analyses of prmt1 proteins in human colon tissues from ... fileanalyses of prmt1 proteins in human...

Analyses of PRMT1 proteins in human colon tissues from

Hirschsprung disease patients

T.-T. WU,*,� T.-W. TSAI,� Y.-T. SHEN,� J.-D. HSU,§ L.-C. YANG§ & C. LI�

*Department of Pediatric Surgery, Chung Shan Medical University Hospital, Taichung, Taiwan

�School of Medicine, Chung Shan Medical University, Taichung, Taiwan

�Department of Biomedical Sciences, Chung Shan Medical University, Taichung, Taiwan

§Department of Pathology, Chung Shan Medical University Hospital, Taichung, Taiwan

Abstract

Background Protein arginine methyltransferase 1

(PRMT1) catalyzes the majority of arginine methyla-

tion in cells and plays important roles in the differ-

entiation and development of neurons. It is also

implicated in the regulation of nitric oxide synthetase

(NOS). Hirschsprung disease (HSCR) is characterized

by the absence of intramural ganglion cells in the

nerve plexuses of the distal gut. Methods Western blot

analyses revealed reduced PRMT1 protein levels in the

aganglionosis segments of HSCR patients. Immuno-

histochemistry detected PRMT1 expression in the

colonic mucosa, the enteric nervous system (ENS) and

endothelial cells. Specific and strong PRMT1 expres-

sion in neuron cell bodies of the plexus was demon-

strated by immunofluorescent double-labeling with

neuron-specific marker HuC/D. Key Results In the

mucosa, PRMT1 was detected at all crypt cells.

Intensive PRMT1 staining was detected in the sub-

mucosal and the myenteric plexuses in normal or

oligoganglionosis segments. Aganglionosis segments

from HSCR patients contain no plexuses, and thus not

labeled with PRMT1. The phenomenon is specific to

the megacolon of HSCR as strong PRMT1 staining was

observed in plexuses of the rectal ectasia segments

(dilated rectum and distal sigmoid not related with

aganglionosis) from anorectal malformation patients.

Furthermore, PRMT1 was also present in the same

neuronal cells expressing neuronal NOS in the plex-

uses. Conclusions & Inferences We suggest that

PRMT1 can be a useful marker for HSCR. This study is

the first illustration of PRMT1 protein expression in

human tissues from non-cancerous disease and set up

the base for further investigations of PRMT1 function

in ENS development and intestinal motility.

Keywords anorectal malformations, Hirschsprung

disease, nitric oxide synthetase, protein arginine

methyltransferase 1.

INTRODUCTION

Protein arginine methyltransferases (PRMTs) are

enzymes that catalyze the transfer of a methyl group

from S-adenosylmethionine (SAM) to guanidine nitro-

gens of specific arginines in proteins.1 Protein arginine

methyltransferase 1 is a predominant type I methyl-

transferase for arginine methylation to form monome-

thylarginine (MMA) and asymmetric dimethylarginine

(ADMA). Proteolytic degradation of arginine methylat-

ed proteins represents the major source of free MMA or

ADMA, which are endogenous inhibitors of nitric oxide

synthases (NOS).2 Besides a well-known endothelial-

derived relaxing factor, nitric oxide (NO) is also an

important neurotransmitter in intestine, mediating

nonadrenergic noncholonergic inhibitory transmis-

sion.3 Protein arginine methyltransferase 1 thus might

play a regulatory role for NO synthesis in intestine

through the formation of ADMA containing proteins

and later free ADMA through protein degradation.

In mammals, PRMT1 has a wide substrate spectrum

including histones, heterogeneous nuclear ribonucleo-

proteins (hnRNPs) and various RNA binding proteins.

Protein arginine methyltransferase 1 and the protein

methylation it catalyzes are involved in various impor-

tant biological processes such as signal transduction,

Address for correspondence

Dr. Chuan Li, Department of Biomedical Sciences, ChungShan Medical University, 110 Sec. 1, Chein-Kuo North Road,Taichung, Taiwan.Tel: 886 4 2473 0022 ext 11807; fax: 886 4 2475 7412;e-mail: [email protected]: 11 August 2009Accepted for publication: 18 April 2010

Neurogastroenterol Motil (2010) 22, 984–e254 doi: 10.1111/j.1365-2982.2010.01523.x

� 2010 Blackwell Publishing Ltd984

RNA processing, transcriptional regulation and even

DNA repair.4 It is also important for early embryogen-

esis,5 and neuronal differentiation.6

Even though ubiquitous expression of PRMT1 RNA

or protein was illustrated in various animal models

including rat,7 mouse,8 and fish,9 direct demonstration

of PRMT1 expression in human tissues are few and are

restricted to tumor samples.10,11 In this study colon

tissue samples from Hirschsprung disease (HSCR)

patients are examined for the expression of PRMT1.

Hirschsprung disease or aganglionic megacolon, the

most common congenital intestinal obstruction, is a

developmental disorder of the enteric nervous system

(ENS) characterized by the absence of intramural

ganglion cells in the nerve plexuses of the distal

gut.12 The ENS derived from migratory neural crest is

the largest subdivision of the peripheral nervous sys-

tem to regulate the gut behaviors, including the

coordination of the smooth muscle contractions of

the gut wall. The ENS neurons and glia cells organized

into ganglia, which are interconnected to form two

types of plexuses: the submucosal (Meissner’s) and the

myenteric (Auerbach’s) plexus between the longitudi-

nal and circular smooth muscle layers. Genes encoding

proteins of the RET signaling pathway (RET, GDNF

and NTN),13–17 participating in the endothelin type B

receptor pathway (EDNRB, EDN3, and ECE-1),18–20

involving transcriptional regulations such as the SRY-

box 10 gene (SOX10),21 and the zinc finger homeobox

1B22 have been reported to be related to the etiology of

HSCR. The importance of signaling pathways guiding

the migration of ENS cells to the terminal region of the

gut can be best exemplified by this disease.

Anorectal malformations (ARM) often associate

with rectal ectasia, a state of massive dilation of the

rectum and distal sigmoid not related with aganglion-

osis as in HSCR. We compared PRMT1 protein

expression in normal and dilated colon segments from

HSCR and ARM patients with rectal ectasia. This

study is the first illustration of PRMT1 protein

expression in human tissues from non-cancerous

disease and initiates the investigation of putative

PRMT1 function in ENS development and intestinal

motility.

MATERIALS AND METHODS

Human tissues

Tissues samples were obtained from eight newborns diagnosed atinfancy (five HSCR and three ARM associated with rectalectasia). Samples from HSCR patients were collected fromaganglionated, oligoganglionated and normal ganglionated

segments. Samples from ARM patients associated with rectalectasia were collected from abnormal distal, dilated and normalsegments. Hirschsprung disease patients received definite pull-through operation at Chung-Shan Medical University Hospitaland ARM patients associated with rectal ectasia were includedin our previous genetic analyses.23,24 The study was approved bythe local Institutional Research Board. Tissue samples werecollected immediately after operation and were stored at )80 �Cuntil use.

Western blot

Colon tissues were weighed, washed two times with phosphatebuffered saline (PBS), and then cut into small slices, resuspendedinto three times (vol/wt) lysis buffer [5% glycerol, 1 mM EGTA,1 mM DTT, 0.5% Triton X-100 and Complete EDTA-freeprotease inhibitor cocktail (Roche, Mannheim, Germany) inPBS] and homogenized with a homogenizer (T10 basic, IKA). Theprotein amount in the extracts was quantified by bicinchoninicacid assay (BCA) (Thermo Scientific, Waltham, MA, USA). Theextracted total proteins (30 lg) was separated by SDS polyacryl-amide gel electrophoresis and transferred to the nylon mem-brane. The membrane was then incubated with primaryantibodies (anti-PRMT1 antibodies; Upstate, 1 : 1000 dilution/anti-b-actin antibodies; Santa Cruz Biotechnology Inc, SantaCruz, CA, USA; 1 : 5000 dilution) at room temperature for60 min. For detection, HRP-conjugated secondary antibody (goatanti-rabbit/mouse-HRP, Santa Cruz Biotechnology Inc) andImmobilon Western (Millipore, Billerica, MA, USA) or VisGlow(Visual Protein Biotechnology Corp, Taiwan) chemiluminescentreagent were used.

Immunohistochemistry

Bowel tissue sections of full thickness and normal segments werefrom typical rectosigmoid HSCR patients and ARM patients withrectal ectasia. Immunohistochemical studies were performed onparaffin-embedded tissue cross sections. The slides were baked at50 �C overnight, soaked in xylene for 10 min three times, 100%ethanol for 5 min, 95% ethanol for 5 min, then 80% and 70%ethanol for one minute each. After rehydration for 5 min, theslides were boiled in citrate buffer (pH 6.1, Target RetrievalSolution; Dako, Carpinteria, CA, USA) for 25 min, washed withwater and PBS, treated with 1–3% H2O2 for 10 min, then washedagain with PBS. The slides were then incubated with the anti-PRMT1 antibodies (Upstate, 1 : 100 dilution) at room temperaturefor 30 min. After PBS wash for 5 min two times, the slides wereincubated with HRP-conjugated secondary antibody (goat anti-rabbit-HRP, Santa Cruz Biotechnology Inc) at room temperaturefor 30 min. Color was developed at room temperature with theSubstrate-Chromogen Solution (Dako LSAB2 System) for 5 minand then counter stained with Mayer hematoxylin, (MERCK,Darmstadt, Germany) for 1 min. The slides were dehydrated in50%, 75% and 100% ethanol for 1 min each, and sealed withCytoseal 60 Mounting medium (Thermo Scientific).

For immunofluorescence, de-paraffined slides were boiled incitrate buffer (pH 6.1, Target Retrieval Solution, Dako) for 25 min.The slides were then incubated with anti-PRMT1 antibodies(Upstate, 1 : 250 dilution) and anti-HuC/D (Molecular Probes,Carlsbad, CA, USA; 1 : 40 dilution) or anti-nNOS (BD Transduc-tion Laboratories, Franklin Lakes, NJ, USA; 1 : 250 dilution)antibodies at 4 �C for 12–16 h. After PBS washing, slides wereincubated with Rhodamine-conjugated anti-mouse IgG (JacksonImmunoResearch, West Grove, PA, USA; 1 : 200 dilution) andFITC-conjugated anti-rabbit IgG (Jackson ImmunoResearch, West

Volume 22, Number 9, September 2010 Analyses of PRMT1 proteins in human colon tissues

� 2010 Blackwell Publishing Ltd 985

Felix

螢光標示

Grove, PA, USA; 1 : 200 dilution) at room temperature for 2 h.Cells were rinsed again, incubated with DAPI (0.5 mg mL)1;Roche) at room temperature for 1 min and mounted in ibidireagent (ibidi GmbH, Germany).

RESULTS

PRMT1 expression in HSCR and ARM bywestern blot analyses

The expression levels of PRMT1 in the colon tissues

were evaluated first by western blot analyses. As

shown in Fig. 1, the amount of PRMT1 protein

expressed in the normal ganglionic and oligoganglionic

colon specimens from HSCR patients were compara-

ble, while that expressed in aganglionic segments was

reduced. Detection of the same blot with anti-b-actin

antibodies confirmed equal protein loading. Fig. 1A

represents the western blot result of colon tissue

samples from a typical short-segment agangliosis

HSCR patient. Similar results were obtained from

samples of other HSCR patients.

Interestingly, similar amount of PRMT1 protein was

expressed in normal, dilated and abnormal distal

segments of ARM patients (Fig. 1B). The results indi-

cate that the reduced PRMT1 expression in the abnor-

mal bowel segments might be specific to HSCR.

Immunohistochemical detection of PRMT1 inHSCR

The histological appearance of the colon specimens

from HSCR patients and the expression of PRMT1 in

the normal ganglionic, oligoganglionic (dilated) and

aganglionic (distal) segments are shown in Fig. 2.

Protein arginine methyltransferase 1 was detected in

mucosa, submucosa and the inner circular and outer

longitudinal smooth muscle layers in sections from

normal, oligoganglionic and even aganglionic segments

of HSCR patients (Fig. 2A, B, C).

In the mucosa, strong PRMT1 staining was observed

in the crypts in all three segments (Fig. 2D, E, F). In the

submucosa, the expression of PRMT1 in human cap-

illary endothelial cells (arrowheads in Fig. 2G, H, I) was

clearly detected. Intensive PRMT1 staining was de-

tected in submucosal plexuses in the normal gangli-

onic and oligoganglionic but not aganglionic segments

(arrows in Fig. 2G, H). Smooth muscle fibers from the

longitudinal and circular muscularis were barely

stained in the cytoplasm but clearly stained in the

nuclei (Fig. 2J, K, L). The most observable difference

between normal and aganglionic tissues is that PRMT1

was significantly detected in the submucosal (Meiss-

ner’s) and the myenteric (Auerbach’s) plexuses only in

normal or oligoganglionic segments (Fig. 2J, K). In

aganglionic segments, the plexuses were not formed

and not labeled with PRMT1 (Fig. 2L). This result

probably can explain the reduced level of PRMT1

detected by western blot in the agangliosis samples.

Immunohistochemical detection of PRMT1in ARM

The tissue samples from ARM patients associated with

rectal ectasia were divided into three segments: nor-

mal, dilated, and distal abnormal. As shown in Fig. 3,

PRMT1 expression patterns in ARM normal (Fig. 3A,

D, G, J), dilated (Fig. 3B, E, H, K) and abnormal distal

(Fig. 3C, F, I, L) segments were similar to those of

normal ganglionic segments from the HSCR patients.

Immunofluorescent analysis of PRMT1expression in ganglions

As PRMT1 stained clearly in the ganglions in the

normal colon segments from HSCR patients, we

further compared its expression pattern with HuC/D

by immunofluorescent double labeling (Fig. 4). HuC/D

is a neuron-specific RNA binding protein and serves as

a specific neuron marker. Protein arginine methyl-

transferase 1 staining fully overlapped with HuC/D

staining in the plexus (Fig. 4C). DAPI is a fluorescent

stain that can bind strongly with DNA and then stains

clearly the nuclei of all cells. Merge of the PRMT1 and

DAPI signals showed that PRMT1 was absent in some

cells that also can not be stained by Hu in the plexus

(Fig. 4D, indicated by arrows; the same region was also

indicated in Fig. 4A, B).

N

Normal

PRMT1

Actin

PRMT1

Actin

O

Dilated

A

Abnormal

A

B

Figure 1 Protein arginine methyltransferase 1 (PRMT1) expression in

Hirschsprung disease (HSCR) and anorectal malformation (ARM) by

western blot analyses. (A) Protein extracts from normal ganglionic,

oligoganglionic and aganglionic (represented as N, O and A) colon

specimens in HSCR were probed by western blot analysis with PRMT1

antibodies. (B) Protein extracts from normal, dilated and abnormal

colon specimens in ARM were probed by western blot analysis with

PRMT1 antibodies. Detection of with an anti-b-actin antibody con-

firmed equal protein loading.

T.-T. Wu et al. Neurogastroenterology and Motility


We then stained the ganglion with neuronal NO

synthase (nNOS) (Fig. 5A). The nNOS and DAPI

staining clearly demonstrated the cytosolic expression

of nNOS in the neurons in the ganglions (Fig. 5C).

Protein arginine methyltransferase 1 appeared to be

present in the same cells expressing nNOS with

cytoplasmic as well as nuclear expression (Fig. 5B, D).

The results further confirm the specific expression of

PRMT1 in the neuron cells. Strong detection of

PRMT1 in the nuclei of neurons and neighboring

muscle fibers also clearly demonstrated its concen-

trated nuclear expression and dilute cytoplasmic stain-

ing. Similar results were obtained in the plexuses from

normal (data not shown) and dilated segments from

ARM patients (Figures S1 and S2).

DISCUSSION

In this study we observed PRMT1 expression in

different cell types of human colon tissues from HSCR

and ARM patients. We showed clear and significant

PRMT1 staining in the submucosal and myenteric

plexuses of normal colon tissues, the first direct

demonstration of PRMT1 expression in human peri-

pheral nervous system. The results are consistent with

PRMT1 functions in neural development and differen-

tiation. In the gut, NO is synthesized mainly by the

nNOS in the submucosal and myenteric plexus.25

Significant expression of PRMT1 in these regions is in

agreement with the possibility that PRMT1 can serve

as a regulator for NOS in intestine through the

A B C

D E F

G H I

J K L

Figure 2 Immunohistochemical detection of protein arginine methyltransferase 1 (PRMT1) in Hirschsprung disease. Overall view of the intestinal

wall in (A) normal ganglionic, (B) oligoganglionic, and (C) aganglonic colon specimens were illustrated with PRMT1staining. Mucosal, submucosal

and muscularis staining in normal ganglionic (D,G,J), oligoganglionic (E,H,K) and aganglonic (F,I,L) colon specimens are shown. Positive stainings in

all crypts, capillary endothelial cells (arrowhead), submucosal and myenteric plexuses (arrows) are indicated. Absence of staining of hypertrophic

nerve bundles is indicated by asterisk. Scale bars: A–C: 500 lm; D–L: 50 lm.



formation of ADMA. Moreover, immunofluorescent

double labeling analysis confirmed concentrated

expression of PRMT1 specifically in the cell bodies of

neurons in the plexus. Co-localization of PRMT1 and

nNOS in the same neuron cells also strengthens their

putative functional correlation.

Furthermore, the absence of submucosal and myen-

teric plexuses in the aganglionic segments from HSCR

patients can be revealed by PRMT1 staining. Identifi-

cation of the ganglions is critical for the diagnosis of

HSCR. Examination of HE-stained sections, acetylcho-

linesterase histochemical analysis or immunochemi-

cal detection with marker genes such as S100 (glia

marker), NeuN (neuron marker) or RET have been

conducted.26–28 As PRMT1 stained most strongly in

both the crypts and plexuses, PRMT1 can serve as a

marker for HSCR. Most of the HSCR markers are

neuron-specific. With these markers, negative staining

of HSCR sections due to aganglionosis can be con-

cluded only after the exclusion of unsuccessful immu-

nohistochemical detection. On the contrary, PRMT1

signals in the mucosa crypts are intense in both normal

and aganglionic segments and can serve as an internal

positive control. Furthermore, we showed that the

effect is specific to the megacolon due to HSCR but not

other bowel disorders with dilated rectum such as

ARM with rectal ectasia. Specifically, both submucosal

and myenteric plexuses can be readily detected by

A B C

D E F

G H I

J K L

Figure 3 Immunohistochemical detection of protein arginine methyltransferase 1 (PRMT1) in anorectal malformation. Overall view of the intestinal

wall in (A) normal, (B) dilated, and (C) abnormal colon specimens were illustrated with PRMT1staining. Mucosal, submucosal and muscularis

staining in normal (D,G,J), dilated (E,H,K) and abnormal distal (F,I,L) colon specimens are shown. Positive staining in all crypts, capillary endothelial

cells (arrowhead), submucosal and myenteric plexuses (arrows) are indicated. Scale bars: A–C: 500 lm; D–L: 50 lm.



PRMT1 in normal and dilated colon tissues from ARM

patients with rectal ectasia.

The expression pattern of PRMT1 in human colon

tissues from HSCR and ARM can be compared with

the PRMT1 expression in colon cancer samples.11

Even though Mathioudaki et al. reported that PRMT1

proteins reside mainly in cytoplasm and rarely in

nucleus in both normal and colon cancer tissues by

immunohistochemistry, we observed concentrated

PRMT1 staining in the nuclei of cells in all layers

of the bowel. Significant cytoplasmic PRMT1 staining

was observed in cells in the crypts and plexuses. On

the other hand, dilute staining in the cytoplasm of

muscle cells was observed with higher magnification.

Immunofluorescent double labeling experiments with

DAPI or nNOS also clearly demonstrated the concen-

trated nuclear expression of PRMT1 in neurons.

Controversial nuclear or cytoplasmic localizations of

PRMT1 have been reported. Protein arginine methyl-

transferase 1 can shuttle between nucleus and cyto-

plasm and inhibition of methylation results in nuclear

accumulation.29 Recently PRMT1 subcellular locali-

zation appeared to be related to the isoforms of

PRMT1,30 the cell-type,31 and might be affected with

interacting proteins.32 It can not be excluded that

different PRMT1 antibodies might recognize different

isoforms of the proteins and thus led to the contra-

dictory results. Nevertheless, our results provide more

detailed information of the localization of PRMT1

protein in different bowel cell types, especially in the

neurons but not other cells of the ganglions by the

immunofluorescent analyses.

A few genes involved in signaling pathways of the

receptor tyrosine kinase RET or endothelin type B

receptor and in transcriptional regulations for ENS

development have been reported to be involved in the

A B

C D

Figure 4 Immunodetection of HuC/D and

protein arginine methyltransferase 1

(PRMT1) in myenteric plexuses in normal

colon sections from an Hirschsprung disease

patient. (A–D) Double labelling with HuC/

D, PRMT1 and DAPI. (A) HuC/D: staining

of ganglion cell bodies, (B) PRMT1: staining

of ganglion cell bodies and nuclei of muscle

cells, (C) HuC/D/PRMT1 Merge, (D)

PRMT1/DAPI Merge: absence of staining in

the non-ganglion cells (arrows). Scale bar:

50 lm.

A B

C DFigure 5 Immunodetection of neuronal NO

synthase (nNOS) and protein arginine

methyltransferase 1 (PRMT1) in myenteric

plexuses in normal colon sections from an

Hirschsprung disease patient. (A–D) Double

labelling with nNOS, PRMT1 and DAPI. (A)

nNOS: staining of ganglion cell bodies, (B)

PRMT1: staining of ganglion cell bodies and

nuclei of muscle cells, (C) nNOS/DAPI

Merge, (D) nNOS/PRMT1 Merge. Scale bar:

50 lm.



etiology of HSCR.12 Nevertheless, mutations in these

genes can only explain a small portion of the HSCR

patients. Genes involved in epigenetic regulations

including DNA methylation and histone modifications

can act as genetic modifiers. As PRMT1 is responsible

for the methylation of histone H4 arginine 3 methyl-

ation,4 and plays critical roles in the differentiation and

development of neuron cells,5,6 protein arginine meth-

yltransferase 1 might be related to the development of

ENS and be a potential modifier of HSCR. Even though

no different subcellular localization or expression

could be observed for PRMT1 in ganglions of HSCR

or ARM patients, illustration of PRMT1 expression

pattern in colon segments can be the basis for further

investigations of putative PRMT1 function in ENS

development and intestinal motility.

ACKNOWLEDGMENT

This study was supported in part by National Science Council(NSC 96-2314-B-040-024), Chung Shan Medical University(CSMU 96-OM-A-020) and Chung Shan Medical UniversityHospital (CSH-2009-C-008) to TTW, and National Science Coun-cil (NSC 93-3112-B-040-001 and 94-2320-B-040-044) to CL. Wethank the patients involved in this study.

AUTHOR CONTRIBUTIONS

TW collected the patients and designed the research andanalyzed the data; TT conducted the western blot and immu-nohistochemical analyses; YS performed immunofluorescentexperiments and helped preparing the manuscript; JH and LYhelped preparing tissue sample sections and immunohistochem-istry; CL designed the research, analyzed the data and wrote thepaper. Competing Interests: the authors have no competinginterests.

REFERENCES

1 Bedford MT, Richard S. Argininemethylation an emerging regulator ofprotein function. Mol Cell 2005; 18:263–72.

2 De Gennaro Colonna V, Bianchi M,Pascale V et al. Asymmetric dime-thylarginine (ADMA): an endogenousinhibitor of nitric oxide synthase anda novel cardiovascular risk molecule.Med Sci Monit 2009; 15: RA91–101.

3 Takahashi T. Pathophysiological sig-nificance of neuronal nitric oxidesynthase in the gastrointestinal tract.J Gastroenterol 2003; 38: 421–30.

4 Bedford MT, Clarke SG. Proteinarginine methylation in mammals:who, what, and why. Mol Cell 2009;33: 1–13.

5 Pawlak MR, Scherer CA, Chen J,Roshon MJ, Ruley HE. Arginine N-methyltransferase 1 is required forearly postimplantation mouse devel-opment, but cells deficient in theenzyme are viable. Mol Cell Biol2000; 20: 4859–69.

6 Cimato TR, Tang J, Xu Y et al. Nervegrowth factor-mediated increases inprotein methylation occur predomi-nantly at type I arginine methylationsites and involve protein argininemethyltransferase 1. J Neurosci Res

2002; 67: 435–42.7 Lin WJ, Gary JD, Yang MC, Clarke S,

Herschman HR. The mammalianimmediate-early TIS21 protein andthe leukemia-associated BTG1 pro-tein interact with a protein-arginineN-methyltransferase. J Biol Chem

1996; 271: 15034–44.

8 Tang J, Frankel A, Cook RJ et al.PRMT1 is the predominant type Iprotein arginine methyltransferase inmammalian cells. J Biol Chem 2000;275: 7723–30.

9 Dong CW, Zhang YB, Lu AJ et al.

Molecular characterisation andinductive expression of a fish proteinarginine methyltransferase 1 gene inresponse to virus infection. Fish

Shellfish Immunol 2007; 22: 380–93.10 Le Romancer M, Treilleux I, Leconte

N et al. Regulation of estrogen rapidsignaling through arginine methyla-tion by PRMT1. Mol Cell 2008; 31:212–21.

11 Mathioudaki K, PapadokostopoulouA, Scorilas A, Xynopoulos D, AgnantiN, Talieri M. The PRMT1 geneexpression pattern in colon cancer.Br J Cancer 2008; 99: 2094–9.

12 Amiel J, Sproat-Emison E, Garcia-Barcelo M et al. Hirschsprung dis-ease, associated syndromes andgenetics: a review. J Med Genet 2008;45: 1–14.

13 Angrist M, Bolk S, Thiel B et al.Mutation analysis of the RET recep-tor tyrosine kinase in Hirschsprungdisease. Hum Mol Genet 1995; 4:821–30.

14 Attie T, Pelet A, Edery P et al.

Diversity of RET proto-oncogenemutations in familial and sporadicHirschsprung disease. Hum Mol

Genet 1995; 4: 1381–6.15 Angrist M, Bolk S, Halushka M,

Lapchak PA, Chakravarti A. Germ-line mutations in glial cell line-derived neurotrophic factor (GDNF)

and RET in a Hirschsprung diseasepatient. Nat Genet 1996; 14: 341–4.

16 Salomon R, Attie T, Pelet A et al.

Germline mutations of the RETligand GDNF are not sufficient tocause Hirschsprung disease. Nat

Genet 1996; 14: 345–7.17 Doray B, Salomon R, Amiel J et al.

Mutation of the RET ligand, neurtu-rin, supports multigenic inheritancein Hirschsprung disease. Hum Mol

Genet 1998; 7: 1449–52.18 Puffenberger EG, Hosoda K, Wash-

ington SS et al. A missense mutationof the endothelin-B receptor gene inmultigenic Hirschsprung’s disease.Cell 1994; 79: 1257–66.

19 Bidaud C, Salomon R, Van Camp Get al. Endothelin-3 gene mutations inisolated and syndromic Hirschsprungdisease. Eur J Hum Genet 1997;5: 247–51.

20 Hofstra RM, Valdenaire O, Arch Eet al. A loss-of-function mutation inthe endothelin-converting enzyme 1(ECE-1) associated with Hirschsprungdisease, cardiac defects, and auto-nomic dysfunction. Am J Hum Genet

1999; 64: 304–8.21 Pingault V, Bondurand N, Kuhlbrodt

K et al. SOX10 mutations in patientswith Waardenburg–Hirschsprung dis-ease. Nat Genet 1998; 18: 171–3.

22 Wakamatsu N, Yamada Y, Yamada Ket al. Mutations in SIP1, encodingSmad interacting protein-1, cause aform of Hirschsprung disease. Nat

Genet 2001; 27: 369–70.23 Wu TT, Tsai TW, Chu CT et al. Low

RET mutation frequency and poly-morphism analysis of the RET and



EDNRB genes in patients with Hir-schsprung disease in Taiwan. J Hum

Genet 2005; 50: 168–74.24 Wu TT, Tsai TW, Chang H et al.

Polymorphisms of the RET gene inhirschsprung disease, anorectal mal-formation and intestinal pseudo-obstruction in Taiwan. J Formos MedAssoc 2010; 109: 32–8.

25 Li ZS, Furness JB, Young HM,Campbell G. Nitric oxide synthaseimmunoactivity and NADPH diaph-orase enzyme activity in neurons ofthe gastrointestinal tract of the toad,Bufo marinus. Arch Histol Cytol

1992; 55: 333–50.26 Taguchi T, Tanaka K, Ikeda K.

Immunohistochemical study of neu-ron specific enolase and S-100 proteinin Hirschsprung’s disease. Virchows

Arch A Pathol Anat Histopathol1985; 405: 399–409.

27 Yang S, Donner LR. Detection ofganglion cells in the colonic plexusesby immunostaining for neuron-spe-cific marker NeuN: an aid for thediagnosis of Hirschsprung disease.Appl Immunohistochem Mol Mor-phol 2002; 10: 218–20.

28 Karim S, Hession C, Marconi S, GangDL, Otis CN. The identification ofganglion cells in Hirschsprungdisease by the immunohistochemi-cal detection of ret oncoprotein.Am J Clin Pathol 2006; 126: 49–54.

29 Herrmann F, Lee J, Bedford MT,Fackelmayer FO. Dynamics of humanprotein arginine methyltransferase1(PRMT1) in vivo. J Biol Chem 2005;280: 38005–10.

30 Herrmann F, Fackelmayer FO. Nu-cleo-cytoplasmic shuttling of proteinarginine methyltransferase 1 (PRMT1)requires enzymatic activity. Genes

Cells 2009; 14: 309–17.31 Herrmann F, Pably P, Eckerich C,

Bedford MT, Fackelmayer FO. Humanprotein arginine methyltransferases invivo – distinct properties of eightcanonical members of the PRMTfamily. J Cell Sci 2009; 122: 667–77.

32 Xie Y, Ke S, Ouyang N et al. Epige-netic regulation of transcriptionalactivity of pregnane x receptor byprotein arginine methyltransferase 1.J Biol Chem 2009; 284: 9199–205.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article:

Figure S1. Immunodetection of HuC/D and protein arginine methyltransferase 1 (PRMT1) in myenteric plexuses

in dilated colon sections from an anorectal malformation patient. (A–D) Double labelling with HuC/D, PRMT1 and

DAPI. (A) HuC/D: staining of ganglion cell bodies, (B) PRMT1: staining of ganglion cell bodies and nuclei of muscle

cells, (C) HuC/D/PRMT1 Merge, (D) PRMT1/DAPI Merge. Scale bar: 50 lm.

Figure S2. Immunodetection of neuronal NO synthase (nNOS) and protein arginine methyltransferase 1 (PRMT1)

in myenteric plexuses in dilated colon sections from an anorectal malformation patient. (A–D) Double labelling with

nNOS, PRMT1 and DAPI. (A) nNOS: staining of ganglion cell bodies, (B) PRMT1: staining of ganglion cell bodies and

nuclei of muscle cells, (C) nNOS/DAPI Merge, (D) nNOS/PRMT1 Merge. Scale bar: 50 lm.

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