Download Bioinformatics analysis of FRG1

Current Status and Future Prospect of FSHD
Region Gene 1
Arman Kunwar Hansda1*, Ankit Tiwari1*, Manjusha Dixit1
Affiliation
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School of Biological Sciences, National Institute of Science Education and Research, PO:
Bhimpur-Padanpur, Via: Jatni, Khurda-752050, Odisha, India.
* These authors contributed equally to this work

Corresponding Author
Address for Correspondence:
Dr. Manjusha Dixit, PhD, School of Biological Sciences, National Institute of Science Education
and Research, PO: Bhimpur-Padanpur, Via: Jatni, Khurda, Odisha, India-752050, Tel.: +91-674249-4195, Fax: +91-674-249-4004, E-mail: [email protected]
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Abstract
FSHD region gene 1 (FRG1), as the name suggests, is the primary candidate gene for
Fascioscapulohumeral muscular dystrophy disease. It seemingly affects muscle physiology in
normal individuals but in FSHD, where it is found to be highly upregulated, might be involved in
disruption of face, scapula and humeral skeletal muscle. Literature on FRG1, reviewed from
1996 to 2016, reveals that it is primarily associated with muscle development and maintenance.
Approximately 75% of FSHD patients also show vascular abnormalities indicating that FRG1
might have some part to play in these abnormalities. Research involving vasculature in X. laevis
larvae shows that FRG1 positively affects normal vasculature. Few of the well-established
angiogenic regulators seem to get affected by abnormal expression level of FRG1. Its primary
localization in sub nuclear structures like Cajal bodies and nuclear speckles indicates regulation
of the above-mentioned factors by transcriptional and post-transcriptional machineries, but indepth studies need to be done to conclude a clear statement. In this review, we have attempted to
present all the work done on FRG1, all the lacunas which need to be unraveled and, hypothesized
a model for our readers to get an insight into its molecular mechanism.
Key words
FRG1; FSHD; RNA Biogenesis; Angiogenesis; Neuromuscular Disorder
Introduction
FRG1 (FSHD Region Gene 1) is the primary candidate gene for Facioscapulohumeral muscular
dystrophy (FSHD). Since its discovery in 1996, through many experimental evidences, it has
been associated primarily with myogenesis in FSHD patients as well as in normal individuals.
Moreover, studies have reported additional functions. Till date the unequivocal molecular
mechanism remains unclear. Exact role of the FRG1 in muscle development is another matter of
discussion and remains poorly understood. FRG1 might affect muscle development directly, by
acting as muscle growth factor or indirectly, by regulating the interplay between development
and vasculogenesis. FRG1 is highly conserved gene in both vertebrates and invertebrates,
suggesting its important role in biological systems. Goal of this review is to recapitulate all the
information known till date about the FRG1 and to look at the possible links among existing
studies to nail down its function. In the end, we have also reviewed the lacunae in the
information needed to build a complete FRG1 story.
History
Pursuit of primary candidate gene for FSHD has been delayed over a century because of the lack
of proper information and technological advancement. Preparation of large number of cDNA
library from FSHD skeletal muscle and screening for target gene had only been fruitful just
about two decades ago, when the cDNA cloning of this novel human gene was completed and
named as FSHD Region Gene 1 (FRG1). It has been compiled under Genebank accession
number L76159. This gene was found to be present 100kb centromeric to the D4Z4 repeats. To
determine the chromosomal distribution of FRG1, its exons were used as probes in fluorescence
in situ hybridization (FISH) analysis. The hybridization signals were found in the short arm of
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acrocentric chromosome- 13, 14, 15, 21 and 22 and at the centromeric region of chromosome 20
(van Deutekom et al. 1996).
Later on, FRG1 was studied in different organisms including C. elegans, X. laevis, mouse, apes
and various cell lines. In C. elegans FRG1 was found to interact with F-actin (Liu et al. 2010).
Mouse overexpressing FRG1 showed FSHD phenotype (Gabellini et al. 2006), and could be
rescued by repressing the level of FRG1 (Wallace et al. 2011). To get an insight into the
molecular machinery of FRG1 mode of action, it has been studied in different cell lines (e.g.,
Hela, C2C12 myoblast, U20S etc) (Chen et al. 2011; Koningsbruggen et al. 2007; Neguembor et
al. 2013). In X. laevis it has been studied in relation with muscle development and vasculature
using ectopic expression and knock down tools (Hanel et al. 2009; Wuebbles et al. 2009).
Nevertheless, even after all these studies its enigma is yet to be unraveled.
Gene
FRG1 gene is present on long arm (q) of chromosome 4, precisely localized at position 35 (Ch.
4q35). It is present 120kb centromeric to the D4Z4 repeats and flanked by ANT1 gene towards
centromeric and FRG2 towards the telomeric region. FRG2 is followed by a double homeobox
promoter region called DUX4 towards the centromere (Gabriels et al. 1999). FRG1 gene spans a
length of 22,386 bp and codes for a mature transcript of length 1042 bp consisting of nine exons
(figure 1) (van Deutekom et al. 1996).
Comparison between the FRG1 homologues of mouse, Fugu rubripes and invertebrates like C.
elegans and Brugia malayi leads us to believe that FRG1 is conserved among vertebrates and
invertebrates (Grewal et al. 1998). During investigation of first gene to be associated with FSHD,
FRG1was also found to be present on chromosome 13, 14, 15, 20, 21 and 22 (van Deutekom et
al. 1996). Supported by analysis of human FRG1 ESTs, these are thought to be pseudogenes
present throughout the genome (Grewal et al. 1999). Detailed analysis of FRG1 sequences from
great apes such as chimpanzee, gorilla, orangutan and an old world monkey Macaca mulatta has
revealed the presence of similar pattern of pseudogenes. All the great apes and human FRG1
sequences contain the Alu-Sx repeat and Alu-J monomer (FRAM), both of which belong to
oldest subfamilies of Alu elements in intron 7, suggesting that different copies must have
originated from a common ancestral sequence. However, in case of old world monkey Macaca
mulatta, only single orthologue of 4q FRG1 and a possible pseudogene, making it total of two
loci of FRG1, was found. Above mentioned data, along with presence of D4Z4-like loci in
hominids and old world monkeys (Clark et al. 1996) and close proximity of these two regions,
led to hypothesize that initial duplication of 4q included FRG1, and was present in common
ancestors of Macaca mulatta and hominoids which were believed to be diverged at least 33
Million years ago. This region underwent further amplification and dispersion in hominidae
lineages and remained localized in heterochromatic regions.
Protein
The open reading frame (ORF) of this transcript codes for a 258 amino-acid long polypeptide
chain of 30kDa molecular weight (van Deutekom et al. 1996). Comparison of FRG1 homologous
genes in vertebrates other than human, have revealed a common, but not well conserved lipocalin
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signature sequence towards the N-terminal end, suggesting that FRG1 may belong to this
superfamily (Grewal et al. 1998). Through computational analysis it was revealed that FRG1p
contains a N-terminal nuclear localization signal (NLS) of around eleven residues
(Koningsbruggen et al. 2004). In the central part, very well conserved among other orthologues
of FRG1, a fascin-like domain (PF06220) has been reported to be present (Liu et al. 2010).
Towards the carboxy terminal end, a bipartite (BP) nuclear localization signal (253-261aa) is
present (Koningsbruggen et al. 2004). There are two common phosphorylation sites conserved in
C. elegans and human FRG1 i.e., T179 and Y209, which match substrate specificity for casein
kinase II and tyrosine kinase respectively (Grewal et al. 1998). Figure 1 shows a schematic of
different domains in FRG1.
Localization
The presence of two nuclear localization signal in FRG1p leads us to believe that it must
primarily be localized in nucleus. Indeed, EGFP tagged endogenous expression of FRG1 in
stably transfected human bone osteosarcoma epithelial cell lines (U20S) was seen in nucleus,
prominently in Cajal bodies and nucleoli. However, transient expression of FRG1 showed
additional staining of nuclear speckles (Koningsbruggen et al. 2004). Immunostaining of X.
laevis oocytes has shown the co-localization of FRG1 with nascent mRNA chain being
transcribed in nucleus. This co-localization of FRG1 with mRNA was further affirmed by RNA
immunoprecipitation (RIP) and RT-PCR (Sun et al. 2011). These data implicate a core role of
FRG1 in RNA biogenesis.
Presence of central fascin-like domain in FRG1 suggests its localization not only in nucleus but
also in cytoplasm (Adams 2004; Edwards & Bryan 1995). Immunocytochemistry and confocal
imaging of FRG1 in human muscle cell lines (TE671) and C. elegans embryo has revealed that it
is both nuclear and cytoplasmic protein. In cytoplasm, it dominantly localizes with dense bodies
in actin filaments (Koningsbruggen et al. 2004; Liu et al. 2010).
Various roles
Candidate gene for FSHD
As the name suggests FRG1 has been primarily associated with FSHD pathophysiology. High
expression levels of FRG1 have been observed in affected skeletal muscle tissues from FSHD
patients. Its expression is seemingly regulated by a highly polymorphic VNTR structure called
D4Z4 repeats. The reduction of these D4Z4 repeats from 11-150 units in healthy individuals, to
less than 10 repeats in FSHD patients (Lunt 1998), might be the cause for overexpression of
FRG1 (Gabellini et al. 2002). In another quest to find its association with FSHD, mouse model
was used (Gabellini et al. 2006). In this particular experiment, they produced transgenic mouse
overexpressing FRG1 (FRG1-high) and observed skeletal muscle dystrophy phenotype similar to
FSHD patients. These FRG1-high mutants, in another experiment, treated with interfering RNAs
(RNAi) targeted at FRG1, improved the myopathic phenotype of the transgenic mice (Wallace et
al. 2011). FRG1 over-expression causes defects in myoblast fusion, leading to deregulated
formation of myotubes. FRG1 over expressing transgenic mice had enriched altered TNNT3
splice variant which affects muscle strength and contractile properties of muscle (Sancisi et al.
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2014). A recent study showed that Four and half Lim Protein 1(FHL1) over-expression in FRG1
over expressing transgenic mice lead to improvement of the myoblast fusion. However, FHL1
over expression could not restore muscle strength and contractile properties (Feeney et al.
2015).The inability to restore muscle strength and contractile properties may be attributed to
altered splice isoforms of TNNT3. FHL1 over-expression does not affect the pathogenic pathway
involving FRG1/Suv-20h/Eid3. FRG1 might affect muscle physiology independent of FHL1
expression explaining inability of FHL1 to rescue FSHD phenotype (Feeney et al. 2015). These
evidences all together point towards a major function of FRG1 in normal physiology of muscle.
In depth search for molecular regulation of FRG1 has established that a transcription factor
named DUX4 directly regulates the expression of FRG1 (Ferri et al. 2015). ChIP-seq
experiments upon ectopic DUX4 overexpression in control human muscle cells (Geng et al.
2012) and bioinformatics analysis of results, (Ferri et al. 2015) revealed that it strongly binds to a
region inside the second intron of FRG1. This region also showed a relative enrichment of
enhancer associated histone marks (H3K4me1 and H3K27Ac), suggesting that it may be an
enhancer that regulates the FRG1 expression. Intriguingly, DUX4 regulates FRG1 expression in
human but not in mouse. The analysis in DUX4 transgenic mice for endogenous FRG1 level
resulted in no significant change (Ferri et al. 2015).
Role in Development
Overpopulation of FRG1 protein in muscles might be the leading cause of FSHD (Gabellini et al.
2002) but a gene evolutionary so conserved (Grewal et al. 1998) can’t possibly have just a single
role. Recently a group has shown the critical role of frg1 on muscle development in Xenopus
laevis (Hanel et al. 2009). Temporal analysis of frg1 mRNA levels by qRT-PCR showed a peak
before midblastula transition (MBT), which is attributed to maternal stores of frg1. But, after
MBT through stage 41, the transcript levels decreased progressively, suggesting an early
requirement in development (Hanel et al. 2009). The spatiotemporal expression pattern of frg1
during different stages of development was studied through whole-mount in situ hybridization.
Frg1 was found diffusely within head, eyes, branchial arches, somites and notochord. These
results were further corroborated by immunohistochemistry using X. laevis frg1 specific antibody
(Hanel et al. 2009). To establish its role in tissue development and differentiation, frg1
morpholinos (FMO) complementary to X. laevis frg1 mRNA, were injected asymmetrically into
two stage blastomeres. Reduced level of frg1 led to smaller myotome and altered epaxial muscle
morphology owing to inability to delaminate by reducing the level of myoD and pax3 expression
levels.
Hanel et al (Hanel et al. 2009) also found that the elevated levels of frg1 disrupted the skeletal
muscle development which is consistent with previous reports. Elevated levels of frg1 lead to
altered epaxial muscle morphology, expansion of myotome and neural plate at neurulation stages
by increasing the expression level of pax3 and myoD, critical muscle transcription factors. In
later stages (stage 36) both myoD and pax3 levels appeared normal but elevated frg1 caused
somite segmentation errors. Similar effects were studied under reduced level of frg1, achieved by
use of morpholinos targeted at frg1. Both overexpression and reduced level of frg1 resulted in
abnormal somite segmentation suggesting that frg1 may be required during the mesenchymal to
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epithelial transition (MET) that occurs during early somite formation (Hanel et al. 2009). Frg1
over-expression in Xenopus somites showed abnormal detachment of muscle cells from
myotome indicating enhanced delamination and cellular migration. Inverse observations were
made with frg1 knockdown in Xenopus somites. The reduction of Vimentin levels with frg1
knockdown in mesenchymal cells of somites noticeably indicated that altered frg1 levels could
lead to abnormal EMT(Hanel et al. 2009). Phenotype of Xenopus embryo with frg1 knockdown
was similar to phenotype with XHas2, a hyalouran biosynthesis enzyme, knockdown(Ori et al.
2006). This hints towards the critical role of frg1 is in maintaining extracellular matrix
components homeostasis (Ori et al. 2006). Additionally, the association of FRG1 with Dab2
strengthens the claims of involvement of FRG1 in MET/EMT process, as DAB2 is a known
regulator of EMT (Wuebbles et al. 2009).
The role of FRG1 was also explored in muscle development in vitro. Differentiation of
myoblasts to myotubes is critical event during muscle development. During differentiation of
primary myoblasts to myotubes, the subcellular localization of FRG1 was enriched at nucleus
compared to the cytoplasm. Further, its localization at Z disc of matured myotubes suggests a
significant role of FRG1 in muscle development in the mammalian system as it’s the first FSHD
candidate gene to be associated with muscle contractile machinery (Hanel et al. 2011).The
localization of FRG1 in nucleus during myoblasts differentiation is crucial as it might lead to
altered expression of various mis-spliced forms. One such protein being Tnnt3 whose misspliced form leads to defect in muscle contractility (Sancisi et al. 2014)
Role in F-actin bundling and mobility in C. elegans
Bioinformatics analysis of FRG1 conserved domains has revealed that it contains a single fascinlike domain (PF06220), which is a signature indicative of all actin-binding proteins (Liu et al.
2010). All fascin family of proteins actively participate in actin bundling by forming
homodimers and heterodimers, thus creating multiple actin-binding sites necessary for actin
bundling (Edwards & Bryan 1995; Kureishy et al. 2002). Immunostaining of C. elegans
endogenous FRG1 showed that it is present in both nucleus and cytoplasm. During larval stage
of C. elegans, FRG1 forms aggregation similar to dense bodies, which is revealed by a striated
and punctate immunostaining pattern. Dense bodies in C. elegans are homologous to vertebrate
focal adhesions which physically anchor the actin cytoskeleton of the muscle cell to the basal
lamina and hypodermis. These dense bodies orient in long rows along the muscle, appearing at
regular intervals alternating with the M-lines (Moerman & Williams 2006).This is further
corroborated by co-immunostaining of FRG1 with PAT3 (β-integrin), an extracellular matrix
receptor, concentrated in dense bodies and M-lines. From analysis of the whole genome for
similar fascin-like-domain containing proteins, it was discovered that FRG1 is the only such
proteins in C. elegans.
The high affinity of FRG1 for F-actin dense bodies and role of dense bodies in C. elegans
motility turns us towards role of FRG1 in C. elegans muscle regulation and motility. Indeed,
recently, a new quantitative assay of C. elegans locomotion with null mutants of different genes,
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has given quite an insight in role of FRG1 (Nahabedian et al. 2012).The FRG1 null mutants were
found to be defective in bending (Nahabedian et al. 2012).
Role in RNA biogenesis and mRNA processing
FRG1p is believed to be involved in RNA processing because of its co-localization in subnuclear structures involved in mRNA and rRNA biogenesis (Koningsbruggen et al. 2004).
Identification of FRG1 in the C-complex of human spliceosome (Rappsilber et al. 2002) and in a
cluster of co-regulated proteins in C. elegans, which is involved in ribosomal and mRNA
biogenesis (Kim et al. 2001) further corroborates its role in RNA processing. These findings
were further supported by the reports about the abnormal splicing of skeletal muscle troponin T
(Tnnt3) and myotubularin-related protein 1 (Mtmr1), in FRG1 transgenic mice. To further
elaborate the role of FRG1p in RNA biogenesis, many FRG1p-interacting proteins were
screened and assessed for their role in RNA processing. Co-immunostaining and Coimmunoprecipitation using VSV tagged FRG1 in U20S cells has revealed formation of
aggregates with PABPN1 and hyper-phosphorylated RNAP-IIO (Koningsbruggen et al. 2007).
PABPN1, which helps in polyadenylation of long non-coding RNAs, has previously been
reported to be present in C-spliceosome complex and RNAP-IIO has been established to be
associated with the elongation complex and assembly of early spliceosome complex. Yeast-two
hybridization screening followed by GST pull down has uncovered FAM71B as a FRG1
interacting protein, which is a conserved Cajal body and nuclear speckles protein
(Koningsbruggen et al. 2007).
Role in Vasculopathy and Angiogenesis
Since FSHD patients have been diagnosed with retinal detachment and deafness, they were
tested for any vascular abnormalities by fluorescein angiography (Padberg et al. 1995). The
findings led to hypothesize that retinal capillary abnormalities are an integral part of the FSH
muscular dystrophy syndrome. Expression profile of FSHD patients and healthy controls using
microarray has revealed 44 upregulated and 84 downregulated genes, of which only 34
upregulated genes were published by Osborne et al in 2007 (Osborne et al. 2007). Out of these
34 genes, 11 were reported to have role in vascular system and 8 of them have established role in
promoting endothelial growth and angiogenesis (e.g., CCN2, CCN3, CD44, ICAM1, melanoma
cell adhesion molecule, MAGP-2, phosphodiesterase 4B, Syndecan 2)(Osborne et al. 2007).
Role of FRG1 in vasculature was experimentally studied in X. laevis larvae. By expression
analysis of EGFP tagged frg1 transgenic larvae, it has been observed that frg1 co-localizes with
capillary marker lectin (Wuebbles et al. 2009), demonstrating its association with capillaries. To
affirm the role of FRG1 in capillaries, 2-cell stage X. larvae embryos were administered with
FRG1 specific morpholinos. The whole mount in situ hybridization of stage 35-36 embryos for
dab2, which encodes for a vascular marker and works as a angiogenic regulator, has shown that
the expression level of dab2 reduced in a dose dependent manner. This result was further
validated by in situ hybridization for msr, which encodes for an early vascular marker for which
similar observation were made (Wuebbles et al. 2009).
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The overexpression of FRG1 in X. laevis through systematic injection of X. tropicalis frg1
mRNA and analysis by in situ hybridization with dab2 and msr probes has revealed an increase
in size and branching of the vasculature structures (Wuebbles et al. 2009).
Bioinformatics analysis of FRG1
Bioinformatics analysis of FRG1 has revealed that it contains conserved nuclear localization
signal peptide, fascin like domain and a bipartite NLS peptide sequence. Though somewhat less
conserved, it also contains a lipocalin motif (Koningsbruggen et al. 2004; Liu et al. 2010; van
Deutekom et al. 1996). To get an insight into the probable distinctive function of FRG1, we
approached it bioinformatically and dominantly focused on its protein sequence and structure.
Firstly, we searched the protein database (PDB; http://www.rcsb.org/pdb/) (Berman et al. 2000),
and found a single NMR (Nuclear Magnetic Resonance) solution structure of mouse homologue
of frg1. Close analysis of the available structure through PyMOL (Chevalier & Stoddard 2001)
revealed that it mainly comprised of 14 beta sheets. These 14 β-sheets are present as pairs of
parallel and anti-parallel sheets of similar length, composing a central motif closing back on
itself similar to beta-barrel folds. A sequence homology analysis through BLAST (Basic Local
Alignment Tool) alignment (Altschul et al. 1990) shows that it belongs to RICIN superfamily of
proteins. This frg1 motif is structurally and sequentially similar to Ricin-type beta-trefoil which
is carbohydrate binding domain formed from presumed gene triplication. The domain is found in
a variety of molecules serving diverse functions such as enzymatic activity, inhibitory toxicity
and signal transduction. Highly specific ligand binding occurs on exposed surfaces of the
compact domain structure. As reported previously it contains a less conserved lipocalin sequence
(Koningsbruggen et al. 2004; Liu et al. 2010; van Deutekom et al. 1996). This motif is also
composed of a highly symmetrical all-β protein, dominated by a single eight-stranded
antiparallel β-sheet closed back on itself to form a continuously hydrogen-bonded β-barrel,
which in cross-section has a flattened or elliptical shape and encloses an internal ligand-binding
site. These proteins are known for their binding of an array of small hydrophobic ligands. The
structural features of the lipocalin fold, a large cup-shaped cavity within the β-barrel and a loop
scaffold at its entrance, are well adapted to the task of ligand binding. This is evident from the
amino acid composition of the pocket and loop scaffold, as well as its overall size and
conformation, determining selectivity. The similarity in both sequence and structure, leads us to
believe that it might belong to lipocalin family of proteins, but functional and biochemical
studies need to be done to assess the proper ligand binding specificity for this class of proteins.
Pairwise comparison and 3D alignment of frg1 (PDB ID: 2yug, (Saito et al. 2008)) by using
PDBeFold of EMBL-EBI (European Molecular Biology Laboratory-European Bioinformatics
Institute) (Arnould et al. 2011), resulted in many proteins above 90% sequence similarity. Out of
them, proteins like Fascin, Microlepiotaprocera Ricin-B like lectin (MPL), Agglutinin, human
FGF 18 are noteworthy. All of them form β-barrel folds closing back on itself with a cup shaped
cavity for ligand binding. Two of the identified proteins, i.e. Fascins and FGF18, are well known
molecules affecting muscle development and angiogenesis. Fascins, a group of actin bundling
protein are well known for their association in tumor progression and angiogenesis. FGF 18
belongs to the FGF family of proteins comprising similar beta trefoil structures as observed in
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FRG1. FGF family of proteins are associated with signal transduction via FGFR, leading to
signaling cascade crucial for skeletal muscle development, tumor progression, and angiogenesis
(Adams 2004; Cross & Claesson-Welsh 2001; Korc & Friesel 2009; Olwin et al. 1994). Thus,
the structural similarity of FRG1 with FGF18 may provide us insights regarding its role in
vasculopathy. This similarity suggests that it might act as a muscle specific growth factor or
possibly an angiogenic regulator previously supported by experimental data.
Current status
At present, research done on FRG1 gives quite an insight on its major functions and molecular
machinery behind it, yet insufficient to draw a proper conclusion. As discussed above, it has
been primarily associated with FSHD and regulated by DUX4 transcription factor. Some reports
have also suggested its role in early muscle development, by regulating the expression levels of
MyoD and Pax3. There are also reports showing evidences of its role in RNA biogenesis. Some
studies support that FRG1 takes part in regulating different factors, those in turn control the
vasculogenesis and angiogenesis. Considering all these aspects, discovered till date, it seems that
FRG1 is a multifunctional protein participating in many pathways in different stages through
several interacting partners. It is highly likely that in initial stages, FRG1 regulates the mRNA
biogenesis of the genes which in turn regulate the muscle development and, in later stages, it
helps in maintenance of muscle cells by stabilizing the F-actins polymerization (Ferri et al. 2015;
Gabellini et al. 2006; Gabellini et al. 2002; Hanel et al. 2009; Koningsbruggen et al. 2007;
Lecroisey et al. 2007; Liu et al. 2010; Nahabedian et al. 2012). In endothelial cells, it may
cause/modulate the transcription of some critical angiogenic/vasculogenic genes, which is
evident from vasculopathy in most cases of FSHD patients, and other studies in model systems
(Koningsbruggen et al. 2004; Osborne et al. 2007; Padberg et al. 1995; Wuebbles et al. 2009).
Lacunae
Till date a lot of information regarding various roles of FRG1 in several fields have been
portrayed, still many voids are yet to be filled. The proper molecular machinery of action of
FRG1 whether in development or in actin bundling, still needs to be looked into. The presence of
N-terminal lipocalin motif is another intriguing fact. Lipocalins are typically small secreted
proteins which bind to hydrophobic molecules, specific cell-surface receptors and have been
associated with transportation of small molecules such as lipids, steroids, retinols. These
molecules also have roles in cryptic coloration, olfaction, pheromone transport, the enzymatic
synthesis of prostaglandins, in the regulation of the immune response and the mediation of cell
homoeostasis (Flower 1996). Does this mean that FRG1 also have roles in cell homeostasis and
transportation of biomolecules? The over expression of angiogenic regulatory genes in FSHD
patients is due to overexpression of FRG1 or the mis-regulation of FRG1 regulatory elements
leading to overexpression of these genes, is another matter that needs to be investigated. Even
though FRG1 is associated with mis-regulation of many genes, its role in cancer biology is not
yet studied. Keeping all the evidences and research done till date we have proposed a
hypothetical model for FRG1 (Figure 2), which also shows the gaps in research.
Conclusion
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We note that the reported roles of FRG1 in both muscle development and F-actin bundling is
very fascinating. It might be possible that FRG1 regulates the development of muscle in
embryonic stages by both regulating the expression level of many muscle growth transcription
factors and by regulating the formation of F-actin filaments towards major growth hotspots. It is
also possible that it helps in transportation of small biomolecules through actin filaments during
muscle development. Overexpression of angiogenic genes and presence of vascular abnormities
in FSHD patients indicate towards the role of FRG1 in angiogenesis. FRG1 might be a major
regulator of angiogenesis by governing the direction of growing blood vessels through F-actin
bundling. At present FRG1 research is not adequate to draw any concluding statements regarding
its function. Hence, further research needs to be done to unravel its mystery.
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Figure 1: Schematic representation for FRG1gene, mRNA and protein (a) shows distribution of
promoter, regulatory elements, exons and introns in FRG1 gene. Numbers show distance in base
pair (b) shows mature transcript of FRG1, it’s length and UTRs (c) shows different domains in
FRG1 protein. aa depicts the amino acid positions of domains.
Figure 2: Hypothetical model showing the biological processes affected by FRG1. Model depicts
that DUX4 regulates FRG1 expression. In turn FRG1 interacts with various proteins associated
with spliceosome complex and regulates splicing of several early muscle development related
genes. FRG1 also interacts with myoD and Pax3, indirectly affecting the expression of muscle
development related genes. FRG1’s interaction with F-actin and PAT3, associates it with muscle
physiology via actin metabolism. FRG1 also affects small hydrophobic molecular transport and
angiogenesis associated genes. Question marks show the gaps in the information.
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