Screening of human cDNA library reveals two differentiation- Title related genes, HHEX and HLX, as promoters of early phase reprogramming toward pluripotency( Dissertation_全文 )
Author(s) Yamakawa, Tatsuya
Citation 京都大学
Issue Date 2016-09-23
URL https://doi.org/10.14989/doctor.k19967
Right 許諾条件により本文は2017-08-01に公開
Type Thesis or Dissertation
Textversion ETD
Kyoto University Page 1 of 58
Running head of the tile
HHEX and HLX facilitate induction of human iPSCs
The Title
Screening of human cDNA library reveals two differentiation related genes, HHEX and HLX,
as promoters of early phase reprogramming toward pluripotency
Names of authors
Tatsuya Yamakawa 1, Yoshiko Sato 1, Yasuko Matsumura 1, Yukiko Kobayashi 1, Yoshifumi
Kawamura 2, Naoki Goshima 3, Shinya Yamanaka 1, 4, Keisuke Okita 1.
Names of institutions at which the work was done
1Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan; 2Japan
Biological Informatics Consortium, Tokyo, Japan; 3Molecular Profiling Research Center for
Drug Discovery, National Institute of Advanced Industrial Science and Technology, Tokyo,
Japan; 4Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA
Author contributions
T.Y.: conception and design, financial support, collection and/or assembly of data, provision of
study material, data analysis and interpretation, and manuscript writing; Y.S., Y.M. and Y.K.:
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collection and/or assembly of data; Y.M. and N.G.: provision of study material; S.Y. and K.O.: conception and design, financial support, provision of study material, data analysis and interpretation, manuscript writing, and final approval of manuscript.
Correspondence information for corresponding author (name, degree, address [including postal code], telephone and fax numbers, and e-mail address)
Correspondence: Keisuke Okita, Ph.D., Department of Life Science Frontiers, Center for iPS
Cell Research and Application, Kyoto University, 53 Kawahara cho, Shogoin, Sakyo ku, Kyoto
606 8507, Japan. Telephone: 81 75 366 7043; Fax: 81 75 366 7098; e mail: okita [email protected] u.ac.jp
A brief acknowledgment of grants, equipment, or drugs for research support
This study was supported in part by a grant from JSPS, JST, AMED and MEXT. We thank all agency for financial support. key words or phrases induced pluripotent stem cell; reprogramming; human cDNA library; screening; HHEX ; HLX
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Abstract
Gene screenings have identified a number of reprogramming factors that induce pluripotency
from somatic cells. However, the screening methods have mostly considered only factors that
maintain pluripotency in embryonic stem cells, ignoring a potentially long list of other
contributing factors involved. To expand the search, we developed a new screening method that
examined 2,008 human genes in the generation of human induced pluripotent stem cells (iPSCs),
including not only pluripotent genes but also differentiation related genes that suppress
pluripotency. We found the top 100 genes that increased reprogramming efficiency and
discovered they contained many differentiation related genes and homeobox genes. We selected
two, HHEX and HLX , for further analysis. These genes enhanced the appearance of premature
reprograming cells in the early phase of human iPSC induction, but had inhibitory effect on the
late phase. In addition, when expressed in human iPSCs, HHEX and HLX interfered with the
pluripotent state, indicating inverse effects on somatic reprograming and pluripotent
maintenance. These results demonstrate that our screening is useful for identifying
differentiation related genes in somatic reprograming.
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Introduction
The original reprogramming of human somatic cells to pluripotency (induced pluripotent stem cells, or iPSCs) was done by forcing the expression of four factors: OCT3/4 and SOX2 with either KLF4 and C-MYC or NANOG and LIN28 [1,2]. Since then, several studies have revealed a number of factors that can enhance or substitute these original factors for reprogramming [3–
5]. These new findings have advanced our understanding of the reprogramming mechanism, including enhancers and barriers of the reprogramming and resulted in better reprogramming efficiency. For example, TP53 induces cell cycle arrest and the apoptosis pathway during the reprogramming and its depletion is associated with a higher number of iPSC colonies and the up regulation of exogenous reprogramming genes [6–8]. GLIS1 is another example and has important roles in the transient primitive streak like mesendodermal state, which may be crucial for the maturation of reprogrammed cells [9,10].
Gene screenings have proven especially useful for the identification of these other factors.
However, in most cases the screenings have used libraries that were limited to genes for embryonic stem cells (ESCs) or epigenetic modifiers [5,11]. Others have employed genome wide scale libraries, but these studies could detect only factors that regulate the
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self renewal or pluripotency of ESCs [12–18]. For comprehensive study of which genes
regulate reprogramming, screenings should move beyond genes that maintain pluripotency and
also consider those involved in differentiation.
Here we report a screening of a human cDNA library that includes 1,361 transcription factors,
187 kinases and 460 other genes by counting iPSC colonies in the setting of human somatic cell
reprogramming. Through this screening, we identified several new genes that increased or
decreased the iPSC generation efficiency. Among the candidate genes, we selected two
homeobox transcription factors, HHEX and HLX , for further analysis, as they are
differentiation related genes that function in multiple developmental processes [19–25]. Mice
lacking HHEX or HLX are embryonic lethal because of visceral hypoplasia around E15 [24,25].
In humans, they are expressed in several tissues, but highly expressed in hematopoietic tissue.
Further, HHEX was reported to promote hepatic differentiation from human ESCs, while HLX is
highly expressed in AML (acute myeloblastic leukemia) patients [26,27]. We found that when
used in somatic cell reprogramming, both factors enhanced the appearance of cells positive for
an early reprogramming marker, TRA 1 60. On the other hand, overexpression in human iPSCs
interfered with the maintenance of the pluripotent state. Our findings suggest that HHEX and
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HLX have inverse effects on the early phase of somatic cell reprogramming and pluripotency and may be useful factors for understanding somatic cell reprogramming. Furthermore, our results demonstrate the resourcefulness of our screening strategy for finding key genes involved in reprogramming.
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Material and Methods
Human cDNA library
The cDNAs obtained from the human proteome expression resource (HuPEX) library were
selected according to the criteria previously described [10]. Among the 43,349 cDNAs, we
mainly chose those known or identified by keyword searches of the Human Gene and Protein
Database (HGPD, http://www.HGPD.jp/) and Entrez gene (http://www.ncbi.nlm.nih.gov/gene)
as transcription factors or kinases [28]. We used cDNAs that covered more than 80% of the
open reading frame reported in RefSeq and had shared identity with the reported protein
sequence of more than 95% at the amino acid level. The selected 2,008 genes were transferred
to pCXLE gw episomal expression vector (Addgene #37626) using the Gateway LR reaction
(ThermoFisher) (Supporting Information Table 1).
Cell culture
Human dermal fibroblasts, HDF1419, HDF1388, HDF1437 and HDF1554 were purchased
from Cell Applications, Inc, and TIG 107, TIG 114 and TIG 120 were obtained from the
Japanese Collection of Research Bioresources. The details of these HDFs were listed in
Supporting Information Table 2. Cryopreserved human peripheral blood mononuclear cells
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(PB MNCs) were purchased from Cellular Technology Limited. Human ESC line H9 was provided by the WiCell Research Institute. Mouse embryonic fibroblasts (MEFs) were isolated from embryonic day 13.5 embryos of C57BL/6 mice, which were bred and killed appropriately following the code of ethics of animal research committee at Kyoto University. HDFs were cultured in DMEM supplemented with 10% fetal bovine serum. MEF and mouse embryonic fibroblast cell line SNL76/7 (SNL) cells were cultured in DMEM supplemented with 7% FBS,
2 mM L glutamine, 50 units/ml penicillin and 50 mg/ml streptomycin [29]. MEF and SNL cells were treated with mitomycin C before use as feeder cells. Established human iPSCs and ESCs were maintained on mitomycin C treated SNL cells in Primate ESC medium (ReproCell) containing 4 ng/ml of bFGF as described previously [1].
Screening for novel factors in iPSC generation
Plasmid DNAs for iPSC generation were prepared using the Qiagen plasmid mini kit (Qiagen).
Each candidate gene from the library was transfected into HDF with basic 5 factors, OCT3/ 4,
SOX2 , KLF4 , L-MYC and LIN28 via episomal plasmid methods at day 0. For 6 days after the transfection, these cells were cultured in DMEM containing 10% FBS. On day 6, the transfected cells were harvested by treatment with 0.25% trypsin/1 mM EDTA and transferred onto
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feeder coated 6 well plates. From the next day, the medium was replaced with human ESC
medium and cultured until iPSC colonies emerged. At day 25, whole well images were taken by
Nikon BioStationCT (Nikon), which is an automated culture incubator that mounts a
microscope with a camera inside. The number of human iPSC colonies was calculated from
these images by CL Quant software for BioStationCT according to the criteria of human
iPSC like morphology. We used an episomal plasmid encoding DsRedExpress as a Mock and
calculated the relative induction efficiency by dividing the number of colonies of each candidate
gene by that of the mock sample.
Generation of human iPSCs
The generation of human iPSCs were carried out as previously described with some
modifications [1,30,31]. iPSC generation from HDFs with episomal plasmid was performed as
described in the screening section above. Cryopreserved PB MNCs were sequentially
transfected with episomal plasmid containing reprogramming factors after thawing and
transferred onto MEF feeder coated plates. These PB MNCs were cultured in two types of
medium that mainly stimulate the proliferation of T cells or hematopoietic stem cells. The
number of iPSC colonies was counted on day 25. For retrovirus reprogramming methods, a
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retrovirus vector encoding each factor was introduced into Plat E packaging cells, and these viruses were collected from the supernatant [32]. We used these viruses to expose
HDF expressing mouse Slc7a1 gene, which is known as a cell surface receptor for ecotropic retrovirus. From the next day, the cells were cultured with DMEM containing 10% FBS and transferred onto feeder coated 6 well plates at day 6. The medium was replaced with human
ESC medium on day 7 and cultured until day 25.
Characterization of human iPSCs
Total RNA was isolated using miRNeasy mini kit (Qiagen). RT PCR of marker gene expression, teratoma formation assay and alkaline phosphatase staining were performed as previously described [1,33]. The primers used for RT PCR are listed in Supporting Information
Table 3.
DNA microarray
Total RNA was labeled with Cyanine 3. Samples were hybridized with the Whole Human
Genome Microarray SurePrint G3 Human GE 8 × 60K v2 (G4851B; Agilent). Each sample was hybridized once using the one color protocol. The arrays were scanned with G2565BA
Microarray Scanner System (Agilent). All microarray results were analyzed using the
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GeneSpring v 13 software program (Agilent). Samples were normalized by a 75th percentile
shift. Probes filtered by percentile were used for the analysis.
Flow cytometry and cell sorting
To analyze TRA 1 60 + cells, we transfected EGFP expressing episomal vector along with
reprogramming plasmids into HDF. The transfected cells were cultured in 10%
FBS supplemented DMEM medium for 8 days. The medium was then replaced with human ES
medium containing 4 ng/ml bFGF. On days 4, 7, 11 and 15, the transfected cells were harvested
by treatment with 0.25% trypsin/1 mM EDTA or accutase and were filtered using a 70 m pore
cell strainer. These cells were stained with anti human TRA 1 60 antibody conjugated with
Alexa 647 (BD Biosciences) for 30 minutes at room temperature. TRA 1 60 + cells in the EGFP
positive fraction were analyzed and sorted by FACS Aria II.
Generation of HDF lines with doxycycline (dox)-inducible factor
To generate lentivirus vectors encoding dox inducible HHEX or HLX , each ORF adjusted to
RefSeq sequence was inserted into pENTR directional TOPO vector (ThermoFisher) and
verified by sequencing. The Gateway cassette (ThermoFisher) under tetracycline responsible
element (TRE) and reverse transactivator rtTA2s M2 were introduced into a pLKO.1 puro
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(Addgene #8453) backbone. Then, HHEX or HLX ORF was inserted into the Gateway cassette in this lentivirus vector (pLV/TRE/rtTA2s M2 gw) by Gateway LR reaction [34]. Lentivirus encoding HHEX or HLX was produced from 293FT cells and infected into HDF as described previously [1]. These HDF were used for the reprogramming with OSKUL episomal vector mixture and cultured with or without dox at the indicated time point. The number of iPSC colonies was counted by BioStationCT on day 30.
Generation of human iPSC lines with dox-inducible factor
To construct dox inducible HHEX or HLX expression piggyBac vector, HHEX or HLX ORF was cloned into PB/TA/ERN using the Gateway LR reaction system [35]. Each plasmid DNA was transfected into human iPSCs with transposase expression plasmid by Neon transfection system (Life technology), and the cells were seeded onto SNL coated 6 well plates. From the next day, stable transformants were selected and maintained by 50 g/ml G418.
Statistical Analyses
All quantitative experiments were biologically repeated at least three times. In all figures, the single and double asterisks indicate P < 0.05 or P < 0.01, respectively, as determined by student’s t test.
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Result
Functional screening of human cDNA library identified novel reprogramming factors
To find novel reprogramming genes that directly influence the reprogramming of human
somatic cells, we used a human cDNA library containing 2,008 genes. Each candidate gene was
transfected separately into human dermal fibroblast (HDF) with 5 other factors ( OCT3/4 , SOX2 ,
KLf4 , L-MYC and LIN28 : OSKUL). The cells were cultured for 25 days, and the number of
iPSC colonies was evaluated by an automatic colony counting system (Fig. 1A). This system
can recognize human iPSC like colonies from whole well pictures using only cell morphologies
and without any reporter gene expression or staining (Supporting Information Fig. S1).
As a result, we could investigate the relative induction efficiency of each of the 2,008 genes
from HDF (Fig. 1B). We have previously shown that the addition of NANOG or GLIS1 or the
inhibition of TP53 enhanced the reprogramming efficiency of human somatic cells with
episomal plasmids [31]. The current screening was consistent with these previous results, as
NANOG and GLIS1 enhanced the efficiency 2.6 and 5.2 fold, respectively, while overexpression
of TP53 decreased it 5 fold. The expression of GLIS1 resulted in the highest induction
efficiency in our screening. However, several novel genes that showed positive or negative
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regulation of somatic reprogramming were also identified (Fig. 1C, D). The functional annotations of these genes were analyzed by DAVID (https://david.ncifcrf.gov) using the entire library as a background [36]. The GO terms enriched in top 100 positive genes were either involved in muscle development or encoding of the homeobox motif. On the other hand, the GO terms enriched in top 100 negative genes were either involved in immune response or metabolic processes or encoding of helix turn helix transcription factors (Supporting Information Fig. S2).
For detailed analysis, we selected two homeobox transcription factors, HHEX and HLX , which share similar developmental function and expression patterns in vivo, from the list of positive genes. Until now, although the effects of HHEX and HLX on differentiation have been explored, their effects on reprogramming have not.
Ectopic expressions of HHEX and HLX enhanced reprogramming efficiency from different types of somatic cells
The ability of these two genes to enhance reprogramming efficiency was confirmed with other dermal fibroblasts from multiple donors and blood cells. HHEX or HLX increased the efficiency of fetal fibroblast (HDF1419) on average 3 fold and of adult fibroblast (HDF1437) more than
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12 fold (Fig. 2A; Supporting Information Fig. S3). HLX increased the induction reprogramming
efficiency of PB MNC more than 2 fold compared with Mock transfected cells in T cell
stimulating medium. On the other hand, HHEX doubled the efficiency in a culture condition that
mainly stimulates the proliferation of hematopoietic stem cells (Fig. 2B). HHEX and HLX were
also effective when incorporated into a retroviral gene delivery method (Fig. 2C), but to a lesser
degree than episomal plasmids. This result could be due to differences in the expression pattern
and intensity of the transduced genes in the two induction methods [30]. Nevertheless, these
data suggested that HHEX and HLX could enhance reprogramming, although the degree of the
enhancement depends on the somatic cell type and gene delivery method.
We investigated the characteristics of the iPSCs induced with HHEX or HLX . These iPSCs
showed typical morphology seen in human ESCs (H9). Expression patterns of pluripotent
marker genes also showed to be at the same level as iPSC line induced with Mock in the same
experiment (Fig. 2D, E). Moreover, the iPSCs could produce all three germ layer cells upon
injection into immunocompromised mice (Fig. 2F; Supporting Information Fig. S4).
HHEX /HLX promoted the initiation of reprogramming
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We next examined the function of HHEX and HLX during different reprogramming processes.
TRA 1 60 is a surface marker of human pluripotent stem cells and is expressed in reprogramming committed cells from the early phase [37]. A part of these committed cells go on to become fully reprogrammed cells. We therefore assessed whether HHEX and HLX can affect the emergence of TRA 1 60 positive cells. The percentage of TRA 1 60 + cells was analyzed using FACS analysis at 4, 7, 11 and 15 days after the transfection of reprogramming genes into HDF1419 (Fig. 3A, B). In samples without HHEX , positive cells occurred at less than 1% on day 4. In contrast, HHEX transfection increased the occurrence of positive cells from day 4 and resulted in more than 37% of cells being TRA 1 60 + by day 7. Notably the percentage of TRA 1 60 + cells decreased after this time point which suggested a possible negative effect of HHEX at late phase of reprogramming. Regarding cells transfected with
GLIS1 , only about 0.7% were TRA 1 60 + by day 4, but this number increased to 45% by day 15.
We then considered the effect of combining HHEX and GLIS. This combination resulted in the highest percentage of TRA 1 60 + cells at day 7 and 11 (Fig. 3B). At day 15, the percentage didn’t denote the same tendency of reduction like HHEX single addition (no significant change from day 7 to 15 in combining HHEX and GLIS ). As distinct from HHEX , HLX transfection
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resulted in only a 1.9 and 2.9 folds increase of TRA 1 60 + cells on days 7 and 11, respectively,
compared with Mock transfected cells. However, on day 15, the percentage of TRA 1 60 + cells
increased to 4 times higher than Mock transfected cells, which is comparable to the increase
with HHEX . These promoting effects at day 7 were also confirmed with multiple HDFs
(Supporting Information Fig. S5). These data suggested that both HHEX and HLX enhanced the
reprogramming efficiency by promoting the emergence of positive cells at the early phase of
reprogramming but at different manners.
To compare the effects of these two genes, we investigated gene expression profiles of the
TRA 1 60 + cells on day 7 (Fig. 3C). HHEX transduced cells had a Pearson correlation with
Mock transfected cells of 0.988 and had 3,383 differentially expressed entities among the
50,599 genes analyzed. To assess whether HHEX brings TRA 1 60 + cells toward the pluripotent
state on day 7, we selected 114 human ESC enriched and 109 HDF enriched entities based on
expression levels that showed at least a 100 fold difference [33]. Among the human
ESC enriched entities, 44 showed a more than 2 fold increase in TRA 1 60 + cells with HHEX
compared with Mock, while only 4 entities showed a more than 2 fold decrease. Comparatively,
20 HDF enriched entities expressed at least a 2 fold decrease with HHEX compared with Mock,
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but only 1 entity with a similar increase. These data suggested that HHEX transfected
TRA 1 60 + cells were closer to the pluripotent state than Mock transfected cells on day 7.
Regarding HLX transfected TRA 1 60 + cells, almost all human ESC and HDF enriched entities were expressed within 2 fold of Mock samples. This result is consistent with HLX increasing the percentage of TRA 1 60 + cells only 1.7 folds on day 7 compared with Mock.
HHEX/HLX interfered with the pluripotent state
Finally, we examined the functions of HHEX and HLX in the late phase of reprogramming by inserting HHEX or HLX into HDF with the dox inducible system. iPSCs were induced from these HDFs with dox treatment at a defined time point (Fig. 4A). When dox was supplemented from day 1 to 10 after transfection of the reprogramming plasmid mixture, the number of iPSC colonies increase compared with the dox ( ) sample (Fig. 4B). However, extension of dox treatment (days 1 to 20 or days 1 to 30) decreased the colony number relative to treatment up to day 10. Furthermore, ectopic expression only on late time points (d11 20, d11 30 or d21 30) decreased the number of iPSC colonies compared with the dox ( ) sample (Supporting
Information Fig. S6). These results suggested that the ectopic expression of HHEX/HLX had
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some negative effect on late phase reprogramming. This inhibitory phenotype was further
examined with iPSC clones having dox inducible HHEX or HLX . Each iPSC was cultured for
one week in ESC maintenance condition with or without dox treatment and was stained based
on the activity of alkaline phosphatase, a marker of pluripotent cells (Fig. 4C). The number of
positively stained colonies was reduced with ectopic HHEX or HLX expression by dox
treatment. In addition, the forced expression of HHEX or HLX reduced the expressions of
pluripotent marker genes although control iPSC encoding dox inducible EGFP did not change
these expressions (Fig. 4D). Microarray analysis also showed differentiation of iPSCs by
ectopic HHEX or HLX expression (Fig. 4E; Supporting Information Fig. S7). Typical lineage
marker genes for each three germ layers were up regulated by HHEX or HLX . Genes that were
up regulated more than 6 folds by HHEX or HLX compared to control iPSC with dox treatment
were selected and used for gene ontology analysis. As a result, these genes were highly
classified to the GO term related to developmental processes (Supporting Information Fig. S7).
These results indicate that HHEX and HLX antagonize the maturation of pluripotency through
negative effects like the induction of differentiation.
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Discussion
Using a new strategy that screens human genes in a reprogramming setting, we identified novel reprogramming factors from a large scale cDNA library with human cells via plasmid based methods. Although many genes have been reported to improve the reprogramming efficiency of mouse somatic cells via retrovirus methods, in many cases these factors do not have the same effect on the iPSC induction of human cells using plasmid methods.
For example, Esrrb was recognized as a substitute gene for Klf4, while subsequent research showed that Esrrb could improve the reprogramming efficiency of Oct3/4 , Sox2 , Klf4 and c-Myc in mouse embryonic fibroblast with retrovirus methods [3,38]. However, we previously found that human ESRRB decreased the reprogramming efficiency of human fibroblasts with plasmid methods [31]. Our approach in the present study should provide a more reliable survey of genes involved in reprogramming from human cells.
Until now, most genome wide screenings employed an experimental system under undifferentiated conditions of ESC to detect which genes regulate ESC self renewal and pluripotency [12–18]. Our approach, however, allows us to evaluate gene effects on somatic reprogramming by counting the number of iPSC colonies. Consequently, we show two new
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candidate factors, HHEX and HLX , enhance early phase reprogramming but inhibit late phase,
indicating they enhance reprogramming but disturb pluripotency in iPSCs. These findings were
in large part because our screening was not restricted to ESC enriched genes and because
plasmids mainly express their transgenes for two weeks after the induction [30].
We found that HHEX and HLX enhanced reprogramming efficiency in fibroblasts by
promoting the emergence of TRA 1 60 + cells in the early phase of reprogramming. Interestingly,
the forced expression of either gene inhibited pluripotency maintenance and induced
differentiation in human iPSCs. These results indicated that HHEX and HLX promote the
initiation of reprogramming but have an inhibitory effect on the late phase. HHEX is reported as
a promoting factor of hepatic differentiation from human ESCs and is thought to be a lineage
specifier [26]. Lineage specifiers are considered determining factors of the differentiated cell
type and antagonists of pluripotency associated genes. Recently, the seesaw model was
proposed to explain how lineage specifiers play an important role in the process of
reprogramming [39,40]. In this model, the somatic state is an off balanced state that inclines
toward specific cell lineages. If the seesaw is balanced between two differentiated cell types
through the addition of other lineage specifiers, the cell can escape from its terminally
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differentiated state and acquire higher potential to return to the pluripotent state. The loss of the off balanced state is thought critical for the early phase of reprogramming. Our microarray analysis showed that TRA 1 60 + cells at day 7 transduced with HHEX and HLX highly expressed differentiation related genes those GO term were mainly classified to stem cell differentiation or regulation of cell differentiation (data not shown). Taken together, our findings suggested that HHEX and HLX facilitate de differentiation of fibroblasts by disturbing their off balanced state. Some lineage specific genes, such as T and MIXL1 , are known to be transiently up regulated in the early phase of reprogramming [9,39,40]. Previously Tanabe et al. reported gene expression profiles of reprogramming cells at day 7, 11, 15 and 28 induced with conventional 4 factors [33]. We investigated their data and found that HHEX and HLX kept on low expression level through the reprogramming process. However it was not clear how these two genes were regulated at earlier time point than day 7 post gene transduction and whether these low expressions have any biological meanings. It is necessarily to perform further analysis to investigate the precise mechanisms of HHEX and HLX on the reprogramming.
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Summary
We used a new screening strategy that investigated the effects of genes from a human cDNA
library on reprogramming, discovering two genes, HHEX and HLX , may enhance the efficiency.
HHEX and HLX showed different function in early and late phase reprogramming, promoting
the former but suppressing the latter. These findings offer new understanding on the induction
of pluripotency. Our screening should welcome the discovery of other genes that improve the
efficiency of reprogramming and also better understanding of the molecular mechanisms
involved.
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Acknowledgement
We thank K. Takahashi, M. Nakagawa, and Y. Yoshida for scientific discussion; M. Narita, K.
Asano, Y. Takase and A. Ichikawa for technical assistance; P. Karagiannis for reading the manuscript; H. Mochizuki (JBIC) for construction of HuPEX clones; and J. Miyazaki (Osaka
Univ.) for the CAG promoter. This study was supported in part by a grant from Funding
Program for World Leading Innovative Research and Development on Science and Technology
(FIRST Program) of Japan Society for the Promotion of Science (JSPS); grants from Core
Center for iPS Cell Research and Projects for Technological Development, Research Center
Network for Realization of Regenerative Medicine in Japan Science and Technology Agency
(JST) and Japan Agency for Medical Research and Development (AMED); Grants in Aid for
Scientific Research of JSPS and the Ministry of Education, Culture, Sports, Science, and
Technology (MEXT) (to S.Y.), Grants in Aid for Scientific Research for Young Scientists B (to
K.O.), and T.Y was a recipient of a JSPS DC1 fellowship.
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Disclosure of Potential Conflicts of Interest
Shinya Yamanaka is a scientific advisor of iPS Academia Japan without salary. The remaining
authors declare no competing financial interests.
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legends
Figure 1. Effects of factors from a human cDNA library on iPSC generation. (A) : Schematic
representation of iPSC induction for the screening. (B) : Relative number of iPSC colonies from
30 000 HDFs transfected with OSKUL and one candidate gene. The number of colonies was
normalized with Mock transfected samples (OSKUL +Mock). (C, D) : List of top 20 genes that
increased (C) or decreased (D) iPSC induction efficiency. Relative induction efficiencies are
shown.
Figure 2. Enhanced reprogramming efficiency by HHEX or HLX . (A, B, C) : Number of iPSC
colonies from human somatic cells. Somatic cells were transfected with basic reprogramming
factors (OSKUL) together with the indicated genes. Control indicates cells transfected with only
EGFP expression vector. As somatic cells, two kinds of HDFs (A) and PB MNCs (B) were used
in the plasmid methods and HDFs expressing mouse Slc7a1 were used in the retrovirus method
(C). For iPSC induction from PB MNCs, two kinds of medium conditions were tested: αβ T cell
stimulating or non T , B cell stimulating conditions. Mean ± SD from at least three independent
experiments. *P < 0.05, **P < 0.01, versus Mock. (D) : Morphology of human ESC and
established human iPSC lines by forced expression of OSKUL + Mock (1456A4), HHEX
33 Page 34 of 58
(1456C1) or HLX (1456D1). Scale bars, 200 m. (E) : Relative mRNA expressions of pluripotent marker genes by real time PCR. Data were normalized to the level of GAPDH. Total
RNA was isolated from a well analyzed human iPSC line (409B2) and human ESC line (H9),
HDF, and human iPSC lines established in this study. Mock , HHEX or HLX iPSCs were induced with OSKUL + Mock, HHEX or HLX , respectively. Mock iPSC line was set at a relative level of 1. Mean ± SD from three different clones. N S means not significant. (F) :
Hematoxylin and eosin staining of teratoma derived from iPSCs with HHEX (1456C1) or HLX
(1456D1) by transplanting iPSCs into the testis of a SCID mouse. Scale bars, 200 m.
Figure 3. HHEX and HLX promoted the emergence of early iPSC committed cells. (A, B) :
Proportion of TRA 1 60 + cells in the population of EGFP + cells on day 7 (A) or from days 4 to
15 (B). Indicated genes were transfected into fetal HDFs (HDF1419) with OSKUL. Mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, versus Mock at each time point. (C) : Scatter plots showing the global gene expression of TRA 1 60 + cells on day 7 between Mock and HHEX or HLX . The blue dots indicate 114 human ESC enriched entities.
The red dots indicate 109 HDF enriched entities. Green lines indicate 2 fold changes (FC =2).
Representative genes are shown in each color.
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Figure 4. HHEX and HLX inhibit late phase reprogramming. (A) : Schematic representation of
iPSC induction with time point expression of HHEX or HLX . OSKUL transfected cells were
transferred onto feeder coated plates after 7 days, and iPSC colonies were counted at 30 days.
The reprogramming period was divided into 3 periods: days (d) 1 10, 11 20 and 21 30. (B) :
Number of iPSC colonies from HDFs inserted with dox inducible HHEX or HLX . Dox was
supplemented on the indicated days. Control indicates cells transfected with only EGFP
expression vector. Mean ± SD from at least from three independent experiments. *P < 0.05, **P
< 0.01, versus dox d1 10. (C) : Alkaline phosphatase (AP) staining of iPSC encoding
dox inducible HHEX or HLX after 7 days culture with dox. Upper panel shows phase contrast
images of iPSCs. Bottom panel shows whole well images after AP staining. Control iPSCs were
integrated with dox inducible EGFP. Scale bars, 100 m for phase contrast images and 10 mm
for whole well images. (D, E): Relative mRNA expression of pluripotent marker genes or
lineage marker genes associated with three germ layer. Total RNA was isolated from iPSCs
encoding dox inducible factor after 7 days culture with or without dox. (D) Data from
quantitative PCR were normalized to the level of GAPDH. The control iPSC without dox was
set at a relative level of 1. Mean ± SD from three independent experiments. *P < 0.05, **P <
35 Page 36 of 58
0.01, versus the iPSC encoding each gene without dox. (E) Data from microarray analysis were normalized to the level of control iPSC with dox. Mean ± SD from three microarray data sets.
*P < 0.05, **P < 0.01, versus control iPSC with dox.
Supporting Information Figure S1. Automatic human iPSC colony counting by BioStationCT and CL Quant. (A) : Representative whole well image taken by BioStationCT. Bottom image is after analysis by CL Quant software. Yellow boxes show human iPSC like morphological colonies. Purple boxes show debris like aggregates of somatic cells. (B) : Comparison between the two colony count methods. Each axis respectively shows the number of colonies by eye under microscope and by BioStationCT with CL Quant software. (C) : Representative examples of phase contrast images of human iPSC like colonies or debris and non human iPSC like colonies. CL Quant software can distinguish each colony by its morphology. Scale bars, 200
m.
Supporting Information Figure S2. GO term analysis of top 100 positive (A) and 100 negative (B) genes from the screening. GO analysis was performed by DAVID. Enriched GO
36 Page 37 of 58
terms that showed p values < 0.05 are listed. The upper graph shows GO terms for biological
process and bottom one shows for protein domain.
Supporting Information Figure S3. Enhancement of reprogramming efficiency by HHEX or
HLX from different donors. Number of iPSC colonies from HDFs were indicated. HDFs were
transfected with basic reprogramming factors (OSKUL) together with the indicated genes.
Control shows cells transfected with only EGFP expression vector. Mean ± SD from at least
three independent experiments. *P < 0.05, **P < 0.01, versus Mock.
Supporting Information Figure S4. Hematoxylin and eosin staining of teratoma derived from
iPSCs with HHEX (1456D4) or HLX (1456C4). Scale bars, 200 m.
Supporting Information Figure S5. HHEX and HLX promoted the emergence of early
iPSC committed cells from different donors. Proportions of TRA 1 60 + cells in the population
of EGFP + cells on day 7 were shown. Indicated genes were transfected into HDFs with OSKUL.
Mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, versus Mock.
Supporting Information Figure S6. HHEX and HLX inhibited late phase reprogramming.
HDFs inserted with dox inducible HHEX or HLX were used in this experiments. After
OSKUL transfection the cells were transferred onto feeder coated plates at day 7, and iPSC
37 Page 38 of 58
colonies were counted at day 30. Dox was supplemented on the indicated days. Control indicates cells transfected with only EGFP expression vector. Mean ± SD from at least three independent experiments. *P < 0.05, **P < 0.01, versus dox ( ).
Supporting Information Figure S7. Enrichment of GO term related to developmental processes by ectopic expression of HHEX or HLX in human iPSCs. Genes that up regulated more than 6 times by HHEX (A) or HLX (B) compared to control iPSC with dox treatment were classified by the gene ontology analyses with GeneSpring v 13 software program (Agilent). Top
12 enriched GO terms are listed. Global gene expressions were analyzed from three microarray data sets.
Graphical Abstract. We used a new screening strategy that investigated the effects of genes from a human cDNA library on reprogramming and discovered two genes, HHEX and HLX, which may offer new understanding on the induction of pluripotency.
38 Page 39 of 58
Figure1 144x262mm (300 x 300 DPI)
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Figure2 220x330mm (300 x 300 DPI)
Evaluation Edition of activePDF DocConverter Visit www.activePDF.com for more details. Page 41 of 58
Figure3 130x115mm (300 x 300 DPI)
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Figure4 206x194mm (300 x 300 DPI)
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human cDNA library
OCT3/4 SOX2, KLF4 + factor α L-MYC, LIN28 Reprogramming somac cell iPS cell
6
5 HLX 4 HHEX 3 2 1 0
Number of iPSNumber colonies/Mock Genes Page 44 of 58
Keisuke Okita, Supporng Informaon Fig. S1. top AB Number of colonies 250 R² = 0.9037 200
150 Eye 100
iPSC 50 debris 0 0 50 100 150 200 Software C human iPSC colony
debris or non-human iPSC colony Page 45 of 58
Keisuke Okita, Supporng Informaon Fig. S2. top
A (-Log2P) 0 2 4 6 8 10 regulaon of transcripon posive regulaon of gene-sepecific transcripon striated muscle ssue development regulaon of gene-specific transcripon skeletal muscle organ development skeltal muscle ssue development muscle ssue development regulaon of transcripon, DNA-dependent regulaon of adapve immune response based on somac recombinaon of immunoglobulin receptors regulaon of adapve immune response regulaon of RNA metabolic process embryonic morphogenesis posive regulaon of specific transcpon from RNA polymerase II promoter
0 2 4 6 Homeodomain-related Homeobox, conseved site Homeobox
(-Log P) B 2 0 2 4 6 8 10 defense response to bacterium B cell acvaon posive regulaon of transcripon posive regulaon of gene expression posive regulaon of transcripon, DNA-dependent posive regulaon of RNA metabolic process posive regulaon of nucleobase, nucleoside, nucleode and nucleic acid metabolic process posive regulaon of nitrogen compound metabolic process posive regulaon of macromolecule metabolic process posive regulaon of macromolecule biosynthec process posive regulaon of biosynthec process posive regulaon of cellular biosynthec process regulaon of transcripon from RNA polymerase II promoter regulaon of RNA metabolic process B cell differenaon leukocyte differenaon response to endoplasmic reculum stress regulaon of transcripon, DNA-dependent regulaon of osteoclast differenaon 0 4 8 12 16 20 RNA recognion mof, RNP-1 Nucleode-binding, alpha-beta plait Ets Winged helix repressor DNA-binding Transcpon factor, fork head, conseved site Transcpon factor, fork head Page 46 of 58
Keisuke Okita, Supporng Informaon Fig. S3. top
HDF1388 HDF1554 160 200 ** ** ** 160 120 ** * cells) cells) 5 5 120 ** 80 ** ** 80
40 40 (from 1.0 × 10 (from 2.0 × 10 Number of iPSCNumber colonies 0 of iPSCNumber colonies 0 ControlMock HHEX HLX HHEX+HLXGLIS1 Control Mock HHEX HLX HHEX+HLXGLIS1
OSKUL OSKUL
TIG-107 TIG-114 TIG-120 140 400 12 * * * * * * 120 * 9 300 cells) cells) 100 cells) *
5 6 ** 5 80 6 200 60
3 40 100
(from 2.0 × 10 (from 1.0 × 10 20 (from 2.0 × 10 Number of iPSCNumber colonies of iPSCNumber colonies of iPSCNumber colonies 0 0 0 ControlMock HHEX HLX ControlMock HHEX HLX GLIS1 Control Mock HHEX HLX HHEX+HLXGLIS1
OSKUL OSKUL OSKUL Page 47 of 58
Keisuke Okita, Supporng Informaon Fig. S4. top
HHEX-iPSC (1456C4) HLX-iPSC (1456D4) Melanocyte Gut-like epithelium Adipose Gut-like epithelium
Muscle Carlage Muscle Page 48 of 58
Keisuke Okita, Supporng Informaon Fig. S5. top
HDF1437 HDF1554 18 ** 12 **
at day 7 at day 7 10
+ 15 + ** 12 ** 8
9 6 ** * cells in EGFP cells in EGFP + 6 + 4 ** ** 3 2
0 Mock HHEX HLX GLIS1 p53 shRNAHHEX+GLIS1 0 Mock HHEX HLX GLIS1 p53 shRNAHHEX+GLIS1 % of% TRA1-60 of% TRA1-60
OSKUL OSKUL
TIG-114 TIG-120 20 35 ** 30 ** at day 7 at day 7
+ 16 + 25 ** 12 20 ** ** 8 15 ** cells in EGFP cells in EGFP + + ** 10 * 4 5 0 0 Mock HHEX HLX GLIS1 p53 shRNA Mock HHEX HLX GLIS1 p53 shRNAHHEX+GLIS1 % of% TRA1-60 of% TRA1-60
OSKUL OSKUL Page 49 of 58
Keisuke Okita, Supporng Informaon Fig. S6. top
HHEX HLX 160 160 * * 140 140
120 120 cells) cells) 5 5 100 100 * 80 80 * * 60 60 * * (from 3.0 × 10 40 (from 1.0 × 10 40 ** Number of iPSCNumber colonies of iPSCNumber colonies 20 20
0 0 11-30 11-30 d1-10 d1-10 dox (-) dox (-) d11-20 d21-30 d11-20 d21-30 Control Control OSKUL OSKUL Page 50 of 58
Keisuke Okita, Supporng Informaon Fig. S7. top
A (-Log2P) 0 5 10 15 20 25 system development mulcellular organismal development anatomical structure morphogenesis organ morphogenesis skeletal system development single-organism developmental process ssue development anatomical structure development organ development regulaon of mulcellular organismal development cell differenaon embryonic skeletal system development B (-Log2P) 0 20 40 60 80 100 single-mulcellular organism process system development anatomical structure development mulcellular organismal development regulaon of mulcellular organismal process ssue development single-organism developmental process response to organic substance extracellular structure organizaon anatomical structure morphogenesis regulaon of cell migraon posive regulaon of mulcellular organismal process Page 51 of 58
Supporting Information Table 1. List of genes in the human cDNA library used in this study. RefSeq RefSeq RefSeq RefSeq Gene Symbol Gene Symbol Gene Symbol Gene Symbol protein ID protein ID protein ID protein ID AATF NP_036270 BNC1 NP_001708 CNOT3 NP_055331 E4F1 NP_004415 ABLIM1 NP_006711 BNC2 NP_060107 CNOT4 NP_037448 EAF2 NP_060926 ABRA NP_631905 BRAF NP_004324 CNOT6L NP_653172 EBF1 NP_076870 ABT1 NP_037507 BRCA1 NP_009229 CNOT7 NP_037486 ECD NP_009196 ACD NP_075065 BRD8 NP_006687 CNOT7 NP_473367 ECOP NP_110423 ACTL6A NP_004292 BRF1 NP_663718 CNOT8 NP_004770 ECOP NP_110423 ACTR5 NP_079131 BRF2 NP_060780 CNTF NP_000605 EED NP_694536 ACVR1 NP_001096 BRF2 NP_060780 COBRA1 NP_056271 EEF1A1 NP_001393 ACVR1B NP_004293 BRMS1 NP_056214 COMMD7 NP_444269 EEF2K NP_037434 ACVR2A NP_001607 BTBD7 NP_060637 COPS2 NP_004227 EFEMP1 NP_001034438 ACVR2B NP_001097 BTF3 NP_001198 COPS3 NP_003644 EGR1 NP_001955 ACVRL1 NP_000011 BTG1 NP_001722 COPS3 NP_003644 EGR2 NP_000390 ADCK4 NP_079152 BUB1 NP_004327 COPS5 NP_006828 EGR2 NP_000390 ADH1A NP_000658 BUB1B NP_001202 COPS7A NP_057403 EGR3 NP_004421 ADNP NP_056154 BUD31 NP_003901 CRABP2 NP_001869 EGR4 NP_001956 ADRBK1 NP_001610 C14orf166 NP_057123 CREB1 NP_604391 EHF NP_036285 AEBP1 NP_001120 C14orf169 NP_078920 CREB1 NP_004370 EHMT1 NP_079033 AEBP2 NP_694939 C17orf42 NP_078959 CREB3 NP_006359 EHMT2 NP_079532 AES NP_945320 C17orf56 NP_653280 CREB3L1 NP_443086 EID1 NP_055150 AES NP_001121 C19orf2 NP_604431 CREB3L2 NP_919047 EID1 NP_055150 AFAP1L2 NP_115939 C19orf33 NP_277055 CREB3L3 NP_115996 EID2B NP_689574 AGT NP_000020 C1D NP_001177194 CREB3L4 NP_570968 EID3 NP_001008395 AHR NP_001612 C1orf129 NP_079339 CREB5 NP_001011666 EIF2AK2 NP_002750 AKNA NP_110394 C20orf20 NP_060740 CREB5 NP_878901 EIF2C1 NP_036331 AKT2 NP_001617 C2orf3 NP_001188263 CREBL2 NP_001301 ELF1 NP_758961 ALS2CR2 NP_061041 C2orf63 NP_689598 CREG1 NP_003842 ELF2 NP_006865 ALX1 NP_008913 C4orf38 Q96NR7 CREG1 NP_003842 ELF3 NP_001107781 ALX4 NP_068745 C5orf41 NP_001161865 CREM NP_001872 ELF4 NP_001120669 ANKRD1 NP_055206 CABC1 NP_064632 CRTC1 NP_056136 ELF5 NP_001413 ANKRD2 NP_065082 CALCOCO1 NP_065949 CRTC3 NP_001036039 ELK1 NP_001107595 ANKRD49 NP_060174 CAMK1D NP_705718 CSDA NP_003642 ELK3 NP_005221 ANKS1B NP_064525 CAMK2B NP_742075 CSDC2 NP_055275 ELK4 NP_001964 ANP32A NP_006296 CAMK2D NP_742126 CSDE1 NP_009089 ELL NP_006523 APBB2 NP_001159526 CAMK2D NP_001212 CSK NP_001120662 ELL3 NP_079441 APEX1 NP_001632 CAMK4 NP_001735 CSNK1A1L NP_660204 ELP3 NP_060561 APITD1 NP_954988 CAMKK1 NP_115670 CSNK1D NP_001884 ELP4 NP_061913 APLP1 NP_001019978 CAND1 NP_060918 CSNK1G2 NP_001310 EMX1 NP_004088 APLP1 NP_005157 CARHSP1 NP_001035941 CSNK2A1 NP_001886 EMX2 NP_004089 APP NP_000475 CASZ1 NP_060236 CSNK2A2 NP_001887 ENO1 NP_001419 ARAF NP_001645 CBFA2T2 NP_005084 CSRNP3 NP_001165644 ENO1 NP_001419 ARHGAP22 NP_067049 CBFA2T3 NP_005178 CTBP1 NP_001319 ENY2 NP_064574 ARID2 NP_689854 CBFB NP_001746 CTCFL NP_542185 EOMES NP_005433 ARL2BP NP_036238 CBX2 NP_116036 CTNNB1 NP_001091679 EPC1 NP_079485 ARMC10 NP_001154483 CBX3 NP_009207 CTNND1 NP_001078931 EPC2 NP_056445 ARNT NP_848514 CBX5 NP_001120794 CUX1 NP_001904 EPHA3 NP_005224 ARNT NP_001659 CBX8 NP_065700 CXXC1 NP_055408 EPHA4 NP_004429 ARNT2 NP_055677 CC2D1A NP_060191 DACH2 NP_444511 EPHB3 NP_004434 ARNTL NP_001025444 CCAR1 NP_060707 DAXX NP_001341 EPHB4 NP_004435 ARNTL NP_001025443 CCDC101 NP_612423 DBP NP_001343 ERCC8 NP_000073 ARNTL2 NP_064568 CCDC17 NP_001108410 DBX2 NP_001004329 ERCC8 NP_001007234 ARRB1 NP_004032 CCDC17 NP_001108410 DCLK1 NP_004725 ERF NP_006485 ASCC1 NP_001185728 CCDC72 NP_057017 DCP1A NP_060873 ERG NP_891548 ASCC2 NP_115580 CCDC96 NP_699207 DDIT3 NP_004074 ERH NP_004441 ASCL1 NP_004307 CCNA1 NP_001104516 DDR2 NP_001014796 ERI1 NP_699163 ASCL2 NP_005161 CCNE1 NP_001229 DDX1 NP_004930 ERN2 NP_150296 ASF1A NP_054753 CCNH NP_001230 DDX20 NP_009135 ESR1 NP_000116 ASF1B NP_060624 CCNL1 NP_064703 DDX5 NP_004387 ESR2 NP_001035365 ASXL1 NP_056153 CCNL2 NP_001034666 DDX54 NP_076977 ESRRA NP_004442 ATF1 NP_005162 CCNT2 NP_490595 DEAF1 NP_066288 ESRRG NP_001429 ATF2 NP_001871 CCRK NP_848519 DEDD NP_001034801 ESRRG NP_996317 ATF3 NP_001025458 CD28 NP_006130 DEDD2 NP_579874 ETS1 NP_005229 ATF4 NP_001666 CD36 NP_001120916 DEK NP_003463 ETS2 NP_005230 ATF5 NP_036200 CD80 NP_005182 DHFR NP_000782 ETS2 NP_005230 ATF6B NP_004372 CD86 NP_787058 DICER1 NP_085124 ETV1 NP_004947 ATF7 NP_006847 CD86 NP_008820 DIDO1 NP_071388 ETV1 NP_004947 ATF7IP2 NP_079273 CDC40 NP_056975 DIP2A NP_996773 ETV2 NP_055024 ATOH7 NP_660161 CDC45 NP_003495 DKK3 NP_001018067 ETV3L NP_001004341 ATOH8 NP_116216 CDC5L NP_001244 DLX1 NP_835221 ETV4 NP_001073143 ATOH8 NP_116216 CDCA7 NP_114148 DLX2 NP_004396 ETV6 NP_001978 AXIN1 NP_003493 CDCA7L NP_001120842 DLX3 NP_005211 ETV7 NP_057219 AXL NP_068713 CDCA7L NP_001120843 DLX4 NP_612138 EVX1 NP_001980 BACH1 NP_001177 CDH1 NP_004351 DLX5 NP_005212 EWSR1 NP_005234 BAG1 NP_004314 CDK2 NP_001789 DLX6 NP_005213 EZH1 NP_001982 BANP NP_524576 CDK4 NP_000066 DMAP1 NP_001029195 EZH2 NP_694543 BANP NP_060339 CDK5 NP_004926 DMRT1 NP_068770 EZH2 NP_694543 BANP NP_060339 CDK7 NP_001790 DMRT2 NP_006548 FADS1 NP_037534 BARD1 NP_000456 CDK9 NP_001252 DMRTA1 NP_071443 FAM136A NP_116211 BARHL1 NP_064448 CDKL2 NP_003939 DMRTB1 NP_149056 FAM90A1 NP_060558 BARX1 NP_067545 CDKL3 NP_001107047 DMRTC2 NP_001035373 FARSA NP_004452 BATF NP_006390 CDX1 NP_001795 DMTF1 NP_001135799 FASTK NP_006703 BATF2 NP_612465 CEBPD NP_005186 DNAJB5 NP_036398 FBXO5 NP_001135994 BAZ1A NP_038476 CEBPE NP_001796 DNAJC1 NP_071760 FES NP_001996 BAZ2A NP_038477 CEBPG NP_001797 DNM2 NP_001005360 FEV NP_059991 BCKDK NP_005872 CEBPZ NP_005751 DNMT1 NP_001370 FEZF1 NP_001019784 BCL10 NP_003912 CENPB NP_001801 DNMT3A NP_072046 FGF2 NP_001997 BCL11A NP_060484 CHD4 NP_001264 DNMT3B NP_787045 FHL2 NP_001034581 BCL11A NP_075044 CHEK2 NP_009125 DNMT3L NP_787063 FHL3 NP_004459 BCL2 NP_000624 CHRAC1 NP_059140 DPF1 NP_001128628 FHL5 NP_065228 BCL6 NP_001697 CHURC1 NP_660148 DPF2 NP_006259 FIZ1 NP_116225 BCL6B NP_862827 CIAO1 NP_004795 DPF3 NP_036206 FLI1 NP_002008 BHLHB2 NP_003661 CIAO1 NP_004795 DPPA2 NP_620170 FLJ23356 NP_115613 Page 52 of 58
BLK NP_001706 CIR1 NP_004873 DR1 NP_001929 FLJ25006 NP_653211 BLZF1 NP_003657 CITED1 NP_004134 DRAP1 NP_006433 FLJ36070 NP_872380 BMI1 NP_005171 CITED2 NP_006070 DYRK1B NP_004705 FLJ44894 NP_001034973 BMP4 NP_570912 CLK1 NP_004062 DYRK2 NP_006473 FLT1 NP_002010 BMP7 NP_001710 CNBP NP_003409 E2F1 NP_005216 FOS NP_005243 BMPR1A NP_004320 CNBP NP_003409 E2F2 NP_004082 FOSB NP_006723 BMPR1B NP_001194 CNOT1 NP_057368 E2F4 NP_001941 FOSL1 NP_005429 BMPR2 NP_001195 CNOT2 NP_055330 E2F5 NP_001942 FOSL2 NP_005244 BMX NP_001712 CNOT2 NP_055330 E2F6 NP_937987 FOXA1 NP_004487
RefSeq RefSeq RefSeq RefSeq Gene Symbol Gene Symbol Gene Symbol Gene Symbol protein ID protein ID protein ID protein ID FOXA2 NP_068556 HEY1 NP_036390 ILK NP_001014794 LOC401898 NP_001013713 FOXA3 NP_004488 HEY1 NP_001035798 ING1 NP_937860 LOC91431 NP_001093246 FOXD4L1 NP_036316 HEY1 NP_036390 ING2 NP_001555 LRPPRC NP_573566 FOXI1 NP_036320 HEY2 NP_036391 ING3 NP_061944 LUC7L3 NP_006098 FOXI2 NP_997309 HEYL NP_055386 ING4 NP_001121055 LUZP4 NP_057467 FOXJ1 NP_001445 HHEX NP_002720 INHBA NP_002183 LYAR NP_060286 FOXK2 NP_004505 HIF1A NP_851397 INSM2 NP_115983 LYK5 NP_001003786 FOXL2 NP_075555 HIF1A NP_001521 INSR NP_001073285 LYL1 NP_005574 FOXM1 NP_068772 HIF1AN NP_060372 INTS12 NP_065128 LZTR1 NP_006758 FOXN1 NP_003584 HIF3A NP_690008 INTS4L1 Q96LV5 LZTR1 NP_006758 FOXN2 NP_002149 HIF3A NP_690007 IRAK4 NP_001107654 MACC1 NP_877439 FOXN3 NP_005188 HIF3A NP_071907 IRF1 NP_002189 MAD2L2 NP_001120797 FOXN4 NP_998761 HIPK4 NP_653286 IRF2 NP_002190 MAEL NP_116247 FOXO3 NP_001446 HLF NP_002117 IRF2BP1 NP_056464 MAF1 NP_115648 FOXO4 NP_005929 HLX NP_068777 IRF3 NP_001562 MAFB NP_005452 FOXP1 NP_116071 HLX NP_068777 IRF4 NP_002451 MAFF NP_036455 FOXP3 NP_001107849 HMG20A NP_060670 IRF6 NP_006138 MAFG NP_002350 FOXR2 NP_940853 HMG20B NP_006330 IRF8 NP_002154 MAFK NP_002351 FOXS1 NP_004109 HMGA1 NP_665908 IRX3 NP_077312 MAML3 NP_061187 FRK NP_002022 HMGB1 NP_002119 IRX3 NP_077312 MAP2K2 NP_109587 FST NP_037541 HMGB2 NP_002120 IRX6 NP_077311 MAP2K3 NP_659731 FUBP1 NP_003893 HMGN1 NP_004956 ISL1 NP_002193 MAP2K3 NP_002747 FUBP3 NP_003925 HMGN2 NP_005508 ISX NP_001008494 MAP2K6 NP_002749 FYN NP_002028 HMGN4 NP_006344 ITGB3BP NP_055103 MAP3K14 NP_003945 GABPA NP_002031 HMOX1 NP_002124 ITK NP_005537 MAP3K15 NP_001001671 GABPB1 NP_005245 HMX2 NP_005510 IWS1 NP_060439 MAP3K5 NP_005914 GABPB2 NP_653219 HN1L NP_653171 JARID1C NP_004178 MAP4K5 NP_006566 GAS7 NP_003635 HNF1B NP_000449 JARID2 NP_004964 MAPK10 NP_620446 GATA2 NP_116027 HNF4A NP_000448 JAZF1 NP_778231 MAPK13 NP_002745 GATA3 NP_001002295 HNF4G NP_004124 JDP2 NP_001128520 MAPK14 NP_620581 GATAD1 NP_066990 HNRNPAB NP_004490 JMJD5 NP_079049 MAPK3 NP_002737 GATAD1 NP_066990 HNRNPAB NP_112556 JMY NP_689618 MAPK6 NP_002739 GATAD2A NP_060130 HNRNPD NP_112737 JRKL NP_003763 MAPK8 NP_002741 GATAD2B NP_065750 HNRNPD NP_112738 JUN NP_002219 MAPK8 NP_620635 GBX1 NP_001092304 HNRNPK NP_002131 JUNB NP_002220 MAPK9 NP_002743 GCLC NP_001489 HNRNPU NP_114032 KAT2B NP_003875 MAPKAPK3 NP_004626 GCN5L2 NP_066564 HNRNPUL1 NP_653333 KBTBD8 NP_115894 MAPKAPK5 NP_003659 GFI1 NP_005254 HNRPDL NP_112740 KCMF1 NP_064507 MATK NP_647612 GFI1B NP_004179 HNRPUL1 NP_008971 KCMF1 NP_064507 MATK NP_002369 GLI4 NP_612474 HOXA1 NP_005513 KCNH6 NP_775115 MAVS NP_065797 GLIS1 NP_671726 HOXA10 NP_714926 KCNIP3 NP_038462 MAX NP_002373 GLIS2 NP_115964 HOXA11 NP_005514 KCTD7 NP_694578 MAX NP_660092 GLP-1 NP_001096637 HOXA2 NP_006726 KDM1B NP_694587 MBD1 NP_001191066 GMCL1 NP_848526 HOXA3 NP_705896 KDM2B NP_001005366 MBD2 NP_003918 GMEB1 NP_077808 HOXA3 NP_109377 KDM4A NP_055478 MBD3L1 NP_660209 GMEB1 NP_006573 HOXA5 NP_061975 KDM4D NP_060509 MBD6 NP_443129 GMEB1 NP_077808 HOXA7 NP_008827 KEAP1 NP_987096 MBNL3 NP_060858 GMEB2 NP_036516 HOXA9 NP_689952 KHDRBS1 NP_006550 MBTD1 NP_060113 GMNN NP_056979 HOXB13 NP_006352 KLF1 NP_006554 MDFI NP_005577 GPBP1L1 NP_067652 HOXB3 NP_002137 KLF10 NP_005646 MEAF6 NP_073593 GRHL1 NP_055367 HOXB3 NP_002137 KLF11 NP_003588 MECOM NP_001098547 GRHL2 NP_079191 HOXB5 NP_002138 KLF12 NP_009180 MECP2 NP_004983 GRHL3 NP_937816 HOXB6 NP_061825 KLF15 NP_054798 MED1 NP_004765 GRIN1 NP_015566 HOXB7 NP_004493 KLF17 NP_775755 MED11 NP_001001683 GRINL1A NP_056347 HOXB8 NP_076921 KLF3 NP_057615 MED12 NP_005111 GRIP1 NP_066973 HOXB9 NP_076922 KLF4 NP_004226 MED14 NP_004220 GRK1 NP_002920 HOXC10 NP_059105 KLF5 NP_001721 MED15 NP_056973 GRK6 NP_001004106 HOXC11 NP_055027 KLF6 NP_001291 MED16 NP_005472 GSC NP_776248 HOXC13 NP_059106 KLF7 NP_003700 MED17 NP_004259 GSC2 NP_005306 HOXC4 NP_055435 KLF7 NP_003700 MED18 NP_001120822 GSK3A NP_063937 HOXC6 NP_710160 KLF9 NP_001197 MED19 NP_703151 GTF2A2 NP_004483 HOXC8 NP_073149 KLHDC2 NP_055130 MED20 NP_004266 GTF2B NP_001505 HOXC9 NP_008828 KRBA2 NP_998762 MED21 NP_004255 GTF2E2 NP_002086 HOXC9 NP_008828 L3MBTL2 NP_113676 MED22 NP_598395 GTF2F1 NP_002087 HOXD1 NP_078777 L3MBTL4 NP_775735 MED23 NP_004821 GTF2F1 NP_002087 HOXD3 NP_008829 LANCL2 NP_061167 MED24 NP_001072986 GTF2F2 NP_004119 HOXD4 NP_055436 LASS2 NP_071358 MED25 NP_112235 GTF2H1 NP_005307 HOXD8 NP_062458 LASS3 NP_849164 MED26 NP_004822 GTF2H1 NP_005307 HSBP1 NP_001528 LASS4 NP_078828 MED27 NP_004260 GTF2H2C NP_001092198 HSF1 NP_005517 LASS5 NP_671723 MED28 NP_079481 GTF2H3 NP_001507 HSF2 NP_001129036 LASS6 NP_982288 MED29 NP_060062 GTF2H5 NP_997001 HSF2BP NP_008962 LBH NP_112177 MED29 NP_060062 GTF2I NP_127492 HSFX1 NP_057237 LCK NP_001036236 MED30 NP_542382 GTF2IRD1 NP_057412 HSFY1 NP_149099 LCOR NP_115816 MED31 NP_057144 GTF3C1 NP_001511 HSPB8 NP_055180 LCORL NP_710153 MED4 NP_054885 GTF3C3 NP_036218 HTATIP NP_006379 LCORL NP_710153 MED6 NP_005457 GTF3C3 NP_036218 HTATIP2 NP_001091991 LDB1 NP_003884 MED7 NP_001094286 GTF3C5 NP_036219 ICK NP_055735 LEP NP_000221 MED8 NP_443109 GTF3C6 NP_612417 ID1 NP_002156 LETMD1 NP_056231 MED8 NP_963836 Page 53 of 58
GZF1 NP_071927 ID2 NP_002157 LGALS3 NP_002297 MED9 NP_060489 GZF1 NP_071927 ID3 NP_002158 LHX2 NP_004780 MEF2A NP_005578 HAND1 NP_004812 ID4 NP_001537 LHX4 NP_203129 MEF2C NP_002388 HBP1 NP_036389 IER5 NP_057629 LHX6 NP_055183 MEF2D NP_005911 HCFC2 NP_037452 IFI16 NP_005522 LHX6 NP_954629 MEIS1 NP_002389 HCLS1 NP_005326 IFNB1 NP_002167 LHX8 NP_001001933 MEIS2 NP_758527 HDAC1 NP_004955 IFNG NP_000610 LIMK1 NP_002305 MEIS2 NP_758526 HDAC11 NP_079103 IFT57 NP_060480 LIN28 NP_078950 MEIS2 NP_733776 HDAC3 NP_003874 IGHMBP2 NP_002171 LIN28B NP_001004317 MEIS3 NP_064545 HDAC4 NP_006028 IGSF1 NP_001164434 LIN54 NP_001108480 MEN1 NP_570711 HDAC5 NP_005465 IKBKAP NP_003631 LIN9 NP_775106 MEOX2 NP_005915 HDAC8 NP_060956 IKBKG NP_001138727 LITAF NP_004853 MESP1 NP_061140 HDAC9 NP_055522 IKZF4 NP_071910 LMCD1 NP_055398 MGC21874 NP_689506 HDGF NP_004485 IKZF4 NP_071910 LMO1 NP_002306 MGC42105 NP_699192 HDX NP_653258 IKZF5 NP_071911 LMO2 NP_001135788 MGC46336 XP_001720872 HES1 NP_005515 IL11 NP_000632 LMO3 NP_001001395 MID1 NP_000372 HES2 NP_061962 IL2 NP_000577 LMO3 NP_061110 MIER2 NP_060020 HES4 NP_066993 IL33 NP_254274 LMO7 NP_005349 MITF NP_937802 HEXIM1 NP_006451 IL6 NP_000591 LMX1A NP_796372 MIZF NP_056332 HEXIM1 NP_006451 ILF2 NP_004506 LOC100290062 XP_002346980 MKL2 NP_054767 HEXIM2 NP_653209 ILF3 NP_036350 LOC152485 NP_849157 MKNK1 NP_003675
RefSeq RefSeq RefSeq RefSeq Gene Symbol Gene Symbol Gene Symbol Gene Symbol protein ID protein ID protein ID protein ID MKRN1 NP_038474 NFXL1 NP_694540 PDCD6 NP_037364 PRAME NP_996839 MKRN1 NP_038474 NFYA NP_068351 PDE8A NP_775656 PRDM1 NP_001189 MKRN2 NP_054879 NFYB NP_006157 PDGFB NP_002599 PRDM11 NP_064614 MKX NP_775847 NFYC NP_055038 PDIK1L NP_690048 PRDM14 NP_078780 MLF1 NP_001182361 NHLH1 NP_005589 PDK1 NP_002601 PRDM4 NP_036538 MLF1IP NP_078905 NHLH2 NP_001104531 PDK2 NP_002602 PREB NP_037520 MLKL NP_689862 NIF3L1 NP_068596 PDK3 NP_005382 PRICKLE1 NP_001138355 MLX NP_937847 NKAP NP_078804 PDK4 NP_002603 PRICKLE3 NP_006141 MLX NP_733752 NKRF NP_060014 PDPK1 NP_002604 PRICKLE3 NP_006141 MLX NP_937847 NKX2-5 NP_004378 PDRG1 NP_110442 PRKACA NP_002721 MLZE NP_113603 NKX2-8 NP_055175 PDS5B NP_055847 PRKACB NP_002722 MNDA NP_002423 NKX3-2 NP_001180 PDX1 NP_000200 PRKAR1A NP_997637 MNT NP_064706 NKX6-3 NP_689781 PELP1 NP_055204 PRKCA NP_002728 MORF4L1 NP_006782 NME2 NP_001018147 PEX14 NP_004556 PRKCB1 NP_002729 MORF4L1 NP_996670 NMI NP_004679 PEX14 NP_004556 PRKCD NP_006245 MORF4L2 NP_036418 NMRAL1 NP_065728 PFDN1 NP_002613 PRKCE NP_005391 MOV10 NP_001123551 NONO NP_001138880 PFDN5 NP_002615 PRKCG NP_002730 MRPL28 NP_006419 NOSTRIN NP_443178 PHB NP_002625 PRKCH NP_006246 MRRF NP_620132 NOTO XP_001719406 PHB2 NP_009204 PRKCZ NP_002735 MSTN NP_005250 NPAS1 NP_002508 PHF10 NP_060758 PRKD1 NP_002733 MSX1 NP_002439 NPAS4 NP_849195 PHF11 NP_001035533 PRKD2 NP_001073351 MSX2 NP_002440 NPM1 NP_002511 PHF12 NP_001028733 PRKX NP_005035 MTA3 NP_065795 NPM1 NP_002511 PHF13 NP_722519 PRMT2 NP_996845 MTERF NP_008911 NR0B1 NP_000466 PHF13 NP_722519 PRMT5 NP_006100 MTERF NP_008911 NR0B2 NP_068804 PHF15 NP_056103 PRMT7 NP_061896 MTERFD3 NP_001028222 NR1D1 NP_068370 PHF17 NP_955352 ProSAPiP1 NP_055546 MTF1 NP_005946 NR1D2 NP_005117 PHF17 NP_079176 PRPF4B NP_003904 MTF2 NP_031384 NR1D2 NP_005117 PHF17 NP_955352 PRPF6 NP_036601 MTPAP NP_060579 NR1H2 NP_009052 PHF19 NP_056466 PRPF6 NP_036601 MXD1 NP_002348 NR1H3 NP_005684 PHF20 NP_057520 PRR13 NP_060927 MXD3 NP_112590 NR1H4 NP_005114 PHF21A NP_057705 PRRX1 NP_073207 MXD4 NP_006445 NR1H4 NP_005114 PHF23 NP_077273 PRRX2 NP_057391 MXI1 NP_569157 NR1I2 NP_003880 PHF5A NP_116147 PSMC3 NP_002795 MYB NP_005366 NR1I3 NP_005113 PHF6 NP_115711 PSMC5 NP_002796 MYBBP1A NP_055335 NR1I3 NP_001070945 PHF7 NP_057567 PSMD9 NP_002804 MYBL2 NP_002457 NR2C1 NP_003288 PHKG2 NP_000285 PSPC1 NP_001035879 MYC NP_002458 NR2C2 NP_003289 PHOX2A NP_005160 PTGES2 NP_079348 MYCBP NP_036465 NR2E1 NP_003260 PHOX2B NP_003915 PTK2B NP_004094 MYD88 NP_002459 NR2F1 NP_005645 PIAS1 NP_057250 PTRF NP_036364 MYEF2 NP_057216 NR2F2 NP_066285 PIAS1 NP_057250 PTRF NP_036364 MYF6 NP_002460 NR2F6 NP_005225 PIAS3 NP_006090 PTTG1 NP_004210 MYLK3 NP_872299 NR3C1 NP_000167 PIAS4 NP_056981 PUF60 NP_510965 MYNN NP_061127 NR3C2 NP_000892 PIBF1 NP_006337 PURA NP_005850 MYOCD NP_001139785 NR4A1 NP_002126 PICALM NP_001008660 PUS1 NP_079491 MYOD1 NP_002469 NR4A2 NP_006177 PIK3R4 NP_055417 PWP1 NP_008993 MYOG NP_002470 NR5A1 NP_004950 PIM1 NP_002639 PYGO2 NP_612157 MYST1 NP_115564 NR5A2 NP_995582 PIM2 NP_006866 RAB18 NP_067075 MYST2 NP_008998 NR5A2 NP_003813 PIM3 NP_001001852 RAB24 NP_001026847 MZF1 NP_932172 NR6A1 NP_001480 PIR NP_001018119 RABGEF1 NP_055319 NAA15 NP_476516 NRBF2 NP_110386 PITX1 NP_002644 RABGEF1 NP_055319 NAA16 NP_078837 NRBP1 NP_037524 PITX1 NP_002644 RAD18 NP_064550 NAB1 NP_005957 NRG1 NP_039258 PITX2 NP_000316 RAD54B NP_036547 NAB2 NP_005958 NRG1 NP_039252 PITX2 NP_700475 RAF1 NP_002871 NACA NP_001106673 NRG1 NP_039253 PITX3 NP_005020 RAN NP_006316 NANOG NP_079141 NRIP1 NP_003480 PIWIL2 NP_060538 RARA NP_000955 NANOGP1 Q8N7R0 NRIP2 NP_113662 PKIA NP_862822 RARB NP_000956 NARFL NP_071938 NRL NP_006168 PKMYT1 NP_872629 RARB NP_057236 NAT14 NP_065111 NSBP1 NP_110390 PKNOX2 NP_071345 RARG NP_001036193 NCOA1 NP_671756 NTRK1 NP_002520 PLAG1 NP_001108106 RARG NP_000957 NCOA3 NP_858045 NUAK2 NP_112214 PLAG1 NP_001108107 RB1 NP_000312 NCOA4 NP_005428 NUP62 NP_001180286 PLAGL1 NP_001074424 RBBP4 NP_005601 NCOA7 NP_001116314 OASL NP_003724 PLAGL1 NP_001074420 RBBP5 NP_005048 NCRNA00288 Q8N7Y1 OGFR NP_031372 PLAGL1 NP_001074420 RBBP7 NP_002884 NDN NP_002478 OLIG1 NP_620450 PLK1 NP_005021 RBBP8 NP_002885 NDNL2 NP_619649 OLIG2 NP_005797 PLK2 NP_006613 RBM14 NP_006319 NDP NP_000257 OLIG3 NP_786923 PLK4 NP_055079 RBM23 NP_060577 NEIL3 NP_060718 ONECUT1 NP_004489 PLRG1 NP_002660 RBM23 NP_001070820 Page 54 of 58
NEK10 NP_689747 OPTN NP_001008212 PMF1 NP_001186582 RBM39 NP_909122 NEK10 NP_001026911 OSM NP_065391 PMFBP1 NP_112583 RBM39 NP_909122 NEK2 NP_002488 OSR1 NP_660303 PMFBP1 NP_112583 RBM9 NP_001076048 NEK3 NP_002489 OSR2 NP_443727 PML NP_150241 RBPJ NP_056958 NEK6 NP_055212 OTP NP_115485 PNRC1 NP_006804 RC3H1 NP_742068 NEURL NP_004201 OTX1 NP_055377 POGK NP_060012 RC3H2 NP_001094058 NEUROD1 NP_002491 OTX2 NP_758840 POGZ NP_665739 RC3H2 NP_061323 NEUROD4 NP_067014 OVOL1 NP_004552 POLE3 NP_059139 RCAN1 NP_981963 NEUROD6 NP_073565 OVOL2 NP_067043 POLR1B NP_061887 RCBTB1 NP_060661 NEUROG1 NP_006152 OXSR1 NP_005100 POLR1D NP_057056 RCOR2 NP_775858 NEUROG2 NP_076924 PA2G4 NP_006182 POLR1E NP_071935 RCOR3 NP_060724 NEUROG3 NP_066279 PAF1 NP_061961 POLR2C NP_116558 REL NP_002899 NFATC1 NP_006153 PAK4 NP_001014831 POLR2K NP_005025 RELB NP_006500 NFATC2 NP_036472 PAK6 NP_064553 POLR3B NP_001154180 REM2 NP_775798 NFATC2IP NP_116204 PARP1 NP_001609 POLR3D NP_001713 REXO4 NP_065118 NFATC4 NP_004545 PARP15 NP_689828 POLR3E NP_060589 RFX2 NP_602309 NFE2 NP_006154 PAWR NP_002574 POLR3H NP_001018060 RFX3 NP_602304 NFE2L1 NP_003195 PAX3 NP_852123 POU2AF1 NP_006226 RFX4 NP_115880 NFE2L2 NP_006155 PAX6 NP_000271 POU2F1 NP_002688 RFX4 NP_115880 NFE2L2 NP_006155 PAX8 NP_003457 POU2F2 NP_001234923 RFX5 NP_000440 NFIA NP_005586 PAX9 NP_006185 POU3F2 NP_005595 RFX6 NP_775831 NFIB NP_005587 PBX1 NP_002576 POU3F4 NP_000298 RFXAP NP_000529 NFIC NP_995315 PBX4 NP_079521 POU4F2 NP_004566 RFXDC2 NP_073752 NFIL3 NP_005375 PBXIP1 NP_065385 POU5F1 NP_002692 RGS14 NP_006471 NFIX NP_002492 PCBD1 NP_000272 POU5F2 NP_694948 RHOA NP_001655 NFKB1 NP_003989 PCGF1 NP_116062 POU6F1 NP_002693 RHOXF2 NP_115887 NFKB2 NP_001070961 PCGF2 NP_009075 PPARA NP_001001928 RHOXF2B NP_001093155 NFKBIA NP_065390 PCGF6 NP_001011663 PPARD NP_006229 RING1 NP_002922 NFKBIB NP_001001716 PCID2 NP_001120674 PPARG NP_056953 RIOK1 NP_113668 NFKBIB NP_002494 PCIF1 NP_071387 PPARG NP_619725 RIOK2 NP_060813 NFKBIL1 NP_004998 PCNA NP_872590 PPARGC1B NP_573570 RIOK3 NP_003822 NFKBIZ NP_001005474 PCTK1 NP_006192 PPIE NP_006103 RNF10 NP_055683 NFRKB NP_006156 PCTK2 NP_002586 PQBP1 NP_001027553 RNF103 NP_005658
RefSeq RefSeq RefSeq RefSeq Gene Symbol Gene Symbol Gene Symbol Gene Symbol protein ID protein ID protein ID protein ID RNF111 NP_060080 SMARCE1 NP_003070 TAF2 NP_003175 TP73 NP_001119712 RNF113A NP_008909 SMURF2 NP_073576 TAF6 NP_001177344 TP73 NP_001119714 RNF113B NP_849192 SMYD1 NP_938015 TAF6 NP_620835 TRAF6 NP_665802 RNF12 NP_057204 SNAI1 NP_005976 TAF6L NP_006464 TRAF7 NP_115647 RNF14 NP_004281 SNAI2 NP_003059 TAF7 NP_005633 TRAPPC2 NP_001011658 RNF141 NP_057506 SNAPC1 NP_003073 TAF9 NP_001015892 TRERF1 NP_277037 RNF168 NP_689830 SNAPC2 NP_003074 TAF9B NP_057059 TRIB2 NP_067675 RNF2 NP_009143 SNAPC5 NP_006040 TAOK1 NP_065842 TRIB3 NP_066981 RNF20 NP_062538 SND1 NP_055205 TAOK2 NP_004774 TRIM16 NP_006461 RNF24 NP_001127809 SNF1LK NP_775490 TAOK2 NP_057235 TRIM24 NP_056989 RNF4 NP_002929 SNIP1 NP_078976 TAOK3 NP_057365 TRIM28 NP_005753 RNF6 NP_005968 SNIP1 NP_078976 TARBP2 NP_599150 TRIM28 NP_005753 RORA NP_599023 SNW1 NP_036377 TARDBP NP_031401 TRIM31 NP_008959 RORB NP_008845 SOHLH1 NP_001012415 TASP1 NP_060184 TRIM42 NP_689829 RORC NP_001001523 SOHLH2 NP_060296 TAX1BP1 NP_001073333 TRIM45 NP_079464 RORC NP_005051 SOLH NP_005623 TAX1BP1 NP_006015 TRIM46 NP_079334 RP6-213H19.1 NP_001035918 SOX12 NP_008874 TAX1BP1 NP_006015 TRIM52 NP_116154 RPAP1 NP_056355 SOX14 NP_004180 TAX1BP3 NP_055419 TRIM62 NP_060677 RPS6KA2 NP_066958 SOX15 NP_008873 TBC1D2B NP_055894 TRIM73 NP_944606 RPS6KA3 NP_004577 SOX17 NP_071899 TBP NP_003185 TRIP4 NP_057297 RPS6KA6 NP_055311 SOX2 NP_003097 TBPL1 NP_004856 TRIP6 NP_003293 RPS6KL1 NP_113652 SOX5 NP_008871 TBR1 NP_006584 TRMT1 NP_060192 RQCD1 NP_005435 SOX5 NP_821078 TBX15 NP_689593 TSC22D1 NP_006013 RRAGC NP_071440 SOX7 NP_113627 TBX21 NP_037483 TSC22D3 NP_932174 RRN3 NP_060897 SOX8 NP_055402 TBX22 NP_001103349 TSC22D3 NP_004080 RRN3 NP_060897 SP1 NP_612482 TBX6 NP_542936 TSFM NP_001166167 RRN3P1 Q2M238 SP100 NP_001193633 TCEA1 NP_006747 TSG101 NP_006283 RSBN1 NP_060834 SP110 NP_536349 TCEA1 NP_958845 TSHZ1 NP_005777 RTF1 NP_055953 SP3 NP_003102 TCEA2 NP_942016 TSHZ2 NP_775756 RUNX1 NP_001116079 SP4 NP_003103 TCEA3 NP_003187 TSSK2 NP_443732 RUNX1T1 NP_004340 SP6 NP_954871 TCEAL1 NP_001006641 TSSK3 NP_443073 RUNX3 NP_001026850 SP7 NP_690599 TCEAL2 NP_525129 TSSK6 NP_114426 RUVBL1 NP_003698 SP8 NP_874359 TCEAL3 NP_001006934 TTF1 NP_031370 RUVBL2 NP_006657 SPDEF NP_036523 TCEAL4 NP_001006938 TTF2 NP_003585 RXRA NP_002948 SPIB NP_003112 TCEAL8 NP_001006685 TTK NP_003309 RXRB NP_068811 SPIC NP_689536 TCEANC2 NP_694580 TTRAP NP_057698 RXRG NP_008848 SPOP NP_001007227 TCEB1 NP_001191790 TULP1 NP_003313 RYBP NP_036366 SPOP NP_001007227 TCEB2 NP_009039 TULP2 NP_003314 S100A1 NP_006262 SPRYD5 NP_116070 TCEB3 NP_003189 TULP3 NP_003315 SAFB NP_002958 SPZ1 NP_115956 TCEB3B NP_057511 TWF2 NP_009215 SALL4 NP_065169 SPZ1 NP_115956 TCERG1L NP_777597 TWIST2 NP_476527 SAP130 NP_001139400 SQSTM1 NP_003891 TCF19 NP_009040 TWISTNB NP_001002926 SAP18 NP_005861 SRA1 NP_001030312 TCF21 NP_003197 TXN NP_003320 SAP30 NP_003855 SREBF1 NP_001005291 TCF4 NP_001077431 TYRO3 NP_006284 SAP30BP NP_037392 SREBF2 NP_004590 TCF4 NP_003190 U2AF1 NP_006749 SATB1 NP_002962 SRFBP1 NP_689759 TCF7 NP_963963 UBA3 NP_937838 SATB2 NP_056080 SRFBP1 NP_689759 TCF7L2 NP_110383 UBE2K NP_005330 SAV1 NP_068590 SRPK1 NP_003128 TCP10L NP_653260 UBE2V1 NP_068823 SAV1 NP_068590 SRPK2 NP_872633 TDGF1 NP_003203 UBE3A NP_570854 SBNO2 NP_001093592 SRSF10 NP_001177934 TEAD2 NP_003589 UBP1 NP_001121632 SCAI NP_775961 SS18 NP_001007560 TEF NP_003207 UHMK1 NP_787062 SCAND1 NP_057642 SS18L1 NP_945173 TEF NP_003207 UHRF1 NP_001041666 SCAND2 Q9GZW5-2 SSBP2 NP_036578 TFAM NP_003192 UIMC1 NP_057374 SCAP NP_036367 SSBP2 NP_036578 TFAM NP_003192 ULK2 NP_055498 SCGB1A1 NP_003348 SSRP1 NP_003137 TFAP2B NP_003212 UNKL NP_075564 Page 55 of 58
SCMH1 NP_001026864 SSX1 NP_005626 TFAP2C NP_003213 USF1 NP_009053 SCML2 NP_006080 SSX2 NP_003138 TFAP2E NP_848643 USF2 NP_003358 SCML4 NP_932347 SSX3 NP_783642 TFAP4 NP_003214 USP2 NP_004196 SCYL3 NP_065156 SSX5 NP_066295 TFB1M NP_057104 USP47 NP_060414 SEC14L2 NP_036561 STAT1 NP_009330 TFB2M NP_071761 UXT NP_004173 SEC14L2 NP_036561 STAT1 NP_644671 TFCP2 NP_005644 VAV1 NP_005419 SERTAD1 NP_037508 STAT3 NP_003141 TFCP2L1 NP_055368 VAX1 NP_954582 SERTAD3 NP_037500 STAT5A NP_003143 TFDP2 NP_006277 VAX2 NP_036608 SET NP_001116293 STAT5B NP_036580 TFE3 NP_006512 VDR NP_000367 SETDB1 NP_001138887 STAT6 NP_001171550 TFEC NP_036384 VEGFA NP_001165097 SETDB2 NP_114121 STK10 NP_005981 TFPT NP_037474 VGLL2 NP_872586 SF1 NP_004621 STK11 NP_000446 TGFB1 NP_000651 VGLL4 NP_055482 SFPQ NP_005057 STK16 NP_001008910 TGFB1I1 NP_057011 VIPAR NP_001180246 SFRP2 NP_003004 STK17B NP_004217 TGFBR2 NP_003233 VPS72 NP_005988 SFRS2 NP_003007 STK24 NP_001027467 TGIF1 NP_775301 VRK2 NP_006287 SFRS2B NP_115285 STK25 NP_006365 TGIF1 NP_733796 WAC NP_057712 SGK1 NP_005618 STK3 NP_006272 TGIF2 NP_068581 WASL NP_003932 SGK3 NP_001028750 STK32A NP_659438 TGIF2LY NP_631960 WBP2NL NP_689826 SHOX2 NP_003021 STK33 NP_112168 TH1L NP_945327 WBP2NL NP_689826 SHOX2 NP_003021 STK36 NP_056505 TH1L NP_945327 WDR13 NP_060353 SIAH2 NP_005058 STK38 NP_009202 THAP1 NP_060575 WDR5 NP_438172 SIRT2 NP_036369 STK38L NP_055815 THOC1 NP_005122 WDR77 NP_077007 SIRT4 NP_036372 STK40 NP_114406 THRA NP_955366 WEE1 NP_003381 SIRT7 NP_057622 SUB1 NP_006704 THRA NP_001177848 WHSC1 NP_015627 SIX1 NP_005973 SUFU NP_057253 THRB NP_000452 WHSC1 NP_579889 SIX6 NP_031400 SUMO1 NP_001005781 THRSP NP_003242 WHSC1L1 NP_060248 SKI NP_003027 SUPT3H NP_852001 TIAL1 NP_001029097 WHSC2 NP_005654 SLA2 NP_115590 SUPT3H NP_003590 TIGD1 NP_663748 WIZ NP_067064 SLC12A8 NP_078904 SUPT4H1 NP_003159 TIGD4 NP_663772 WNT3A NP_149122 SLC26A3 NP_000102 SUPT5H NP_001104490 TIGD6 NP_112215 WNT7A NP_004616 SLC30A9 NP_006336 SUPT7L NP_055675 TIGD7 NP_149985 WT1 NP_077742 SLTM NP_079031 SUV39H2 NP_001180354 TLE1 NP_005068 WWP1 NP_008944 SMAD1 NP_001003688 TADA2L NP_597683 TLE3 NP_065959 WWTR1 NP_056287 SMAD2 NP_001003652 TADA2L NP_001479 TLE6 NP_079036 XBP1 NP_001073007 SMAD2 NP_001003652 TADA3L NP_006345 TLK1 NP_036422 XRCC6 NP_001460 SMAD3 NP_005893 TAF10 NP_006275 TLX2 NP_057254 XRN2 NP_036387 SMAD5 NP_001001419 TAF11 NP_005634 TLX3 NP_066305 YAF2 NP_001177909 SMAD6 NP_005576 TAF12 NP_005635 TMX1 NP_110382 YBX1 NP_004550 SMAD7 NP_001177752 TAF13 NP_005636 TNF NP_000585 YBX2 NP_057066 SMAD9 NP_001120689 TAF15 NP_631961 TNFAIP3 NP_006281 YEATS4 NP_006521 SMARCA4 NP_001122316 TAF15 NP_003478 TNIP2 NP_077285 YES1 NP_005424 SMARCA5 NP_003592 TAF1A NP_647603 TOX2 NP_001092268 YWHAB NP_003395 SMARCD1 NP_003067 TAF1A NP_001188465 TP53 NP_000537 YWHAH NP_003396 SMARCD2 NP_003068 TAF1C NP_647610 TP53INP1 NP_150601 YY1 NP_003394 SMARCD3 NP_001003802 TAF1D NP_077021 TP53RK NP_291028 YY2 NP_996806
RefSeq RefSeq RefSeq RefSeq Gene Symbol Gene Symbol Gene Symbol Gene Symbol protein ID protein ID protein ID protein ID ZAK NP_598407 ZNF154 NP_001078853 ZNF395 NP_061130 ZNF616 NP_848618 ZAP70 NP_001070 ZNF155 NP_003436 ZNF396 NP_665699 ZNF619 NP_775927 ZBED2 NP_078784 ZNF155 NP_003436 ZNF397 NP_115723 ZNF620 NP_787084 ZBED3 NP_115743 ZNF16 NP_001025147 ZNF398 NP_065832 ZNF621 NP_001091884 ZBTB1 NP_001116801 ZNF165 NP_003438 ZNF408 NP_079017 ZNF622 NP_219482 ZBTB10 NP_001264074 ZNF17 NP_008890 ZNF41 NP_700359 ZNF623 NP_001075949 ZBTB11 NP_055230 ZNF175 NP_009078 ZNF410 NP_067011 ZNF626 NP_001070143 ZBTB12 NP_862825 ZNF177 NP_001166122 ZNF414 NP_115746 ZNF627 NP_660338 ZBTB16 NP_001018011 ZNF18 NP_653281 ZNF415 NP_060825 ZNF630 NP_001032824 ZBTB17 NP_003434 ZNF180 NP_037388 ZNF415 NP_060825 ZNF639 NP_057415 ZBTB17 NP_003434 ZNF180 NP_037388 ZNF418 NP_597717 ZNF641 NP_689533 ZBTB20 NP_056457 ZNF182 NP_008893 ZNF419 NP_001091964 ZNF645 NP_689790 ZBTB22 NP_001138810 ZNF184 NP_009080 ZNF425 NP_001001661 ZNF648 NP_001009992 ZBTB22 NP_005444 ZNF185 NP_001171586 ZNF426 NP_077011 ZNF649 NP_075562 ZBTB25 NP_008908 ZNF187 NP_001018854 ZNF433 NP_001073880 ZNF655 NP_001009958 ZBTB26 NP_065975 ZNF187 NP_689949 ZNF434 NP_060280 ZNF655 NP_001009960 ZBTB3 NP_079060 ZNF189 NP_003443 ZNF438 NP_877432 ZNF658B NP_001027468 ZBTB33 NP_006768 ZNF19 NP_008892 ZNF441 NP_689568 ZNF660 NP_775929 ZBTB37 NP_115911 ZNF19 NP_008892 ZNF442 NP_110451 ZNF662 NP_997287 ZBTB37 NP_115911 ZNF193 NP_006290 ZNF443 NP_005806 ZNF664 NP_689650 ZBTB4 NP_065950 ZNF195 NP_009083 ZNF444 NP_060807 ZNF665 NP_079009 ZBTB40 NP_001077090 ZNF197 NP_008922 ZNF446 NP_060378 ZNF667 NP_071386 ZBTB43 NP_054726 ZNF2 NP_066574 ZNF449 NP_689908 ZNF668 NP_078982 ZBTB45 NP_116181 ZNF20 NP_066966 ZNF454 NP_872400 ZNF669 NP_079080 ZBTB46 NP_079500 ZNF200 NP_003445 ZNF461 NP_694989 ZNF670 NP_149990 ZBTB48 NP_005332 ZNF202 NP_003446 ZNF467 NP_997219 ZNF671 NP_079109 ZBTB7B NP_056956 ZNF205 NP_001035893 ZNF480 NP_653285 ZNF672 NP_079112 ZBTB8 NP_001035531 ZNF207 NP_003448 ZNF483 NP_001007170 ZNF673 NP_001123370 ZBTB8B NP_001139192 ZNF211 NP_006376 ZNF484 NP_001007102 ZNF675 NP_612203 ZBTB9 NP_689948 ZNF212 NP_036388 ZNF485 NP_660355 ZNF678 NP_848644 ZC3H10 NP_116175 ZNF212 NP_036388 ZNF488 NP_694579 ZNF680 NP_848653 ZC3H12A NP_079355 ZNF213 NP_001128127 ZNF491 NP_689569 ZNF681 NP_612143 ZC3H15 NP_060941 ZNF214 NP_037381 ZNF493 NP_787106 ZNF682 NP_001070817 ZC3H7B NP_060060 ZNF22 NP_008894 ZNF497 NP_940860 ZNF682 NP_149973 ZCCHC12 NP_776159 ZNF221 NP_037491 ZNF500 NP_067678 ZNF683 NP_001108231 ZDHHC12 NP_116188 ZNF222 NP_037492 ZNF501 NP_659481 ZNF684 NP_689586 ZDHHC12 NP_116188 ZNF223 NP_037493 ZNF506 NP_001092739 ZNF688 NP_660314 ZDHHC15 NP_659406 ZNF225 NP_037494 ZNF509 NP_660334 ZNF689 NP_612456 ZDHHC16 NP_115703 ZNF226 NP_001027544 ZNF510 NP_055745 ZNF691 NP_056995 ZDHHC21 NP_848661 ZNF227 NP_872296 ZNF511 NP_665805 ZNF691 NP_056995 ZDHHC23 NP_775841 ZNF227 NP_872296 ZNF512 NP_115810 ZNF692 NP_060335 ZDHHC4 NP_060576 ZNF228 NP_037512 ZNF513 NP_653232 ZNF696 NP_112157 ZDHHC5 NP_056272 ZNF23 NP_666016 ZNF514 NP_116177 ZNF699 NP_940937 Page 56 of 58
ZDHHC6 NP_071939 ZNF233 NP_861421 ZNF517 NP_998770 ZNF7 NP_003407 ZDHHC7 NP_060210 ZNF239 NP_001092754 ZNF521 NP_056276 ZNF70 NP_068735 ZDHHC8 NP_037505 ZNF24 NP_008896 ZNF524 NP_694951 ZNF700 NP_653167 ZEB1 NP_110378 ZNF248 NP_066383 ZNF526 NP_597701 ZNF701 NP_060730 ZEB1 NP_110378 ZNF25 NP_659448 ZNF527 NP_115829 ZNF703 NP_079345 ZEB2 NP_055610 ZNF250 NP_066405 ZNF529 NP_066002 ZNF704 NP_001028895 ZFAND3 NP_068762 ZNF251 NP_612376 ZNF530 NP_065931 ZNF705A NP_001004328 ZFAND5 NP_001095890 ZNF253 NP_066385 ZNF532 NP_060651 ZNF707 NP_776192 ZFAND6 NP_061879 ZNF257 NP_258429 ZNF540 NP_689819 ZNF709 NP_689814 ZFAT NP_065914 ZNF26 NP_062537 ZNF543 NP_998763 ZNF710 NP_940928 ZFP1 NP_710155 ZNF263 NP_005732 ZNF545 NP_597723 ZNF713 NP_872439 ZFP1 NP_710155 ZNF275 NP_001073954 ZNF547 NP_775902 ZNF714 NP_872321 ZFP161 NP_003400 ZNF277 NP_068834 ZNF549 NP_694995 ZNF720 NP_001124385 ZFP2 NP_085116 ZNF280A NP_542778 ZNF550 NP_001034743 ZNF730 NP_001264332 ZFP3 NP_694563 ZNF280B NP_542942 ZNF551 NP_612356 ZNF74 NP_003417 ZFP36 NP_003398 ZNF281 NP_036614 ZNF552 NP_079038 ZNF74 NP_003417 ZFP36L1 NP_004917 ZNF285 NP_689567 ZNF553 NP_689865 ZNF740 NP_001004304 ZFP36L2 NP_008818 ZNF286A NP_065703 ZNF554 NP_001096121 ZNF746 NP_689770 ZFP37 NP_003399 ZNF295 NP_001091872 ZNF556 NP_079243 ZNF75A NP_694573 ZFP41 NP_776193 ZNF296 NP_660331 ZNF557 NP_001037853 ZNF75D NP_009062 ZFP42 NP_777560 ZNF3 NP_116313 ZNF557 NP_001037852 ZNF763 NP_001012771 ZFP64 NP_060667 ZNF3 NP_116313 ZNF558 NP_653294 ZNF764 NP_219363 ZFP64 NP_071371 ZNF300 NP_443092 ZNF560 NP_689689 ZNF764 NP_219363 ZFP64 NP_955459 ZNF302 NP_001012320 ZNF561 NP_689502 ZNF766 NP_001010851 ZFP91 NP_444251 ZNF32 NP_001005368 ZNF562 NP_060126 ZNF768 NP_078947 ZFPL1 NP_006773 ZNF321 NP_976052 ZNF563 NP_660319 ZNF77 NP_067040 ZGPAT NP_115916 ZNF322A NP_078915 ZNF564 NP_659413 ZNF771 NP_057727 ZIC4 NP_115529 ZNF322A NP_078915 ZNF565 NP_001035939 ZNF773 NP_940944 ZIC4 NP_001161850 ZNF323 NP_665916 ZNF566 NP_116227 ZNF774 NP_001004309 ZIK1 NP_001010879 ZNF324 NP_055162 ZNF567 NP_689816 ZNF776 NP_775903 ZKSCAN1 NP_003430 ZNF324B NP_997278 ZNF569 NP_689697 ZNF780A NP_001136051 ZKSCAN2 NP_001012999 ZNF329 NP_078896 ZNF57 NP_775751 ZNF780B NP_001005851 ZKSCAN3 NP_077819 ZNF331 NP_001073375 ZNF572 NP_689625 ZNF782 NP_001001662 ZKSCAN4 NP_061983 ZNF333 NP_115809 ZNF573 NP_689573 ZNF784 NP_976308 ZKSCAN5 NP_055384 ZNF334 NP_060572 ZNF574 NP_073589 ZNF785 NP_689671 ZMAT1 NP_115817 ZNF334 NP_060572 ZNF575 NP_777605 ZNF785 NP_689671 ZMAT5 NP_001003692 ZNF334 NP_955473 ZNF576 NP_077303 ZNF79 NP_009066 ZMYND11 NP_006615 ZNF33A NP_008905 ZNF579 NP_689813 ZNF790 NP_996777 ZMYND11 NP_006615 ZNF34 NP_085057 ZNF580 NP_057286 ZNF790 NP_996777 ZMYND8 NP_898868 ZNF341 NP_116208 ZNF581 NP_057619 ZNF791 NP_699189 ZMYND8 NP_898868 ZNF343 NP_077301 ZNF581 NP_057619 ZNF793 NP_001013681 ZMYND8 NP_898869 ZNF345 NP_003410 ZNF582 NP_653291 ZNF799 NP_001074290 ZNF10 NP_056209 ZNF347 NP_115973 ZNF583 NP_689691 ZNF8 NP_066575 ZNF101 NP_149981 ZNF347 NP_115973 ZNF585A NP_954577 ZNF821 NP_060000 ZNF114 NP_705836 ZNF35 NP_003411 ZNF585B NP_689492 ZNF83 NP_001099019 ZNF12 NP_057349 ZNF350 NP_067645 ZNF586 NP_060122 ZNF83 NP_001099023 ZNF121 NP_001008727 ZNF354A NP_005640 ZNF587 NP_116217 ZNF84 NP_001120844 ZNF131 NP_003423 ZNF354B NP_478137 ZNF593 NP_056955 ZNF844 NP_001129973 ZNF132 NP_003424 ZNF354C NP_055409 ZNF596 NP_001035880 ZNF846 NP_001071092 ZNF133 NP_001076799 ZNF358 NP_060553 ZNF597 NP_689670 ZNF92 NP_689839 ZNF134 NP_003426 ZNF366 NP_689838 ZNF599 NP_001007249 ZNRD1 NP_055411 ZNF135 NP_003427 ZNF37A NP_001007095 ZNF605 NP_899061 ZSCAN1 NP_872378 ZNF136 NP_003428 ZNF382 NP_116214 ZNF607 NP_116078 ZSCAN10 NP_116194 ZNF138 NP_006515 ZNF383 NP_689817 ZNF610 NP_775801 ZSCAN16 NP_079507 ZNF140 NP_003431 ZNF384 NP_001035005 ZNF613 NP_079116 ZSCAN18 NP_076415 ZNF143 NP_003433 ZNF385A NP_056296 ZNF614 NP_079316 ZSCAN18 NP_076415 ZNF148 NP_068799 ZNF394 NP_115540 ZNF615 NP_940882 ZSCAN2 NP_870992
RefSeq Gene Symbol protein ID ZSCAN2 NP_060364 ZSCAN20 NP_660281 ZSCAN21 NP_666019 ZSCAN22 NP_862829 ZSCAN29 NP_689668 ZSCAN30 NP_001159484 ZSCAN4 NP_689890 ZSCAN5 NP_077279 Page 57 of 58
Supporting Information Table 2 List of human dermal fibroblasts (HDF) used in this study. Name Age Sexa Raceb HDF1388 36 F C HDF1419 22w F unknown HDF1437 56 M C HDF1554 77 F C TIG-107 81 F J TIG-114 36 M J TIG-120 6 F J aM, male; F, female bC, Caucasian; J, Japanese Page 58 of 58
Supporting Information Table 3 List of primers used in this study. Primer Sequence (5' to 3') Purpose hOct4-S1165 GAC AGG GGG AGG GGA GGA GCT AGG qRT-PCR for endogenous OCT4 expression hOct4-AS1283 CTT CCC TCC AAC CAG TTG CCC CAA AC hSox2-S1430 GGG AAA TGG GAG GGG TGC AAA AGA GG qRT-PCR for endogenous SOX2 expression hSox2-AS1555 TTG CGT GAG TGT GGA TGG GAT TGG TG hNANOG-S1678 TGG CTG CCG TCT CTG GCT ATA GAT qRT-PCR for endogenous NANOG expression hNANOG-AS1797 AAG CCT CCC AAT CCC AAA CAA TAC G3PDH-F ACC ACA GTC CAT GCC ATC AC qRT-PCR for GAPDH expression G3PDH-R TCC ACC ACC CTG TTG CTG TA