FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells.

Abstract

Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line.
One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members.
The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.

Full text

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BioMed Central
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BMC Genomics
Open Access
Research article
FAM20: an evolutionarily conserved family of secreted proteins
expressed in hematopoietic cells
Demet Nalbant1, Hyewon Youn1,3, S Isil Nalbant1, Savitha Sharma1,
Everardo Cobos2,3, Elmus G Beale1, Yang Du1 and Simon C Williams*1,3
Address: 1Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA, 2Department
of Internal Medicine, Texas Tech University Health Sciences Center, Lubbock, Texas 79430, USA and 3Southwest Cancer Center at University
Medical Center, Lubbock, Texas 79430, USA
Email: Demet Nalbant - demet-nalbant@uiowa.edu; Hyewon Youn - youn.hyewon@ttuhsc.edu; S Isil Nalbant - sisil@su.sabanciuniv.edu;
Savitha Sharma - savitha.sharma@ttuhsc.edu; Everardo Cobos - everardo.cobos@ttuhsc.edu; Elmus G Beale - elmus.beale@ttuhsc.edu;
Yang Du - cbbyd@yahoo.com; Simon C Williams* - simon.williams@ttuhsc.edu
* Corresponding author
Abstract
Background: Hematopoiesis is a complex developmental process controlled by a large number of factors
that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the
hematopoietic growth factors, play critical roles in these processes and have important biological and
clinical significance. We have employed representational difference analysis to identify genes that are
differentially expressed during experimentally induced myeloid differentiation in the murine EML
hematopoietic stem cell line.
Results: One identified clone encoded a previously unidentified protein of 541 amino acids that contains
an amino terminal signal sequence but no other characterized domains. This protein is a member of family
of related proteins that has been named family with sequence similarity 20 (FAM20) with three members
(FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a
single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis
elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20
family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the
zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and
found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal
sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during
hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also
expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A
was expressed in more limited set of human tissues than the other two family members.
Conclusions: The FAM20 family represents a new family of secreted proteins with potential functions in
regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only
expressed during early stages of hematopoietic development and may play a role in lineage commitment
or proliferation. The expansion in gene number in different species suggests that the family has evolved as
a result of several gene duplication events that have occurred in both vertebrates and invertebrates.
Published: 27 January 2005
BMC Genomics 2005, 6:11doi:10.1186/1471-2164-6-11
Received: 19 August 2004
Accepted: 27 January 2005
This article is available from: http://www.biomedcentral.com/1471-2164/6/11
© 2005 Nalbant et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Background
Hematopoietic differentiation is a complex process
whereby multiple functionally and morphologically dis-
tinct cell types arise from a population of pluripotent
hematopoietic stem cells (PHSCs) [1]. The accurate and
efficient regulation of hematopoietic development is con-
trolled by a large number of regulatory proteins that have
been identified over the past few decades. These regula-
tory molecules include the hematopoietic growth factors
(HGFs), soluble proteins that recognize specific receptors
on the surface of sub-populations of hematopoietic cells,
thereby initiating signal transduction pathways that mod-
ulate the differentiation, proliferation, and/or survival of
target cells [2]. The identification of regulators of hemat-
opoiesis has been an ongoing effort for many years and
has benefited from the existence of accessible cell line
models as well as the characterization of genes affected by
somatic mutations associated with specific human leuke-
mias [3].
We have used a pair of factor-dependent murine cell lines
to identify novel genes expressed within distinct hemat-
opoietic lineages as an approach to the identification of
novel candidate genes for development of diagnostic and
therapeutic approaches to leukemia. The EML and MPRO
cell lines were both established by infecting murine bone
marrow cells with a retrovirus expressing a dominant neg-
ative retinoic acid receptor α (RARα) protein [4,5]. The
infected cells were selected in the presence of either stem
cell factor (SCF) or granulocyte/macrophage colony stim-
ulating factor (GM-CSF). EML are SCF-dependent and
resemble uncommitted hematopoietic progenitor cells.
They can be induced to differentiate to the promyelocyte
stage of granulopoiesis in the presence of interleukin-3
(IL-3) and high doses of all trans retinoic acid (atRA)
[4,6]. Terminal neutrophil differentiation of EML cells can
be induced by replacement of IL-3 and SCF with GM-CSF.
MPRO cells are GM-CSF-dependent and can be induced to
differentiate to neutrophils by adding high doses of atRA
to the culture medium. The expression patterns of a
number of genes expressed during hematopoiesis have
been examined in EML and MPRO cells and generally
agree with the patterns observed in other cell systems and
in primary hematopoietic cells. Thus, EML and MPRO
provide a powerful system for the identification and char-
acterization of novel genes expressed within the hemat-
opoietic lineage.
We have employed the representation difference analysis
technique [7] to identify cDNAs representing genes
expressed at higher levels in EML cells 72 hours after
induction of differentiation than in uninduced cells. We
describe the identification of a clone derived from an
uncharacterized putative secreted protein. We have per-
formed a comparative genomics analysis and determined
that this protein is the founding member of an extended
family of highly related proteins. This family contains
three members in mammalian species, one or two mem-
bers in invertebrate or simple vertebrate species and five
or six members in fish. We have determined that one fam-
ily member is a secreted glycoprotein and describe the
expression pattern of the human and mouse genes in tis-
sues and during hematopoietic differentiation.
Results
Identification of differentially expressed genes by
representational difference analysis (RDA)
Total RNA was prepared from EML cells grown in the pres-
ence of SCF alone (0 hour) or in medium supplemented
with IL-3 and atRA for 72 hours. The RNA was converted
to cDNA and subjected to three rounds of RDA as previ-
ously described [6]. Six differentially-expressed clones
were identified [6,8]. Clone number 1623 was chosen for
further analysis and the differential expression of this gene
was confirmed by Northern blot analysis. 1623 mRNA
was essentially undetectable in the 0 hour sample but
readily detectable in the 72 hour sample (data not shown
and see figure 10).
Sequence analysis of clone 1623
The initial cDNA isolated by RDA was a 273 bp fragment
that appeared to contain the coding sequence of the C-ter-
minus of a protein that was not present at that time in
public databases. To identify the full open reading frame
of this cDNA, we first performed rapid amplification of
cDNA ends (RACE) in both the 5' and 3' directions. Exten-
sion in the 3' direction revealed the presence of a consen-
sus polyadenylation signal located 154 nucleotides
downstream of the putative translation stop codon. Exten-
sion in the 5' direction yielded an additional 443 bp of
sequence containing a contiguous ORF. Comparison of
this extended sequence to public databases identified a
cDNA (NM_017565) that was identical to clone 1623 in
the region of overlap. This cDNA was isolated from a
mouse mammary tumor but no functional analysis had
been performed. We designed PCR primers based on the
published sequence and confirmed that the cDNA iso-
lated from EML cells was identical to the published
sequence. The full length ORF was 1623 bp in length and
encoded a protein of 541 amino acids (Figure 1A). The
protein did not contain any recognizable motifs when
examined using domain mapping software such as
SMART or Profilescan (see Methods). However, a putative
amino terminal signal sequence was identified using the
SignalP analysis program. The cDNA mapped to an 11
exon gene located on mouse chromosome 11E1 (Figure
1B). The gene spanned approximately 60,000 bp of
genomic sequence and the relatively large size of the gene
is primarily due to the fact that the first intron is greater
than 44,000 bp in length (Figure 1C).

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Characterization of the full length mouse 1623 (Fam20a) cDNA and genomic sequence
Figure 1
Characterization of the full length mouse 1623 (Fam20a) cDNA and genomic sequence. A. The full length cDNA
derived from the original RDA clone was isolated using a combination of 5' and 3' rapid amplification of cDNA ends (RACE)
procedures, comparisons to public databases, and amplification of putative full length clones by PCR. The full open reading
frame was 1623 bp in length and encoded a 541 amino acid protein. The locations of regions conserved within the subse-
quently identified FAM20 family are indicated using underlines. Eight cysteine residues that are also conserved within the family
are indicated in bold and four putative N-glycosylation sites are indicated in red type. B. The distribution of the 11 exons of the
mouse Fam20a gene is shown with the exons indicated using numbers. A consensus polyadenylation signal is located down-
stream of the terminal exon. C. The sizes of the 11 exons and 10 introns of the Fam20a gene are shown.
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Identification of a family of related genes
The full length cDNA and the encoded protein were com-
pared to sequences in public databases. We reported pre-
viously a weak similarity to a protein named Fjx1, which
is the mouse orthologue of a Drosophila protein named
four-jointed [8]. However, the degree of sequence identity
between these proteins was low (16%) and thus the
search was extended to include uncharacterized proteins.
We first identified two other mouse proteins that dis-
played significant similarity to, but were distinct from, the
query sequence. One protein (Accession number
NP_663388 aka Riken C530043G21) was 409 amino
acids in length and displayed 27% identity to the query
sequence while the second (NP_085042) was a truncated
version of a 579 amino acid protein that displayed 40%
identity to the query sequence. We have subsequently dis-
covered that these proteins are members of a highly
related family and this family has received the official
name "family with sequence similarity 20" (FAM20) from
the Human Genome Organization Gene Nomenclature
Committee. The protein derived from our original 1623
cDNA is named Fam20a in mouse and the other two fam-
ily members are named Fam20b and Fam20c, respec-
tively. Continued searches of public databases revealed
the existence of related proteins in several other species.
Each mammalian genome contains genes encoding three
members that are orthologous to the three mouse pro-
teins mentioned above. The accession numbers for the rel-
evant cDNAs in human and rat are listed in Table 1 and
we have also identified the same number of related
sequences in other mammalian genomes, including the
pig, cow and dog (data not shown). However, most of
these sequences are incomplete and will not be described
in detail here.
In order to gain further information concerning the origin
of the FAM20 family, we searched for related sequences in
several other invertebrate and vertebrate organisms. The
ascidian Ciona intestinalis is a model for a basal chordate
organism and has emerged as a powerful model for evolu-
tionary and developmental studies [9,10]. In particular,
many gene families or subfamilies are represented by sin-
gle members in C. intestinalis and thus the identification
of an orthologue in this organism can provide useful
information for evaluating the evolutionary origin of the
members of a gene family. Consistent with this concept,
we identified a single cDNA and the corresponding
genomic locus in C. intestinalis that displayed significant
sequence similarity to the mammalian FAM20 genes and
proteins (Table 1). Complete genome sequences are also
available for several invertebrate species and two related
sequences were identified in Drosophila melanogaster and
Anopheles gambiae with one family member in Caenorhab-
ditis elegans (Table 2). Finally, analysis of genomic cDNA
and protein databases for the pufferfish (Fugu rubripes)
and zebrafish (Danio rerio) revealed the presence of six
and five family representatives, respectively (Table 1). The
gene numbers in these various species are listed on an ide-
alized evolutionary tree in Figure 2 and suggest that the
FAM20 gene family has undergone a complex set of gene
duplications in both the invertebrate and chordate
lineages.
Assignment of subfamily relationships
To elucidate the nature of these putative gene duplica-
tions, we sought to assign the individual sequences from
Table 1: Accession numbers for vertebrate FAM20 family members1
Family
Member
HumanMouse RatFugu ZebrafishCiona
FAM20A
FAM20B
FAM20C
NM_017565
NM_014864
NM_020223
NM_153782
NM_145413
NM_030565
XM_221067 (BK001521)
XM_222770
XM_221975 (BK001522)
SINFRUP00000146987 (BK001515)
SINFRUP00000138548 (BK001520)
SINFRUP00000140879 (BK001516)
SINFRUP00000141732 (BK001518)
SINFRUP00000163431 (BK001517)
BK001519
ND
CAI11712
ENSDARP00000009272
ENSDARP00000005688
ENSDARP00000028589
ND
AK115425
ND
1. Accession numbers refer to nucleotide sequences in GenBank except in the Fugu and Zebrafish listing where the accession numbers refer either
to predicted peptides in the Ensembl database (entries beginning SINFRUP or ENSDARP) or Genbank (entry beginning CAI).
Entries beginning with BK refer to predicted sequences in the Third Party Annotation database arising from this study. ND: None detected.
Table 2: Accession numbers for invertebrate FAM20 family
members1
Family Member DrosophilaMosquito C. elegans
FAM20A
FAM20B
ND
NM_170079
NM_206490
ND
ND
EAA08010
EAA13434
ND
ND
NM_078126
Fam20CND
1. Accession numbers refer to nucleotide sequences in GenBank. ND:
None detected.

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the various species into subfamilies based on protein
sequence and gene structure, specifically using the
number and size of exons in the latter case. Initially, the
exon distribution of each of the Fam20 members in three
mammalian species (human, mouse and rat) was com-
pared and revealed obvious inter and intra orthologue
similarities (figure 3A). Each FAM20A gene contained 11
exons and exon sizes were identical in these three species.
Likewise, each FAM20B gene contained 7 exons that were
identical in size in human, mouse and rat. The FAM20C
genes each contained 10 exons and only exon 1 displayed
any variation in size amongst these three species. In intra-
orthologue comparisons, the exons in FAM20B and
FAM20C genes clearly aligned with exons in the FAM20A
genes, with small variations (in multiples of three bases)
in the size of the internal exons in FAM20B. FAM20B lacks
exons corresponding to exons 2–4 of FAM20A while
FAM20C lacks exon 3. In addition, exons 8 and 9 in
FAM20A and FAM20C are represented by a single exon in
FAM20B that is identical in size to the combined exons in
the other two genes. Thus, the three mammalian genes are
highly evolutionarily related and presumably are derived
from a common ancestral gene.
To assign the genes identified in other species to these
three subfamilies, we performed a global comparison of
the peptide sequences derived from 25 of the identified
family members listed in Tables 1 and 2. One zebrafish
protein (from FAM20A) was omitted as its sequence is
incomplete. A dendrogram showing the results of this
Evolutionary distribution of FAM20 gene number
Figure 2
Evolutionary distribution of FAM20 gene number. An idealized evolutionary tree (modified from [10]) is shown with
the number of FAM20 genes identified in several genomes as described in the text. The gene numbers are supportive of a single
gene duplication event occurring in invertebrates (at least in insects) and multiple gene duplication events occurring in higher
vertebrates.
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