The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing
Nucleic Acids Research
The chromatin scaffold protein SAFB1 localizes SUMO-1 to the promoters of ribosomal protein genes to facilitate transcription initiation and splicing
Hui-wen Liu 1 2
Tapahsama Banerjee 1 2
Xiaoyan Guan 0 1
Michael A. Freitas 0 1
D. Parvin 1 2
0 Department of Molecular Virology, Immunology, and Medical Genetics, Comprehensive Cancer Center, The Ohio State University , Columbus, OH 43210 , USA
1 Present addresses: Hui-wen Liu, Oregon Health and Science University , Portland, OR 97239 , USA. Xiaoyan Guan, Florida State University , Tallahassee, FL , USA
2 Department of Biomedical Informatics, Comprehensive Cancer Center, The Ohio State University , Columbus, OH 43210 , USA
Early steps of gene expression are a composite of promoter recognition, promoter activation, RNA synthesis and RNA processing, and it is known that SUMOylation, a post-translational modification, is involved in transcription regulation. We previously found that SUMO-1 marks chromatin at the proximal promoter regions of some of the most active housekeeping genes during interphase in human cells, but the SUMOylated targets on the chromatin remained unclear. In this study, we found that SUMO-1 marks the promoters of ribosomal protein genes via modification of the Scaffold Associated Factor B (SAFB) protein, and the SUMOylated SAFB stimulated both the binding of RNA polymerase to promoters and premRNA splicing. Depletion of SAFB decreased RNA polymerase II binding to promoters and nuclear processing of the mRNA, though mRNA stability was not affected. This study reveals an unexpected role of SUMO-1 and SAFB in the stimulatory coupling of promoter binding, transcription initiation and RNA processing.
Small Ubiquitin-related Modifier (SUMO) proteins are
highly conserved among eukaryotes, and protein
SUMOylation has a critical role in a variety of cellular signaling
pathways including control of cell cycle progression, DNA
repair, gene expression and nuclear architecture (1). Among
various SUMO substrates that have been identified,
transcription factors and co-regulators comprise one of the
largest groups. Studies have provided strong evidence for the
involvement of SUMOylation in transcriptional regulation
(2). SUMOylation of those transcription factors in general
is repressive, and current models suggest that
SUMOylation leads to the recruitment of transcriptional co-repressor
complexes and histone deacetylases (HDACs) to the
promoters (3,4). However, there is also evidence that
SUMOylation of transcription factors can lead to gene activation
(5–7). In a previous study, we found that SUMO-1 modifies
chromatin-associated proteins located at the promoter
regions of highly active genes in human cells, including those
that encode ribosome protein subunits (8). SUMO
association on active promoters has also been observed in yeast and
in human fibroblasts (9,10). These studies have suggested
that SUMOylation of transcription factors is not merely
acting as a switch for gene silencing; rather, it also plays
an important role for modulating transcription activation.
However, the role of how SUMOylation modulates
chromatin structure, and further participates in transcriptional
control of constitutive genes is largely unknown.
In this study, we first sought to identify the SUMOylated
protein bound to the chromatin at active promoters, and we
found that Scaffold Associated Factor-B (SAFB), a DNA
and RNA binding protein, is one of the SUMO-1 targets.
Two homologs (SAFB1 and SAFB2) have been found with
74% similarity at the amino acid level, and up to 98%
similarity in some functional domains and display redundant
activity (11). SAFB1 interacts with the carboxy-terminus of
RNA polymerase II (RNAPII) and RNA processing
proteins such as SR proteins (12–15), suggesting a potential
role in RNA splicing. SAFB binds AT-rich scaffold/matrix
attachment regions (S/MAR) on DNA, which are found
close to regulatory loci and mediate chromatin looping to
coordinate distant chromatin interactions and higher order
chromatin structure (16,17). SAFB proteins interact with
RNA through the RNA recognizing motif (RRM), which
suggests a role in mRNA processing. Together, this
suggests that SAFB may be part of a ‘transcriptosome
complex’ to couple transcription, splicing, and polyadenylation
(13). This hypothesis is supported by a study that SAFB1
interacts with CHD1, a chromatin modifying protein that
also possesses activities in RNA splicing (18,19). In
addition, SAFB has been found to function as a co-repressor
of estrogen-dependent transcription (20), and participates
the repression of immune regulators and apoptotic genes
(21). Recent studies suggest that it may be involved in a
more widespread manner by functioning as a positive
regulator for permissive chromatin of the myogenic
differentiation (22), and in response to DNA damage (23).
Here, we provide evidence that both SAFB1 is a
SUMO1 substrate bound to the chromatin during interphase in
a region centering on 100 bp upstream of the
transcription start site. Like SUMO-1, depletion of SAFB
diminished RNAPII binding to promoters and decreased RNA
expression of these ribosomal protein genes, revealing an
unexpected role of SAFB linking transcription initiation to
RNA processing of the highly active ribosomal protein (RP)
MATERIALS AND METHODS
Chromatin affinity purification (ChAP) for mass
ChAP was based on the ChIP method except that the
immunoprecipitation was replaced by a two-step affinity
purification from HeLa-SUMO1 cells, a HeLa-derived cell
line that expresses a SUMO-1 protein that includes on its
amino-terminus a hexa-histidine tag and a biotin binding
domain (8). Cells were synchronized in S phase or in
mitosis. 108 HeLa-SUMO1 cells were lysed in lysis buffer
I (50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid–potassium hydroxide (HEPES–KOH), pH 7.5, 140
mM NaCl, 1 mM Ethylenediaminetetraacetic acid (EDTA),
10% glycerol, 0.5% NP-40, 0.25% Triton-X-100), and the
cell pellet was resuspended in lysis buffer II (10 mM Tris,
pH 8, 80 mM NaCl, 1 mM EDTA, 0.5 mM ethylene
glycol tetraacetic acid (EGTA)), and, following centrifugation
(1400 x g), the chromatin was recovered from the pellet and
resuspended in lysis buffer III (50 mM Tris pH 8; 0.01%
SDS; 1.1% Triton X-100; 80 mM NaCl). The isolated
chromatin was sheared to 200–300 bp by sonication, incubated
with 375 l of Ni-NTA beads (Qiagen) for 16 h at 4◦C.
After washing in wash buffer I (50 mM Tris pH 8; 0.01% SDS;
1.1% Triton X-100; 150 mM NaCl), chromatin fragments
were eluted in 6 ml elution buffer (washing buffer I with 300
mM imidazole). The nickel eluate was incubated with 375
l of streptavidin beads (Invitrogen) for 6 h at 4◦C. After
three stringent washes, wash buffer II (50 mM Tris pH 8; 10
mM EDTA; 1% SDS; 1M NaCl), and three times of 10 mM
Tris buffer, pH 8. The chromatin was then trypsinized (ratio
1:120 w/w) in solution for 2 h at 37◦C. Following
lyophilization, the digested solution was dissolved in 50 l high
performance liquid chromatography (HPLC) grade water and
subjected to LC–MS/MS; protein identification was
analyzed using Massmatrix 2.4.2 (24). Environmental
contaminants were removed from the identified proteins.
Antibody used in this study
The rabbit polyclonal SUMO-1 and 8WG16 monoclonal
antibodies were described previously (8,25). Other
antibodies used included the RNAPII phospho-serine 5 antibody
(Abcam cat. no. ab5131), SAFB antibody (Millipore cat.
no. 05-588) and -tubulin (Sigma).
Isolation of nuclear and cytoplasmic RNA, RT-qPCR
Nuclear and cytoplasmic RNAs were purified by lysis of
HeLa-SUMO1 cells in 200 l of lysis buffer (10 mM Tris–
HCl, pH 8, 1.5 mM MgCl2, 0.5% NP-40, 140 mM NaCl
and 0.5 U of RNase inhibitor) and loaded onto 200 l of
cushion buffer (10 mM Tris–HCl, pH 8, 1.5 mM MgCl2,
1% NP-40, 140 mM NaCl and 0.4 M sucrose). The samples
were centrifuged for 10 min at 800 × g. The cytoplasmic
supernatant was treated with proteinase K, extracted with
phenol–chloroform and precipitated with ethanol. The
nuclear pellet was resuspended in 100 l of DNAse I buffer
(50 mM Tris–HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl2
and 0.5 U RNase inhibitor) and treated 20 U of DNase
I (Invitrogen) for 60 min at 37◦C. The samples were then
extracted with phenol–chloroform and precipitated with
ethanol. The mRNA levels were determined by reverse
transcriptase quantitative polymerase chain reaction
(RTqPCR) using the iScript reverse transcription and iQSYBR
Green supermix (Bio-Rad). The results were normalized to
Immunoprecipitation, ChAP-qPCR and ChIP-qPCR
Immunoprecipitation was performed following established
procedures (26). Chromatin immunoprecipitation or
affinity purification was done following the protocol that has
been reported previously (8). Briefly, 2 × 107 HeLa cells (if
ChIP) or HeLa-SUMO1 cells (if ChAP) were fixed with 1%
formaldehyde, lysed in lysis buffer, sonicated to size ranging
from 200 to 1000 bp, chromatin was diluted 4-fold, after
removal of a control aliquot, incubated at 4◦C overnight with
antibody against 8WG16 (1:100), 5 g Ser-5p antibodies, or
5 g IgG antibody was used as a negative control.
Immunocomplex was precipitated with 50 l protein A sepharose
beads (GE Healthcare), washed with
Radioimmunoprecipitation assay buffer, 10 mM Tris pH 7.5, 300 mM NaCl,
1% NP-40, 1% deoxylcholate, 0.1% SDS (RIPA) buffer
followed by crosslink reversal. The immunoprecipitated DNA
was purified by PCR purification kit (Qiagen). All the
experiments included at least three independent replicates. The
primer sequences are available in Supplementary Table S2.
SUMOylation facilitates RNAPII recruitment on
constitutively active promoters
We have previously shown that SUMO-1 is enriched on the
chromatin proteins bound to promoter regions of some of
the most active genes during interphase, such as ribosomal
protein (RP) encoding genes (8), which are highly
abundant and constitutively transcribed by RNA polymerase
II (RNAPII). Depletion of SUMO-1 caused a decrease in
mRNA production, suggesting that the presence of
SUMO1 on the active promoter regions had a positive role for
SUMOylation in transcriptional regulation (8). It had not
been shown in human cells whether the decrease in mRNA
abundance following depletion of SUMO-1 was due to a
decrease in RNAPII binding. In yeast, which have only one
SUMO isoform, SUMO protein is required for RNAPII
recruitment on the constitutive genes during transcription
initiation (9). We tested whether the SUMO-1 mark on the
chromatin bound to a housekeeping promoter stimulates
active RNAPII recruitment to promoters in mammalian
cells. We investigated RNAPII occupancy under defective
SUMOylation in HeLa cells by siRNA transfection
targeting UBC9, the only SUMO-specific E2 ligase found in cells,
and testing for RNAPII binding to promoter regions of
ribosomal protein genes such as RPL3, RPL7A, RPL10A,
RPL26, which were found enriched with SUMO-1 (8). In
addition, we analyzed the -actin promoter, which was not
bound by SUMO-1, as a negative control. The enrichment
of SUMO-1, RNAPII with phosphorylated serine-5
(pSer5) of the carboxy-terminal domain (CTD) and RNAPII
that is mainly unphosphorylated (8WG16) bound to
promoters, was analyzed by ChIP-qPCR. The results were
normalized to the control siRNA in each experiment to
correct for a modest amount of variation in the percent of
input obtained in each quantitative PCR assay. The results
showed that upon UBC9 siRNA depletion, the occupancy
of SUMO-1 on the promoters of RP genes significantly
decreased 4- to 5-fold compared to controls (Figure 1C).
We found that depletion of UBC9 and consequent defect
in SUMOylation caused a decrease in the occupancy at
these promoters of both unphosphorylated and
phosphorylated form of RNAPII. Binding of the unphosphorylated
RNAPII was decreased 2.5- to 5-fold. Binding of the
pSer5 form of RNAPII was decreased 4- to 5-fold compared to
controls (Figure 1A and B). By contrast, the binding of
either form of RNAPII to the -actin promoter was not
affected by depletion of UBC9 (Figure 1A and B). It is
noteworthy that the effect on the pSer-5 form of RNAPII was
of somewhat higher magnitude than the unphosphorylated
form of RNAPII, suggesting that SUMOylation impacted
the initiation process. The same results, shown in
Supplementary Figure S1 but as individual experiments, clearly
indicate the reduction in RNAPII and phospho-RNAPII
associated with these promoters after depletion of the UBC9.
SAFB is SUMO-1 modified and associated with RP gene
Since SUMOylated chromatin-associated proteins and
transcription factors are low in abundance, and
SUMOylated targets are highly dynamic and rapidly reversed by
SUMO proteases, a cell line called HeLa-SUMO1 that
stably expresses SUMO-1 fused with a hexahistidine and
biotinylated (HB) tag was used for isolation of
SUMO-1labeled chromatin proteins. SUMO-1 modified chromatin
associated proteins were shown to be present during
interphase and absent during mitosis (8). To identify the
SUMO1 substrates that mark the promoters during interphase, we
purified the chromatin fraction from cells in either S phase
or mitosis. The SUMOylated, and tagged, chromatin
proteins were purified by metal ion affinity purification
followed by avidin affinity purification with stringent washes.
Purified proteins were then analyzed by mass spectrometry
in order to identify proteins covalently bound to
SUMO1 during S phase and not during mitosis. Fifty-two
proteins were identified by MS analysis, and the gene ontology
(GO) term analysis showed that the top functions of those
chromatin-associated proteins purified by virtue of binding
to SUMO-1 in interphase were in RNA metabolism and
protein synthesis pathways (Supplementary Table S1). For
example, many RNA processing related factors, such as
hnRNPs, PTB-associated splicing factor (SFPQ) and CPSF7,
were detected as chromatin associated factors that were
covalently SUMOylated. We suggest that SUMOylation of
these splicing related factors on the promoters may serve
as a link between transcriptional initiation and pre-mRNA
splicing (Supplementary Table S1).
The top 20 proteins identified by mass spectrometry are
listed in order of highest score in Table 1. SAFB2 and
SAFB1 were the proteins with the fifth highest score and
the seventh highest score and had zero peptides detected in
the mitotic chromatin but a high number of peptides in the
S-phase chromatin. The peptide coverage for each protein is
shown in Supplementary Figure S2. Interestingly, SAFB1/2
binding sites in promoter regions have been shown in
another study (21), and two of the identified protein peaks
representing SAFB binding sites in the pS2 and Hsp27
promoters (21) from that study coincided with genomic loci we
observed to be bound by SUMO-1 in HeLa cells (data not
shown). In another study, it had been found that SAFB was
SUMOylated (26). We confirmed that SAFB was
SUMOylated by affinity purification and immunoblot analysis,
revealing a prominent SAFB protein in the input sample
migrating at a position consistent with unmodified protein
plus several bands of slower migration and lighter intensity.
The unbound fraction contained a band of similar mass as
the unmodified SAFB. By contrast, the bound, SUMO-1
tagged eluate contained multiple polypeptides that bound
to SAFB specific antibody and that were shifted to slower
migration (Figure 2A, lane 3), which we interpret to be
consistent multiple SUMOylations of the two isoforms of the
SAFB protein. In addition, we tested whether SAFB
localized to the RP gene promoters by transfecting HeLa cells
with SAFB-1 gene fused to the his6-biotin (HB) tag, and
followed by ChIP-qPCR analysis. Transfection into control
cells of a HB-tag only plasmid was used to control for
nonspecific binding. SAFB-1 was found to associate with all
eight of the RP promoters tested (Figure 2B).
SAFB localizes SUMO-1 to promoters
To determine if SAFB was responsible for the recruitment
of SUMO-1 binding on the specific promoters, we tested
whether depletion of SAFB affected the recruitment of
SUMO-1 to promoters that we had previously
characterized to be SUMO-1 bound (8). Since there are two highly
related isoforms of SAFB, we depleted both homologs with
siRNAs targeting SAFB1/2. Following siRNA
transfection, immunoblot analysis showed that SAFB protein was
depleted by >90% (Figure 3C). Consistent with earlier
results, ChIP-qPCR analysis showed that under control
conditions SUMO-1 and RNAPII were enriched on the RP
gene promoter regions analyzed; IL2 was included as a
negative control since it is not expressed in HeLa cells and
its promoter had no detected SUMO-1. SAFB depletion
caused a significant decrease in the SUMO-1 marks on
the RP gene promoters, down to 40–50% compared to the
controls (Figure 3A). Depletion of SAFB also caused a
decrease in RNAPII occupancy on these promoters
(Figure 3B). By contrast, when testing active genes that are not
labeled by SUMO-1, such as -actin, depletion of SAFB
did not affect RNAPII occupancy on its promoter (Figure
3B), suggesting that SAFB facilitates RNAPII binding on
the SUMO-1 labeled active genes. We further asked whether
this phenomenon was caused by SAFB1. To this end, a
second set of siRNAs for depletion of SAFB targeted the
3 UTR of SAFB1 and a second site in the ORF of SAFB2.
Transfection of this second set of siRNAs also decreased
ChIP specific for SUMO-1 at RP gene promoters, and
expression of SAFB1 from a cotransfected plasmid rescued
SUMO-1 binding to these promoters (Figure 4). These
results clearly indicate that SAFB1 is a functionally relevant
SUMO-1 target bound to the promoters, and these results
support a model whereby the SUMOylation of SAFB
stimulates RNAPII binding to target gene promoters.
SAFB depletion caused down regulation of mRNA processing
of RP genes
We have shown previously that SUMO-1 marks the
chromatin just upstream of the transcription start site of
constitutive housekeeping genes and that the SUMO-1 mark
stimulated transcription. In addition, the SUMO-1 mark was
also found enriched on exons in the human genome,
suggesting a potential role for facilitating splicing (8). Given
that SAFB interacts with the CTD of RNAPII (13), that
SAFB1 is involved in recruitment of SUMO-1 and RNAPII
on the promoters (Figures 3 and 4), and that our mass
spectrometry results indicated multiple SUMOylated splicing
factors (Supplementary Table S1), we tested whether SAFB
depletion may affect mRNA expression of the RP genes at
the level of pre-mRNA splicing. We investigated the RNA
processing of two RP genes, RPL26 and RPL7a, by
quantifying RNA containing the exon-exon junction for spliced
mRNA in the nucleus, and we quantified the abundance of
the intron-exon junctions for measuring pre-mRNA
concentration. We are confident that the PCR product from the
unspliced pre-mRNA did not result from contamination of
genomic DNA since the samples were thoroughly treated
with DNase and since the pre-mRNA decreased over time
following actinomycin D treatment (Figure 6), and such a
decrease over time is inconsistent with genomic DNA
contamination. The RT-qPCR analysis showed that depletion
of either SUMO-1 or SAFB did not affect the abundance
of the primary transcripts relative to the control in the
nucleus (Figure 5A), but the spliced mRNA purified from the
nucleus was less abundant in SUMO-1 or SAFB depleted
cells. This result suggested that SUMO-1 and SAFB are
involved in mRNA processing (Figure 5B). It was surprising
that the decrease in splicing did not result in an excess
accumulation of nuclear pre-mRNA. We suggest that the
decrease in initiation (Figure 1) was balanced by the decrease
in RNA processing (Figure 5B) to yield little change to the
unspliced pre-mRNA (Figure 5A). We also tested the
mature mRNA in the cytosol, and the RP genes were reduced
due to either SUMO-1 or SAFB depletion (Figure 5C).
To further investigate the effect of SAFB on RNA
metabolism, we asked whether depletion of SAFB affected
mRNA stability. Actinomycin D was included in media to
block new mRNA transcription, and we harvested RNA
species at different time points. We found that there is no
change of mature mRNA, suggesting the RP RNA is fairly
stable for at least 2 h, independent of SUMOylation or of
SAFB (Figure 6A). The precursor mRNA decreased at a
similar rate from cells that were SAFB depleted or control
siRNA transfected, and the difference in splicing rate for
SAFB depleted is not detected when in the presence of
actinomycin D, indicating that RNA stability is not affected by
SAFB depletion (Figure 6B). It was noted that following
addition of actinomycin D to the medium, there was a rapid
and reproducible spike in the abundance of the RPL26
prespliced RNA and spliced RNA. This spike was observed in
all three replicates of the control depleted cells as well as the
SAFB depleted cells, though the timing of the spike was
perhaps delayed in the SAFB depleted cells. Though the cause
of this spike in RNA concentration was not clear, its
presence did not affect the interpretation that the RNA stability
was not affected by SAFB depletion. Taken together with
the other experiments, the newly transcribed mRNA in the
nucleus was down regulated in SAFB depleted cells
(Figure 4B), and we suggest that SUMOylated SAFB stimulates
mRNA splicing, and the down regulation of mRNA in the
nucleus was not due to the RNA stability.
Since ribosomal protein expression is highly sensitive
to cell proliferation rates, we tested whether depletion of
SAFB or SUMO-1 affected cell proliferation. We depleted
SAFB and SUMO-1 from HeLa cells as usual (Figure 6E),
and seeded 10 000 cells per well and measured the cell
number each day for 4 days. The proliferation rates were not
statistically different (Figure 6D), indicating that the changes
in RP RNA abundance observed in Figure 5 were not
secondary to significant changes in growth rates.
In this study, we discovered that SUMO-1 binds to the
chromatin at promoters of ribosomal protein genes via the
scaffold attachment factor, SAFB. We found that SAFB is
SUMOylated, and depletion of SAFB caused a decrease
of SUMO-1 association with the promoters on the
chromatin. These promoters, encoding RP genes and
translation factors, are among the most active RNAPII
promoters in the cell, and SUMO-1-tagged SAFB stimulates both
the recruitment of RNAPII and the splicing of the product
RNAs. In addition, a number of RNA processing factors
were found to be SUMO-1 targets, suggesting that
SUMO1 marks on the promoter during the transcription cycle can
be important for determining the efficiency of pre-mRNA
splicing. The SUMO-1-SAFB axis revealed in this study,
defines a fast track for gene expression; blocking this pathway
via RNAi mediated depletion did not cause a total
inhibition of transcription and processing but rather a decrease
toward the expression levels of most protein-encoding genes
The SAFB1 protein has been reported to be covalently
modified by SUMO-1 as well as SUMO-2/3, and this
modification is associated with transcriptional repression
activity (26). The lysine acceptors for the SAFB1-mediated
corepressor function were identified to be K231 and K294,
and these modifications were important for the SAFB1
recruitment of HDAC3 to these promoters and
transcriptional repression (26). In the current study, only SUMO-1
was tested, and it remains to be tested if SUMO-2/3
modification has similar effects on stimulating transcriptional
initiation and RNA processing. In addition, the SUMO E3
ligase PIAS1 and the SENP1 SUMO protease were found
to regulate the conjugation and removal of SUMO proteins
on to SAFB1 (26). It will be of great interest to determine
whether these enzymes similarly affect the ribosomal
protein transcription and RNA process characterized in the
current study. It was striking that SUMOylation of SAFB1
was associated with transcriptional repression of estrogen
receptor regulated genes (21,26), but in the current study
this modification resulted in transcriptional stimulation of
RP genes. It will be of great interest to characterize the
genespecific activities of the SUMO1 modification of SAFB.
SUMOylation of multiple proteins in a single
pathway that stimulate the DNA repair process has been
described (27), and we suggest that a similar synergy exists
for SUMOylation of SAFB and splicing factors for
coupling transcription initiation with processing. The concept
of linking transcription initiation with splicing has been
described; a previous study showed that the strength of a
promoter-bound activator could also affect the efficiency
of constitutive splicing and 3 -end cleavage of different
reporter pre-mRNAs (28). This activator-dependent increase
in pre-mRNA processing efficiency required the RNAPII
CTD (29). In this study, we found that the link between
initiation and mRNA processing of RP genes depended on the
SUMOylation of SAFB.
Nuclear function depends on organizing platforms for
establishing structural and functional domains in the
nucleus. In this study, we found that SUMOylation facilitates
RNAPII recruitment on the constitutive promoters, which
is consistent with the observation in yeast (9). In
addition, SAFB depletion caused down-regulation of
SUMO1 and RNAPII binding on those highly active promoters,
and the mature mRNA expression, suggesting that SAFB
links transcription initiation and splicing for the most
active RNAPII transcripts in the cell. There are emerging
studies showing the importance of SAFB on regulation of
chromatin architecture. For example, SAFB1 participates in
chromatin remodeling by interacting with ATP-dependent
chromatin modifying proteins such as CHD1 (18). The
CHD1 protein binds to a histone mark in promoters and
regulates early transcription events including splicing (19).
It is possible that SAFB interaction with CHD1 is in part
regulated by SUMO-1 and is interacting with the
CHD1spliceosome complex. A recent study reported that SAFB1
regulates chromatin accessibility in response to genotoxic
stress, and it is transiently recruited to DNA damage sites
for efficient signaling and the downstream phosphorylation
of chromatin (23). Another study showed that SAFB1 is
associated with the activation of skeletal muscle gene
expression during myogenic differentiation by facilitating the
transition of promoter sequences from a repressive
chromatin structure to one that is transcriptionally active (22).
It is well-established that the localization of the splicing SR
proteins coincides with the sites of active RNAPII during
transcription (29). Considering that both SAFB and
splicing SR proteins interact with the RNAPII CTD, and the
concept of a transcriptosome complex has been suggested
to link transcription initiation and splicing (13), we suggest
that the SUMOylation of SAFB participates in this
process. Interestingly, SAFB interacts with SF2/ASF in vivo
(13), and it has been reported recently that SF2/ASF
functions as a cofactor to enhance SUMOylation through the
E3 ligase PIAS1 (30). Therefore, it is possible that SAFB
and SF2/ASF may serve as the functional link between the
RNA processing and SUMOylation machinery.
Taken together, the results of this study suggest a novel
function for SAFB in regulation of gene expression by
coordinating transcriptional initiation and RNA processing.
It is possible that SUMOylated SAFB associates with the
CTD of the initiating RNAPII and travels with RNAPII as
it synthesizes nascent pre-mRNA, and subsequently
facilitates the assembly of splicing complexes and splicing on the
first intron to emerge, and further coordinates transcription
and pre-mRNA processing levels.
Supplementary Data are available at NAR Online.
We thank Dr S. Oesterreich (University of Pittsburgh) for
providing plasmids for the expression of SAFB1.
Author contributions: H.-W.L. and J.D.P. designed research;
H.-W.L, X.G. and T.B. performed research; H.-W.L. and
J.D.P. analyzed data; and H.-W.L. M.A.F. and J.D.P. wrote
Pelotonia Predoctoral Cancer Research Training Fellowship (to H-w. Liu). Funding for open access charge: Departmental funds.
Conflict of interest statement. None declared.
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