The Primary open-angle glaucoma gene WDR36
functions in ribosomal RNA processing and
interacts with the p53 stress–response pathway
Jonathan M. Skarie and Brian A. Link?
Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI, USA
Received February 25, 2008; Revised and Accepted May 7, 2008
Primary open-angle glaucoma (POAG) is a genetically complex neuropathy that affects retinal ganglion cells
and is a leading cause of blindness worldwide. WDR36, a gene of unknown function, was recently identified
as causative for POAG at locus GLC1G. Subsequent studies found disease-associated variants in control
populations, leaving the role of WDR36 in this disease unclear. To address this issue, we determined the
function of WDR36. We studied Wdr36 in zebrafish and found it is the functional homolog of yeast Utp21.
Utp21 is cell essential and functions in the nucleolar processing of 18S rRNA, which is required for ribosome
biogenesis. Evidence for functional homology comes from sequence alignment, ubiquitous expression, sub-
cellular localization to the nucleolus and loss-of-function phenotypes that include defects in 18S rRNA pro-
cessing and abnormal nucleolar morphology. Additionally, we show that loss of Wdr36 function leads to an
activation of the p53 stress–response pathway, suggesting that co-inheritance of defects in p53 pathway
genes may influence the impact of WDR36 variants on POAG. Although these results overall do not provide
evidence for or against a role of WDR36 in POAG, they do provide important baseline information for future
Glaucomas are a heterogeneous group of blinding neuropa-
thies that result in retinal ganglion cell (RGC) death, visual
field deficits and optic nerve atrophy (1). They are currently
a leading cause of blindness worldwide, and estimates
predict 60 million people will be diagnosed with the disease
by 2010 (2). The most prevalent form of glaucoma is
primary open-angle glaucoma (POAG), the incidence of
which increases with advancing age (3). POAG is a complex
disease, with multiple genetic and environmental factors inter-
acting to cause disease (1,4,5). High intraocular pressure (IOP)
is the greatest risk factor for developing glaucoma, and is
thought to result from defects in aqueous humor drainage
within the anterior segment of the eye. It is unknown
exactly how elevated IOP impacts POAG. Currently, the
most accepted hypothesis is that elevated pressure in the
anterior chamber of the eye is translated to the posterior
chamber, creating stress at the optic nerve head and, ulti-
mately, resulting in death of RGCs (6–11). Though high
IOP is the most common risk factor, it is not required for
the development of POAG. About one-third of cases occur
in normal tension patients (IOP , 22 mmHg) (12). Together,
this suggests that glaucoma can result from insults to the drai-
nage structures in the anterior chamber and/or from intrinsic
defects in RGCs (1).
Determining the genetic risk factors of POAG has been an
area of intense study, but the complex nature of the disease
has made the search very difficult (13). Through human
genetic screens, at least 20 loci have been found that link to
POAG, but causative genes have only been reported for
three (13). These genes are MYOCILIN, OPTINEURIN and
WDR36. Genetically, both MYOCILIN and OPTINEURIN
have been well established in influencing POAG, but the
endogenous functions of these proteins as well as the exact
mechanism by which they cause disease are unknown (1).
MYOCILIN is the causative gene at locus GLC1A, and is
associated with high IOP cases of POAG (13,14). MYOCILIN
is expressed at high levels in trabecular meshwork cells, and it
is thought that disease causing mutations result in cellular
?To whom correspondence should be addressed at: Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, 8701
Watertown Plank Road BSB 405, Milwaukee, WI 53226, USA. Tel: þ1 4144568072; Fax: þ1 4144566517; Email: email@example.com
# The Author 2008. Published by Oxford University Press. All rights reserved.
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Human Molecular Genetics, 2008, Vol. 17, No. 16
Advance Access published on May 10, 2008
dysfunction as a result of blocked secretion and ectopic
meshwork cells result in raised IOP, which then contributes
to glaucoma. OPTINEURIN is the causative gene at locus
GLC1E and was found to cause normal-tension POAG (21).
Genetic studies in a variety of populations revealed that
some initially described disease-causing mutations are also
found in normal individuals (22). This indicates that OPTI-
NEURIN causes POAG when in permissive backgrounds,
and acts as a key modifier in others (1). In cultured cells,
OPTINEURIN can act to regulate apoptosis through inter-
actions with tumor necrosis factor-alpha, but it is unknown
how this occurs and whether this contributes to the apoptotic
RGC cell loss found in POAG (23–26).
WDR36 encodes a protein of unknown function that was
recently described as a causative gene for adult-onset POAG
andnarrowed agenetic linkage to5q22.1(GLC1G) (27–29).In
segregate with all affected family members and not with any
unaffected individuals (27). Further analysis of 17 unrelated
patients with either high- or low-tension POAG revealed a
total of four amino acid changes that were not found in 200
control individuals (27). When other groups extended the study
of WDR36 to different populations, the relationship between
WDR36 and glaucoma appeared more complex. Originally
described disease-causing variants have been found in control
individuals with an equal frequency as patients with POAG
(30–33). Although these data indicate that WDR36 is not causa-
tive for POAG in all populations, recent studies suggest that
WDR36 may act as a modifier of the disease. Hauser et al. (30)
found correlations between WDR36 variants and POAG severity
in POAG pedigrees. Additional genetic association studies have
also found a higher frequency of rare WDR36 variants in glau-
coma patients compared with controls, and have suggested that
WDR36 variants may contribute to a small number of unrelated
glaucoma cases (32,34,35). Taken together, the current genetic
data suggest that WDR36 may act as a modifier of POAG, and/
or as a causative gene for POAG in certain populations. Alterna-
tively, WDR36 variants may simply mark certain disease haplo-
types and not be directly involved in POAG pathology. To
better understand the role of WDR36 in POAG, determining its
function and manipulating the gene in animal models are
crucial. Although the function of WDR36 is unclear, it is
known that it is a 100 kDa protein containing four conserved
protein domains: a guanine nucleotide-binding protein (G-beta)
WD40 repeat, an AMP-dependant synthetase and ligase, a cyto-
specific WD40-associated domain (27). In human and mouse
tissues, WDR36 is widely expressed, and it was also found to be
upregulated during human T-cell proliferation (27,36).
In this study, we use zebrafish to determine the function of
Wdr36, the homolog of human WDR36, and investigate the
affected cellular signaling pathways for its involvement in
POAG. We show that Wdr36 is functionally homologous to
the yeast Utp21, a component of the rRNA processome, and
is involved in 18S rRNA processing and nucleolar homeosta-
sis. We further show that wdr36 loss of function results in acti-
vation of the p53 stress–response pathway, consistent with
disrupted nucleolar function.
WDR36 shares sequence homology to yeast Utp21
To gain insight into the cellular function of vertebrate
WDR36, BLASTp analysis using human WDR36 was
carried out against Drosophila melanogaster (Dm), Caenor-
habditis elegans (Ce) and Sacchaormyces cerevisiae (Sc)—
organisms with extensive protein function data. The closest
related proteins in each species were: Dm, AAK93538
(NP_650284); Ce, Y45F10D.7 (NP_502661); Sc, Utp21
(NP_013513) (37,38). Of the homologous proteins identified,
only the putative yeast ortholog, Utp21, was studied. It was
found that this protein is part of the yeast rRNA small-subunit
(SSU) processome and is essential for 18S rRNA maturation
(38,39). Human WDR36 shares 24% identity with yeast
Utp21. Subsequent protein alignments revealed that Utp21
and zebrafish Wdr36 share 25% identity, whereas human
WDR36 and zebrafish Wdr36 share 64% identity. To test
the hypothesis that vertebrate Wdr36 is the functional
homolog of yeast Utp21, we explored the expression and cel-
lular consequences of loss of Wdr36 function in zebrafish.
wdr36 is ubiquitously expressed
hybridization. Similar to that reported for human and mouse,
RT–PCR analysis suggested that zebrafish wdr36 is ubiquitous
as transcripts were detected in all embryonic and adult tissues
assayed (Fig. 1) (27). It also indicated that wdr36 is maternally
contributed (Fig. 1). Zebrafish embryos do not begin zygotic
transcription until the 512-cell stage, and instead rely on a
large supply of maternal protein and transcripts for early devel-
hybridization when the alkaline phosphatase reporter reaction
was allowed to develop for longer periods of time (data not
shown). Interestingly, shorter reporter reaction times showed
enrichment of wdr36 transcripts within tissues undergoing
high levels of proliferation (Fig. 1). Tissues with the greatest
levels included the developing eye cup, proliferative zones
within the CNS and cells throughout the gut (Fig. 1). Within
the eye, as differentiation progressed, high levels of gene
expression were maintained in the lens and retinal periphery
where growth is ongoing. These results indicate that wdr36 is
ubiquitously expressed, but is enriched in proliferative cells
Wdr36 fusion proteins localize to the nucleolus
To investigate the sub-cellular localization of Wdr36, we
expressed various epitope-tagged versions of Wdr36 within
multiple cell types in zebrafish embryos. Fusion protein
expression was driven by an inducible heat shock promoter
(hsp70) in an attempt to control the protein levels. Following
heat shock induction, epitope-tagged Wdr36 localized to both
the cytoplasm and nucleoli (Fig. 2). Multiple epitope tags (N-
and C-terminus 6X-myc or YFP) all showed equivalent localiz-
ations. Nucleolar localization was confirmed by co-expressing
with either Histone2A fusions, which localize to chromatin
but not the nucleolus, or with B23, which specifically labels
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