(2024) 25:2
Chen et al. BMC Molecular and Cell Biology
https://doi.org/10.1186/s12860-023-00498-x

BMC Molecular andCell Biology

Open Access

RESEARCH

Sumoylation of SAP130 regulates its
interaction with FAF1 as well as its protein
stability and transcriptional repressor function
Chang‑Han Chen1,2, Hung‑Wei Lin3, Meng‑Fang Huang3, Chi‑Wu Chiang5, Kuen‑Haur Lee6,
Nguyen Thanh Phuong3, Zong‑Yan Cai3, Wen‑Chang Chang4,7 and Ding‑Yen Lin3,4*

Abstract
Background Fas-associated factor 1 (FAF1) is a multidomain protein that interacts with diverse partners to affectnumerous cellular processes. Previously, we discovered two Small Ubiquitin-like Modifier (SUMO)-interacting motifs(SIMs) within FAF1 that are crucial for transcriptional modulation of mineralocorticoid receptor. Recently, we identi‑
fied Sin3A-associated protein 130 (SAP130), a putative sumoylated protein, as a candidate FAF1 interaction partnerby yeast two-hybrid screening. However, it remained unclear whether SAP130 sumoylation might occur and function‑
ally interact with FAF1.
Results In this study, we first show that SAP130 can be modified by SUMO1 at Lys residues 794, 878 and 932
both in vitro and in vivo. Mutation of these three SUMO-accepting Lys residues to Ala had no impact on SAP130 asso‑
ciation with Sin3A or its nuclear localization, but the mutations abrogated the association of SAP130 with the FAF1.
The mutations also potentiated SAP130 trans-repression activity and attenuated SAP130-mediated promotion of cell
growth. Additionally, SUMO1-modified SAP130 was less stable than unmodified SAP130. Transient transfection experi‑ments further revealed that FAF1 mitigated the trans-repression and cell proliferation-promoting functions of SAP130,
and promoted SAP130 degradation by enhancing its polyubiquitination in a sumoylation-dependent manner.
Conclusions Together, these results demonstrate that sumoylation of SAP130 regulates its biological functions
and that FAF1 plays a crucial role in controlling the SUMO-dependent regulation of transcriptional activity and protein
stability of SAP130.
Keywords SAP130, FAF1, Sumoylation, SUMO-interacting motifs (SIMs)*Correspondence:Ding‑Yen Lin
lindy@mail.ncku.edu.tw
1
Department of Medical Research, Taichung Veterans General Hospital,
Taichung 407219, Taiwan, ROC
2
Department of Applied Chemistry, and Graduate Instituteof Biomedicine and Biomedical Technology, National Chi Nan University,
Nantou 545301, Taiwan, ROC
3
Department of Biotechnology and Bioindustry Sciences, College
of Bioscience and Biotechnology, National Cheng Kung University,
Tainan 70101, Taiwan, ROC
4
Department of Pharmacology, College of Medicine, National Cheng
Kung University, Tainan 70101, Taiwan, ROC
5
Institute of Molecular Medicine, College of Medicine, National Cheng
Kung University, Tainan 70101, Taiwan, ROC

6
Institute for Cancer Biology and Drug Discovery, College of Medical
Science and Technology, Taipei Medical University, Taipei 11031, Taiwan,
ROC
7
Graduate Institute of Medical Sciences, College of Medicine, Taipei
Medical University, Taipei Medical University, Taipei 11031, Taiwan, ROC© The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecom‑mons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Chen et al. BMC Molecular and Cell Biology(2024) 25:2

Introduction
Small ubiquitin-like modifiers (SUMOs) are membersof the ubiquitin-like superfamily that can be covalently
conjugated to various target proteins. This conjugation(sumoylation) is a common, reversible posttranslational
modification that serves as a crucial regulatory mechanism in many important biological processes, includingtranscription, DNA damage response, cell cycle progression, cellular localization, proteostasis, and nuclear
body formation (reviewed in [1]). Similar to ubiquitination, sumoylation is an enzymatic reaction catalyzed bya three-enzyme cascade (E1, E2, and E3). Sumoylationof proteins involves the covalent attachment of SUMO
to a Lys residue within a short consensus sequence ina target protein (ΨKXD/E, where Ψ is a large hydrophobic residue). In addition to covalent attachment of
SUMO to substrate proteins, noncovalent interactions
may occur between SUMO and specialized protein
domains called SUMO-interacting motifs (SIMs). The
most well characterized SIM consists of a hydrophobic
core (V/I-X-V/I-V/I or V/I-V/I-X-V/I/L) that is typicallyflanked by a stretch of acidic residues [2–4]. These noncovalent interactions between SUMO-modified proteinsand SIM-containing binding partners may be key contributors to SUMO-dependent functions. For example,
SUMO-targeted ubiquitin ligases (STUbLs) are SIM-containing RING finger ubiquitin ligases that recognize polysumoylated proteins via a SUMO/SIM interaction andtrigger the ubiquitylation and subsequent degradation of
the sumolyated targets [5]. Furthermore, recent studies
have shown that proteins with both SUMO and ubiquitin conjugates (SUMO-Ub chains) can be recognized bya unique SUMO-Ub chain receptor containing tandem
SUMO- and ubiquitin-interacting motifs (tSIM-UIMs) tomediate specific biological functions, such as DNA damage response [6–8]. Thus, defining the functional effectsof sumoylation for key protein targets is valuable.
The mSin3A-histone deacetylase corepressor is amultiprotein complex utilized by a wide variety of transcriptional repressors [9, 10]. Sin3A-associated protein
130 (SAP130) was initially identified as a component of
the mSin3A corepressor complex by MALDI-TOF mass
spectrometry experiments [11]; however, very little is
known about the protein. The only predicted motifs
in the SAP130 protein are two proline-rich regions,which provides little insight into its potential function
within the mSin3A complex. Both mSin3A and HDAC1
are known to bind the C-terminus of SAP130 between
amino acids 836 and 1047, but SAP130 lacks a recognizable DNA binding domain. Still, its intrinsic transcriptional repression activity has been demonstrated
by utilizing a Gal4-SAP130 chimera to inhibit a Gal4driven luciferase reporter gene. Additionally, SAP130

Page 2 of 16

has been reported to associate with the Cullin4B-Ring
E3 ligase complex (CRL4B), which suggests a role for
Sin3A-HDAC complex in CRL4B-mediated transcriptional regulation [12]. Importantly, SAP130 has beenfound to be modified by SUMO1 [13], but the functional impacts of SAP130 sumoylation remain unclear.
Fas-associated factor 1 (FAF1) was originally identified as a Fas binding partner that potentiates Fasinduced apoptosis [14]. Subsequent research revealed
that FAF1 interacts with diverse proteins and is
involved in a variety of biological processes. For
instance, FAF1 can regulate immune response, as it
suppresses NF-kB activity by disrupting IκB kinase(IKK) complex assembly and interfering nuclear translocation of RelA (p65) [15, 16]. Furthermore, FAF1 acts
as a positive regulator of type 1 interferon in response
to RNA virus infections through its targeting of NLRX1[17]. Faf1 gene-disrupted mice show embryonic lethality at the two-cell stage [18]. Meanwhile, FAF1 wasshown to play a pivotal role in oxidative stress-induced
necrosis during the pathogenesis of Parkinson’s disease [19]. Importantly, FAF1 interacts with polyubiquitinated proteins and valosin-containing protein(VCP), which may serve as a scaffold protein, regulating ubiquitin-mediated proteasomal degradation [20].
This FAF1-mediated modulation of protein stability is
known to regulate several key cellular processes. For
example, FAF1 inhibits Wnt signaling by promoting
the proteasomal degradation of β-catenin [21]. Furthermore, FAF1 destabilizes TβRII on the cell surface byrecruiting VCP/E3 ligase complex, thereby suppressing
TGF-β-mediated cancer metastasis [22]. Along theselines, reduced FAF1 expression was found in somehuman tumors (reviewed in [23]), making the protein acandidate tumor suppressor.
In previous work, we identified two SIMs within FAF1
and demonstrated that FAF1 interacts with sumoylated
mineralocorticoid receptor to represses its transactivation [24]. Notably, two recent studies revealed
that C. elegans UBXN-3/FAF1 is an important regulator of DNA replication, as it controls the dynamics of
SUMO- and ubiquitin-modified DNA replication factors on chromatin [25, 26]. These studies highlight thecritical role of FAF1 for modulating both the SUMO
and ubiquitin-modified proteins. Identifying potential
novel binding partner(s) of FAF1 may help elucidate therole it plays in controlling protein homeostasis; to do
this, we performed yeast two-hybrid screening using
FAF1 as bait. Here, we identified SAP130 as a FAF1interacting protein. In the current study, we sought todetermine whether and how the putative interaction
between sumoylated SAP130 and FAF1 might occur
and affect cellular function.

Chen et al. BMC Molecular and Cell Biology(2024) 25:2

Materials and methods
Plasmids and antibodies

Yeast constructs expressing wild-type (WT) and SIM
mutated (DM) Gal AD-FAF, mammalian vectors expressing EGFP-SUMO1, HA-FAF1, Flag-FAF1 WT and DM
were described previously [24]. Mammalian expression vectors encoding HA-tagged human SAP130
and HDAC1 (HG17174-NY and HG11486-NY) werepurchased from Sino Biological Inc. (Beijing, China).
pCS2 + MT-mSin3A was a gift from Bob Eisenman(Addgene plasmid # 30,452; http://​n2t.​net/​addge​ne:​
30452; RRID: Addgene_30452). Myc-tagged ubiquitin
was cloned into the pCMV-Tga3C vector (Stratagene).
A polymerase chain reaction (PCR) fragment encoding
the C-terminal of human SAP130 from HA-SAP130 was
subcloned into the pBTM116 vector in frame with the
LexA domain to generate the LexA-SAP130 bait. SAP130
mutations at three potential SUMO-conjugated Lys residues were created in the pBTM116 and pCMV3 using the
Quikchange site-directed mutagenesis kit (Stratagene)with LexA-SAP130, and pCMV3-SAP130 as templates.
All constructs were verified by DNA sequencing. The
constructs pRS-hMR and pMMTV-Luc were generous
gifts from Dr. Ron M. Evans (The Salk Institute, La Jolla,
CA). Plasmid M50 Super 8 × TOPFlash was a gift from
Randall Moon (Addgene plasmid # 12,456; http://​n2t.​net/​addge​ne:​12456; RRID: Addgene_12456). Plasmid pFRLuc with five Gal4-binding sites upstream of the minimal
promoter driving the luciferase reporter gene and the
NF-κB luciferase reporter construct (pNF-κB-Luc) werepurchased from Stratagene. The following antibodies
were purchased: HA (HA.11; Babco/Covance, Berkley,CA), HA (Biolegend, San Diego, CA), FLAG (M2; Sigma),c-Myc (9E10; GeneTex, Taiwan), GFP (JL-8; BD Biosciences Clontech), FAF1 (C1C3; GeneTex, San Antonio,
TX); SAP130 (12130–1-AP, Proteintech), HDAC1 (10E2,
Cell Signaling Technology), SIN3A (D9D6, Cell Signaling
Technology) and actin (clone AC-74; Sigma, St. Louis,MO).
Yeast two‑hybrid screening and β‑Gal assay

Yeast two-hybrid library screening with full-length
human FAF1 bait was performed as described previously [24]. Briefly, we transformed the L40 yeast strain
with the LexA-FAF1 plasmid followed by transformation
with 200 μg of the human testis cDNA library (Clontech).
Yeast transformants were then selected on medium containing 5 mM 3-amino-(1,2,4) triazole (Sigma) lacking
histidine, leucine, and tryptophan. Histidine protrotophic(His +) colonies were further tested for β-galactosidase
activity. The plasmids from both His + and X-gal + colonies were isolated by the curing process of MC1066 bacterial strain and sequenced. The interaction strength wasPage 3 of 16

determined according to the appearance of blue color on
X-Gal plates, or by the quantitative liquid β-galactosidase
assays using lysates from three separate yeast cultures
according to the instructions of the Galacto-Light Pluskit (Applied Biosystems).
Cell culture, transfection and lentivirus‑based short hairpin(sh)RNA transduction

HEK293, HEK293T, HeLa, COS-1, HCT116 andL-Wnt3a cells were cultured in Dulbecco’s modified
Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA)supplemented with 10% fetal bovine serum (FBS). NB4cells were maintained in RPMI 1640 medium (Gibco,
Grand Island, NY, USA) containing 10% FBS. The
NB4 cell line was kindly gifted from Dr. Tsai-Yun Chen(National Cheng Kung University, Tainan, Taiwan). Allother cell lines were obtained from the American Type
Culture Collection (Manassas, VA). All cells were incubated at 37 °C in a 5% CO2 atmosphere. Plasmids weretransfected using the PolyJet™ reagent (SignaGen Laboratories, Gaithersburg, MD). For stable SAP130 transfection of HEK293 cells, the plasmid constructs ofpCMV3-HA-SAP130 WT or 3KA mutant were transfected into HEK293 cells and selected with hygromycin(100 μg/ml). Human FAF1 shRNA (TRCN0000004244)
MISSION® shRNA Lentiviral Transduction Particles
were purchased from Sigma-Aldrich (St. Louis, MO).
To generate lentivirus-shRNA FAF1 knockdown cells,
HEK293T, HCT116 and NB4 cells were infected at low
confluence (20%) for 24 h with lentiviral supernatants
diluted 1:1 with normal culture medium in the presenceof 8 μg/ml of polybrene (Sigma). Forty-eight hours after
infection, cells transduced by lentiviruses were selectedunder 2 μg/ml puromycin for 1 week and then passaged
before use.
Growth curves and colony formation assays

The growth rate of cells was monitored by seeding 2 × ­105
cells in 60-mm dishes containing 5% FBS. Cells werecounted at 24-h intervals using a hemocytometer over a
period of seven days. For the colony formation assay, cells
were plated at a low density onto 60-mm dishes, and the
medium was changed every 3 days. Two weeks later, thenumber of colonies was counted using the Sigmascan
software program after staining with 2% methylene blue.
Immunoprecipitation and western blotting

For testing the protein–protein interactions in mammalian cells, the expression construct encoding HA-tagged
wild-type SAP130 or SAP130 3KA mutant, along with
the Flag-tagged FAF1 or myc-tagged mSin3A expression construct, was transfected into COS-1 cells. At 48 h
after cotransfection, cells were solubilized in modified

Chen et al. BMC Molecular and Cell Biology(2024) 25:2

RIPA buffer consisting of 50 mM Tris–HCl (pH 7.8),
150 mM NaCl, 5 mM EDTA, 0.5% Triton X-100, 0.5%
Nonidet-P40, 0.1% sodium deoxycholate, and a protease inhibitor mixture (Roche Molecular Biochemicals,Indianapolis, IN). To measure the levels of sumoylation,
various SAP130 point mutants or wild-type SAP130 and
EGFP-SUMO1 constructs were transfected into COS-1
cells. The cells were then lysed with modified RIPA buffer
containing 20 mM N-ethylmaleimide (NEM; Sigma). For
detection of ubiquitinated SAP130 proteins by immunoprecipitation, HEK293 cells were co-transfected withthe plasmid encoding Myc-Ubiquitin and wild-type HASAP130, along with plasmids encoding Flag-FAF1 or DM
mutant, followed by the treatment with MG132 (5 μM)
for another 6 h. Whole cell lysates were mixed with antiserum against HA (Babco/Covance) antibody, and theimmunocomplexes were mixed with protein G Plus/protein A-agarose beads (Merck Millipore). After overnightincubation at 4
­0C, the immunocomplexes were thengently washed three times with the PBS buffer followedby Western blot analysis. For Western blot analysis,
immunoprecipitated molecules or total cell lysates wereseparated by SDS-PAGE, transferred onto nitrocellulose membranes (Amersham, GE Healthcare, Germany),
blocked with 5% milk, and probed with anti-FLAG or
anti-GFP or anti-Myc antibodies. Specific blot signals
were visualized on an X-ray film by incubating with ECL
chemiluminescence kit (Amersham Biosciences). The
specific intensity of each protein band on X-ray film wasmeasured by Image J software (NIH, USA) and results
were normalized to corresponding actin band densities.
Dual‑luciferase reporter assay

For reporter gene assays, cells were transfected in 6-wellplates with 2 μg of DNA, including the indicated reporter
constructs and SAP130 or FAF1 expression vectors, andthe constitutive Renilla luciferase control reporter vector
pRL-TK (Promega, Madison, WI). The total amount ofplasmid per well was kept constant by adding pcDNA3
empty vector. For the experiments of MR activation, cells
were treated with either vehicle or ­10−6 M of aldosterone at 24 h post-transfection, and reporter gene activity was measured after an additional 24 h. To monitor
NF-κB transcriptional activity, cells were treated with
TNF-α (20 ng/ml) for 6 h at 36 h post-transfection. To
stimulate Wnt signaling in HEK293 cells, cells were cotransfected with the TopFlash-Luc reporter, pRL-TK,
and HA-FAF1 or SAP130 WT or 3KA mutant plasmids.
Cells were treated with Wnt3A conditioned medium(CM) or left untreated and lysed for a luciferase assay at
48 h post-transfection. CM from cells expressing Wnt3A
was prepared from L-Wnt3A cells (CRL-2647; American
Type Culture Collection Manassas, VA) after 4 days andPage 4 of 16

an additional harvest following 3 days in culture. CM
were combined, sterile filtered and mixed 1:1 with normal media for use. Luciferase activity was determinedusing the dual-luciferase reporter assay system (Promega,
Madison, WI).
Immunofluorescence

HeLa or COS-1 cells were transfected with HA-tagged
FAF1, SAP130 WT or 3KA construct by the lipofection
method. At 48 h after transfection, the cells were fixedfor 10 min with 4% paraformaldehyde in phosphatebuffered saline (PBS) and then permeabilized with cooledacetone for 1 min at -20 °C. The permeabilized cells werethen incubated with anti-HA monoclonal antibody alone
or combined with SIN3A or HDAC1 antibody for 1 h atroom temperature. Subsequently, cells were washed threetimes with PBS and then incubated with fluorescein isothiocyanate-conjugated anti-mouse or rabbit IgG (Jackson ImmunoResearch) alone or combined with Texas
red-conjugated anti-mouse IgG at room temperature forone hour. Nuclei were stained by DAPI (4′,6′-diamidino-2-phenylindole,10 μg/ml). After washing again with
PBS, the coverslips were inverted and mounted with
GEL/Mount (biomeda corp) to prepare the fluorescence
images for analysis with an Olympus BX51 microscope.
In vitro sumoylation assays

Expression constructs of HA-tagged wild-type or KA
mutant SAP130 were transiently transfected into COS-1
cells. Cell extracts were harvested 48 h later for immunoprecipitation with anti-HA antibody and followed byin vitro sumoylation assays. In vitro sumoylation assays
were performed as described previously [24]. A typicalsumoylation reaction was performed in 20 μl of reaction
mixture containing 150 ng of SUMO E1 recombinant
proteins (LAE Biotechnology, Taichung, Taiwan), 1 μg of
SUMO-1, 1 μg of Ubc9, and proteins bound to beads in areaction buffer (2 mM ATP, 20 mM HEPES pH 7.5, and5 mM ­MgCl2). The reactions were carried out at 37 °C for
2 h, and the reactions were stopped by washing with PBS
for three times. Beads of samples were analyzed by SDSPAGE followed by Western blot analysis using anti-HA
antibody.
RNA extraction and real‑time quantitative PCR (qPCR)

Total RNA was isolated from control, HEK293 SAP130
WT and 3KA stable cells (approximately 2 × 107) using
an RNeasy mini kit (Qiagen, Valencia, CA) and thenreverse-transcribed using the ThermoScript RT-PCR system (Invitrogen). The real-time qPCR analysis was performed using the SYBR Green Advantage qPCR Premix(Clontech) and C1000™ Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). PCRs were performed using theChen et al. BMC Molecular and Cell Biology(2024) 25:2

Page 5 of 16

following conditions for 30 cycles: 95 °C for 15 s, 60 °C quantitative β-gal assays (Fig. 1B). To further verify
for 15 s, and 72 °C for 20 s. The following primer pairs that SAP130 interacts with FAF1 in mammalian cells,
were used. SAP130, forward (5’-CGG​
GTC​
AAA​
GAG​ COS-1 cells were co-transfected with expression conGAG​AAG​AAA-3’) and reverse (5’-CAG​CAC​AGA​GGT​ structs encoding Flag-FAF1 and HA-SAP130. Cell
GGA​
CTT​
T-3’), and GAPDH, forward (5’-CCC​
ACT​ lysates were subjected to immunoprecipitation with
CCT​CCA​CCT​TTG​AC-3’) and reverse (5’- TCT​CTC​ an anti-HA antibody followed by Western blot analysis
TTC​CTC​TTG​TGC​TCTTG-3’). Relative amounts of the with an anti-Flag antibody. As shown in Fig. 1C, FAF1
SAP130 transcripts were determined using the compara- was detected in the immunoprecipitated complexes oftive CT method and were normalized to the GAPDH SAP130. Furthermore, we noted that the C-terminal
fragment of SAP130 isolated by the yeast two-hybrid
housekeeping control.
system contains three putative SUMO conjugation sites
at lysine residues K794, K878 and K932, according to
cBioPortal analysis
cBioportal website (http://​www.​cbiop​ortal.​org/) is an the consensus sequence (I/L/V)KXE. While SAP130
open-access resource for the interactive exploration of was previously reported to be modified by SUMO1 [13],multidimensional cancer genomics data [27, 28]. Using the biological significance of its sumoylation remains
the cBioPortal database, the correlations between mRNA unknown. We thus examined whether SAP130 could
expression of SAP130 and the FAF1 in various cancer be modified by SUMO1 in cultured cells transiently
expressing HA-SAP130 and EGFP-SUMO1. Immunotypes were investigated.
precipitation of HA (SAP130) followed by immunoblotting for either HA or EGFP revealed an additional
Statistical analysis
Statistical analyses were performed using two-tailed Stu- slower migrating band corresponding to sumoylated
dent’s t-test. P values were calculated using Prism v. 3.03 SAP130 (Fig. 1D).
We further generated SAP130 mutants in which each
(GraphPad Software, San Diego, CA).
lysine residue within the SUMO1 acceptor sites were
replaced by alanine (A) or arginine (R), and we evaluResults
ated the sumoylation pattern of the mutated proteins
Identification of SAP130 as an FAF1‑interacting protein
by in vitro and in vivo analyses. Our data showed thatand a SUMO substrate
We previously conducted a yeast two-hybrid screen SAP130 K794A or K794R markedly reduced the extent
using human FAF1 as bait and isolated several candi- of SAP130 sumoylation both in vitro and in vivo, sugdate FAF1-interacting proteins, including ubiquitin, gesting that K794 is a major sumoylation site of SAP130
Ubc9, and SUMO-1 [24]. Among the identified can- (Fig. 1E, F and Supplemental Figure S1). Simultaneous
didates, one clone encoding the C-terminal region of mutation of all three lysines in SAP130 (K794A/K878A/
SAP130 spanning amino acid residues 760–1048 was K932A; 3KA or K794R/K878R/K932R; 3KR) almost
also isolated (Fig. 1A). Interestingly, SAP130 was first completely abolished the sumoylation of SAP130. These
identified as a protein that binds to the mSin3A core- data confirmed that SAP130 is a sumoylation substrate,pressor complex [11]. The specificity of FAF1 asso- leading us to hypothesize that FAF1 may interact withciation with the clone in yeast cells was validated by sumoylated SAP130.

(See figure on next page.)
Fig. 1 Identification of SAP130 as an FAF1-interacting protein and a SUMO substrate. A Schematic representation of the SAP130 clone (F23)that interacted with LexA-FAF1 bait, including the positions of three potential SUMO conjugation sites. B Yeast co-transformed as indicatedwith bait and prey were analyzed with quantitative β-Gal assays and on X-Gal-containing plates. LexA-lamin served as a negative control. C COS-1
cells were transiently transfected with the indicated combinations of expression constructs encoding Flag-tagged FAF1 and HA-tagged SAP130,
respectively. After 48 h, whole cell lysates (WCL) were used for immunoprecipitation (IP), followed by Western blot (WB) analysis with the indicatedantibodies. The arrow indicates the position of the co-precipitated FAF1. Star indicates the heavy chain of IgG. D SAP130 is sumoylated in vivo.
A plasmid expressing HA-tagged SAP130 was co-transfected with EGFP control or EGFP-SUMO1 plasmid into COS-1 cells. Cell lysates were used
for IP with an anti-HA antibody, followed by WB analysis using antibodies against HA and GFP. The black asterisk and white asterisks indicate
EGFP-SUMO-1-modified and unmodified SAP130 proteins, respectively. The blue and red arrowheads indicate the EGFP and EGFP-SUMO1 proteins,respectively. E SUMO1 modification of SAP130 occurs at Lys-794, 878, and 932. HA-tagged wild-type SAP130 and various mutant SAP130 proteins
were subjected to an in vitro sumoylation assay as described in Materials and Methods. SUMO-1 modified and unmodified SAP130 proteins wereanalyzed by immunoblotting with anti-HA antibody. The bracket and arrow indicate SUMO-modified and unmodified SAP130 proteins, respectively.
F In vivo sumoylation analyses of wild-type and mutants of SAP130. The assays were performed as described in Fig. 1D. The bracket and arrow
indicate EGFP-SUMO-1-modified and unmodified SAP130 proteins, respectively. The blue and red arrowheads indicate the EGFP and EGFP-SUMO1
proteins, respectively. The immunoblots were cropped for clarity. Full length blots are presented in Supplemental Figure S8

Chen et al. BMC Molecular and Cell Biology

Fig. 1 (See legend on previous page.)(2024) 25:2

Page 6 of 16

Chen et al. BMC Molecular and Cell Biology(2024) 25:2

SUMO‑dependent interaction between FAF1 and SAP130

In our previous study, we identified two SIMs within
FAF1 and showed that the motifs are crucial for binding
for sumoylated mineralocorticoid receptor [24]. Thus,
our findings that SAP130 interacts with FAF1 and that
SAP130 is sumoylated implied that SUMO might berequired for the FAF1-SAP130 interaction. To test thispossibility, we constructed WT and 3KA or 3KR mutant
SAP130 C-terminal regions (amino acids 760–1048)
fused to LexA. Interactions of these constructs with Gal4
AD-FAF1 WT or SIM double mutation (DM) were thenassessed for the ability to activate lacZ and HIS3 reporter
genes in the yeast two-hybrid system. Since the C- terminal region of SAP130 is also known to bind mSin3A andHDAC1 [11], plasmids expressing full-length mSin3A
and HDAC1 fused to the Gal4 AD were also tested in
the experiment. Yeast co-transformed with GalADFAF1 WT, and LexA-SAP130 C-terminal fragment wereable to grow on media lacking histidine and had strong
β-gal activity (Fig. 2A), indicating a positive interaction between FAF1 and SAP130. These interactions
were further verified by a liquid β-galactosidase assay(Fig. 2B). The interaction between SAP130 and mSin3A
was weak as evidenced by the low β-galactosidase activity in the X-gal assay, and no significant interaction could
be observed between SAP130 and HDAC1. Remarkably,
the SAP130 3KA and 3KR mutants displayed significantly
reduced interaction with FAF1 compared to SAP130 WT(Fig. 2A, B and Supplemental Figure S2). Meanwhile,
the SAP130 3KA interaction with mSin3A was slightly
increased, suggesting that the three lysine residues
involved in this interaction. Additionally, the FAF1 DM
mutant exhibited markedly reduced binding to SAP130.
Together, these results imply that both the sumoylationof SAP130 and SIMs of FAF1 are largely responsible forthe interaction between the two proteins.
Next, we performed a reciprocal co-immunoprecipitation assay for SAP130 and FAF1. To do so, we co-transfected Flag-tagged FAF1 and analyzed its association
with HA-tagged SAP130 WT or 3KA in COS-1 cells. As
shown in Fig. 2C, FAF1 could be coimmunoprecipitatedwith SAP130 WT, whereas the SAP130 3KA mutant

Page 7 of 16

did not readily associate with the FAF1 in this cellular
system, indicating that sumoylation is required for theprotein–protein interaction. Notably, mutations of theconsensus SUMO conjugation sites in SAP130 did notaffect the association with mSin3A (Fig. 2D). Together,
these results strongly suggest that sumoylation of SAP130
is crucial for binding FAF1.
Sumoylation of SAP130 protein modulates its
transcription‑suppressing function, protein stability
and cell proliferation‑promoting effect

SAP130 is a subunit of the mSin3A-histone deacetylase
corepressor complex that acts as a transcriptional repressor, and its intrinsic transcriptional repression activity has
been demonstrated by testing a Gal4-SAP130 chimera
in a Gal4-Luc reporter assay [11]. To assess the effect of
SAP130 sumoylation on its transcriptional repressionfunction, we used a similar reporter gene assay in which
the DNA binding domain of Gal4 is fused to SAP130 WT
or 3KA mutant. COS-1 cells were co-transfected with aluciferase reporter construct (Gal4-Luc) and plasmids
encoding Gal4-SAP130 WT or Gal4-SAP130 3KA. The
luciferase assay results showed that Gal4-SAP130 3KA
exhibited a stronger repression activity than SAP130 WT(Fig. 3A).
The mSin3A corepressor complex has been implicatedin transcriptional repression by nuclear hormone receptors and NF-κB signaling pathway [29, 30]. Similarly,
studies have shown that FAF1 inhibits mineralocorticoid
receptor-mediated transactivation, as well as NF-κB and
Wnt/β-catenin signaling. Therefore, we next assessed
whether SAP130 sumoylation might play a role in regulating these signaling processes. Our data showed thatoverexpression of FAF1, SAP130 WT, or SAP130 3KA
significantly attenuated transcription of luciferase reporters driven by mineralocorticoid receptor-stimulated
MMTV, TNF-α-stimulated NF-κB and Wnt-induced
TOPFlash promoters. In all cases, we found that SAP130
WT exhibited a weaker repression activity than FAF1
(Fig. 3B-D). Notably, the SAP130 3KA or 3KR mutant significantly inhibited the reporter activities compared with
SAP130 WT, suggesting that the 3KA or 3KR mutant has(See figure on next page.)
Fig. 2 SUMO-dependent interaction between FAF1 and SAP130. A Yeast strain L40 was co-transformed with bait (human WT SAP130 or mutant
at three sumoylation residues (3KA), or the control protein lamin fused to the LexA DNA-binding domain) and prey constructs [human FAF1 WT,
FAF1 with SIM mutant (DM), mSin3A or HDAC1 fused to the Gal-activation domain (AD)]. Yeast transformants were spotted on plates with histidine(-TULL) or without histidine (-TULLH), and with X-Gal (+ X-Gal) media. Schematic presentation of FAF1 DM and SAP130 C-terminal 3KA mutants
analyzed in a yeast two-hybrid assay (top). B Yeast co-transformed with the indicated bait and prey were analyzed by quantitative β-Gal assays.
C Coimmunoprecipitation assays. COS-1 cells co-transfected with Flag-FAF1 and HA-SAP130 WT or 3KA mutant of expression constructs were
subjected to IP experiments followed by WB analysis with the indicated antibodies. WCL indicates whole cell lysate. D COS-1 cells co-transfectedwith myc-tagged mSin3A and HA-SAP130 WT or 3KA expression constructs were subjected to IP and WB analysis with the indicated antibodies. The
immunoblots were cropped for clarity. Full length blots are presented in Supplemental Figure S9

Chen et al. BMC Molecular and Cell Biology

Fig. 2 (See legend on previous page.)(2024) 25:2

Page 8 of 16

Chen et al. BMC Molecular and Cell Biology(2024) 25:2

enhanced repressor activity compared to SAP130 WT(Fig. 3B-D and Supplemental Figure S3). These results
imply that sumoylation contributes to the transrepressional function of SAP130. To determine the effects of
FAF1 on SAP130-mediated transcriptional repression,
we examined whether SAP130-mediated transrepression could be affected by knockdown or overexpressionof FAF1. As shown in Fig. 3E, we demonstrated that FAF1shRNA significantly attenuated SAP130-mediated suppression of TNF-α-stimulated NF-κB promoter. Additionally, overexpression of FAF1 in the FAF1 knockdown
background strongly enhanced SAP130-mediated transrepression of NF-κB promoter (Fig. 3E). These results
indicate that SAP130 and FAF1 work in concert to regulate gene transcription. Furthermore, the transcriptional
repression by SAP130 and FAF1 were not associated withchanges in the expression levels and subcellular localization of Sin3A and HDAC1 proteins. We observed thatoverexpression of FAF1 or SAP130 does not alter theendogenous Sin3A and HDAC1 protein levels and their
nuclear localization (Supplemental Figure S4).
It is known that sumoylation is linked to transcriptional repression and events such as changes in subcellular localization, protein half-life and interaction
with binding partners, so we tested whether any ofthese above mentioned events could be involved inaltering SAP130 repressor activity. An immunofluorescence analysis in HeLa cells revealed that SAP130

Page 9 of 16

WT, 3KA and 3KR mutant proteins were localized innucleus (Fig. 3F and Supplemental Figure S5). Thus,
SAP130 sumoylation does not appear to be involved in
its nuclear targeting. We next tested whether sumoylation controls the stability of SAP130 protein. COS-1
cells were transfected with HA-tagged SAP130 WT or
3KA mutant, followed by treatment with the translation inhibitor cycloheximide (CHX). Cells were thenharvested at different time-points and immunoblotted for HA. Interestingly, both proteins showed very
similar turnover rate within 3 h (Supplemental FigureS6). However, because only a very small proportion of
the SAP130 in the cell is sumoylated at steady state,
this method may not reveal the change of stability ofsumoylated SAP130. To solve the issue, alternatively,
we transfected COS-1 cells with HA-tagged SAP130
WT or 3KA mutant together with EGFP-SUMO1
and then treated with MG132, a proteasome inhibitor. Cells were further lysed and immunoblotted for
HA (Fig. 3G). Analyzing and comparing the modified and unmodified SAP130 forms in several experiments revealed that the amount of SUMO1-modified
SAP130 was increased to a greater extent in the presence of MG132 (7- to 12-fold increase) than unmodified SAP130 (1.5- to threefold increase). Nevertheless,
SAP130 3KA showed an increase in protein levels
upon MG132 treatment similar to that of SAP130 WT.
These results suggest that SUMO1 modification may(See figure on next page.)
Fig. 3 Functional analysis of SAP130 sumoylation. A Repression of a Gal4-dependent luciferase reporter gene relative to Gal4 control by Gal4
fusions to SAP130 WT and 3KA mutant. B FAF1, SAP130 WT and 3KA mutant efficiently inhibited mineralocorticoid receptor (MR) transactivation.
The MMTV-Luc reporter plasmid, the internal control plasmid pRL-TK (Renilla), pRS-hMR, and two different doses of the HA-tagged-FAF1, SAP130
WT or 3KA mutant plasmids were co-transfected into COS-1 cells. The total DNA amount was 2 μg (including addition of empty vector). At 24 hpost-transfection, cells were treated with either vehicle or 1 μM aldosterone (Aldo.), and reporter gene activities were measured after another24 h period. C Both SAP130 WT and 3KA mutant could inhibit Wnt signaling. HEK293 cells were co-transfected with the TopFlash-Luc reporter,pRL-TK, and HA-FAF1 or SAP130 WT or 3KA mutant plasmids. Cells were treated with Wnt3A conditioned medium (CM) or left untreated and lysed
for a luciferase assay at 48 h post-transfection. D Both SAP130 WT and 3KA mutant could efficiently inhibit NF-κB activation. HEK293T cells wereco-transfected with pNF-κB-Luc reporter plasmid and pRL-TK plasmid together with two different doses of HA-tagged-FAF1, SAP130 WT or 3KA
mutant plasmid. At 36 h post-transfection, cells were treated with tumor necrosis factor (TNF)-α (20 ng/ml) for 6 h or left untreated, and thenwere subjected to a luciferase assay. E SAP130 and FAF1 cooperate in repressing gene transcription. HEK293T cells were transiently transfectedwith pNF-κB-Luc, SAP130, and FAF1 expression plasmids or infected with lentiviruses expressing control shRNA or FAF1 shRNA as indicated. At 36 h
post-transfection/post-transduction, cells were treated with TNF-α (20 ng/ml) for 6 h or left untreated, and followed by a luciferase assay. F The
SUMO mutation does not alter SAP130 subcellular localization. Immunofluorescence images of HeLa cells transiently expressing HA-tagged SAP130
WT or 3KA as indicated. Immunostaining was performed with an anti-HA antibody (green). DAPI staining shows the position of the nucleus. Scalebars represent 10 μm. G Effect of the proteasome inhibitor MG132 on the accumulation of SUMO1-modified and unmodified forms of SAP130.
HA-SAP130 WT and 3KA SAP130 mutant were co-transfected with EGFP control or EGFP-SUMO1 plasmid into COS-1 cells. Twenty-four hours
after transfection, the cells were cultured in the presence and absence of 5 μM MG132 for another 8 h. SAP130 accumulation was detected by WB
with an anti-HA antibody. Cell lysates were also immunoblotted with anti-EGFP antibody to confirm the expression of EGFP and EGFP-SUMO1.
The bracket and arrow indicate EGFP-SUMO-1-modified and unmodified SAP130 proteins, respectively. The blue and red arrowheads indicate
the EGFP and EGFP-SUMO1 proteins, respectively. H Cell growth curves of HEK293 cells stably transfected with constructs expressing control
vector, HA-SAP130 WT or 3KA. SAP130 expression was analyzed using immunoblotting. I Colony formation assays were performed in stable vector(control), SAP130 WT and SAP130 3KA-overexpressing cells. Colonies were stained with 2% methylene blue 14 days later. Quantitative results in A-E,
H and I represent the mean ± SD of three independent experiments. Statistical analyses were performed using two-tailed Student’s t-test. *p < 0.05;**p < 0.01. The immunoblots were cropped for clarity. Full length blots are presented in Supplemental Figure S10

Chen et al. BMC Molecular and Cell Biology

Fig. 3 (See legend on previous page.)(2024) 25:2

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Chen et al. BMC Molecular and Cell Biology(2024) 25:2

potentiate the proteasomal degradation of SAP130,
supporting the idea that sumoylation of SAP130 plays arole in its protein stability.
While SAP130 protein has not yet been reported toregulate cell proliferation, we also examined whether
sumoylation of SAP130 might regulate cell proliferation. HEK293 cells were stably transfected with SAP130
WT or 3KA mutant and subjected to cell growth analysis. Compared to control cultures, SAP130 WT-overexpressing HEK293 cells showed a significantly increased
growth rate over a 7-day period, whereas SAP130 3KA
mutant had lower growth-promoting effect than SAP130WT (Fig. 3H). Additionally, overexpression of SAP130
WT resulted in an increase in colony numbers and sizes
compared with vector-transfected cells (Fig. 3I). Consistently, in contrast to SAP130 WT, SAP130 3KA was unable to significant increase the colony formation (Fig. 3I).
Together these results suggest that the sumoylation of
SAP130 is crucial for regulating its transcription-suppressing function, protein stability and its promotion ofcell proliferation.
FAF1 mitigates SAP130‑mediated transrepression
and suppresses SAP130‑mediated promotion of cellproliferation by promoting SAP130 protein degradation

Up to this point, we had shown that sumoylation of
SAP130 is required for its interaction with FAF1 andmodulates its protein stability. Since FAF1 acts as a scaffold and to regulate protein degradation, we next tested
whether FAF1 affects the steady-state level of SAP130.
We first overexpressed FAF1 and observed a consequent
decrease in SAP130 protein level (Fig. 4A). Additionally,

Page 11 of 16

the FAF1-mediated degradation of SAP130 could be
inhibited by pretreatment with the proteasome inhibitor
MG132, suggesting that FAF1-mediated SAP130 degradation occurs through a proteasome-dependent pathway(Fig. 4A). In line with these results, knockdown of FAF1
expression in human HEK293T, HCT116 and NB4 celllines consistently increased endogenous SAP130 protein levels (Fig. 4B and Supplemental Figure S7). Next,
we wanted to understand how FAF1 could affect SAP130
protein levels. First, we assessed the impact of FAF1 on
the SAP130 mRNA level. Co-transfection of FAF1 WT or
FAF1 DM mutant with the SAP130 did not alter SAP130mRNA levels, suggesting that FAF1 did not affect SAP130mRNA stability (Fig. 4C). Then, we determined theeffects of FAF1 on SAP130 protein half-life. COS-1 cells
were co-transfected with either FAF1 WT or DM mutant
and then treated with CHX. The SAP130 protein levels
at various time-points after CHX treatment were determined by immunoblotting. The results showed that overexpression of FAF1 WT significantly reduced the SAP130
half-life from 3 h to 1.5 h (Fig. 4D). However, overexpression of the DM mutant did not cause a significant change
in SAP130 levels. To further test whether and how FAF1
regulates the proteasomal degradation of SAP130, we
examined the effects of FAF1 on SAP130 ubiquitination.
Overexpression of FAF1 WT significantly increased theubiquitinated forms of SAP130, but the DM mutant didnot (Fig. 4E), suggesting that FAF1 promotes SAP130
degradation by enhancing its polyubiquitination upon
SUMO/SIM-mediated binding.
Furthermore, we examined the effects of modulating FAF1 on SAP130-mediated repression of(See figure on next page.)
Fig. 4 FAF1 motigates SAP130-mediated transrepression and suppresses SAP130-upregulated cell proliferation by promoting SAP130 protein
degradation. A FAF1 decreases the level of SAP130. HEK293 cells were co-transfected with HA-tagged SAP130 and the Flag-tagged FAF1 WT. At
24 h post-transfection, cells were treated with or without MG132 (5 μM) for 6 h. Cell lysates were immunoblotted with antibodies for HA, Flagand actin. B HEK293T, HCT116 and NB4 cells were infected with lentiviruses expressing control non-targeted shRNA or FAF1 shRNA as indicated.
Cell lysates were immunoblotted with antibodies for SAP130, FAF1 and actin. C FAF1 does not affect the mRNA level of SAP130. The abundanceof SAP130 mRNA was measured by RT-qPCR with GAPDH as the internal control. Results represent the mean ± standard deviation of three
independent experiments. D. COS-1 cells were co-transfected with the HA-SAP130, control vector, Flag-FAF1 WT, or Flag-FAF1 DM. After 48 h, cells
were treated with 10 μg/ml cycloheximide (CHX) for the indicated time. Cell lysates were subjected to immunoblotting with an anti-HA or anti-Flag
antibody, with actin as a loading control. The SAP130 expression level was quantified, and values were normalized to actin. E HEK293 cells wereco-transfected with myc-Ub, HA-SAP130 and Flag-FAF1 WT or Flag-FAF1 DM plasmids. After 48 h, cells were treated with MG132 (5 μM) for another6 h. Cell lysates were used for IP with anti-HA antibody, and bound proteins were detected by WB with anti-HA antibody or anti-myc antibody. The
SAP130 and FAF1 levels were determined by immunoblotting. WCL indicates whole cell lysate. F Inhibition of SAP130-mediated transcriptional
repression by FAF1. COS-1 cells were transiently transfected with Gal4-SAP130 WT or Gal4-SAP130 3KA expression vector in the absence or presenceof the indicated FAF1 expression vector together with a Gal4-dependent luciferase reporter. After 48 h, the luciferase activities were measured
for each sample. Quantitative results represent the mean ± SD from three independent experiments. **p < 0.01; NS, not significant. G and H FAF1
suppressed SAP130-mediated cell growth ability. HEK293 cells stably expressing the SAP130 WT or 3KA were transiently transfected with control
empty vector or Flag-FAF1 plasmid for 48 h and then split for the cell growth curve and colony formation assays. G For the colony formation assay,
cells were plated at a low density on dishes. After 2 weeks, cell colonies were stained with methylene blue. H Cell growth curve assay demonstrated
that overexpressing of FAF1 WT, but not FAF1 DM, inhibited the SAP130-mediated cell proliferation capability on the indicated time points.
Quantitative results represent the mean ± SD from three independent experiments. *P < 0.05, **p < 0.01; NS, not significant. The immunoblots were
cropped for clarity. Full length blots are presented in Supplemental Figure S11

Chen et al. BMC Molecular and Cell Biology

Fig. 4 (See legend on previous page.)(2024) 25:2

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Chen et al. BMC Molecular and Cell Biology(2024) 25:2

Gal4-responsive luciferase reporter activity. We reasoned that if FAF1 participates in the SAP130 degradation, overexpression of FAF1 should attenuate therepressive effect. As shown in Fig. 3A, co-transfection
with the Gal4 luciferase reporter construct with an
expression construct for Gal4-SAP130 WT or Gal4SAP130 3KA mutant could both significantly decreased
the reporter activity (Fig. 4F). As expected, co-expression of FAF1 relieved Gal4-SAP130 WT-mediatedrepressive activity, but it did not affect the activity of
Gal4-SAP130 3KA (Fig. 4F). We next explored if FAF1
was involved in regulating SAP130-mediated biological
functions, the cell growth ability was assessed. Our data
revealed that overexpression of FAF1 WT remarkably
attenuated the SAP130 WT-upregulated cell growth bycolony formation and cell growth curve assays (Fig. 4G
and H). However, FAF1 WT did not have a significant
effect on SAP130 3KA-mediated cell growth. Consistently, in contrast to FAF1 WT, FAF1 DM was unable
to significant inhibit SAP130 WT and 3KA-mediatedcell growth. These findings suggest that FAF1 relieves
SAP130-mediated transcriptional repression andpro-proliferative effect in a manner dependent on the
SUMO-SIM interactions.

Page 13 of 16

To further study the relationship between expressionof SAP130 and FAF1 in clinical samples, we analyzed thecancer datasets via cBioPortal database. The results indicated that the mRNA level of SAP130 was negatively correlated with that of FAF1 in several types of cancer, suchas diffuse large B-cell lymphoma (Person correlation:-0.45; p < 0.001), prostate adenocarcinoma (Person correlation: -0.37; p < 0.001), thyroid carcinoma (Person correlation: -0.17; p < 0.001) and colorectal adenocarcinoma(Person correlation: -0.13; p < 0.001) (Fig. 5). These results
are consistent with our in vitro data and also suggest thatthe interaction between SAP130 and FAF1 may play a
critical role in cancer development.

Discussion
In our previous study, we defined two SIMs within FAF1
that are crucial for binding for sumoylated mineralocorticoid receptor [24]. Additionally, we showed that FAF1
can promote the degradation of mineralocorticoid receptor, thereby inhibiting the its transactivation of target
genes. Here, we further show that FAF1 physical andfunctional interaction with SAP130 depends on SIM/
SUMO. We first show that SAP130 can be sumoylated at
Lys residues 794, 878 and 932. Then, we demonstrate that

Fig. 5 The mRNA levels of SAP130 and FAF1 were negatively correlated in tumor sample from different different types of cancer. Data shown are
correlation of the mRNA levels between SAP130 and FAF1 in tumor samples of various types of cancer via cBioPortal database

Chen et al. BMC Molecular and Cell Biology(2024) 25:2

SAP130 interacts with FAF1 in a SUMO/SIM-dependent manner, but binding of SAP130 to mSin3A/HDAC1
is largely unaffected by sumoylation. We also show thatsumoylation of SAP130 inhibits its transrepressive activity, and protein stability but does not affect its nuclear
localization. Furthermore, our data suggest that sumoylation is crucial for SAP130-induced promotion of HEK293
cell proliferation and that FAF1 promotes SAP130
ubiquitination and degradation, while downregulating
SAP130-mediated transcriptional repression and cellproliferation-promoting function. Thus, our study provides compelling evidence that FAF1 plays an important aregulatory role in SUMO-mediated transcriptional activity and protein stability of its interacting partner SAP130.
Sumoylation is known to affect many characteristics
of proteins, including their protein–protein interactions,transcriptional regulation functions, subcellular localization and stability. We defined three SUMO conjugationsites within the SAP130 C-terminal domain and showedthat these sites are crucial for interactions with the FAF1.
Of note, the C-terminal portion of SAP130 also interacts
with mSin3A and HDAC1 [11]. Interestingly, we did notobserve evidence for sumoylation-induced changes in thebinding of mSin3A to SAP130 (Fig. 2). The association ofmSin3A with the SAP130 3KA mutant may also explain
why the mutant still exhibits repression activity (Fig. 3AD). Thus, our findings suggest a conformational or spatial
separation between SAP130 structural features involved
in its association with mSin3A/HDAC1 complex andthose involved in sumoylation-mediated recruitment of
FAF1.
We also investigated whether SUMO1 modifications
might play a role in modulating the transrepression activity and stability of SAP130. Our results showed that when
all three SAP130 SUMO conjugation sites are mutatedto Ala, the transcriptional repressive activity is increased
compared with WT protein, suggesting that conjugation of SUMO1 to SAP130 attenuates its transcriptional
repressor activity (Fig. 3A-D). The mechanism of thischange in SAP130 is likely due to sumoylation-mediatedalterations in protein stability. Along these lines, wefound that SUMO1-modified SAP130 is stabilized to agreater extent than unmodified SAP130 after treatment
with MG132 (Fig. 3G), suggesting that SUMO1 modification of SAP130 potentiates proteasomal degradation. We
also demonstrated that the interaction between SAP130
and FAF1 promotes proteasome-mediated degradationof SAP130 (Fig. 4E). Consistent with these findings, overexpression of FAF1 significantly inhibited the intrinsic
repression ability of SAP130 (Fig. 4F). It is conceivablethat a sumoylation-induced interaction with FAF1 may
facilitate SAP130 degradation and decrease the transcriptional repressive activity of SAP130. While we provide

Page 14 of 16

evidence that SAP130 is a sumoylation target, the physiological determinants regulating its sumoylation remain
unknown. It is possible that diverse stimuli participate
in this process, and may thereby fine-tune SAP130-regulated transcriptional activity.
Although SAP130 is known to associate with mSin3A/
HDAC complex during transcriptional repression, little
information exists about the role of SAP130 in the control
of cell growth and signaling pathways. Here, we reportthat sumoylation of SAP130 contributes to transcriptional repression of mineralocorticoid receptor, NF-κB
and WNT signaling pathways (Fig. 3B-D). As such, weobserved that the SAP130 3KA or 3KR mutant exhibits
greater inhibition of these reporter activities than WT
SAP130, which suggests a role for SAP130 sumoylationin the regulation of gene transcription. Furthermore,
HEK293 cells that stably express SAP130 show enhanced
cell proliferation, whereas expression of the SAP130 3KA
mutant does not enhance proliferation to the same extent(Fig. 3H and I). Up to now, no role for SAP130 protein in
the regulation of cell proliferation has yet been reported,and it still remains unclear how or whether the degrees
of SAP130 expression and sumoylation may be associated with tumorigenesis. It will therefore be of interest
to further clarify whether SAP130 has tumor-promoting potential. Given that FAF1 has been implicated as atumor-suppressor and we demonstrated that FAF1 inhibits SAP130-mediated transcriptional function, stability and cell proliferation-promoting ability, it is possiblethat FAF1 would attenuate any oncogenic potential of
SAP130. Notably, analysis of the cancer datasets via cBioPortal database revealed that the mRNA level of SAP130
was negative correlated with FAF1 mRNA level in several types of cancers (Fig. 5). Thus, our findings warrant
new studies to expand our understanding of the roles of
SAP130 and FAF1 in carcinogenesis.
FAF1 participates the proteasomal degradation ofubiquitinated proteins, as it is thought to act as a scaffold protein or ubiquitin receptor for multiple ubiquitinrelated domains, including ubiquitin-associated (UBA),
ubiquitin-like 1 and 2 (UBL1, UBL2), and ubiquitinregulatory X (UBX) domains [20]. Our work provides
novel mechanistic insights into the role of FAF1/SIMs
in SUMO-dependent transcriptional modulation andproteolysis ([24] and this study). Recent studies haverevealed the importance of SUMO-Ub chain receptors that contain tandem SIMs and UIMs (tSIM-UIMs)[6–8]. In addition, two very recent studies reported
that C. elegans UBXN-3/FAF1 is an important coordinator of DNA replication, as it controls the turnover of
SUMO- and ubiquitin-modified DNA replication factors[25, 26]. Taken together, these studies and ours highlightthe importance of FAF1 in modulating the stability ofChen et al. BMC Molecular and Cell Biology(2024) 25:2

SUMO- and ubiquitin-modified proteins. Further work
will be required to clarify how the SIMs and ubiquitinrelated domains of FAF1 differentially contribute to its
regulation of diverse biological processes.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12860-​023-​00498-x.
Additional file 1: Supplementary Figure S1. Characterization of SUMO
target lysine residues in SAP130. Western blot showing HA-tagged
SAP130 modified by EGFP-SUMO-1. A plasmid expressing HA-tagged
wild-type SAP130 or various mutant SAP130 was co-transfected with
EGFP control or EGFP-SUMO1 plasmid into COS-1 cells. Cell lysates wereused for WB analysis using antibodies against HA and EGFP. The bracket
and arrow indicate EGFP-SUMO-1-modified and unmodified SAP130
proteins, respectively. The blue and red arrowheads indicate the EGFP and
EGFPSUMO1 proteins, respectively. Notably, the mutation of Lysine (K)794, 878, and 932 to alanine (A) or arginine (R) showed the similar results.
Supplementary Figure S2. SUMO-dependent interaction between FAF1
and SAP130. (A) Yeast strain L40 was co-transformed with bait (human
WT SAP130 or mutant at three sumoylation residues (3KA or 3KR), or
the control protein lamin fused to the LexA DNA-binding domain) andprey constructs [human FAF1 WT or FAF1 with SIM mutant (DM) fusedto the Gal-activation domain (AD)]. Yeast transformants were spotted onplates with histidine (-TULL) or without histidine (-TULLH), and with X-Gal(+X-Gal) media. Schematic presentation of FAF1 DM, SAP130 C-terminal
3KA and 3KR mutants analyzed in a yeast two-hybrid assay (top). (B) Yeast
co-transformed with the indicated bait and prey were analyzed by quan‑titative β-Gal assays. Supplementary Figure S3. Sumoylation of SAP130
protein modulates its transcription-suppressing function. The SAP130 3KA
or 3KR mutant exhibited similar transcriptional repression activity on MRstimulated MMTV and TNF-α-stimulated NF-κB luciferase reporters. (A) The
MMTV-Luc reporter plasmid, the internal control plasmid pRL-TK (Renilla),pRS-hMR, and HA-tagged-SAP130 WT, 3KA or 3KR mutant plasmids
were co-transfected into COS-1 cells. The total DNA amount was 2 μg(including addition of empty vector). At 24 h post-transfection, cells weretreated with either vehicle or 1 μM aldosterone (Aldo.), and reporter geneactivities were measured after another 24 h period. (B) HEK293T cells wereco-transfected with pNF-κB-Luc reporter plasmid and pRL-TK plasmid
together with HA-tagged-SAP130 WT, 3KA or 3KR mutant plasmid. At 36 h
post-transfection, cells were treated with tumor necrosis factor (TNF)-α (20ng/ml) for 6 h or left untreated, and then were subjected to a luciferase
assay. Results represent the mean ± SD of three independent experiments.
Statistical analyses were performed using two-tailed Student’s t-test. *p< 0.05. Supplementary Figure S4. Overexpression of FAF1 or SAP130
does not alter the endogenous Sin3A and HDAC1 protein levels and their
nuclear localization. (A) HEK293T cells were transfected with the control
vector or two different doses of the HA-tagged-FAF1, SAP130 WT or 3KA
mutant plasmid. At 48 h post-transfection, cell lysates were subjected toimmunoblotting with an anti-HA , Sin3A or HDAC1 antibody, with actin asa loading control. The immunoblots were cropped for clarity. Full length
blots are presented in Supplemental Figure S12. (B) COS-1 cells were tran‑siently transfected with HA-tagged FAF1, SAP130 WT or 3KA expression
plasmid. After 48 h, cells were subjected to immunostaining analyses with
anti-HA antibody (green) and anti-Sin3A or HDAC1 antibody (red). The sec‑
ondary antibodies for these studies were fluorescein isothiocyanatecon‑jugated anti-rabbit IgG and Texas red-conjugated anti-mouse IgG. DAPI
staining shows the position of the nucleus. The open arrowhead indicatesthe HA-FAF1 or SAP130-transfected cells. Supplementary Figure S5. The
SUMO mutation does not alter SAP130 subcellular localization. Immu‑
nofluorescence images of HeLa cells transiently expressing HA-tagged
SAP130 WT, 3KA or 3KR as indicated. Immunostaining was performed
with an anti-HA antibody (green). DAPI staining shows the position of thenucleus. Supplementary Figure S6. COS-1 cells were transfected with
the HA-SAP130 WT or HA-SAP130 3KA. After 48 h, cells were treated with
10 μg/ml cycloheximide (CHX) for the indicated time. Cell lysates were
subjected to immunoblotting with an anti-HA antibody, with actin as aPage 15 of 16

loading control. The SAP130 expression level was quantified, and values
were normalized to actin. The immunoblots were cropped for clarity. Fulllength blots are presented in Supplemental Figure S13. Supplementary
Figure S7. HEK293T, HCT116 and NB4 cells were infected with lentiviruses
expressing control non-targeted shRNA or FAF1 shRNA as indicated.
Cell lysates were immunoblotted with antibodies for SAP130, FAF1 andactin as in Fig. 4B. Quantification of endogenous SAP130 protein levels in
shFAF1 lentiviruses infected NB4 (A), HCT116 (B) and HEK293T cells (C).
Three independent Western Blots were quantified by densitometry using
Image J software. The protein expression of SAP130 was normalized withrespect to the corresponding actin band densities. *p < 0.05; **p < 0.01.
Supplementary Figure S8. Full length blots used in Fig. 1. The cropped
areas used in Fig. 1 are shown in red boxes. Supplementary Figure S9.
Full length blots used in Fig. 2. The cropped areas used in Fig. 2 are shownin red boxes. Supplementary Figure S10. Full length blots used in Fig. 3.
The cropped areas used in Fig. 3 are shown in red boxes. Supplementary
Figure S11. Full length blots used in Fig. 4. The cropped areas used inFig. 4 are shown in red boxes. Supplementary Figure S12. Full length
blots used in supplementary Figure S4. The cropped areas used in supple‑
mentary Figure S4 are shown in red boxes. Supplementary Figure S13.
Full length blots used in supplementary Figure S6. The cropped areas usedin supplementary Figure S6 are shown in red boxes.
Acknowledgements
We thank Dr. Marcus Calkins for English editing. We thank Dr. Tsai-Yun Chenfrom National Cheng Kung University (Tainan, Taiwan) for kindly providing
NB4 cells.
Authors’ contributions
CHC and DYL designed this study. CHC carried out the majority of the experi‑ments. HWL, MFH, NTP and ZYC contributed to performing the experiments.
CWC, KHL and WCC supervised the work and critically read the manuscript.
DYL wrote the draft and revised the manuscript. All of the authors read andapproved the final manuscript.
Funding
This work was supported by the grant 110–2320-B-006–040 from the Ministryof science and technology of Taiwan (to D.-Y. L.).
Availability of data and materials
The reagents used and data generated in this study are available from thecorresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 20 August 2023 Accepted: 20 December 2023

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