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Supplementary Materials

Construction of Synthetic Nucleoli in Human Cells Reveals

How a Major Functional Nuclear Domain is Formed

and Propagated Through Cell Divisision

Authors: Alice Grob, Christine Colleran and Brian McStay*

Centre for Chromosome Biology, School of Natural Sciences,

National  University  of  Ireland  Galway,  Ireland. Supplemental Materials contains: Supplemental Figures and Legends 1-12 Supplemental Fig. 1 relates to Materials and Methods Supplemental Fig. 2 and 3 relates to Fig. 1 Supplemental Fig. 4 relates to Fig. 2 Supplemental Fig. 5 and 6 relates to Fig. 3 Supplemental Fig. 7 relates to Fig. 4 Supplemental Fig. 8 and 9 relates to Fig. 4 and 5 Supplemental Fig. 10 relates to Fig. 5 Supplemental Fig. 11 relates to Fig. 6 Supplemental Fig. 12 and 13 relates to Fig. 7 Supplemental Table 1 relates to Materials and Methods Supplemental Video 1 relates to Fig. 6

The neo-NOR cassette (20.4kb)!Human promoter!

XEn elements!

Mouse coding sequences!

18S! 5.8S! 28S!

Transcriptional! terminator!

5ʼETS!ITS1! ITS2! 3ʼETS!

Mouse 5ʼETS probe!2.9kb AcuI-SalI fragment !

from 5ʼ external transcribed spacer!(+120/+3100)!

XEn probe! Mouse ITS2 probe!1.1kb PCR fragment!

(+7036/+8123)!

Mouse 3ʼETS probe!0.5kb PCR fragment!

(+12854/+13404)!

Mouse 28S probe!35-mer oligonucleotide!

(+11028/+11063)!

DJ BAC probe!(CT476834)!

IGS probe!11.9kb Eco RI fragment from intergenic spacer!

(+31497/+43464)!!

Human 5ʼETS probe!2.9kb NotI fragment !

from 5ʼ external transcribed spacer!(+270/+3170)!

q-arm BAC probes!13q11 (AC018739)!

15q11.2 (AC068446)!22q11.2 (AC013360)!

Supplemental Figure S1. Schematic representation of hybridization probes. (A) Hybridization sites of probes used to visualize and identify individual acrocentric chromosomes (q-arm and DJ BAC), endogenous NORs (IGS) and endogenous pre-rRNA (human 5’ETS). The 43972bp sequence used as a reference rDNA repeat corresponds to nucleotides 105424-149395 of BAC clone RP11-337M7 (Acc. No. AL592188). Next generation sequencing of nucleolar DNA has confirmed that this sequence is a representative rDNA repeat (McStay unpublished data). (B) Location of hybridization probes used to identify neo-NORs (XEn) by DNA-FISH and their derived transcripts (mouse 5’ETS, ITS2, 28S and 3’ETS) by RNA-FISH. The coordinates of mouse probes correspond to the sequence of a complete mouse rDNA repeat (Acc. No. BK000964).

A!

B!

centromere!

2!

Supplemental Figure S2. UBF depletion induces the appearance of a sub-G1 cell population. (A) Western blot of proteins extracted from HT1080 cells and UBF-KD cells cultured with 1µg/ml Dox for 48h, 96h and 144h. Molecular weight markers in kDa are indicated on the left. Anti-UBF antibodies revealed a ~50-fold depletion of UBF1 (97kDa) and UBF2 (94kDa) while anti-RPA43 antibodies reveal the conserved level of pol I subunit RPA43 (43kDa). (B) FACS analysis of HT1080 cells and UBF-KD cells cultured with 1µg/ml Dox for 24h, 48h, 72h and 96h. Following 72h of culture with 1µg/ml Dox, G2/M cells decrease while sub-G1 cells accumulate (red *).

*  

3!

Supplemental Figure S3. UBF down-regulation releases rDNA repeats from the nucleolus. Combined 3D-immuno FISH performed on HT1080 and UBF-KD cells cultured with 2ng/ml Dox reveals that dissociated rDNA repeats identified using an IGS probe (arrowheads) are devoid of UBF.

UBF

-KD

!+2

ng/m

l Dox!

HT1

080!

α-UBF! IGS! Merge! Merge with DAPI!

4!

α-Fibrillarin!

Con

t. si

RN

A!U

BF s

iRN

A!

α-UBF! Merge! Merge with DAPI!A! B!

C! D!

Cont.!UBF siRNA!

- UBF!

DAPI! Silver!C

ont.

siR

NA!

UBF

siR

NA!

Con

trol

siR

NA!

DAPI! rDNA!

UBF

siR

NA!

DAPI! rDNA!

Supplemental Figure S4. Formation of 2° constriction is UBF-dependent in Ptk-2 cells. (A) A cDNA clone encoding Ptk UBF was cloned and sequenced in order to design 3 siRNAs. Western blot of proteins extracted from Ptk-2 cells transfected with control or UBF siRNAs shows a 20-fold UBF depletion. (B) Immuno-staining of siRNA transfected Ptk-2 cells identifies the nucleolar remnants after UBF depletion. (C) Metaphase chromosome spreads prepared from control and UBF siRNA transfected Ptk-2 cells were hybridized with a human 18S and 28S rDNA probe. Arrowheads indicate the NOR. UBF depletion induces the loss of the 2° constriction associated with the single NOR in these cells. (D) Silver-staining (arrowheads) of metaphase spreads from Ptk-2 cells indicates that UBF depletion results in the loss of NOR silver-staining.

5!

Supplemental Figure S5. Chromosomal mapping and characterization of neo-NORs. (A) FISH experiments performed on metaphase chromosome spreads prepared from each neo-NOR line classify them into acrocentric (a1-a3) and metacentric (m1-m3) clones. XEn probe labels neo-NORs, while a human IGS probe identifies endogenous NORs. BAC clones RP11-42OH1 (AC18739), RP11-32B5 (AC068446) and RP11-278E23 (AC013360) identify respectively the q-arms of acrocentric chromosomes 13, 15 and 22. Chromosome painting with chromosome paints prepared from chromosome specific DOP-PCR products (kind gift from M. and T. Cremer) identify metacentric chromosome 4. (B) The size and integrity of each neo-NOR was determined by Southern blotting. Genomic DNA from HT1080 and neo-NOR cell lines were digested to release neo-NOR transcription units and probed with sequences derived from the mouse 5’ETS. Similarly digested pNeo-NOR plasmid served as a size marker. Note the high degree of sequence rearrangement. (C) Table indicating the chromosomal location and the estimated copy number of intact transcription units integrated in neo-NOR lines. (D) Silver-staining of metaphase spreads from pseudo-NOR clone 3D and neo-NOR clone m1. Pseudo-NOR and neo-NOR are indicated by arrowheads. Note the silver-staining of an endogenous NOR (*) in the lower right panel.

*!

6!

Supplemental Figure S6. Transcription across the neo-NOR cassette. (A) RNA FISH experiments performed on mouse 3T3 and human HT1080 cells demonstrate the specificity of mouse (green) and human (red) 5’ETS probes and identify neo-NOR-derived transcripts in neo-NOR clones a1-a3, m2 and m3. (B) RNA FISH using mouse ITS2 probe identifies neo-NOR-derived transcripts in clone m1. (C) RNA FISH using mouse 3’ETS probe identifies neo-NOR-derived transcripts in clones a1-a3 and m1-m3.

7!

Supplemental Figure S7. Neo-NORs recruit FC, DFC and GC components to build neo-nucleoli. Combined 3D-immuno RNA FISH reveals that FC/DFC factors coupling rDNA transcription to pre-rRNA processing, t-UTP10 and Treacle, together with DFC factors, Nap57 and U3-55K, and GC factor Nucleolin, colocalize with neo-NOR-derived transcripts in clone m1.

8!

Supplemental Figure S8. Efficient Maturation of neo-NOR-derived pre-rRNA. (A) Quantitation of the S1 nuclease protection assays presented in Fig. 5A. Note the similar ratio between cleaved and uncleaved transcripts in mouse and neo-NOR lines. (B) Table indicating the estimated percentage of neo-NOR-derived 28S rRNAs among different RNA fractions extracted from the 6 neo-NOR clones.

9!

Supplemental Figure S9. Probing strategy for detection of neo-NOR-derived 18S and 28S rRNAs. (A) The upper panel correspond to a schematic representation of the species specific primers used for RT-PCR based identification of mouse 18S rRNA. The gel illustrates the specificity of these primers for mouse 18S rRNA in RT-PCR. (B) Schematic representation of the oligonucleotide used to probe neo-NOR-derived 28S rRNAs.

10!

Supplemental Figure S10. Ribosome and polysome purification. (A) Coomassie-stained SDS protein gel of whole cell extract (WCE), nuclear (N), post mitochondrial (PMT) and ribosome (R) fractions prepared from HT1080 cells. The low-molecular-weight histone bands (*) and the size range of ribosomal proteins are indicated. (B) Polysomal fractions were identified by electrophoresis of RNA aliquots extracted from polysome sucrose gradient fractions. The example shown is from clone m1. Polysome, small 40S and large 60S ribosome subunit fractions are indicated.

A!

62!

49!

38!

28!

14!

WCE! N! PMT!R!

HT1080!

Purification steps!

*  *  *  

Rib

. pro

tein

s!

B!

Polysomes!

28S!18S!

60S! 40S!

direction of sedimentation!

11!

Supplemental Figure S11. Nucleolar segregation upon Actinomycin D (AMD) treatment. AMD treatment reveals the compartmentalized structure of nucleoli as illustrated in cartoon form (upper panel). FC/DFC regions are visualized with UBF antibodies (green), while GC regions are visualized with Nop52 antibodies (red).

12!

Supplemental Figure S12. UBF depletion induces neo-NOR condensation and loss of neo-nucleoli. Combined 3D-immuno FISH experiments performed on clone m1 cells transfected with control or UBF siRNAs reveal the condensation of neo-NORs (XEn probe) and their failure to form neo-nucleoli recruiting UBF and Fibrillarin (Fib) upon UBF siRNA knockdown. Images shown are restricted to the nuclear region containing neo-NORs.  

XEn!

Control siRNA!

UBF siRNA!

α-U

BF!

XEn !

XEn!

α-F

ib!

XEn !

Control siRNA!

UBF siRNA!

XEn!

α-U

BF!

XEn !

XEn!

α-F

ib!

XEn !

13!

Supplemental Figure S13. UBF depletion silences neo-NOR transcription. (A) RNA FISH performed on clone m1 cells transfected with control or UBF siRNAs. Mouse and human 5’ETS probes (M- and H-5’ETS respectively) reveal the lost of neo-NOR transcription upon UBF siRNA knockdown. (B) A graph representing the percentage of cells with transcriptionally active neo-NORs in clone m1 cells transfected with control or UBF siRNA.

Con

trol

siR

NA!

UBF

siR

NA!

M-5ʼETS! H-5ʼETS!A!

B!

Perc

enta

ge o

f cel

ls !

with

act

ive

neo-

NO

R!

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!"#$%&'()*+!,-*./0#!+1(+-02.)3(

:"-;"<=112">3/?"/0@.2<03A3.B".=-C5D4"

14!

Supplemental Table S1. Oligonucleotide probes. List and sequences of the oligonucleotide probes used for 3D-immuno RNA FISH, S1 nuclease protection assays and Northern blotting. Note that the probes testing endogenous human and ectopic neo-NOR rDNA transcription start (+1) correspond respectively to nucleotides -18/+42 and -19/+41.

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Supplemental Video S1: neo-NOR territory. A video showing the 3D arrangement of neo-NOR organization within large mature nucleoli. 3D-RNA FISH was performed on neo-NOR clone m1. Neo-NOR derived transcripts were detected using a mouse 5’ETS probe (green) transcripts from endogenous NORs were detected using a human 5’ ETS probe (red). This video was prepared from 3D images of deconvolved Z-stacks rotated to create a series of bookmarks. The movie is an animation of the transition between these bookmarks.

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