Genes Genet. Syst. (2021) 96, p. 1–11 Roles of DSCR4 gene in human cell migration

1

Possible roles for the hominoid-specific DSCR4 genein human cells
Morteza M. Saber1,2,3,4, Marziyeh Karimiavargani3,5, Takanori Uzawa3,
Nilmini Hettiarachchi1, Michiaki Hamada4,6,
Yoshihiro Ito3 and Naruya Saitou1,2,7,8*
1

Population Genetics Laboratory, National Institute of Genetics, Mishima, Shizuoka 411-8540, Japan2
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Bunkyo-ku,
Tokyo 113-0033, Japan
3
Nano Medical Engineering Laboratory, RIKEN, Wako, Saitama 351-0198, Japan
4
Department of Electrical Engineering and Bioscience, Faculty of Science andEngineering, Waseda University, Shinjuku-ku, Tokyo 169-8555, Japan
5
Graduate School of Science and Engineering, Saitama University,
Saitama, Saitama 388-0825, Japan
6
Computational Bio Big-Data Open Innovation Laboratory (CBBD-OIL),
National Institute of Advanced Industrial Science and Technology(AIST), Shinjuku-ku, Tokyo 169-8555, Japan
7
Department of Genetics, School of Life Science, Graduate University for
Advanced Studies, Mishima, Shizuoka 411-8540, Japan
8
Faculty of Medicine, University of the Ryukyus, Nishihara-cho,
Okinawa 903-0215, Japan(Received 24 February 2020, accepted 28 August 2020; J-STAGE Advance published date: 24 March 2021)

Down syndrome in humans is caused by trisomy of chromosome 21. DSCR4(Down syndrome critical region 4) is a de novo-originated protein-coding genepresent only in human chromosome 21 and its homologous chromosomes in
apes. Despite being located in a medically critical genomic region and an abundance of evidence indicating its functionality, the roles of DSCR4 in human cells
are unknown. We used a bioinformatic approach to infer the biological importance and cellular roles of this gene. Our analysis indicates that DSCR4 is likely
involved in the regulation of interconnected biological pathways related to cell
migration, coagulation and the immune system. We also showed that these predicted biological functions are consistent with tissue-specific expression of DSCR4
in migratory immune system leukocyte cells and neural crest cells (NCCs) thatshape facial morphology in the human embryo. The immune system and NCCs
are known to be affected in Down syndrome individuals, who suffer from DSCR4
misregulation, which further supports our findings. Providing evidence for thecritical roles of DSCR4 in human cells, our findings establish the basis for further
experimental investigations that will be necessary to confirm the roles of DSCR4
in the etiology of Down syndrome.
Key words:
tion

Down syndrome, DSCR4, orphan gene, cell migration, human evolu-

INTRODUCTION
Down syndrome is a human genetic disease, with thehigh incidence of 1 out of 700 live births making thisdisorder the leading genetic cause of mental retardaEdited by Yoko Satta* Corresponding author. saitounr@nig.ac.jp
DOI: https://doi.org/10.1266/ggs.20-00012

tion and congenital heart disease (Hassold and Jacobs,
1984). The major phenotypes characterizing Down
syndrome are variable in age of onset, frequency andseverity, and include immune deficiency, heart disease,dysmorphology of facial characters and underlying skeleton, Hirschsprung’s disease, alterations of brain structure, early onset of Alzheimer’s pathology and increased
risk of leukemia. Mental retardation, characterized bycertain behavioral and cognitive deficits, is also a com-

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M. M. SABER et al.

mon feature in Down syndrome individuals (Cohen, 2002;
Epstein, 2002; Wiseman et al., 2009; Lana-Elola et al.,2011).
Down syndrome is caused by the inheritance of an
extra copy of chromosome 21q. Since the completion of
the human genome project, it is known that this regionspans ~33.5 million base pairs (Mb) of DNA and contains~300 genes (Hattori et al., 2000). So far, mouse transgenic models have been the main tool for the analysis ofgene–phenotype correlation in Down syndrome (Kahlem
et al., 2004; Lyle et al., 2004). Studies of trisomic mousemodels containing an extra copy of the mouse genome
segment that is orthologous to the q arm of chromosome
21 imply that all trisomic genes are up-regulated by ~50%
across multiple tissues (Kahlem et al., 2004; Lyle et al.,2004). Although the underlying mechanism accounting
for how the relatively small increment in transcription
results in any of the commonly observed phenotypes in
Down syndrome is yet unknown, the correspondence ofthe shortest genomic region shared by Down syndrome
individuals with the same Down syndrome characteristics has led to the hypothesis that a critical chromosomal region called Down syndrome critical region(DSCR) contains a dosage-sensitive gene or set of genes
whose misregulation is responsible for the emergence of
Down syndrome features (Jiang et al., 2015). The previously defined DSCR spans ~5.4 Mb ranging from a
proximal boundary between DS21S17 and D21S55 to a
distal boundary between MX1 and BCEI (Korbel et al.,2009). This region harbors about 33 genes that are conserved between human and mouse (Nikolaienko et al.,2005) and has been associated with multiple Down syndrome characteristics such as craniofacial abnormalities,
joint hyperlaxity and mental retardation (Lana-Elola etal., 2011). Another study of partial trisomy 21 further
defined a 34-kb region as critical to the Down syndrome
phenotype (Pelleri et al., 2016).
Down syndrome studies using multiple transgenic
mouse models in which the DSCR-orthologous region isoverexpressed or underexpressed, in particular chromosome 16 segmental trisomies such as Ts1Cje, Ts65Dn and
Ts1Rhr and segmental monosomies including Ms1Rhr,
have so far provided valuable information in constructing
the phenotypic map of DSCR (Olson et al., 2007). However, investigations on functional annotation of orthologous genes located in the DSCR revealed that, first, themouse orthologous genes are actually dispersed within
the genome and do not have the same synteny as their
human counterparts (Gardiner et al., 2003), and, second,
not all the genes present in the human DSCR region
have orthologs in mouse, which illustrates the inability ofmouse models of Down syndrome to accurately simulate
the human Down syndrome condition (Saber et al., 2016).
Down syndrome critical region 4 (DSCR4) is a novel denovo-originated protein-coding gene in the DSCR region,and is present only in humans and apes (superfamily
Hominoidea), without a known ortholog in non-hominoidorganisms (Fig. 1) (Kumar et al., 2017). This gene was
first discovered during the human chromosome 21 genome
sequencing effort, and was found to have no homolog inthe mouse genome (Saber et al., 2016). DSCR4, previously called DSCRB (Nakamura et al., 1997), shares abidirectional promoter with the DSCR8 gene and is positively and negatively regulated by multiple transcription
factors (Toyoda et al., 2002; Dunn et al., 2006; Asai etal., 2008). DSCR4 encodes an experimentally verified
protein with 118 amino acids (Uhlén et al., 2015). The
amino acid sequence of DSCR4 can potentially form protein secondary structure; however, this protein shows nodiscernible homology to any known or putative protein
domains (Saber et al., 2016). This lack of homology isprobably due to the de novo origin of DSCR4 and to thefact that two of the three coding exons of this gene arederived from retrotransposons (Saber et al., 2016). A
detailed discussion of the transposon-derived origin of
DSCR4 exons can be found in Saber et al. (2016). Hypomethylation of the DSCR4 promoter has been shown
to be an epigenetic marker for Down syndrome (Du etal., 2011), which is another confounding factor in using
mouse models to simulate the Down syndrome conditionin humans. However, no study has so far investigated
the functionality or subcellular interactions of DSCR4
inside human cells.
In this study, to systematically interrogate the cellularroles and gene regulatory networks involving DSCR4, we
overexpressed wild-type DSCR4 in human non-cancerous
cells and measured the consequences on the cell transcriptome by differential gene expression analysis. It

Fig. 1. De novo evolution of DSCR4 and its surrounding
genomic region. Schematic depiction of the evolution of DSCR4
in common ancestors of hominoids (human, chimpanzee, gorilla,orangutan and gibbons). Evolution of DSCR8 and the bidirectional promoter that drives the expression of both genes are also
depicted. Divergence time estimates were taken from Kumar etal. (2017). mya, million years ago.

Roles of DSCR4 gene in human cell migration
has been shown that chromosome 21 transcripts are
increased in proportion to gene dosage, i.e., 50% moreabundant than normal in Down syndrome cells (Kahlem
et al., 2004). Therefore, overexpression of DSCR4 is the
optimal approach to simulate the Down syndrome condition. Through functional profiling, we investigated thebiological processes affected by overexpression of DSCR4
to unravel the likely role of DSCR4 in cellular pathways and its contribution to the etiology of Down syndrome, along with how this gene might contribute to theunique characteristics displayed by the family Hominidae(Saber et al., 2016). Our analysis provides evidence forroles of DSCR4 in the regulation of cell migration andthe immune system, which are also biological pathways
affected in Down syndrome individuals.
MATERIALS AND METHODS
Cell culture The human papillomavirus 16 E6/E7-transformed cell line HS-27A, an immortalized noncancerous cell type with epithelial morphology, waspurchased from ATCC (CRL-2496) and cultured in RPMI1640 medium (Invitrogen) supplemented with 10% fetal
bovine serum (FBS) (Invitrogen) according to ATCC protocols.
Plasmid DNA vector and control vector construction The PTCN-DSCR4 expression vector (BC096162),
which contains the full-length cDNA sequence of DSCR4
including upstream and downstream untranslated
regions (UTRs), was purchased from transOMIC technologies (Supplementary Fig. S1A). No marker wasadded to DSCR4 cDNA, to ensure that the function of theextra copy of DSCR4 was not affected. Cytomegalovirusderived promoter and enhancer sequences placed before
DSCR4 cDNA in the vector ensure proper expression in
eukaryotic cells. For production of a control plasmid(PTCN-control), we removed the DSCR4 cDNA sequence
along with UTR elements from PTCN-DSCR4 using a
GeneArt Seamless Cloning and Assembly kit (Supplementary Fig. S1B). With DSCR4 cDNA being the only
difference between PTCN-DSCR4 and PTCN-control, wesought to minimize the confounding elements in our differential gene expression analysis.
Kill curve assay The first critical step for generating
stably transfected cells is determining the optimal concentration of the selection reagent for selecting stable cellcolonies. The purpose of the kill curve assay is to determine the minimum antibiotic concentration needed to kill
all the cells over the course of one week. Since the optimal concentration is cell type-dependent, we performed
this assay for HS-27A cells using the Cell Counting Kit-8(Sigma). The HS-27A cells were treated with G418
(Geneticin; Sigma) in a concentration gradient between 0

3(negative control) and 1,500 μg/ml. The number of cells
in each well was counted after a week and the optimum
concentration of G418 for treatment of HS-27A cells wasdetermined as 1,400 μg/ml.
DNA transfection and stable cell line selection For
transfection of HS-27A cells with PTCN-DSCR4 plasmid,
first, the optimal concentration of OMNIfect transfection
reagent was determined using a concentration gradientof OMNIfect and a pcDNA3 vector containing the GFPmarker. In the next step, using the optimal concentration of OMNIfect (2 μl/ml), HS-27A cells were transfectedwith the PTCN-DSCR4 and PTCN-control vectors. Single cell colonies successfully transfected with DSCR4
were selected by incubation with RPMI-1640 containing
10% FBS and 1,400 μg/ml G418 over approximately 21
days.
Gene expression analysis using microarray Total
RNA was extracted from three PTCN-DSCR4-transfected
and three PTCN-control-transfected HS-27A samples
along with one normal HS-27A sample using a PureLink
RNA Mini Kit (Invitrogen), and RNA quality was assessedand confirmed using an Agilent 2100 Bioanalyzer (Agilent
Technologies). The isolated RNA was then carriedthrough the Agilent preparation protocol and each sample was hybridized to one SurePrint G3 Human 8 × 60K
v3 GeneChip (Agilent Technologies). Raw data were processed and analyzed using GeneSpring GX software alongwith the RobiNA package (Lohse et al., 2012). Quality
assessment was conducted by normalized expression
value correlation analysis between the three sample
groups (Supplementary Fig. S2). Normalized expression
values (log base 2) for each chip were calculated using
quantile normalization after background correction by
the RMA method (Irizarry et al., 2003), which has been
proven to measure expression levels reliably. The gene
expression matrix was filtered to exclude probe sets thatare either not positive or significant, not uniform, notabove background or population outliers. Finally, saturated probe sets defined as “Class P” by GeneSpring GX
software in PTCN-DSCR4-transfected HS-27A samples
with at least a two-fold change in expression comparedwith PTCN-control-transfected HS-27A samples were
determined and used for further gene set enrichment
analysis. False discovery rate in all analysis was controlled by applying a Bonferroni adjustment.
Confirmation of DSCR4 gene perturbation using
qPCR Quantitative real-time PCR (qPCR) was conducted by synthesizing cDNA from 1 mg DNA-free RNA
using random hexamer primers and a PrimeScript First
Strand cDNA Synthesis kit (Takara). qPCR was performed using the CFX96 Touch Real-Time PCR Detection System. Expression of DSCR4 and the GAPDH

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M. M. SABER et al.

housekeeping gene was measured using a SYBR Greenbased gene expression assay (Applied Biosystems). The
primers for qPCR analysis were designed to amplify
intron–exon boundaries so as to avoid amplification of
any potential DNA contamination. The GAPDH housekeeping gene was used as an endogenous control fornormalization. As expected, no significant difference
between the cycle threshold (Ct) values was observedfor GAPDH across all the samples. Melt curve analysis also confirmed strictly specific amplifications in qPCR
assays. The comparative Ct approach was employedwith a CFX96 Touch Real-Time PCR machine to quantify DSCR4 expression levels, and confirmed that DSCR4
is overexpressed approximately 9-fold in PTCN-DSCR4transfected cells compared with PTCN-control-transfected cells (Supplementary Fig. S3).
Functional profiling Differentially expressed probeset IDs in PTCN-DSCR4-transfected HS-27A samples
were mapped to ENTREZ IDs and Kyoto Encyclopediaof Genes and Genomes (KEGG) IDs, and successfully
mapped IDs were submitted to ClusterProfiler software(Yu et al., 2012). Gene Ontology (GO) terms and Gene
Regulatory Networks (GRNs) that were significantly overrepresented in the set of differentially expressed genes(DEGs) were identified using ClusterProfiler. Up- anddown-regulated gene sets were analyzed independently
using SurePrint G3 Human 8 × 60K v3 gene sets as areference. GO terms and GRNs with significant enrichment (false discovery rate (FDR) < 0.05) were consideredin the analysis. Biological processes enrichment analysis was conducted using data from the Reactome curated
pathway database, and pathway enrichment analysis was
performed by an enrichment test in the set of manuallydrawn pathway maps provided by KEGG (Kanehisa etal., 2016). FDR in all analysis was controlled by applying a Bonferroni adjustment.
To investigate the hypothesis that the DSCR4 gene hasa unique expression pattern in human tissues and cells,
RNA-Seq data of human tissues from the Roadmap Epigenome project (Roadmap Epigenomics Consortium et al.,2015) were retrieved and analyzed. The log-transformed
average RPKM (reads per kilobase per million mapped
reads) expression scores for the DSCR4 gene and the adjacent DSCR8 gene were calculated across human tissues
along with cell lines for which genome-wide gene expression data were available. The YuGene tool was used toscale the gene expression data derived from different
platforms for the purpose of performing integrated analyses (Lê Cao et al., 2014). This application also provides
a normalized gene expression database using available
microarray data across human cells. Using the provided
database, YuGene was also employed for further evaluation of tissue-specific expression of the DSCR4 gene.

RESULTS
Computational analysis There is considerable biological evidence indicating functionality of the DSCR4
gene such as its possessing epigenetic marks for an active
regulatory region within the promoter, and potential secondary structure in the encoded protein, as well as its
harboring a fetal epigenetic marker for Down syndrome
and being the binding site for multiple transcription factors. To verify the functionality of DSCR4 from an evolutionary point of view, we first performed derived allele
frequency (DAF) analysis using 1000 Genomes Project
data (The 1000 Genomes Project Consortium, 2015),
which revealed that DSCR4 protein-coding sequences
have higher rates of polymorphisms with low-frequency
derived alleles compared with DSCR4 intronic sequences(Fig. 2A).
Intrinsic structural disorder (ISD) is the degree towhich a given peptide folds as a stable three-dimensional
protein, i.e., an ordered protein, versus a flexible andunstructured (disordered) entity. Natural protein
sequences are more intrinsically disordered than translated random sequences (Yu et al., 2016; Wilson et al.,2017). Therefore, ISD can be used as a criterion to distinguish functional genes from random ORFs erroneously
identified as novel genes. ISD analysis of DSCR4 using
IUPred (Dosztányi et al., 2005) revealed that the protein
encoded by this gene has a higher degree of ISD comparedwith its proximate but older protein-coding gene, DSCR8,
and with the conserved housekeeping genes GAPDH and
ACTB (Fig. 2B). Translated DSCR4 introns, on the other
hand, have very low ISD scores.
Differential gene expression analysis We performedexperimental gene perturbation analysis for in vivo interrogation of DSCR4 functionality. To enhance the reliability of the analysis, data cleaning was performed bydiscarding DEGs with unsaturated probe signals, whichmight represent false positives. We found that a total of
253 probes targeting protein-coding genes among 36,427
probes for protein-coding genes ( < 0.5%) were differentially expressed between DSCR4-overexpressing HS-27A
cells and control samples (Supplementary Fig. S4). Of
these 253 DEGs, which were used for further investigations, 131 represent down-regulated and 122 represent
up-regulated protein-coding genes (see Supplementary
Table S1).
Predicting DSCR4 functions by Gene Ontology
analysis After identification of DSCR4 overexpressionmediated DEGs, we performed GO analysis to identify
interconnected cellular pathways that were enriched in
the DEGs. Functional annotation tools were used toarrange genes in associated categories based on associated GO terms and participation in biological pathways.

Roles of DSCR4 gene in human cell migration5

the six enriched pathways are directly involved in theregulation of movement, migration and motility of cells
or cell compartments. On the other hand, the genes upregulated by DSCR4 overexpression are enriched mainlyin the regulation of coagulation and hemostasis processes
along with maintenance of apical/basal cell polarity (Figs.
3C and 3D).
If these predictions are correct, we would expect DSCR4
to be expressed mainly in cells with migratory characteristics. We investigated this hypothesis by quantifying
the expression of DSCR4 across all human cell transcriptome data available in the Roadmap Epigenomics Project (Roadmap Epigenomics Consortium et al., 2015) and
YuGene database (Lê Cao et al., 2014). Only a small
portion of highly specialized cells actively migrate within
the human body, including leukocytes, stem cells andimmune system cells (Entschladen et al., 2005). In linewith our predictions, Roadmap data analysis revealed
that DSCR4 has significant expression only in K562 cells(Fig. 4). K562 is a human immortalized myelogenous
leukemia cell line with erythroleukemia type. Consistent with this finding, analysis of the YuGene databasealso revealed that monocytes, which are a type of leukocyte, display the highest expression level of DSCR4(Supplementary Fig. S5).

Fig. 2. Functional analysis of DSCR4. (A) Derived allele frequency (DAF) analysis of DSCR4. DSCR4 coding sequences(CDSs) have significantly higher rates of polymorphisms with
low-frequency derived alleles compared with DSCR4 intronic
sequences (chi-square test, P = 0.0132), indicating the action of
purifying selection on the CDSs of DSCR4. (B) Intrinsic structural disorder (ISD) characteristics of DSCR4. DSCR4 protein
shows a higher degree of intrinsic structural disorder comparedwith its proximate but older protein-coding gene, DSCR8, andthe housekeeping genes GAPDH and ACTB. The ISD score of
DSCR4 is also significantly higher than that of DSCR4 translated introns (t-test, P = 0.002).

The six GO terms significantly enriched in downregulated DEGs represent interconnected pathways in
the GRNs of human cells (Figs. 3A and 3B). Four of

DSCR4-associated cellular pathways GO and GRN
analysis suggested that DSCR4 is involved mainly inthe regulation of cell migration, hemostasis and coagulation. What, though, are the cellular pathways throughwhich DSCR4 accomplishes these functions? To answer
this question, we reanalyzed the up- and down-regulated
DEGs using the Reactome database and KEGG. The
Reactome database catalogs a reductionist model whichasserts that all biological activity can be represented as
events located in subcellular compartments (Fabregat etal., 2016). Consistent with GO and GRN results, theseanalyses revealed enrichment for several sub-processes
involved in cell migration, coagulation and the immune
system (Supplementary Fig. S5). Down-regulated DEGs
are specifically enriched for the function of semaphorins
and integrins, which are important families of proteins
involved in the regulation of cell migration (Supplementary Fig. S5A). Strict guidance cues (either repulsive,
inhibitory or attractive) normally precede the processof cell migration, and semaphorin receptors are known
to govern cell migration mainly by regulating integrinbased cell–substrate adhesion and cytoskeleton dynamics(Huttenlocher and Horwitz, 2011). Semaphorins havealso been shown to guide NCC migration in zebrafish (Yuand Moens, 2005). Integrins are also a family of membrane-bound molecules that are important in cell migration, and integrin-based adhesion has served as a model
for studying the central role of adhesion in migration(Huttenlocher and Horwitz, 2011). The up-regulated

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Fig. 3. Gene regulatory network and Gene Ontology analysis of DSCR4 overexpression-mediated DEGs. (A) GRN and (B) GO analysis of DEGs down-regulated by DSCR4 overexpression revealed enrichment of six interconnected pathways involved in the regulationof migration, locomotion and remodeling processes. (C) GRN and (D) GO analysis of up-regulated DEGS, on the other hand, showedenrichment for three interconnected pathways involved in the regulation of blood coagulation and hemostasis along with maintenanceof apical/basal cell polarity. Gene Ratio: number of genes within that list which are annotated to the node/size of the list of genes ofinterest.

DEGs, on the other hand, revealed enrichment for theprocesses of fibrin clot formation and the complement
cascade, which are major components in coagulationand innate immunity, respectively (Supplementary Fig.
S5B). From the evolutionary perspective, the two pathways of coagulation and the innate immune system havecommon ancestry and are highly integrated (Delvaeye
and Conway, 2009), which is consistent with our results
and supports the hypothesis of DSCR4 being simultaneously involved in functions of the innate immune systemand hemostasis.
The KEGG pathway database is a collection of manuallydrawn pathway maps representing existing knowledge
on molecular interaction, reaction and relation networks(Kanehisa et al., 2016). KEGG analysis further corroborated the results we obtained with the Reactome database by showing that DSCR4 overexpression-mediatedDEGs are enriched in coagulation and the complement
cascade, and it also revealed that coagulation and thecomplement cascade are tightly interconnected not onlyRoles of DSCR4 gene in human cell migration

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Fig. 4. Expression profile of DSCR4 across human cell lines and tissues. According to Roadmap Epigenomics Project data, DSCR4
and DSCR8, which share a bidirectional promoter, are highly expressed only in K562 cells, a type of leukemia cell. Analysis of transcriptome data provided by Prescott et al. (2015) showed that DSCR4 and DSCR8 also display high expression in human and chimpanzee neural crest cells, which are critical migratory cells involved in facial morphogenesis in the embryo. (1) Data from Prescott et al.
(2015). (2) Samples also include esophagus, lung, spleen and fetal large intestine. (3) Samples also include brain germinal matrix,
hippocampus, fetal small intestine, stomach, left ventricle, small intestine, sigmoid colon, HEPG2 cells and HMEC cells.

Fig. 5. Interconnection of DSCR4 overexpression-mediated perturbed pathways. KEGG analysis of DSCR4 overexpression-mediatedDEGs shows enrichment for the tightly interconnected pathways of the coagulation cascade and the complement cascade (highlighted
in red) and further confirm the connection of these cascades with cell adhesion, migration and proliferation (red circle).

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with each other but also with processes of cell migration,adhesion and proliferation (Fig. 5). In summary, theseresults indicate that all the pathways affected by DSCR4
gene perturbation are closely connected and are mainly
involved in the processes of cell migration, the immune
system and coagulation.
DISCUSSIONDSCR4 is an orphan gene, present only in humans andapes, whose evolution has occurred in multiple steps overthe past 100 million years mainly as a result of the actionof retrotransposons (Fig. 1). Detailed information on theevolutionary steps by which the three coding exons of
DSCR4 arose can be found in Saber et al. (2016). Youngde novo-originated genes tend to be small in size withrelatively low expression levels and weak conservation,
which also holds true for DSCR4 and raises doubts about
the functionality of this young, experimentally verified,protein-coding gene; recent investigations of ISD, however, support the preadaptation hypothesis of de novo
gene evolution, indicating that young genes show extreme
levels of gene-like traits. These findings also reject thecontinuum view of de novo gene birth, which suggests a
series of intermediate stages, or ‘proto-genes’, between
non-coding DNA sequences and a fully functional gene(Wilson et al., 2017). The same results were also foundin our ISD analysis of DSCR4 (Fig. 2B), which is consistent with the results of DAF analysis (Fig. 2A) and previous findings by Saber et al. (2016) and further supports
the functionality of DSCR4.
The present study consistently indicates the likely
roles of DSCR4 in cell migration, coagulation and theimmune system (Fig. 3). Regulation of cell migration
is defined as any process that modulates the frequency,
rate or extent of cell migration, and regulation of cellular component movement is defined as any process thatmodulates the frequency, rate or extent of the movementof a cellular component (The Gene Ontology Consortium,2000). Tissue and bone remodeling processes aredefined as the reorganization or renovation of existingtissues and bones, respectively. Tissue remodeling caneither change the characteristics of a tissue, such as inblood vessel remodeling, or result in the dynamic equilibrium of a tissue, such as in bone remodeling (The Gene
Ontology Consortium, 2000). Remodeling processes are
critical during development, wound repair and metastatic
invasion and are driven by coordinated migration ofcells through the three-dimensional extracellular matrix(Gjorevski et al., 2015). The other two pathways inwhich down-regulated DEGs are enriched are also indirectly involved in the regulation of cell migration. The
first of these pathways, positive regulation of catenin
import into the nucleus, is defined as any process that
increases the rate, frequency or extent of the directedmovement of a catenin protein from the cytoplasm into
the nucleus (The Gene Ontology Consortium, 2000). The
import of β-catenin from cytoplasm to nucleus with theassistance of Wnt protein leads to activation of a signaling pathway named Wnt/β-catenin signaling (Cai et al.,2013; Jang et al., 2015). It has been shown that the Wnt/
β-catenin signaling pathway is involved in migration ofbreast cancer cells and metastasis (Cai et al., 2013; Janget al., 2015). Negative regulation of JUN kinase is theother enriched pathway in down-regulated DEGs and is
described as any process that stops, prevents, or reduces
the frequency, rate or extent of JUN kinase activity. JUN
N-terminal kinases are a group of enzymes that bind andphosphorylate c-JUN proteins. c-jun is a proto-oncogene
and is the homolog of the viral oncoprotein v-jun (Zada etal., 2003). In breast cancer cells, c-jun is known to play a
key role in migration and invasion of mammary epithelial
cells (Jiao et al., 2010). On the other hand, hemostasis
and coagulation, which are the main processes enrichedin up-regulated DEGs, are critical for the function of theblood and innate immune systems (Esmon et al., 2011;
Degen and Palumbo, 2012). Regulation of hemostasis
and coagulation are respectively defined as the processes
leading to stopping of bleeding (loss of body fluid) or to
the arrest of the circulation to an organ and the processes
that modulate the frequency, rate or extent of coagulation. Among the other enriched genes involved in themaintenance of apical/basal cell polarity are wnt11 andankyrin1, which are directly involved in the regulationof cell migration, i.e., a biological function that is alsoenriched in down-regulated DEGs. Ankyrin1 is a cytoskeleton adaptor protein, and it is affected by p53 andalters cell migration (Hall et al., 2016); and, by interacting with silberblick, Wnt11 is involved in controlling cell
migration and morphogenesis in zebrafish (Ulrich et al.,2003). In summary, the enriched pathways and processes within the DEGs down-regulated by DSCR4 overexpression consistently indicate a likely role for DSCR4
in the regulation of cell migration. These predictions
were further validated by comprehensive analysis of the
Roadmap and YuGene expression databases.
T cells and dendritic cells are two other immune systemcell types with proven migratory behavior that notably
express DSCR4 (Supplementary Fig. S6). Three typesof stem cells and their derivatives, including embryonicstem cells (ESCs), ESC-derived neurons and induced
pluripotent stem cells (iPSCs), with migratory features
are also among the top DSCR4-expressing cells. Blastocysts, the structure formed early in the developmentof all mammals, contain another type of cell with migratory characteristics that highly expresses DSCR4. NCCs
are an embryonic cell population that emerges early inthe development of vertebrates and is most relevant tounique human facial traits (Bronner and LeDouarin,2012). These cells arise from the dorsal part of the neural

Roles of DSCR4 gene in human cell migration
tube ectoderm and migrate into the branchial arches andwhat will later form the embryonic face structure, consequently establishing the central plan of facial morphology(Prescott et al., 2015). Analyses of the YuGene database(Supplementary Fig. S6) and the NCC transcriptome inhuman and chimpanzee (Prescott et al., 2015) (Fig. 4),
indeed revealed high expression of DSCR4 in migratory
NCCs as expected. Nakamura et al. (1997), in their original discovery of the DSCR4 gene, also detected abundant
expression of this gene in placenta as well as in skeletaland heart muscles. Trophoblasts, which are specialized
cells of the placenta, possess migratory behavior (Burrows
et al., 1996). Additionally, stem cell migration has beenshown to be an essential component of muscle regeneration (Kowalski et al., 2017). These observations further
indicate the likely role of DSCR4 in cell migration. Insummary, the expression profile of DSCR4 across humancells and tissues supports the predicated functions of thisgene revealed by our gene perturbation analysis, andindicates likely roles for DSCR4 in the regulation of cell
migration and in the immune system (Fig. 4).
If DSCR4 is involved in the regulation of cell migration,and in coagulation and the immune system, we might
expect such features to be observed in human disorders
associated with DSCR4 overexpression. Down syndrome
is a human disorder characterized by an average 50%
overexpression of genes located in this region. Acuteleukemia is associated with Down syndrome 10–20-foldmore frequently than in the general population, and thisdisorder is known as the most leukemia-predisposing
syndrome (Fong and Brodeur, 1987; Xavier and Taub,2010). Leukocytes are the human cells that express
DSCR4 to the highest level (Fig. 4 and Supplementary
Fig. S6); they display active migratory characteristics
and have a prominent role in the coagulation process(Bouchard and Tracy, 2003), both of which are biological
functions that are enriched in DSCR4 overexpressionmediated DEGs. Another major characteristic of Down
syndrome individuals is immune deficiency, especiallyaffecting the innate immune system (Bloemers et al.,2010). This symptom is consistent with our finding thatthe innate immune system is enriched with genes affectedby DSCR4 overexpression (Fig. 5 and Supplementary Fig.
S5). In addition, multiple immune system cells including
monocytes (leukocytes), T cells and dendritic cells wereshown to highly express the DSCR4 gene (Supplementary
Fig. S6). Migratory behavior is also essential for the function of immune system cells. Besides highly migratory
innate immune cells such as neutrophils, which act as thefirst line of defense at the site of inflammation, T lymphocytes can also achieve rapid movement (Goldberg et al.,2016). These results indicate an active role for DSCR4
in immune system cells and also in migratory behaviorof human cells. Dysmorphology of facial characteristics
is another main feature of Down syndrome individuals

9that can be explained, at least in part, by our findingsthat DSCR4 is involved in the regulation of cell migrationand that NCCs have notable expression of DSCR4 (Fig.
4 and Supplementary Fig. S6). Cell migration is essential for the function of NCCs, which establish the facialcharacteristics of the embryo; hence, imbalanced DSCR4
expression may affect the migratory behavior of NCCs,
which could in turn affect facial morphology of Down syndrome individuals.
Because there was no previous information on thefunctions of the DSCR4 gene, we were obliged to use abrute-force approach to identify its functions, which isa statistically challenging objective considering that thecellular effects of short and young genes are usually moderate. The results of our gene perturbation and differential expression investigations, however, are consistent
with the transcription profile of human cells and withhuman diseases in which DSCR4 gene expression is perturbed. This suggests likely roles for DSCR4 in interconnected pathways of cell migration, the immune systemand coagulation.
This study is limited by its lack of experimental confirmation of the computational findings. Of great importance is the requirement for in vitro analysis to validate
the proposed difference in migratory behavior between
NCC-differentiated bone marrow cells overexpressing DSCR4 and control cells. Performing this analysis
was not feasible for us due to technical limitations withregard to differentiating DSCR4-overexpressing bonemarrow cells into NCCs or migratory immune systemcells. A second major limitation is the requirement forexperimental verification of the possible role of DSCR4 in
the formation of the characteristic upward-slanting eyelids and flat nasal bridge in Down syndrome individuals. Investigating this hypothesis was also beyond ourreach due to the requirement for in vivo experiments in
an ape model organism. Notably, this analysis is challenging because of the hominoid-specific occurrence of
DSCR4, which precludes the use of common model organisms such as the mouse for such experiments. Whileacknowledging these limitations, our computational analysis provides a solid hypothesis for future experimental
investigation of the hominoid-specific DSCR4 gene. Further experimental research is indeed required to address
the above-mentioned limitations and to unravel the rolesof this orphan and still-unclassified gene in the etiology
of Down syndrome.
DATA AVAILABILITY
All microarray data have been deposited in the ArrayExpress
database with accession number E-MTAB-8779, in accordancewith the MIAME guidelines published by the Functional Genomics Data Society (FGED).

10

M. M. SABER et al.

This work was supported by a research support grant fromthe Sasakawa Foundation and a foreign student fellowship from
the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan to M. M. S. and by a Grant-in-Aid for
Scientific Research from MEXT to N. S. Some of the analyses
were performed on the National Institute of Genetics of Japansupercomputer.

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Supplementary Fig. S1. DSCR4 plasmid vector and control vector structure.
(A) PTCN-DSCR4 plasmid carrier includes the full-length cDNA sequence of DSCR4 and its
upstream and downstream untranslated regions (UTRs), along with a CMV-derived enhancer andpromoter for strong expression in eukaryotic host cells, and eukaryotic and bacterial selection
markers. (B) PTCN-control vector was constructed from the PTCN-DSCR4 vector by removing
the DSCR4 coding sequence and UTR elements. Supplementary Fig. S2. Expression correlation analysis between DSCR4-overexpressing HS-27A
cells and control samples.
Scatter plots of expression correlation between DSCR4-overexpressing cells (PTCN-DSCR4transfected HS-27A cells) and control samples (PTCN-control-transfected HS-27A cells andnormal HS-27A cells) confirmed that microarray experiments and the normalization process were
properly conducted. PTCN-DSCR4-transfected HS-27A samples also showed more uniform
expression values against PTCN-control-transfected samples than against normal HS-27A
samples, as expected because PTCN-DSCR4- and PTCN-control-transfected cells underwent thesame transfection treatment.

Supplementary Fig. S3. Confirmation of DSCR4 overexpression in DSCR4-transfected
HS-27A cells.
qPCR expression analysis of DSCR4 normalized by GAPDH revealed approximately 9-foldoverexpression in PTCN-DSCR4-transfected HS-27A cells compared with control-transfected
samples, which is consistent with microarray results and confirms the accuracy of ouroverexpression procedures. The analysis also showed a 4-fold increase in DSCR4 expression
compared with normal (non-transfected) HS-27A cells.

Supplementary Fig. S4. Distribution of DSCR4 overexpression-mediated DEGs across the genome.
(A) 131 and (B) 122 protein-coding genes are respectively down-regulated and up-regulated in
DSCR4-overexpressing HS-27A cells compared with control samples. Supplementary Fig. S5. DSCR4-associated cellular pathways.
(A) Cellular pathways enriched in genes down-regulated by DSCR4 overexpression.
Semaphorins and integrins are two important classes of membrane proteins that are involved in
the regulation of cell migration. (B) Cellular pathways enriched in genes up-regulated by DSCR4
overexpression. Regulation of the complement cascade and the clotting cascade plays criticalroles in the innate immune system and hemostasis, respectively.

Supplementary Fig. S6. Expression profiles of DSCR4 in the YuGene database.
DSCR4 has the highest expression value in monocytes, which are a type of leukocytes, followed by twoother immune system cells with migratory characteristics. Stem cells, tumor cells, neural crest cells andblastocyst components are the other types of cells with migratory behavior that highly express DSCR4.