1 Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA
2 Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
3 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical
School, Boston, MA 02115, USA
4 Department of Pathology, Brigham & Women's Hospital, 75 Francis Street,
Boston, MA 02115, USA
5 Department of Pediatric Cardiology, Johns Hopkins University School of
Medicine, Baltimore, MD 21287, USA
6 Department of Pediatrics, Harvard Medical School, Boston, MA 02115, USA
Author for correspondence (e-mail:
ramesh_shivdasani{at}dfci.harvard.edu)
Accepted 5 November 2004
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SUMMARY |
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Key words: Developmental gene expression, Epithelial-mesenchymal interaction, Oncofetal genes, Hepatoma-derived growth factor, Heterogeneous nuclear ribonucleoprotein, Mouse
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Introduction |
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The fetal mouse gut endoderm, which appears as a pseudostratified
epithelium between embryonic days (E) 12 and 13, undergoes a pivotal cellular
transition over the ensuing 2 days to form the first rudimentary intestinal
villi (Fig. 1A) and to initiate
expression of lineage-specific genes
(Maunoury et al., 1992;
Simon et al., 1993
). We
reasoned that accurate transcriptional profiles over this developmental
interval could provide fundamental information about underlying mechanisms,
and characterized intestinal gene expression using serial analysis of gene
expression (SAGE) (Velculescu et al.,
1995
). Coupled with localization of gene products, the resulting
mRNA profiles
(http://genome.dfci.harvard.edu/GutSAGE)
identify candidate regulators of cell interactions and mucosal
differentiation. We also applied this resource to address longstanding
questions about reactivation of fetal genes in cancer.
|
HDGF was first isolated from conditioned fibroblast media as a factor that
stimulated proliferation of heterologous cultured cells
(Nakamura et al., 1994;
Oliver and Al-Awqati, 1998
).
Although it is hence regarded as a growth factor, the amino acid sequence
lacks a signal peptide but includes a region homologous to the high-mobility
group (HMG) box of nuclear proteins
(Nakamura et al., 1994
) and a
nuclear localization signal that is essential for its activities
(Everett et al., 2001
;
Kishima et al., 2002
). Two
groups have characterized a related protein LEDGF (lens epithelium-derived
growth factor) as a nuclear protein and apparent transcriptional regulator
(Fatma et al., 2001
). To date,
insight into HDGF functions derives largely from study of cell lines, and its
physiological roles in development or cell differentiation are obscure.
Prompted by our demonstration of its significant regulation in the developing
intestine and frequent re-expression in DNA mismatch repair (MMR)-proficient
colon cancers, we identified HDGF-associated nuclear proteins and investigated
HDGF functions in gut epithelial differentiation.
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Materials and methods |
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RT-PCR
Pooled RNA samples, different from those used to construct SAGE libraries,
were extracted with Trizol (Invitrogen), treated with RNase-free DNaseI
(Ambion) and reverse transcribed with oligo-(dT) primer. First-strand cDNA was
used as the template for PCR amplification in the presence of 0.01 mCi/ml
[32P] dCTP with Tm of 62°C. Cycle numbers were
adjusted to ensure linear amplification, and PCR products were resolved by
non-denaturing polyacrylamide gel electrophoresis and detected by
autoradiography. PCR primers are listed in Tables S2A,B in the supplementary
material, unless reported previously (Tou
et al., 2004). Glyceraldehyde 3-phosphate dehydrogenase (GAPD),
human ß-amyloid and mouse hypoxanthine phosphoribosyl transferase (HPRT)
mRNAs verified equal sample loading.
In situ hybridization
RNA in situ hybridization was performed on 6-10 µm paraffin sections of
paraformaldehyde-fixed whole embryos or isolated fetal or adult intestine,
using digoxigenin-labeled (Roche) riboprobes. Rehydrated tissue sections were
treated successively with 50 µg/ml proteinase K, 0.2 M HCl, 0.1 M
triethanolamine in 0.25% acetic anhydride and 2 xSSPE. Slides were
incubated in hybridization buffer containing 50% formamide for 4 hours at
60°C, then hybridized overnight with the probe at 60°C, washed in 0.2
xSSC at 60°C, blocked for 2 hours at room temperature with 10% goat
serum and 2 mg/ml bovine serum albumin diluted in PBS containing 0.1%
Triton-X100 (PBT), and incubated overnight at 4°C with alkaline
phosphatase-conjugated anti-digoxigenin antibody (Roche). After washing,
slides were treated with 5 mM levamisole (Sigma), rinsed in alkaline buffer,
treated with NBT/BCIP solution (Roche), postfixed in 4% paraformaldehyde,
counterstained with Methyl Green, and examined by light microscopy. Images
were captured on a CCD camera (QCapture) with Photoshop7.0 software
(Adobe).
Immunostaining
Trp53 (DO-1, Santa Cruz), Ki67 (NovoCastra), HDGF
(Everett et al., 2001), hnRNPK
(gift of K. Bomsztyk, University of Washington), TLS/Fus (gift of D. Goodman,
Vollum Institute, Oregon) and other antibodies were used to stain 7 µm
paraffin wax embedded sections of human colorectal tumors, mouse fetal
intestine or cultured HeLa cells. Sections were rehydrated, fixed further in
4% paraformaldehyde in PBS, and treated successively with warm 0.01 M citrate
(pH 6), 40% methanol in 0.3% hydrogen peroxide, and PBS containing 10% goat
and 10% fetal bovine sera or 5% milk. Slides were incubated sequentially with
antibodies diluted in PBS containing 5% goat serum, horseradish
peroxidase-conjugated anti-mouse (Amersham Pharmacia) or anti-rabbit (Santa
Cruz Biotechnology) IgG and diaminobenzidine peroxidase substrate (Vector
Laboratories). Mounted sections were examined for image capture as described
above. Cultured cells were fixed in 4% paraformaldehyde for 15 minutes,
permeabilized in 0.1% NP-40 for 5 minutes, and blocked with 3% bovine serum
albumin (BSA) in PBS containing 0.1% Triton (PBST). Antibody incubation was in
PBST with 3% BSA and fluorophore-conjugated secondary antibody (Jackson
ImmunoResarch Laboratories) incubation in PBST for 30 minutes. Cells were
washed in PBST, mounted in aqueous medium containing DAPI, and confocal images
were captured on the LSM510 imaging system (Zeiss).
Organ explant and cell culture
Intact mouse fetal intestines were dissected and cultured as described
(Tou et al., 2004), over
filter disks in Fitton-Jackson-modified BGJb medium (Life Technologies)
containing 0.1 mg/ml ascorbic acid. Explants were maintained in 5%
CO2 atmosphere, with medium changed every 2 days. pK7 plasmid DNA
(2 mg/ml) encoding green fluorescent protein (GFP)-tagged full-length or
truncated HDGF (Everett et al.,
2001
) was flushed into the gut lumen by capillary injection,
followed by placement between platinum electrodes and delivery of three 10 ms
pulses of 80 V each using a BTX830 squarewave electroporator (BTX, San Diego,
CA), washing and culture.
HeLa cells were cultured in Dulbecco's modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 50 µg/ml
streptomycin. Apoptosis was induced by treating the cells for 16 hours with 2
ng/ml human TNF and 35 µM cycloheximide (Sigma). Necrosis was
induced by treatment for 16 hours with ionomycin (Sigma) in serum-free DMEM.
For permeabilization, cells was washed with ice-cold buffer containing 20 mM
HEPES (pH 7.3), 110 mm K acetate, 5 mM Na acetate, 2 mM Mg acetate, 1 mM EGTA,
2 mM DTT and protease inhibitors, incubated on ice in the same buffer with
0.1% NP-40, washed twice with PBS and continued for immunostaining.
Isolation of HDGF-associated proteins
The detailed purification procedure has been described previously
(Nakatani and Ogryzko, 2003).
Briefly, HeLa cells stably expressing Flag and HA epitope-tagged HDGF were
propagated in suspension. Nuclear extracts were isolated from 12 L cultures
and HDGF complexes were purified successively with anti-Flag and anti-HA
antibodies (Sigma). Purified complexes were separated by 4-20% gradient
SDS-PAGE and stained with silver stain. Individual protein bands were excised,
digested with trypsin and analyzed by tandem mass spectrometry (Taplin
Biological Mass Spectrometry Facility, Harvard Medical School). Tryptic
peptides were matched to proteins by the Sequest database-searching program.
For glycerol gradient sedimentation, concentrated HeLa or mouse E13.5
intestine nuclear extracts were loaded over 5 ml of a 10-40% glycerol gradient
and centrifuged for 10 hours at 236,000 g (Beckman SW 55 Ti
rotor). Individual 100 µl fractions were resolved by SDS-PAGE and analyzed
by immunoblotting.
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Results |
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Thirty-eight out of 40 transcripts tested independently by reverse
transcription (RT)-PCR showed expression changes concordant with the SAGE data
(represented in Fig. 1B), as
did eight out of nine independent genes by northern analysis (represented in
Fig. 1E), attesting to the
fidelity of SAGE results. As an example, Gli1 mRNA, a target and
effector of hedgehog signaling (Marigo et
al., 1996), is significantly downregulated after E13
(Fig. 1B), consistent with
reported expression dynamics
(Ramalho-Santos et al., 2000
)
and with prior evidence for obligate attenuation of hedgehog signaling in
vertebrate gut differentiation (Zhang et
al., 2000
). Similarly, the levels of Fabpi
(Fabp2 Mouse Genome Informatics), Fabpl
(Fabp1 Mouse Genome Informatics), Cdx2 and other
mRNAs vary in agreement with published results (see Table S1 in the
supplementary material; and data not shown). Our complete dataset
(http://genome.dfci.harvard.edu/GutSAGE),
annotated with comprehensive functional and genetic information, thus provides
a unique resource to outline the composition of molecular pathways active in
mammalian intestine organogenesis.
Insights into gut development derived from SAGE expression profiles coupled with transcript localization
We record 703 significant (P<0.015) temporal changes in
transcript abundance, revealing notable modulation of gene activity as the
epithelium differentiates
(http://genome.dfci.harvard.edu/GutSAGE).
A representative amount of this analysis (see Table S1 in the supplementary
material) outlines prominent transcriptional alterations that accompany the
villous transition in intestine development. Expression of selected adult
epithelial markers is known to occur by E15
(Maunoury et al., 1992;
Simon et al., 1993
). Numerous
transcripts associated with mucosal architecture (e.g. cadherin 17 and
transgelin), metabolic or secretory functions (e.g. lipid-binding proteins and
cryptdin peptides), and surface markers of cell maturation (e.g.
galactose-binding lectins) are detected concomitantly. To capture changes
occurring in all major cellular compartments, we prepared SAGE libraries from
unfractionated tissue. Moreover, the intestinal long axis undergoes regional
patterning during development, whereas our profiling considered the small
bowel as a single organ and excluded the cecum and colon. Consequently, the
expression data alone do not identify sites of gene expression within the
developing gut. For genes of particular interest, this limitation is readily
overcome by complementary approaches, including RNA in situ hybridization, to
localize transcripts. Fig. 1C
illustrates the value of these methods to map spatiotemporal patterns of fetal
gene expression.
Transcripts that increase significantly at E15 are usually present at high
levels in the adult organ (three examples shown in
Fig. 2, `Increasing') and some
of them can serve as molecular markers of tissue differentiation. Examples
include the pyrin-CARD domain caspase-recruiting protein ASC and proline-rich
acidic protein 1 (Prap1). The latter was originally described as a selective
marker of the pregnant uterus (Kasik and
Rice, 1997) and later found to be expressed at high levels in the
adult small bowel (Bates et al.,
2002
). Detailed examination of Prap1 mRNA expression
confirms its activation after E13 in a regionally restricted manner that
anticipates its distribution in the adult organ
(Fig. 1D). We have also shown
Prap1 to be a useful in vitro marker of intestinal epithelial
cytodifferentiation (Tou et al.,
2004
).
|
|
Significance of transcripts that decline in abundance with progressive epithelial differentiation
Genes with reduced expression past E13 potentially serve functions that are
restricted to the period of organogenesis. Indeed, the proportion of the
genome that is dedicated to developmental functions is unclear. We assessed
expression of 32 transcripts, selected arbitrarily from a total of 254 genes
that showed higher SAGE representation at E12 or E13 than at E15. Seven of
these mRNAs were undetectable past E15
(Fig. 2, `Decreasing', four
examples shown), whereas 25 transcripts could be detected again at E17
(`Dynamic', seven examples). Half of the Dynamic, and all the Decreasing,
transcripts are weakly or not detected in any segment of the adult intestine
with a sensitive RT-PCR assay (Fig.
2). Because analysis of RNA isolated from the whole organ may
underestimate expression that is confined to minor cell populations, we
confirmed absence of most of these transcripts in the adult mouse gut by in
situ hybridization (data not shown). Transient expression of many genes during
organogenesis suggests that a substantial fraction of mammalian genes may
serve dedicated functions in development, although some mRNAs in this class
(e.g. Pthr1) may have separate roles in other sites
(Urena et al., 1993).
Moreover, groups of fetally expressed genes may be subject to common molecular
mechanisms of gene silencing concomitant with tissue differentiation.
Silencing of genes during normal development may be especially relevant to
cancer, a disease in which cells display immature morphology and gene
expression is broadly dysregulated. Some proteins expressed exclusively in
tumors and in developing embryos, such as carcinoembryonic antigen and
-fetoprotein, are oncofetal markers of clinical utility
(Carr et al., 1997
;
Uriel, 1975
); however, the
extent to which tumor cells recapitulate embryonic gene expression and the
significance of this phenomenon are unknown. We therefore attempted to examine
expression of all 32 Dynamic or Decreasing transcripts in seven samples of
malignant and adjacent normal human colon tissue. For this purpose, we regard
the small intestine and colon to have highly similar biology and gene
expression profiles, as shown or suggested in previous studies
(Bates et al., 2002
;
Potten, 1998
); colon cancer is
the second leading cause of cancer death in many developed nations, whereas
primary tumors of the small bowel are rare
(http://seer.cancer.gov).
First, we identified the known or presumptive orthologous human gene and
designed suitable primers for RT-PCR; 27 primer pairs for the corresponding
human genes amplified the predicted fragment reliably. Second, we rigorously
established two independent standards (GAPD and ß-amyloid) to ensure
equal loading of RNA from each matched pair of tumor and normal tissue
(Fig. 3A,B) and we amplified
Tgfbi, a TGFß-inducible gene whose expression is known to be
elevated in colon cancers (Zhang et al.,
1997
), as a positive control.
|
|
HDGF expression in mammalian fetal gut development and colorectal cancer
HDGF mRNA is downregulated concomitantly with transition of the gut
endoderm into a villous epithelium and there is a corresponding, albeit
delayed, decline in protein levels (Fig.
4A). In the fetal gut, HDGF mRNA and protein localize in the
developing mucosa and are excluded from the mesenchyme
(Fig. 4B); HDGF protein is
restricted to cell nuclei. In the adult intestine, weak residual HDGF
expression is confined to nuclei in the lower half of the villous projections
(Fig. 4C), the site of
temporary residence of undifferentiated epithelial cells. HDGF expression thus
correlates with undifferentiated states in the developing and adult gut
mucosa; there is no variation along the rostrocaudal axis of the digestive
tract (data not shown).
In Fig. 3B, we show elevated
HDGF mRNA in 2 of 7 human colon cancers. HDGF protein was
overexpressed in 14 of 28 colorectal cancers we tested independently by
immunohistochemistry (Fig. 4D). Again, HDGF was present only in nuclei and overexpressed in the tumor
compartment; cells at the invasive front, particularly isolated invasive foci
and large cells with undifferentiated morphology, were often the most strongly
stained (Fig. 4E). This finding
may be pertinent to the role of HDGF-containing complexes discussed below, and
staining throughout the cancerous tissue argues against HDGF simply marking
rapidly dividing cells. Colon cancers with deficient or intact DNA mismatch
repair (MMR) express distinctive genetic, biological, and prognostic features
but there is limited understanding about the molecular basis for these
differences (Peltomaki, 2003).
Notably, HDGF was overexpressed in 11 out of the 16 tumors proficient for MMR,
compared with three out of 12 MMR-deficient colon cancers
(Fig. 4F; P=0.027 by
the Fisher exact test). Thus, although HDGF is expressed weakly in normal
adult gut mucosa, our findings reveal oncofetal properties and especially
elevated expression in MMR-proficient colon tumors.
Isolation and characterization of an HDGF-containing nuclear protein complex
HDGF has few sequence motifs that suggest possible cellular functions,
except for partial homology with a subfamily of high-mobility group (HMG)-box
proteins exemplified by HMGB1. The latter is also a nuclear protein that may
be found in culture media, owing to its weak association with chromatin, which
is disrupted in necrotic cells but enhanced when cells die by apoptosis
(Scaffidi et al., 2002).
Indeed, as noted for HMGB1, native HGDF could be extracted from HeLa cell
nuclei within 1 minute of mild NP-40 detergent treatment and completely within
5 minutes, whereas the chromatin-associated histone 2Az protein resisted such
extraction (Fig. 5A). In HeLa
cells treated with ionomycin to induce necrosis, HDGF was lost from the cell
nucleus, whereas after treatment with tumor necrosis factor
and
cycloheximide, which induces apoptosis, HDFG remained in the nucleus (data not
shown). These findings affirm nuclear expression of HDGF and might explain its
appearance in conditioned cell media.
|
Available antibodies against the putative HDGF-associated proteins lack immunoprecipitating activity, which precludes independent assessment of protein interactions. Instead, we resolved nuclear extracts from HeLa cells and from E13 mouse intestine by glycerol gradient sedimentation. Specific antibodies against hnRNPK and TLS/Fus (hnRNPI antibody is not readily available) revealed partial co-sedimentation with HDGF, mainly in fractions 6-9 (Fig. 5C). These results support the idea that nuclear HDGF associates, probably sub-stoichiometrically, with these factors. Immunostaining for HDGF, TLS/Fus and hnRNPK confirmed diffuse nuclear localization of each within innumerable discrete foci (Fig. 5D), although the subnuclear resolution is insufficient to verify co-localization of these abundant proteins. However, expression dynamics for the HDGF-associated proteins provide independent support for their participation in a common developmental pathway: mRNA and protein levels for both hnRNPK and TLS/Fus parallel that of HDGF in the fetal mouse gut (Fig. 5E). The three HDGF-associated factors we studied are RNA-binding proteins. Our observations thus combine to place previously obscure HDGF mechanisms within a RNA metabolic pathway that is developmentally controlled and pertinent to cancer. Although the two novel HDGF-associated proteins (Fig. S1) lack discernible functional motifs, their identification also should enable further study of HDFG function.
Differentiation and cellular functions of HDGF
The strong correlation between HDGF expression and undifferentiated
epithelial states (Fig. 4) led
us to hypothesize that HDGF regulates mucosal cell differentiation negatively.
We reasoned that if declining HDGF levels during gut development enable
expression of maturation-associated genes, then forced HDGF expression may
prevent or delay their activation. We introduced DNA by luminal injection and
electroporation in intestines explanted from E14 mouse fetuses, when
endogenous mRNA levels are dropping and prior to native expression of
differentiation genes (Tou et al.,
2004). Epithelial expression of GFP-tagged HDGF fusion protein
(Fig. 6A,B) reduced mRNA levels
of several intestine-specific differentiation markers: Apo1a, liver
(Fabpl)- and intestine (Fabpi)-specific fatty acid-binding
proteins, metallothionein 2 (Mt2), and Prap1
(Fig. 6C). Villin RNA levels
were unchanged, which implies that HDGF influences differentiation genes
selectively, and expression of the inhibited markers recovered partially after
additional culture, indicating that HDGF overexpression did not compromise
explant viability. An inactive HDGF form lacking the NLS
(Everett et al., 2001
) did not
affect differentiation-related mRNAs (Fig.
6C); levels of the intact and mutant proteins were comparable
(Fig. 6B). We have previously
shown good correlation between molecular and histological maturation in fetal
gut explants (Tou et al.,
2004
). However, the conditions for DNA electroporation affect
tissue morphology adversely and preclude evaluation of HDGF effects on
epithelial cytodifferentiation.
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Discussion |
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Experimental tests reproduce sample results from our analysis with more than 90% accuracy. Building on the quantitative aspects of SAGE, our web resource (http://genome.dfci.harvard.edu/GutSAGE) provides primary data on numbers of sequence tags and enumerates the statistical significance of differences measured across developmental stages. A versatile search function permits users to parse genes according to expression or functional criteria, with immediate access to the primary data. Tag and gene annotations are continually updated, as each SAGE entry is linked to three public resources: LocusLink, GeneOntology and SAGEmap; LocusLink will soon be replaced by its versatile successor Entrez Gene (http://www.ncbi.nlm.nih.gov/entrez/query/static/help/genehelp.html). These features assure the currency of the data and will enable investigators to apply a developmental dataset to address many outstanding problems. In particular, the basis for important interactions between epithelial and mesenchymal cells may be revealed through signaling components that vary over the E12-E15 interval. Although the data presented in Table 1 do not imply a vital role for every listed product, they represent an enriched gene set for targeted investigation. Such studies could be facilitated by localizing gene expression using in situ hybridization and an array of stage-specific molecular markers identified in this study. These advances enhance the collective ability to address issues such as the relationship between fetal and adult mucosal stem cells and how or why certain classes of genes, such as the stress-response group, are co-regulated during intestinal differentiation.
Genetic pathways that initiate intestinal tumors are well characterized
(Batlle et al., 2002;
Kinzler and Vogelstein, 1996
),
whereas most alterations that accompany disease progression
(Stoler et al., 1999
) are not.
Yet, it is the latter changes that probably underlie especially ominous
features of colonic and other epithelial tumors, including tissue invasion,
viability at distant sites and drug resistance. Each of these features is
strongly correlated with the degree of tumor cell differentiation
(Deans et al., 1994
), but the
basis for this correlation is unknown. Although it is commonly assumed that
poor differentiation of tumor cells reflects reversion to a primitive state,
the definition of such states is vague. By contrast, in some leukemias and
lymphomas, where relationships between tumor cells and steps in normal
development or cell differentiation are defined precisely, the resulting
insights guide both understanding and treatment of the disease
(Tenen, 2003
). We have
therefore begun to ask to what extent a common carcinoma might recapitulate
the gene expression program associated with its developmental origins. Current
cancer treatments, even when directed against molecular carcinogenic
mechanisms, frequently are confounded by toxicities that limit the dose or
discourage drug development. Such toxicity typically reflects expression of
the drug target in non-tumor tissues. Oncofetal proteins that are essential
for malignant behaviors thus represent attractive targets for cancer treatment
because their absence or reduced levels in normal adult tissues may confer
especially wide therapeutic windows. An approach to identify oncofetal
proteins could thus find useful therapeutic applications.
Our findings implicate the oncofetal protein HDGF in regulation of normal
and pathologic gut mucosal differentiation. In the mammalian gut, HDGF
localizes in epithelial cells, peak expression coincides with the
developmental villous transition and the protein is overexpressed in human
colorectal cancers, especially those with intact DNA mismatch repair. HDGF is
a nuclear protein rather than a classical, secreted growth factor and forced
expression in the fetal mouse intestine retards epithelial development. HDGF
shares discernible sequence similarity and cellular properties with the
ubiquitous and abundant nuclear protein HMGB1
(Nakamura et al., 1994), which
may regulate gene transcription but also has differentiation and pathological
roles when released from necrotic cells
(Melloni et al., 1995
;
Muller et al., 2001
;
Scaffidi et al., 2002
).
Presumably, both HDGF and HMGB1 are released passively from necrotic cells
following lysis. More important from a mechanistic standpoint is our isolation
of nuclear complexes wherein HDGF associates with well-characterized
RNA-binding proteins.
Not only does HDGF associate with two hnRNPs and TLS/Fus but the four mRNAs
are regulated in tandem in intestine organogenesis and both HDGF and TLS/Fus
are commonly overexpressed in human colon cancers. TLS/Fus, which can interact
with DNA and RNA, regulates transcription and, in the context of a pathogenic
fusion oncoprotein, confers a transcriptional activation domain
(Rabbitts et al., 1993).
Besides having a role in preserving genomic integrity
(Baechtold et al., 1999
;
Hicks et al., 2000
;
Kuroda et al., 2000
), TLS/Fus
binds pre- and processed mRNAs, and engages in nucleocytoplasmic RNA transport
(Iko et al., 2004
;
Lerga et al., 2001
;
Zinszner et al., 1997
). It
contains both RNA-binding domains and functions shared with hnRNPs and is
known to interact with hnRNPI (Meissner et
al., 2003
). hnRNPI binds intronic polypyrimidine tracts, and both
hnRNPI and hnRNPK associate in a protein complex
(Kim et al., 2000
) and
interact with Ro ribonucleoprotein-associated Y RNAs
(Fabini et al., 2001
); both
factors are implicated in gene transcription and pre-RNA splicing, nuclear
export of mature mRNAs, translation and regulation of mRNA stability
(Dreyfuss et al., 2002
;
Reed and Hurt, 2002
). Their
interaction and developmental co-regulation with HDGF thus strongly suggests
related cellular functions for the latter and represents a novel insight into
putative HDGF mechanisms. Recently, cytosolic TLS/Fus, hnRNPs K and I, other
RNA-binding proteins, and RNA were found within novel focal adhesion-related
structures called spreading initiation centers
(de Hoog et al., 2004
). Our
studies do not address the possibility of similar roles for HDGF, although
notably, HDGF expression in colon tumors is especially prominent in isolated
foci of large, invasive cells (Fig.
5E).
In summary, HDGF is one of several genes we identify whose peak expression coincides with fetal gut epithelial morphogenesis and in carcinomas of the same developmental origin. HDGF acts to limit epithelial differentiation, which is significant in light of its expression pattern, and associates with a group of nuclear proteins that share biochemical properties and functions in RNA metabolism. Our approach can be extended to identify other oncofetal proteins, which could present safer therapeutic targets, and to better understand molecular mechanisms common to development and cancer.
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ACKNOWLEDGMENTS |
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![]() |
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/2/415/DC1
* These authors contributed equally to this work
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