25oC at 2500 rpm. 72 Hours post infection, infected viable

cells were FACS sorted (7AAD-, GFP+ and/or CFP+) to give one single cell per well in 96 well

round bottom plates with Serum Free Expansion Medium containing, 1X Pen/Strep, 3U/ml Epo,

SCF25 ng/ml, -merceptoethanol 10nM, Dexamethasone 4g/ml. Clones were FACS analyzed for

GPA expression at day 8 post sorting, and half of the culture was taken for genotyping and MYC

expression analysis.

Cell culture of cell lines

All cell lines were obtained from commercial sources (www.dsmz.de). UKE-1 and HNT-34 are

derived from 20q deleted ET patient transformed into AML and MDS patient transformed into AML

respectively (10); (11). K562, HEL, SET2, CMK, and UKE-1 cells were cultured in RPMI 1640

(Sigma-Aldrich), supplemented with 10% heat inactivated fetal bovine serum (PAA Biotech) and 1%

Penicillin and streptomycin (Invitrogen). HNT-34 cells were cultured in RMPI 1640 (Sigma Aldrich),

20% heat inactivated fetal bovine serum (PAA), and 1% penicillin and streptomycin (Invitrogen).

293T cells were cultured in DMEM (PAA) supplemented with 10% heat inactivated fetal bovine

serum (PAA Biotech) and 1% Penicillin and streptomycin (Invitrogen).

shRNA generation and viral infection

shRNA viruses were purchased from Sigma-Aldrich®, L3MBTL1-shRNA1 (TRCN0000016864),

L3MBTL1-shRNA2 (TRCN0000016866), L3MBTL1-shRNA3 (TRCN0000016867), SGK2-

shRNA1 (TRCN0000002111) SGK2-shRNA2 (TRCN0000002112), SGK2-shRNA3

(TRCN0000002113), and for murine targets, mL3-shRNA1 (TRCN0000226027), mL3-shRNA2

(TRCN0000226028), mL3-shRNA3 (TRCN0000226091), mS2-shRNA1 (TRCN0000022879) and

mS2-shRNA2 (TRCN0000022881) were sub-cloned in lentiviral vector pLKO1.3G co-expressing

22

GFP (Kindly gifted Dr J Larsson, Lund University Sweden) as described before (18). GFP was

replaced with CFP from pMSCV-ires-CFP to generate pLKO1.3G CFP vector. To produce lentiviral

particles shRNA vectors with psPAX2 and pMD2-G coat plasmids co-transfected in 293T cells using

GeneJuice® (Millipore). Medium was changed 24 hours after transfection and viral supernatants were

collected 48 hours and 72 hours after transfection and viral particles were concentrated by

centrifuging the supernatant at 28000rpm for 3 hours, at 4C. Concentrated viral particles were re-

suspended in 500µl of PBS and 50µl of this concentrated virus suspension was coated with Polybrene

(4µg/ml) and used to infect 200,000 K562 or CD34+ cells in a fibronectin-coated 24 well dish by spin

inoculation. Cells counted and analyzed by FACS every 4th day. For CD34+ cells infection, virus was

first coated on 24 well, fibronectin coated plates by spinning the concentrated virus on a dish with

medium for 15 minutes at 4C at 3000rpm. After coating with virus, CD34+ cells were added to the

plate. Scrambled shRNA infected and uninfected cord blood CD34+ cells were used as controls for off

target effects of shRNA viruses. In order to make sure there were no off-target effects, these cells

were monitored in the same way as the knock-down cells and found to be consistent with each other

for growth and differentiation kinetics.

Retrovirus generation and transduction

L3MBTL1 cDNA was cloned between EcoRI and XhoI sites in pMSCV-ires-GFP and SGK2 cDNA

was cloned between EcoRI and BamHI sites in pMSCV-ires-CFP (A kind gift of Dr BJ Huntly,

Cambridge, UK (19)). Amphotropic retroviruses with VSV-G coat were generated as described

(http://www.stanford.edu/group/nolan/protocols/pro_helper_free.html). Briefly, 293T cells were

transiently transfected with pMSCV-L3MBTL1-ires-GFP and/or pMSCV-SGK2-ires2-CFP, pCMV

8.91 and pVSV-G and medium was changed 24 hours after transfection. Virus containing

supernatant was harvested 48 hours and 72 hours post-transfection. Viral supernatant was diluted with

fresh RPMI supplemented with 10% heat inactivated FCS and 1% penicillin and streptomycin, and

used for infecting HNT-34 cells in 6-well fibronectin coated plates. Two rounds of spin infections

23

were carried out for 12 hours at 2500rpm at 32C. Medium was changed 12 hours after infection then

cells induced with 50µM hemin in RPMI supplemented with 10% FCS (heat inactivated) and 1%

penicillin/streptomycin. Cell counts and FACS analysis for GPA expression was performed every 24

hours starting 48 hours from infection. RNA was extracted at the day 4 of culture for globin gene

expression analysis.

Human SNP genotyping for expressed alleles

Genomic DNA sequencing for each SNP (SGK2 SNP rs3827067 and GDAP1L1 SNP rs1247963) was

performed from buccal or whole blood DNA from informative individuals and their parents. Two

independent samples were taken for each individual shown to confirm genotype, except for the

parents of the GDAP1L1 families. cDNA sequencing in the probands was performed using the

primers described below. For SGK2, CD2+ T cells and for GDAP1L1, GPA+ erythroblasts were found

to express a moderate level of transcript, so these were used for further expression analysis. Multiple

PCR products were sequenced from independently derived cDNAs of the original cell sample, to

confirm the allelic expression pattern.

Mutation and sequencing analysis

SSCP/HA analysis of all candidate genes except SGK2 and JPH2 was performed as described

previously (6). Direct sequencing of SGK2 and JPH2 was performed as described previously (12).

Primers used for each fragment are listed below.

Bisulphite sequencing and Quantitative Pyro-sequencing

Bisulphite sequencing was performed as described previously (13). Quantitative pyrosequencing was

done as described before (14) using the primers described by Woodfine et al (15).

24

Colony genotyping

BFU-E and CFU-GM colonies were picked into 50µl RLT buffer (Qiagen) in 96-well plates and

stored at -80oC. 20ul RLT lysate per colony was transferred to a new plate, and 40µl Isopropanol was

added to each well. DNA was precipitated by centrifugation for 1 hour at 3600rpm at room

temperature in an Eppendorf 3810R centrifuge with the A-2- DWP rotor. Pellets were washed twice

with 70% ethanol, dried by brief centrifugation upside-down and re-suspended in 50µl sterile dH2O.

2.5µl of DNA was used per PCR reaction for each relevant marker. Patient genotypes were

determined using a panel of 16 microsatellites across a 5Mb region of 20q encompassing both the

previously described MDS and MPN CDRs (38315925-43320563). Constitutional material, either T

cells isolated using CD2 Dynabeads (Invitrogen) or buccal DNA isolated using the Epicentre DNA

extraction kit (CamBio, Cambridge, UK) was compared to granulocyte DNA. PCR primers for

markers with high reported heterozygosity (60 to 85%) were taken from the GenLoc database

(http://genecards.weizmann.ac.il/geneloc/index.shtml). One primer was labeled with 6-FAM and PCR

products were diluted 1 in 10 before running on an ABI 3700XL. Markers used were: D20S107,

D20S170, D20S108, D20S858, D20S466, D20S46, D20S899, D20S96, D20S721, D20S150,

D20S169, D20S861, D20S911, D20S119, D20S481 and D20S1151. Microsatellite PCR products

were analyzed on an ABI 3730XL analyzer to quantify loss of chromosome 20q as indicated by loss

of marker alleles in the tumor sample, compared to constitutional material. The ratio of the areas

under each peak in the tumor sample, once normalized to the areas under the corresponding peaks in a

constitutional sample, is expressed as a fraction, then subtracted from 1 and converted to a percentage.

Colonies were scored for deletion by the retention of just one allele, or no deletion by the presence of

both alleles. Colonies not meeting these criteria were excluded from further analysis. At least 3

informative microsatellites were obtained for each colony. Colonies with clear genotypes for all

microsatellites analysed were tested by quantitative RT-PCR for expression of L3MBTL1, GDAP1L1,

MYBB and C20orf111.

25

Strand-specific cDNA synthesis and PCR

Strand specific cDNA for mRNA for L3mbtl1 and Sgk2 was synthesized from C57BL6 x 129S

reciprocal cross F1 mice and Gdap1l1 strand specific cDNA was synthesized from C57BL6 x

CAST/EiJ F1 mouse tissue. Strand specific cDNA for murine L3mbtl1, Sgk2 and Gdap1l1 were

synthesized using primer L3MBTL-R1671, SGK2-R1462 and GDAP1L1-R1448 respectively.

For human L3MBTL1, the cDNA for the sense transcript was primed using a primer in exon 14 and

exon 24 (primer L3MBTL1-SS-P3 in the table below) for mRNAs in the coding region of the gene,

(primer L3MBLT1-SS-P2) for mRNAs extending into the non-coding region of the gene and in the P5

region, chr20:41614055-74 (see Figure S3A) as a control where no mRNAs are expected to be

transcribed (primer L3MBTL1-P1). For the antisense transcript, cDNA was primed from exon 24

(primer L3MBTL1-AS-P2) and the exon 17 region (L3MBTL1-SS-P3) where Ensembl ESTs predict

antisense expression; and from exon 12 (L3MBTL1-AS-P4) and P5 region (L3MBTL1-AS-P1) which

are not predicted to have antisense expression. In Figure S3B sense RNAs were primed from exon 24

to make the sense cDNA, and the antisense RNAs were primed from either exon 12 region (for exon

13 PCR analysis), exon 24 region (for P3 and P5 analysis), and exon 17 (for analysis of the other

exons studied).

Chromatin Immuno-Precipitation (ChIP)

CD34+ cells differentiated in early erythroid cells using phase I of bi-phasic erythroid differentiation

with fresh media change every 2 days. 12 hours before the harvest of cells, cells were stimulated with

fresh media containing erythropoietin 2U/ml. Harvested cells were sorted for infected cells and 1

million infected day 8 erythroblasts used per condition of chromatin immune precipitation as

described before (16). Briefly, infected cells were twice washed with cold PBS, and cross linked using

0.4% formaldehyde solution in PBS and cross linking stopped by adding with 2M glycine solution to

a final concentration of 0.125M glycine, cells were subsequently washed with cold PBS, and lyzed

26

with Cell Membrane lysis solution (10mM Tris/HCl pH 8.0, 10mM NaCl, 0.2% NP40 and protease

inhibitor cocktail ) and then with nucleus lysis solution (50mM Tris/HCl pH 8.1, 10mM EDTA, 1%

SDS and protease inhibitor cocktail), nuclear material was sonicated and any debris removed by

centrifugation. Chromatin material extracted was then diluted with ChIP dilution buffer (20mM

Tris/HCl pH 8.1, 2mM EDTA, 150mM NaCl, 1% Triton X100, 0.01%SDS and protease inhibitor

cocktail) and cleaned using pre-immune serum and then incubated with respective antibody (4ug for

H3K4me3 and 12ug for all others). Antibodies used in ChIP were anti-L3MBTL1 (SantaCruz

Biotech, sc-50038) anti-BRG1 (AbCam ab4081), anti-H3K4me3 (Millipore Clone MC315) and anti-

phospho-S2-PolII (AbCam ab5095). Chromatin was incubated with respective antibodies overnight

(18 hours) at 4oC with constant rotation. Next chromatin and antibody mixture was incubated with

25ul Dynabeads labelled with either protein G or protein A (Invitrogen, 100-04D and 100-01D) for

two hours at 4oC with content rotation. Labelled beads were then isolated and washed in a magnetic

rack (Invitrogen, 123-21D) and washed using wash buffer 1 (20mM Tris/HCl pH 8.1, 2mM EDTA,

50mM NaCl, 1% Triton X100, and 1% SDS) twice, wash buffer 2 (10mM Tris/HCl pH 8.1, 1mM

EDTA, 0.25M LiCl, 1% NP40, and 1% Sodium deoxycholate monohydrate) once and then twice with

Tris/EDTA (pH 7.8, 50mM). Precipitated chromatin extracted from beads using extraction buffer

(0.1M NaHCO3, 1% SDS) and precipitated DNA isolated by adding 5M NaCl and incubating at 67oC

overnight. Samples were then incubated with 3ul Proteinase K (20mg/ml) and 1ul of RNAse at 55oC

for two hours and DNA was purified using a PCR purification kit (Qiagen).

Immunoprecipitation and western blot

pMSCV-L3MBTL1-ires-GFP or pMSCV-SGK2-ires-GFP (described above) were used to transfect

HNT-34 cells with control empty vector (pMSCV-ires-GFP) using Amexa transfection reagent ‘R’

and using transfection protocol T-027 (Amexa®). After 96 hours of transfection HNT34 cells were

lysed and BRG1 was immunoprecipitated using anti-BRG1 (Abcam ab4081) and analysed for

phosphoserine phosphorylation using antibody against pan-phosphoserine (Abcam, ab6639) as

27

described before (17). Part of cell lysate was used for expression analysis of L3MBTL1, SGK2 and

Actin. Western blot analyses were performed on total cell lysates using the following antibodies: anti-

L3MBTL1 (Santa Cruz sc-50038), anti-SGK2 (Santa Cruz sc-98972) and anti-Actin (Sigma).

Study Approval

The study of human samples was approved by the Cambridge and Eastern Region Ethics Committee

(REC reference number 07/MRE05/44). Patients gave written informed consent, and research was

carried out in accordance with the Declaration of Helsinki. The macaque studies were approved by

SingHealth Institutional Animal Care and Use Committee (IACUC #2009/SHS/509). All the mouse

work was performed under UK home office project license numbers 80/2376 and 80/2567. All

experiments in tammar wallaby were approved by institutional ethics committees at Department of

Zoology, University of Melbourne, Australia and specimen transfer agreement reference number is

GB107A.

Statistical Methods

An unpaired Student's t-test was used for all analyses, within the GraphPad Prism4 software

(GraphPad Software, Inc). Significance was determined by p-vlaues <0.05, ‘*’; <0.01, ‘**’; and

<0.001, ‘***’.

28

Primers list:

Quantitative PCR primers:

Primer Sequence

Human quantitative RT-PCR primers

ABL ABL-F 5-GCGTGAGAGTGAGAGCAG-3 ABL-R 5-TCTCGGAGGAGACGTAG-3

SFRS6 SFRS6E1F1 5-GTCTACATAGGACGCCTGAG-3 SFRS6E2R1 5-TTGCCGTTCAGCTCGTAAAC-3

L3MBTL1 L3MBTLE10F2 5-TGTACTTCATCCTCACCGTG-3 L3MBTLE11R1 5-AGTTGGCATTGACCCAGAAG-3

SGK2 SGK2E9F 5-CTGAAGTGCTTCGGAAAGAG-3 SGK2E10R2 5-CGGCTGGTGCAGAATGTTC-3

IFT52 IFT52E6F2 5-TGCCTGGGATCATTGATGAG-3 IFT52E7R1 5-ACAGAACCTGTAGACAGAAC-3

MYBB MYBBE4F1 5-AATGCCAGTACAGGTGGCTG-3 MYBBE5R1 5-TTCAGGTGCTTGGCAATCAG-3

JPH2 JP2E3F1 5-CTGGGCATAGAGACCAAG-3 JP2E4R1 5-TCCCTCCATCAGCATAGGTC-3

C20orf111 CT111E4F2 5-GGATGCATCAGGGTCTGTAG-3 CT11E5R1 5-TGTCTTTAGATGCACATGTGG-3

GDAP1L1 GD1L1E2F2 5-AAGGTGCGGCTGGTGATC-3 GD1L1E2R2 5-ATGAACCAGGGCTCCTTCTG-3

HBA HBA-F 5-TGGACAAGTTCCTGGCTTCT-3 HBA-R 5-CCGCCCACTCAGACTTTATT-3

HBB HBB-F 5-GAAGGCTCATGGCAAGAAAG-3 HBB-R 5-CACTGGTGGGGTGAATTCTT-3

MYC MYC-1F 5-CAGCTGCTTAGACGCTGGATT-3 MYC-1R 5-GTAGAAATACGGCTGCACCGA-3

RUNX1 RUNX1-F 5-ACTTCCTCTGCTCCGTGCTG-3 RUNX1-R 5-GCGGTAGCATTTCTCAGCTC -3

CCNE1 CCNE1-F 5-TGCCACCCGGGTCCACAG-3 CCNE1-R 5-GCACGTTGAGTTTGGGTAAAC-3

Murine quantitative RT-PCR primers. L3mbtl1 Forward 5-CTTTCCAGAAGCGGTCAGTC-3

Reverse 5-GGCTCTGACTCCTCTGATGG-3 Sgk2 Forward 5-ACGTGCTGTTGAAGAACGTG-3

Reverse 5-CCCGCTGTAGATGGAAGAAG-3 GapdH Forward 5-TCAACGACCCCTTCATTGAC-3

Reverse 5-ATGCAGGGATGATGTTCTGG-3 Human quantitative ChIP-PCR primers:

MYC Promoter

Forward 5-GGTGGTGGAGGGAGAGAAAA-3 Reverse 5-CTGTATGTAACCCGCAAACG-3

MYC TSS

Forward 5-GATCCTCTCTCGCTAATCTC -3 Reverse 5-TGCCTCTCGCTGGAATTACT-3

29

Human strand specific cDNA synthesis primers

Primer name Sequence

mRNA specific primers

L3MBTL1-SS-P1 5-CTGCTCTTCCAGAGCTGCTT-3 (P5) L3MBTL1-SS-P2 5-TGAATTTTCTGCCCTTGACC-3 (E24) L3MBTL1-SS-P3 5-GTCAATGCCTTCCAACTTCA-3 (E14)

Antisense specific primers

L3MBTL1-AS-P1 5-CAGTGAAGACAGGGACAGCA-3 ( chr20: 41622153) L3MBTL1-AS-P2 5-ATCTGCATGTCTCCTCCCAC-3 (Exon 24 region) L3MBTL1-AS-P3 5-CTGCTTCCCTCTCCACTGAC-3 (Exon 17) L3MBTL1-AS-P4 5- GTTCAGCTGGAGCCAGTACC-3 (Exon 12)

ABL ABL-mRNA-P 5- GATGAGCCCGTCGGCCACCG-3 (Exon 4)

Human L3MBTL1 expression primers for antisense cDNA

Region Primer name

Sequence Annealing temperature

Product size

Intergenic region

P5F 5-TGCTGTCCCTGTCTTCACTG-3 60C 905bp

P5R 5-CGTCACCAATTCACATGAGG-3 P3F 5-CTAAAGCCCCCAGACCCTAC-3

60C 681bp P3R 5-GGTCAAGGGCAGAAAATTCA-3

Exon 24 region

E24-F 5-AGCGAAGGTTGGGTTTACAA-3 60C 581bp

E24R 5-GCCAGGGGCCAAAATATAAG-3 Exon 21 - 19 region

E19F 5-AAGAAGCCTCGCCATCACG-3 59C 318bp

E21R 5-GACATGAAGAGGGACTGGTGC-3 Exon 18 region

E18F 5-CCATGTCACAGGCAAGTTCA-3 58C 160bp

E18R 5-GTGATGGCGAGGCTTCTTC-3

Exon 13 region

E13F 5-CTTCCAGGTGGGCATGAAG-3 58C 130bp E13R 5-GTCATAAGTATCATCCCAGT-3

Human SNP genotyping primers

L3MBTL1 P5 5AGCGAAGGTTGGGTTTACAA 3

rs2664519 P6 5-TGCCAGGGGCCAAAATATAAG-3

SGK2 SGK2intron1F 5-CCGCGTGACATCAGCTAG-3

rs3827067 SGK2Intron2R 5-ACGTGTGTGCTCCTGCAAAC-3

IFT52 IFT52intron2F 5-TCCCTTCATCTCTGAGCCTC-3

rs1883790 iFT52intron3R 5-AGGCCACACAGTAAGTGGTG-3

JPH2 JPH2Intron1F 5-TTGTCAGGGGCTATGATGAG-3

rs12479863 JPH2Intron2R 5-GTCCCTTGAAGCCATGTGTC-3

GDAP1L1 GD-2F 5-ACTATGTGGAGCGCACCTTC-3

rs12479863 GD-6R 5-GTGGATGTCACCCAGGACTT-3

30

Wallaby SNP genotyping primers

L3MBTL Genomic

DNA

MeL3UTR_F 5- CACCCACTTTTCTCTCGTCAG-3 60oC MeL3MEx22R2 5- GGTCAGAAGTGACCCCACAT-3

L3MBTL cDNA

MeL3cDNAF1 5 – CGTTGACCCACCCATTTACT-3 60oC

MeL3cDNAR1 5- AGGAAGGCAATGAGGGATTT-3

Macaque cDNA expression primers

Gene Primer name


L3MBTL1

F 5- GCACCAGTCCCTCTTCATGT-3 59C R 5- TTGTGTTCCCACCATATCAGTC-3

SGK2 F 5- CTCCACCCTTCAACCCAAA-3

60C R 5- GGGCAGAAATACAGCCTCTG-3

Macaque SNP genotyping

Gene Primer name


SNP location

L3MBTL1 Genomic DNA

F 5-CATCTTTGGTTTCCAAGGTCTTCGG-3 60C chr10 (-) 20910951 R 5- CACTGCACCCGGCTGACAAAA-3

SGK2 Genomic DNA

F 5-TAGGAAGCATGGGGCACTCACAG -3 60C chr10 (-) 20867268 R 5-CAAAATCCAAACACCAGGAGGCC -3

L3MBTL1 cDNA

F 5-GCACCAGTCCCTCTTCATGT-3 59C R 5-TTGTGTTCCCACCATATCAGTC-3

SGK2 cDNA

F 5-CTCCACCCTTCAACCCAAA-3 60C R 5-GGGCAGAAATACAGCCTCTG-3

Macaque bisulphite sequencing primers

Location Primer name


Location

L3MBTL DMR

F 5-TGTTGGTGTTGGAGTTGGT-3 59C chr10: 20937937 -20938069

R 5- ACCCTAAATATATCTTACTTTCCC-3

31

Mouse genotyping and SNP analysis

Primer Sequence mRNA specific primers

L3mbtl1-R1671 5-TCGTAAGTATCGCCCCAGTC-3 Sgk2-R1462 5-CTCCGAGCCACTGTGTCTTA-3 Gdap1l1-R1448 5-ATGCTAACCAGTCCCCACAG-3

Human bisulphite sequencing primers

Primer name Sequence Tm oC

SGK2 Promoter 1 1st

Round SGK2P1BiF3 5-TTGGAAGTTTTTATGAAATGATTGA-3

55C SGK2P1BiR3 5-CCTACTCACAAATAAACCTCTCACC-3

2nd Round

SGK2P1BiF4 5-AGGGTGTTGTGTGAGATTAATTTTT-3 55C

SGK2P1BiR4 5-AAATCCTAAAAAACAACCCCATAC-3 SGK2 promoter 2

1st Round

SGK2P2BiF1 5-TTTTAAGAAGGTTATGAGGTTGGTG-3 55C

SGK2P2BiR1 5-AAATATAACCCCTCCCAAACTAATT-3 2nd

Round SGK2P2BiF2 5-TGGGTTAAGATTAAGGTTTTGAGAT-3

55C SGK2P2BiR2 5-AAAACATAAAATCTAAACCCATCCC-3

Intergenic CpG island 1st

Round ASBisF3 5-ATTTGGAAGGATTGTTATTTTTTTT-3

55C ASBisR3 5-CCACCAACACACATAACAACATATAT-3

2nd Round

ASBisF4 5-AGGTTGGAGTGTAGTGGTATGATTT-3 55C ASBisR4 5-TACTATACCACCAAACTCCCTCTC-3

L3MBTL1 Exon 5b 1st

Round L3Ex5bBisF2 5-GTTTGTGGGGATTGGTTAAGTT-3

60C L3Ex5bBisR1 5-AATAACCTCCTCCAACCTTCTC-3

2nd Round

L3Ex5bBisF1 5-AGTTGGTTTAGATGATGGGTTTTAG-3 60C

L3Ex5bBisR2 5-AAACCCAACTCAAAACCTAAAAAA-3

Primer name Sequence TmoC

Product length

SNP

L3mbtl1 L3mbtl1-P2 5-TCAGGCTTCTGGATTGGAC-3

59C 415bp rs27347041 L3mbtl1-P4 5-CTGGCTTCTGCTCCACTCTT-3

Sgk2 Sgk2 F359 5-GTTGGAGTTCCTAGCCCACA-3

60C 880bp rs27331555 Sgk2 R1162 5-CATCCCAGTTTATGGGACTG-3

Gdap1l1 Gdap1l1 F964 5-CCAACCTGCAGTCCTTCTTT-3

61C 192bp rs28243099 Gdap1l1 R1134 5-AGTAGGCAAAGTAGCCCATCC-3

32

Human mutation screening primers

PTPRT1

Exon Forward primer Reverse primer Tm °C

1 GTTAGGACTCGGGGGACAC GCCCACACAACTTTCTCCTC 59 2 AGCCGACGAGACAGAGGTAA TGCCATCTCAGAGAGCTCAA 60 3 GATCTCTGGCCACTCCTCTG AGCACCTGTAGGGAGAGCAA 59 4 CACCAAAGTGTGGCCTTTTT TTGGGAGGAAGGGAAAGACT 59 5 CTGAGCCGGGCTACTTCTTA CTCACAAGCCCAGACCTCTC 59 6 TGGATATCCGTGTTGGGAGT GTTTGGGGAGTTTGTGTTGG 60 7 CCTTGTCGTCATGTGCTTGT CATAATGGAGCCTGGGAAAA 60 8 TTTCTTGCCTGCATGTTTTG ACTCCCTGGAGTTGTGCAAT 60 9 AACGTACAGCCCATCAGACC TTTGTTCTTGGGGCTACCAG 60 10 GTATTTGGAGGCTGGGATCA AGTGGGGGTGAAACAACCTT 60 11 CCCCTTTCCTAAACGTCCTC CCATGTGGCACAGAGAAGAA 60 12 CGAACCAATGCTTCCACATA AAAATGCAAACAAGGCAAGG 60 13 GTCATCCGATGGGGAAAAA TGGCTGAAGAACAGGTGAAG 60 14 CAGTTTTGTTCACCGTGCTG GTGTGGGTTGATGGGTGAAT 60 15 TGCCTGGCACATAGTTAGCA CCCTTCAAACAGCAACACAA 60 16 CTTTTTCCCCCATTTTGGAC CAGTGCACTTTCAAATGTAACACA 60 17 CATGGTTTGTTCTGCCTTGA TTAGGATGAACTGCCCCAAG 60 18 TTGAGTCCCAAGTTGGTTCC GCTCCCAGGTGATACTGAGG 60 19 TTCCACTAGGAGTCCCATCG AAGCTTCCATCTTGGCATGT 60 20 TCAACCATCCCCTTGATTTC TGGATGCAGTGGTAGATGGA 60 21 TGTTCTCATTTTGCCCATGA TGAAGCCTCTCTGAGGCACT 60 22 TCAGGCTCACATGTCTCAGG CGATGCATGGAACAAAGAGA 60 23 AACCCTGTGGACTGAAATGC AACAAGCTGGCTCTCATGGT 60 24 GTTCCTCAGTGCAGCAGCTA TCTCGAACTCCTGACCTCGT 60 25 TGTGGTTTGCACATGCATTA CTCCTTGGTCAGGGCTACAG 60 26 TAATTCCCAGGCCACTGTTC CTTGATGCTGGGCTTCTCA 60 27 AGGGTGGAAATAGGCGAGTT CACCTCCACCTCAGGAAGAA 60 28 CAGGGCGTGGAGAGATAAAA GGGATCTCCCTCCAGGTTTA 60 29 GCCTTTGAGCTCCTTCTGTG CACAGGCATCACTTCCTCAA 60 30 TGGTCTGTCTTCCACCATGA AGCATCTGCAAGGATGCTCT 60 31 GGACCTAGGAACATGGCTCA GCATACCAAGGGCACAGAAT 60 32 TGCATTTTCCCTTTCCTTTTT CAACAGGAGACCCCTCAGAA 60

SFRS6


1 GCTTCTTTCCTTGGAGAGTTCC CCCCATCAAAACGAGCATCAAC 59 2 CCCCCAGGGTCCCCAAG CGTCCTGGCTAACGACTCC 59 3 ACTGATTACATGCATTTCTACATTTT GGGGGATTTTTGGTTATGTT 59 4 GCATTAGTCATATCATTTCTATCTTGA AGGCATCTAATTGTTCCCTTCA 56 5 ATTGTTTCCTTCCCACAGTGTC GATTTCCGAGTCTGACCAATCT 59 6 TTCACAGTGACTCAACACAACG GGCACGGATGTATTTAAGTTGG 59 7 TGCCTAAGTAGGAAAGTGTTCCAT CTCACTAAGTATTGAGAAAATCGGTC 59

33

SGK2


1 GAAGCCAGCTTCCAGCAG GGTAACTAGTCATGCTGGAGACCA 58 2 CAGTCCCATCTTAAGCTCTG TGCAGAACTGCTAAGGAAGC 58 3 TTGTGGCAAGGAGATCGTAG ATTTCCTTAGCCTCGGGAAG 60 4 GTTGTTGGAGAAGGGATCAG GAATGCTAGACCAGAGAATCTG 58 5 CTCCTGGAAGGCTCTCTG TAGGCTGCGGCTTGAGAAG 60 6 GAGGCCTTGTACTGCTGTTG TGTTGTGACCACTTCTGCAC 60 7 CGGGATAAAAGAGGCTGTTG CATTCCAGAACTTGGCATGC 59 8 GGAAGAATGCTGCAGGTCAG GGTGTCTCAGCCATTTAGTG 59 9 GAATAGTTCTCCTGCCAGGAC GTACACAGACTCATGGTCAG 56 10 TAGGCCTGGCCATACCCTTG TATAGCCCACTCAGGAGAAGTG 62 11 GTCAGCTCTTGGTCATCTTC GAGCTTGTGTAGACCACATG 59 12 CTGCCATGCGAGCCATTG TTTAGAAGGCAGCAGGATGC 60 13 CAGTTGCTTGTGGAGCTCTG TTGGCAAGCATAGCAAGCTC 56 14 GACATCCCTCTCTGAGGATC GGTTCTCTGGAGACAAGAAG 54 14a GCTCCTTTGGCAGCTCTG AGGGATAGTCACGTACCCAG 60 14b AATGTTTCGGAGTCCAGGAC GGTTCTCTGGAGACAAGAAG 60

IFT52


1 GGGACCCTGGATGTTCTATGAC TGGGTTTATTTTTGGTGAAAAG 562 CAGCAGGACAATATCATTTAGAAGG GGGTCTTTGGAATATTGTTTATTGA 563 CCGGTATTTCAAAGCTCAGACT GGCCTCTGCTTCCAAATAGTTA 594 CACTGGACCTAGAGGGCTATGAT TCGGGCAAAAGTCTTTCAAGTAT 565 GATTACAGGTGTGAGCGACGAT CTTCAGGTCAAAATTGGTATGAGTC 596 CTCCAAAATGATACATCTTCCTCA TGCTGTACTAATGAATGCAGTGTG 567 CATGTTTTTAAAATTTGAATGTGTTTC AAGCCTTCTGGAAATGGTAAGG 598 GAGATGCACTTTCGGATTTGAG AGCTGCTTTTAAATGAGCAAAT 569 CCTGACCTTCAGCTTTTTCAAAT TGAGAAGCTGGACTCATTTTTCA 10 GCTCTTGCTGTGCTAAAAGGAAC ACACTCAGAATGGAGTGCAGTGT 5911 GTGTGGTCAGTTAGACGTGCTG GCTGACAAGATAAGGGCAACAG 5912 TTTCCTTTACCTTATCCTCCCTCA CCTGGGCAACAAGAGTGAAACT 5613 TGGGAAGGGCAGAAGTTAAGAA GAGGGGCTCCCACTACACTGT 5914 GTCCAAAGCACTGAAGAGTTTACA ATCCAGAACTGGGGTTGAGAAA 56

MYBB

Region Forward primer Reverse primer Tm °C

Promoter GGCTCTAGGGACCCAGTAG GAAGGCGTCAGCGTGTCAG 59 Exon 1 GCGGGAGATAGAAAAGTGCTTC GGGGAGGGGTGAGTTAAAGG 56 Exon 2 ATGGACACACCATCCTTGACC ACTCCAGGCTCAGTTCCTCTG 59 Exon 3 CCCTGAGGTTTTCTGCACGTA AGGCACACTGTTCTCCCAGAG 59

34

Exon 4 CCCTGAGCCTAGTACTTAAC AACACAAGGCAATCTCACAG 56 Exon 5 TCAGGTGGATGTGAAGGGCTAT AACCCTCCTCCATCAGAAACAC 56 Exon 6 ACAGAGCTGGGGTTCAAAGG GTGAAATCAAACCAAGCCAAAG 59 Exon 7 TATCTCAGCGAAATGCAAATGG TGTGCTCACTGTGAAGTGTTGTG 56 Exon 8 CCCGTAATGAATGAGTCCTCTTG TGTCTGTACCGACCCAATAAGCA 59 Exon 9 GGGATACTCATGCAGGTCATCA GTCTGGTGTTGCTGAGGGAGA 59 Exon 10 ATCCCACTGTGCAGAGATCC GCCCCTATCCTGTCACTAGTTC 59 Exon 11 TTTCTACAACCTGTCCCCAGAC CTGGGTTGGTCCCACAGTC 59 Exon 12 AGGGTCCTCTCCAAAACTCAAC GGAATCCAGACACTCACCCTAA 56 Exon 13 CCTCTTTCAGGTCTCAGCAG CCTATGCAAAGGCCCTGAG 59 Exon 14 TAGTCCCTGCCTGGATGGTAAC TAGGTCACCAGGGAACCATGAG 59 JPH2


1a AGGGCATGTGAGTGGTGATG TTACAAGGCCATTGGATCTC 60 1d TCCTTCCTCATGCCTCCAG GTCCCCAGCCTTTTCAAAGA 60 1b CTGACCTTTCCGTCCCAG CTCCATCATCAAAGTCGAAG 60 1c TTGTCAGGGGCTATGATGAG TTCTGTGCCAATTGCCGGTC 60 2a TTGCACTACCATGCGAACTC TGAGGTCGCTCTTAAGGAAG 60 2b GCCATGGCTACGGAGTAC TCCTTGACCAGCACGTTGTG 60 2c CTTCGAGGCCGATATCGAC CGAAGAGCCTCCAATTAACC 60 3 ATTCATTGACTGCCTGCGTG GCATCTCAGATTCCAGTAAGC 60 4a CGACTGAGCCCATGAATGAG CTTTGGGGATGATGGGCTTC 60 4b CTTTACCAGGGCTACCACAG TGCTCTATCCTGCTTCGGTC 60 5 CTCCTGGAAGGCTCTCTG TAGGTCTTGGCTTCTGCAG 60 6a GATGCAGAGGTGGGATCTTG AGACAGAGCACTGTGTTCTG 60 6b AATCCTTTGGCTGTGGGCTG TTCCATGGGCTTCTGTGCTC 64 6c CCTGTGAAATGGCTTGTCTC GAAGGTTCCCAAGCATTGAAG 60 6d TTGGAGGCTTTGGCTTTGTG TCGTGAGAACAAGGACACAG 60

C20orf111


1 GGAGGCAGAGGACTACTGTGAA CAGTGGAAGCTGAGACGCAGT 61 2 CCCACCTGTTTTTACTTTGCTC GAGCTTTTTAGGGAAAAGGGACT 59 3 GAACAATATGTTCTCCTCATTTTACAG GGACACTCAGAAAGGAAAATCC 56 4 CTCCATATCCCAACATCTTCTTG CCACAAGGTCTGCCATTAACTAAA 59

GDAP1L1


1 AACTGCCCGGTGAGTAATGA TGAGCAGTCCTGGAAGGAAC 60

35

2 CCCTCCTGTGTGTGACCTCT ACCCCTGAGGATCTGTGGTA 60 3 GAGCCCAGTGAGGAGAGATG GAAAGCAGAGGGGCTGTCTC 60 4 GGGAGCAACCAAGGTATCAG GTTCATTTCCGTGGAAGAGC 60 5 TCCTTCCATCCCTGAACATC TCTGACTCCATAGCCCATCC 60 6A TTGCCTCCTCTTTCTTCCAC TGGGACTCACCTCAGATCCT 60 6B TCTGAGGTGAGTCCCAGGAT CAGCACTTGGCAATGAGAAC 60 3' UTR1 GTCTCTGTGCTGTGTGATTC TAGTCCATGTCCAGGAGAAG 56 3' UTR2 AGGTCCCTGAAGATCAGAAG CAGCACTTGGCAATGAGAAC 56

SUPPLEMENTARY REFERENCES

1. Bench, A.J., Li, J., Huntly, B.J., Delabesse, E., Fourouclas, N., Hunt, A.R., Deloukas, P., and

Green, A.R. 2004. Characterization of the imprinted polycomb gene L3MBTL, a candidate

20q tumour suppressor gene, in patients with myeloid malignancies. Br J Haematol 127:509-

518.

2. Swerdlow, S.H., Jaffe, E.S., International Agency for Research on Cancer., and World Health

Organization. 2008. WHO classification of tumours of haematopoietic and lymphoid tissues.

Lyon: International Agency for Research on Cancer. 439 p. pp.

3. Bench, A.J., Aldred, M.A., Humphray, S.J., Champion, K.M., Gilbert, J.G., Asimakopoulos,

F.A., Deloukas, P., Gwilliam, R., Bentley, D.R., and Green, A.R. 1998. A detailed physical

and transcriptional map of the region of chromosome 20 that is deleted in myeloproliferative

disorders and refinement of the common deleted region. Genomics 49:351-362.

4. Asimakopoulos, F.A., White, N.J., Nacheva, E., and Green, A.R. 1994. Molecular analysis of

chromosome 20q deletions associated with myeloproliferative disorders and myelodysplastic

syndromes. Blood 84:3086-3094.

5. Asimakopoulos, F.A., and Green, A.R. 1996. Deletions of chromosome 20q and the

pathogenesis of myeloproliferative disorders. Br J Haematol 95:219-226.

6. Bench, A.J., Nacheva, E.P., Hood, T.L., Holden, J.L., French, L., Swanton, S., Champion,

K.M., Li, J., Whittaker, P., Stavrides, G., et al. 2000. Chromosome 20 deletions in myeloid

malignancies: reduction of the common deleted region, generation of a PAC/BAC contig and

identification of candidate genes. UK Cancer Cytogenetics Group (UKCCG). Oncogene

19:3902-3913.

7. Fiedler, W., Henke, R.P., Ergun, S., Schumacher, U., Gehling, U.M., Vohwinkel, G., Kilic,

N., and Hossfeld, D.K. 2000. Derivation of a new hematopoietic cell line with endothelial

features from a patient with transformed myeloproliferative syndrome: a case report. Cancer

88:344-351.

36

8. Hamaguchi, H., Suzukawa, K., Nagata, K., Yamamoto, K., Yagasaki, F., and Morishita, K.

1997. Establishment of a novel human myeloid leukaemia cell line (HNT-34) with

t(3;3)(q21;q26), t(9;22)(q34;q11) and the expression of EVI1 gene, P210 and P190 BCR/ABL

chimaeric transcripts from a patient with AML after MDS with 3q21q26 syndrome. Br J

Haematol 98:399-407.

9. Ailles, L.E., Gerhard, B., and Hogge, D.E. 1997. Detection and characterization of primitive

malignant and normal progenitors in patients with acute myelogenous leukemia using long-

term coculture with supportive feeder layers and cytokines. Blood 90:2555-2564.

10. van den Akker, E., Satchwell, T.J., Pellegrin, S., Daniels, G., and Toye, A.M. 2010. The

majority of the in vitro erythroid expansion potential resides in CD34(-) cells, outweighing

the contribution of CD34(+) cells and significantly increasing the erythroblast yield from

peripheral blood samples. Haematologica 95:1594-1598.

11. James, C., Ugo, V., Le Couedic, J.P., Staerk, J., Delhommeau, F., Lacout, C., Garcon, L.,

Raslova, H., Berger, R., Bennaceur-Griscelli, A., et al. 2005. A unique clonal JAK2 mutation

leading to constitutive signalling causes polycythaemia vera. Nature 434:1144-1148.

12. Baxter, E.J., Scott, L.M., Campbell, P.J., East, C., Fourouclas, N., Swanton, S., Vassiliou,

G.S., Bench, A.J., Boyd, E.M., Curtin, N., et al. 2005. Acquired mutation of the tyrosine

kinase JAK2 in human myeloproliferative disorders. Lancet 365:1054-1061.

13. Arnaud, P., Monk, D., Hitchins, M., Gordon, E., Dean, W., Beechey, C.V., Peters, J., Craigen,

W., Preece, M., Stanier, P., et al. 2003. Conserved methylation imprints in the human and

mouse GRB10 genes with divergent allelic expression suggests differential reading of the

same mark. Hum Mol Genet 12:1005-1019.

14. Messerschmidt, D.M., de Vries, W., Ito, M., Solter, D., Ferguson-Smith, A., and Knowles,

B.B. 2012. Trim28 is required for epigenetic stability during mouse oocyte to embryo

transition. Science 335:1499-1502.

15. Woodfine, K., Huddleston, J.E., and Murrell, A. 2011. Quantitative analysis of DNA

methylation at all human imprinted regions reveals preservation of epigenetic stability in

adult somatic tissue. Epigenetics Chromatin 4:1.

16. Delabesse, E., Ogilvy, S., Chapman, M.A., Piltz, S.G., Gottgens, B., and Green, A.R. 2005.

Transcriptional regulation of the SCL locus: identification of an enhancer that targets the

primitive erythroid lineage in vivo. Mol Cell Biol 25:5215-5225.

17. Pannu, J., Nakerakanti, S., Smith, E., ten Dijke, P., and Trojanowska, M. 2007. Transforming

growth factor-beta receptor type I-dependent fibrogenic gene program is mediated via

activation of Smad1 and ERK1/2 pathways. J Biol Chem 282:10405-10413.

18. Karlsson, C., Larsson, J., and Baudet, A. 2010. Forward RNAi screens in human stem cells.

Methods Mol Biol 650:29-43.

37

19. Dawson, M.A., Prinjha, R.K., Dittmann, A., Giotopoulos, G., Bantscheff, M., Chan, W.I.,

Robson, S.C., Chung, C.W., Hopf, C., Savitski, M.M., et al. 2011. Inhibition of BET

recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature

478:529-533.

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