A Transcriptional Regulatory Role of the THAP11-HCF-1 Complex in Colon Cancer Cell Function

Article (PDF Available)inMolecular and Cellular Biology 32(9):1654-70 · February 2012with62 Reads
DOI: 10.1128/MCB.06033-11 · Source: PubMed
Abstract
The recently identified Thanatos-associated protein (THAP) domain is an atypical zinc finger motif with sequence-specific DNA-binding activity. Emerging data suggest that THAP proteins may function in chromatin-dependent processes, including transcriptional regulation, but the roles of most THAP proteins in normal and aberrant cellular processes remain largely unknown. In this work, we identify THAP11 as a transcriptional regulator differentially expressed in human colon cancer. Immunohistochemical analysis of human colon cancers revealed increased THAP11 expression in both primary tumors and metastases. Knockdown of THAP11 in SW620 colon cancer cells resulted in a significant decrease in cell proliferation, and profiling of gene expression in these cells identified a novel gene set composed of 80 differentially expressed genes, 70% of which were derepressed by THAP11 knockdown. THAP11 was found to associate physically with the transcriptional coregulator HCF-1 (host cell factor 1) and recruit HCF-1 to target promoters. Importantly, THAP11-mediated gene regulation and its chromatin association require HCF-1, while HCF-1 recruitment at these genes requires THAP11. Collectively, these data provide the first characterization of THAP11-dependent gene expression in human colon cancer cells and suggest that the THAP11–HCF-1 complex may be an important transcriptional and cell growth regulator in human colon cancer.

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Available from: Jian-Jun Wei
A Transcriptional Regulatory Role of the THAP11–HCF-1 Complex in
Colon Cancer Cell Function
J. Brandon Parker,
a,d
Santanu Palchaudhuri,
a
Hanwei Yin,
a
Jianjun Wei,
b
and Debabrata Chakravarti
a,c
Division of Reproductive Biology Research, Department of OB/GYN,
a
Department of Pathology,
b
and Robert H. Lurie Comprehensive Cancer Center,
c
Feinberg School of
Medicine, Northwestern University, Chicago, Illinois, USA, and Biomedical Graduate Studies, Pharmacology Graduate Group, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania, USA
d
The recently identified Thanatos-associated protein (THAP) domain is an atypical zinc finger motif with sequence-specific DNA-
binding activity. Emerging data suggest that THAP proteins may function in chromatin-dependent processes, including tran-
scriptional regulation, but the roles of most THAP proteins in normal and aberrant cellular processes remain largely unknown.
In this work, we identify THAP11 as a transcriptional regulator differentially expressed in human colon cancer. Immunohisto-
chemical analysis of human colon cancers revealed increased THAP11 expression in both primary tumors and metastases.
Knockdown of THAP11 in SW620 colon cancer cells resulted in a significant decrease in cell proliferation, and profiling of gene
expression in these cells identified a novel gene set composed of 80 differentially expressed genes, 70% of which were derepressed
by THAP11 knockdown. THAP11 was found to associate physically with the transcriptional coregulator HCF-1 (host cell factor
1) and recruit HCF-1 to target promoters. Importantly, THAP11-mediated gene regulation and its chromatin association require
HCF-1, while HCF-1 recruitment at these genes requires THAP11. Collectively, these data provide the first characterization of
THAP11-dependent gene expression in human colon cancer cells and suggest that the THAP11–HCF-1 complex may be an im-
portant transcriptional and cell growth regulator in human colon cancer.
The Thanatos-associated protein (THAP) domain is an evolu-
tionarily conserved C2-CH (C-X
2-4
-C-X
35-50
-C-X
2
-H) zinc
finger motif with sequence-specific DNA-binding activity (5, 33–
35). Twelve THAP proteins, each containing an N-terminally lo-
cated THAP domain, have been identified in humans (THAP0 to
THAP11), and a subset of these (THAP0, -1, -2, -4, -7, and -11) are
also conserved in mice and rats (7).
THAP domains are approximately 80 to 90 amino acids in
length and, in addition to zinc-coordinating residues, contain sev-
eral conserved or invariant residues necessary for proper domain
folding and DNA-binding activity (4, 5, 7, 35). The majority of
conserved THAP proteins also contain a coiled-coil protein inter-
action domain adjacent to a host cell factor 1 (HCF-1)-binding
motif (HBM) (26). The tetrapeptide HBM (E/DHXY, where X is
any amino acid) facilitates the interaction of THAP proteins and
other DNA-binding factors with the Kelch domain of HCF-1, a
transcriptional coregulator and cell proliferation factor associated
with a variety of enzymatic and histone-modifying activities, in-
cluding SIN3/HDAC histone deacetylase, SET1/MLL histone
methyltransferase, and MOF histone acetyltransferase (11, 22, 23,
26, 30, 39, 42).
Individual THAP proteins have been implicated in a diverse
array of physiological processes, including cell proliferation, reg-
ulation of transcription, apoptosis, and maintenance of embry-
onic stem (ES) cell pluripotency (2, 3, 6, 9, 12, 24, 33, 45). The
DNA- and HCF-1-binding properties of THAP proteins naturally
suggest that these proteins may regulate normal or disease-specific
physiological processes in a DNA- and chromatin-dependent
manner. Indeed, mutations in the THAP1 gene which disrupt
DNA binding have recently been identified as a genetic determi-
nant of the neurological disorder DYT6 dystonia, suggesting that
this disease may be a result of the perturbation of a THAP1-de-
pendent gene expression program (12, 38). In addition, THAP1
has been shown to regulate the proliferation and cell cycle pro-
gression of vascular endothelial cells through HCF-1-dependent
transcriptional regulation of RRM1 (ribonucleotide reductase 1),
a gene known to be required for S-phase DNA synthesis (6, 26).
The murine homolog of human THAP11, termed RONIN, has
recently been shown to be required for ES cell proliferation (9).
Homozygous deletion of Ronin was found to be embryonically
lethal to mice. The inner cell mass of Ronin null blastocysts failed
to proliferate when the cells were cultured in vitro, while forced
overexpression of RONIN in ES cells promoted teratocarcinoma
formation in immunocompromised mice and also prevented
spontaneous ES cell differentiation upon culture in the absence of
leukemia inhibitory factor (9). The ability of RONIN/THAP11 to
exert strong antidifferentiation effects in mouse ES cells was ini-
tially suggested to result from RONIN-dependent global tran-
scriptional repression concomitant with the deposition of tran-
scriptionally repressive histone modification (9). However, recent
work in the same laboratory suggests that RONIN/THAP11 may
activate, as well as repress, transcription (10). Contrasting with the
role of THAP11 in ES cells, Zhu et al. have recently reported that
THAP11 functions as a negative regulator of cell growth in human
HepG2 hepatoma cells through transcriptional repression of the
proto-oncogene MYC (45).
These findings suggest that THAP proteins likely function in
DNA- and chromatin-dependent processes, including transcrip-
Received 29 July 2011 Returned for modification 23 August 2011
Accepted 17 February 2012
Published ahead of print 27 February 2012
Address correspondence to Debabrata Chakravarti, debu@northwestern.edu.
Supplemental material for this article may be found at http://mcb.asm.org/.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
doi:10.1128/MCB.06033-11
1654 mcb.asm.org 0270-7306/12/$12.00 Molecular and Cellular Biology p. 1654 –1670
tion. However, the transcriptional regulatory properties of most
human THAP proteins and their role in physiological processes
remain largely unknown. In this report, we identify a previously
uncharacterized role for THAP11 as a transcription and cell
growth regulator in human colon cancer cells. THAP11 was found
to be differentially expressed in cell culture models of human
colon cancer progression, and immunohistochemical analysis of
tissue microarrays (TMAs) similarly revealed increased THAP11
expression in primary and metastatic tumors. Using microarray-
based profiling of gene expression in SW620 THAP11 knockdown
cells, we have determined that the majority of THAP11-regulated
genes are derepressed upon THAP11 knockdown. We have per-
formed extensive molecular characterization of THAP11-medi-
ated transcriptional regulation and determined that THAP11 not
only recruits but requires HCF-1 for stable chromatin association.
Collectively, these data provide the first characterization of a di-
rectly regulated, THAP11-dependent gene expression program in
human cancer cells and suggest that the THAP11–HCF-1 complex
may be an important transcriptional and cell growth regulator in
human colon cancer.
MATERIALS AND METHODS
Plasmids and cloning. A Mammalian Gene Collection-verified full-
length cDNA clone of human THAP11 was purchased from Open Biosys-
tems, PCR amplified, and inserted into expression vectors pGEX-4T1 (GE
Life Sciences), p3xFLAG-CMV14 (Sigma), and pCMX-Gal4
1-147
using
standard molecular cloning procedures. The retroviral expression vector
pBABE-puro has been described elsewhere (28). The vector pBABE-EGFP
was generated from pBABE-puro by replacing the puromycin resistance
cassette (HindIII/ClaI excised) with the enhanced green fluorescent pro-
tein (GFP) coding sequence, which was PCR amplified from pEGFP-C2
(Clontech).
The retroviral short hairpin RNA (shRNA) expression vector pSuper-
.Retro.Puro, here abbreviated pSRP, was purchased from Oligoengine, as
was the nonsilencing control shRNA pSRP MAMM-X (designated shNS).
THAP11 and HCF-1 shRNA targeting sequences were designed using
Dharmacon siDESIGN Center (32). Synthetic oligonucleotides corre-
sponding to shRNA targeting sequences were cloned into BglII/HindIII-
linearized pSRP according to the manufacturer’s instructions. An addi-
tional nonsilencing control shRNA (designated shNS2) targeting GFP was
also cloned into pSRP. A summary of the shRNA targets and the corre-
sponding synthesized oligonucleotide sequences used in this study is pro-
vided in the supplemental material.
An expression construct for nonsilenceable THAP11 (p3xFLAG-
THAP11-Rescue) resistant to shRNAs was generated by two successive
rounds of site-directed mutagenesis using p3xFLAG-THAP11 and the
QuikChange site-directed mutagenesis kit (Stratagene). Each mutagene-
sis reaction introduced silent mutations into three consecutive codons
within the shRNA targeting sequence using primers listed in the supple-
mental material. The p3xFLAG-THAP11-Rescue construct was further
subcloned by PCR amplifying the nonsilenceable THAP11 coding se-
quence, including a 3xFLAG tag, and inserted into the EcoRI/SalI sites of
pBABE-EGFP, generating pBABE-EGFP-THAP11-Rescue-3xFLAG. The
sequence correctness of all constructs was verified by automated DNA
sequencing.
Cell culture and treatment. Cell lines 293T/17 (CRL-11268), HT-29
(HTB-38), HCT-116 (CCL-247), SW480 (CCL-228), SW620 (CCL-227),
LoVo (CCL-229), DLD-1 (CCL-221), and Colo-320HSR (CCL-220.1)
were purchased from the American Type Culture Collection. HT-29 and
HCT-116 were maintained in McCoy’s 5A medium with 10% fetal bovine
serum. DLD-1 and Colo-320HSR cells were grown in RPMI 1640 medium
containing 10% fetal bovine serum. SW480, SW620, and 293T/17 cells
were maintained in Dulbecco’s modified Eagle medium (high glucose)
containing 10% fetal bovine serum. LoVo cells were grown in F12-K
with 10% fetal bovine serum. All cells were grown without supplemen-
tal antibiotics in a humidified 37°C incubator containing 5% CO
2
.
Generation and purification of THAP11 antibody. A custom rabbit
polyclonal antibody was generated against the carboxyl terminus of hu-
man THAP11 (amino acids 132 to 313). Recombinant glutathione S-
transferase (GST)–THAP11(132-313) was produced in Escherichia coli
strain Rosetta-2 BL21(DE3) (Novagen) and purified using glutathione
Sepharose as previously described (24). Protein was subjected to prepar-
ative-scale SDS-PAGE and Coomassie stained, and gel bands correspond-
ing to GST–THAP11(132-313) were excised for use as an immunogen.
Animal immunizations and serum collection were performed by a com-
mercial facility (Covance). GST-specific antibodies were depleted by pass-
ing crude serum over a cross-linked GST-glutathione Sepharose column.
Further affinity purification of anti-THAP11 antibodies was performed
using the immunogen immobilized on nitrocellulose as described else-
where (36). Briefly, GST–THAP11(132-313) was subjected to single-well
SDS-PAGE, transferred to nitrocellulose, identified by Ponceau S stain-
ing, and excised. The nitrocellulose strip was blocked for 1 h with 3%
bovine serum albumin (BSA) in phosphate-buffered saline (PBS) and
then incubated overnight at 4°C with 1 ml of anti-THAP11 antiserum
diluted 1:10 in PBS with 3% BSA. The antibody solution was discarded,
and the nitrocellulose strip was washed with several changes of PBS at
room temperature. The THAP11 antibody was eluted by incubating the
nitrocellulose with 0.2 M glycine (pH 2.5) and immediately neutralized
with 0.1 volume of 1 M Tris-HCl, pH 8.0. One-tenth volume of 10PBS
was added to the affinity-purified anti-THAP11 antibody, which was then
concentrated by filtration using Microcon centrifugal filter devices ac-
cording to the manufacturer’s instructions (Millipore). The specificity of
the affinity-purified THAP11 antibody was confirmed by immunoblot-
ting.
Immunoblotting. Whole-cell extracts were prepared from subconflu-
ent cells using modified radioimmunoprecipitation assay (RIPA) buffer
(20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1 mM EDTA, 1 mM EGTA,
1% IGEPAL CA-630, 1% sodium deoxycholate, 0.25% SDS). Extracts
were clarified by centrifugation at 20,000 gfor 15 min at 4°C, and
protein concentrations determined by bicinchoninic acid (BCA) assay
(Pierce).
Thirty micrograms of whole-cell extract was separated on precast 8 to
16% polyacrylamide gels (Invitrogen), transferred to nitrocellulose mem-
brane, and stained with Ponceau S to confirm equal protein loading.
Membranes were blocked in PBS– 0.05% Tween 20 (PBST) containing
5% nonfat dry milk and incubated overnight at 4°C with primary anti-
body diluted in PBST–5% nonfat dry milk. Membranes were subse-
quently washed with PBST, incubated with the appropriate horseradish
peroxidase (HRP)-conjugated secondary antibody diluted in PBST–5%
nonfat dry milk, and developed using enhanced chemiluminescence
(ECL) plus detection reagent (GE Life Sciences). Immunoblotting for
THAP11 was performed using our custom-generated THAP11 antibody.
The commercially available primary antibodies used for immunoblotting
included antibodies to
-actin (Sigma A5441), FLAG M2 (Sigma F1804),
and HCF-1 (Bethyl Laboratories A301-399A). HRP-conjugated anti-
mouse and anti-rabbit secondary antibodies were purchased from Sigma.
Coimmunoprecipitation and immunoblot analysis. Cells in 15-cm
2
tissue culture dishes were rinsed three times with ice-cold PBS, scraped
into PBS, and collected by centrifugation at 500 gfor 5 min at 4°C. Cells
were then resuspended in 5 pellet cell volumes (PCV) of buffer A (10 mM
HEPES-KOH [pH 7.6], 10 mM KCl, 1.5 mM MgCl
2
) containing Com-
plete protease inhibitors (Roche) and allowed to swell for 10 min on ice.
Cytoplasmic membranes were lysed by dropwise addition of IGEPAL CA-
630 from a 10% stock solution to a final concentration of 0.5% while the
cells were being gently mixed by vortexing at a half-maximum setting.
Cells were incubated on ice for 5 min, and plasma membrane lysis was
verified by trypan blue staining. Nuclei were isolated by centrifugation at
2,000 gfor 5 min at 4°C, washed once with buffer A, and resuspended in
1 PCV of buffer C (20 mM HEPES-KOH [pH 7.6], 420 mM NaCl, 1.5 mM
Transcriptional Regulation by THAP11–HCF-1 Complex
May 2012 Volume 32 Number 9 mcb.asm.org 1655
MgCl
2
, 0.2 mM EDTA, 25% glycerol) supplemented with Complete pro-
tease inhibitors (Roche). Nuclei were extracted for 45 min at 4°C with
gentle inversion. Nuclear extracts were clarified by centrifugation
(20,000 gfor 15 min at 4°C), diluted with 1 volume of buffer C without
glycerol or NaCl, and adjusted to 0.5% IGEPAL CA-630. Nuclear extracts
were reclarified by centrifugation to remove precipitates formed by dilu-
tion, and protein concentration was determined by BCA assay. Immuno-
precipitations were performed with 0.5 to 1 mg of nuclear extract and 1 to
2
g of either affinity-purified rabbit polyclonal anti-HCF-1 (Bethyl Lab-
oratories A301-399A) or normal rabbit IgG (Sigma) overnight at 4°C with
inversion. Protein G Dynabeads (20
l) were added, and immunoprecipi-
tation was continued for an additional 2 h. Beads were then washed four
times with binding buffer (20 mM HEPES-KOH [pH 7.6], 150 mM NaCl,
1.5 mM MgCl
2
, 0.2 mM EDTA, 0.5% IGEPAL CA-630), and bound pro-
teins were eluted by boiling in 2Laemmli buffer. Immunoprecipitated
proteins were resolved by SDS-PAGE and immunoblotted with HCF-1
and THAP11 antibodies as described above. Blots were developed by ECL
using an anti-rabbit light-chain-specific, HRP-conjugated secondary an-
tibody (Jackson ImmunoResearch 211-032-171) to minimize obscuring
of the THAP11 signal by comigrating IgG heavy chains.
Immunofluorescence analysis. For indirect immunofluorescence
analysis, cells grown on glass coverslips were fixed with 4% paraformal-
dehyde in PBS for 10 min at room temperature. Fixed cells were rinsed
three times with PBS and permeabilized with 0.2% Triton X-100 in PBS
for 5 min at room temperature. Coverslips were washed twice with PBS,
blocked with PBS–3% BSA for 30 min at 37°C, and incubated with affin-
ity-purified anti-THAP11 antibody in PBS–3% BSA for1hat37°C. Cov-
erslips were then washed three times with PBS and incubated with Alexa
Fluor 488-conjugated anti-rabbit IgG (1:1,000; Invitrogen) in PBS–3%
BSA for 1 h at 37°C. Coverslips were again washed three times in PBS,
counterstained with 4=,6-diamidino-2-phenylindole (DAPI), and
mounted in Prolong antifade reagent (Invitrogen). Samples were ana-
lyzed using a Zeiss LSM 510 META laser scanning confocal microscope.
Immunohistochemistry and TMAs. TMA slides prepared from for-
malin-fixed, paraffin-embedded samples were obtained from US Biomax.
TMAs contained 33 normal colonic mucosa, 7 benign tubular adenoma,
and 133 colon cancer samples. In addition, 3 liver samples and 37 samples
from lymph nodes with metastatic diseases were also included. All cases
contained tissue cores in triplicate. TMAs were deparaffinized in two
changes of xylene and rehydrated in a graded alcohol series using standard
procedures. Slides were subjected to heat-induced antigen retrieval by
microwaving in citrate buffer (10 mM sodium citrate, 0.05% Tween 20,
pH 6.0), followed by blocking of endogenous peroxidases with 3% hydro-
gen peroxide. Immunohistochemical staining for THAP11 was per-
formed using affinity-purified anti-THAP11 antibody (1:400 dilution),
Vectastain Elite ABC detection reagents, and 3,3=-diaminobenzidine tet-
rahydrochloride substrate (Vector Laboratories). Hematoxylin-counter-
stained slides were evaluated using a semiquantitative dual-scoring sys-
tem as described elsewhere (41). Briefly, the intensity of THAP11
immunoreactivity in cell nuclei was scored numerically (0 negative, 1
weak, 2 moderate, 3 strong), as was the percentage of THAP11-
immunopositive nuclei (0 0%, 1 1 to 10%, 2 11 to 50%, 3 51 to
100%). The intensity and percent immunopositivity scores were added,
and samples were categorized into low/weak expression (combined score,
3) and high/strong expression (combined score, 3) groups. The sta-
tistical significance of differences between the combined scores of normal
and primary cancer samples or normal and metastatic cancer samples was
determined by the chi-square test.
Retrovirus production. Vesicular stomatitis virus G glycoprotein
(VSVG)-pseudotyped retrovirus was produced in 293T/17 cells (70%
confluent in 10-cm
2
dishes) by cotransfection with pCMV-VSVG (4
g),
pMLV-GagPol (8
g), and a retroviral construct (12
g) using Lipo-
fectamine 2000 (Invitrogen) according to the manufacturer’s instruc-
tions. Following overnight transfection, cells were given fresh medium
(Dulbecco’s modified Eagle’s medium [DMEM]–10% fetal bovine serum
[FBS]) and allowed to equilibrate for several hours in a 37°C cell culture
incubator prior to being shifted to a 32°C 5% CO
2
humidified cell culture
incubator for an additional 24 to 30 h. Retroviral supernatants were har-
vested 48 h from the start of transfection, cleared of residual 293T/17
cells by centrifugation (1,000 g, 5 min), and either used immediately or
aliquoted and stored at 80°C for future use.
Retroviral transduction and stable cell production. To generate
pools of SW620 and Colo-320HSR cells stably expressing either control or
THAP11 shRNA, cells in six-well plates (20% confluent) were trans-
duced with a 1:1 mixture of viral supernatant and fresh medium, adjusted
to 8
g/ml Polybrene, and spin infected at 500 gfor2hat32°C. Fol-
lowing spin infection, cells were returned to the 37°C incubator for2hand
then given fresh growth medium. Two days postransduction, cells from
individual wells of the six-well plate were split into 10-cm
2
dishes contain-
ing 2
g/ml puromycin. Cells were grown under selection for an addi-
tional 2 days, after which mock-transduced cells exhibited complete cell
death. For rescue experiments using nonsilenceable THAP11, SW620 cells
were first transduced with pSRP shRNA virus and transduced 24 h later
with the indicated pBABE-EGFP virus. Doubly expressing cells were then
selected with puromycin and sorted for GFP expression using fluores-
cence-activated cell sorting (FACS).
RNA isolation and quantitative reverse transcription (RT)-PCR.
Total RNA was isolated by using the Qiagen RNeasy minikit according to
the manufacturer’s instructions. Total RNA (1
g) was reverse tran-
scribed using qScript cDNA synthesis mix (Quanta Biosciences) contain-
ing both random hexamer and oligo(dT) primers. Quantitative PCR
(qPCR) was performed with diluted cDNA using an ABI PRISM 7900HT
384-well real-time PCR machine (Applied Biosystems) in a final volume
of 20
l using SYBR green PCR master mix (Applied Biosystems) and
gene-specific primers. Fold changes in mRNA levels were determined us-
ing the ⌬⌬C
T
method normalized to
-actin.
Microarray gene expression analysis. Analysis of gene expression in
SW620 cells stably expressing either control (pSRP-shNS, pSRP-shNS2)
or THAP11 (pSRP-T11A, pSRP-T11C) shRNAs was done using Nimble-
gen Homo sapiens 385K oligonucleotide microarrays. Total RNA from
two independent pools of SW620 cells per shRNA were isolated using
Qiagen RNeasy minikits as described above. RNA quality was verified
using an Agilent 2100 Bioanalyzer and provided to Nimblegen for subse-
quent cDNA synthesis, labeling, and microarray hybridization.
Nuclear run-on assay. Modified nuclear run-on assays using 5-bro-
mouridine (BrU)-labeled nascent RNA were performed as previously de-
scribed, with minor modifications (8). SW620 cells (110
8
) expressing
either pSRP-shNS, pSRP-shT11A, or pSRP-shT11C were rapidly cooled
by rinsing in ice-cold PBS, scraped into ice-cold PBS, and collected by
centrifugation (500 g, 5 min, 4°C). Cell pellets were resuspended in
hypotonic lysis buffer (10 mM Tris-HCl [pH 7.6], 10 mM NaCl, 3 mM
MgCl
2
) and immediately centrifuged as before. Cell pellets were loosened
by gentle vortexing (setting 6), resuspended in hypotonic lysis buffer con-
taining 0.5% IGEPAL CA-630, and incubated on ice for 5 min. Cell lysis
was routinely 90%, as determined by trypan blue staining and hemacy-
tometer counting. Nuclei were recovered by centrifugation (1,000 g,5
min, 4°C), washed once in hypotonic lysis buffer containing 0.5%
IGEPAL CA-630, and centrifuged as before. Recovered nuclei were resus-
pended in 1 ml of freezing buffer (50 mM Tris-HCl [pH 8.0], 40% glyc-
erol, 5 mM MgCl
2
, 0.1 mM EDTA, 40 U RNasinPlus [Promega]) and
aliquoted into 1.5-ml tubes at 200
l(10
7
nuclei) per tube, snap-frozen
in liquid nitrogen, and stored at 80°C.
For run-on transcription, thawed nuclei were mixed with an equal
volume (200
l) of 2run-on buffer (10 mM Tris-HCl [pH 8.0]; 5 mM
MgCl
2
; 300 mM KCl; 1% Sarkosyl; 160 U RNasinPlus; 5 mM dithiothre-
itol [DTT]; 1 mM each ATP, CTP, GTP, and BrUTP) and incubated at
30°C for 30 min. Following run-on transcription, samples were digested
with DNase I and proteinase K. Total RNA was extracted using acid phe-
nol-chloroform-isoamyl alcohol (25:24:1), ethanol precipitated, and re-
suspended in nuclease-free water. Total RNA was then further purified
Parker et al.
1656 mcb.asm.org Molecular and Cellular Biology
using Qiagen RNeasy minispin columns according to the RNA cleanup
procedure described by the manufacturer. Anti-BrdU monoclonal anti-
body (Sigma; 5
l per immunoprecipitation, 15
l total) was preincu-
bated with 90
l of protein G Dynabeads in 1 ml binding buffer (10 mM
Tris-HCl [pH 7.6], 100 mM NaCl, 1 mM EDTA, 0.05% Tween 20, 0.1%
IGEPAL CA-630, 15
g yeast tRNA) for1hat4°C. Dynabeads were
washed three times in binding buffer without supplemental tRNA and
resuspended in 90
l of binding buffer. To immunoprecipitate BrU-la-
beled nascent RNA, 25
g of nuclear run-on RNA, 2.5
g of yeast tRNA,
and 40 U of RNasinPlus were added to 500
l of binding buffer on ice.
Anti-BrdU antibody-bound Dynabeads (30
l per immunoprecipitation)
were added, and immunoprecipitations was continued for1hat4°Cwith
end-over-end rotation. Beads were subsequently washed three times in
binding buffer and eluted by the addition of 300
l of buffer RLT (Qiagen
RNeasy kit). Immunoprecipitated RNAs were purified using the Qiagen
RNeasy minikit, reverse transcribed, and analyzed by quantitative RT-
PCR as described above.
ChIP. Chromatin immunoprecipitation (ChIP) was performed essen-
tially as previously described, with minor modifications (21). SW620 cells
(210
7
) or those stably expressing the indicated retroviral constructs
were cross-linked by the addition of 1/10 volume of freshly prepared
formaldehyde cross-linking buffer (11% formaldehyde, 10 mM HEPES-
KOH [pH 7.6], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA) to tissue
culture dishes and incubated for 10 min at room temperature. Cross-
linking was terminated by the addition of 1/20 volume of 2.5 M glycine.
Cross-linked cells were washed three times with ice-cold PBS, scraped into
PBS, and recovered by centrifugation (1,500 g, 10 min, 4°C). Cell pellets
were resuspended in 1 ml of lysis buffer 1 (50 mM HEPES-KOH [pH 7.6],
140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IGEPAL-CA630, 0.25%
Triton X-100, 1protease inhibitors) and incubated for 10 min at 4°C
with gentle rocking. Nuclei were recovered by centrifugation, resus-
pended in 1 ml of lysis buffer 2 (10 mM Tris-HCl [pH 8.0], 200 mM NaCl,
1 mM EDTA, 0.5 mM EGTA, 1protease inhibitors), and extracted for
10 min at room temperature with gentle inversion. Nuclei were again
recovered by centrifugation, resuspended in 1 ml of lysis buffer 3 (10 mM
Tris-HCl [pH 8.0], 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1%
sodium deoxycholate, 0.5% Sarkosyl, 1protease inhibitors), and soni-
cated in an ice-water bath using a Misonix microtip-equipped sonicator at
setting 6 (6 W [root mean square] output power) for 12 cycles of 15 s of
sonication followed by a 1-min cooling interval. The sonicated chromatin
was adjusted to 1% Triton X-100 from a 10% stock solution, and debris
was removed by centrifugation at 20,000 gat 4°C for 20 min. The
protein concentration of solubilized chromatin was determined by BCA
assay, and approximately 300
g of chromatin was immunoprecipitated
overnight at 4°C with the indicated antibodies. Protein G Dynabeads (20
to 30
l) were added, and immunoprecipitations were continued for an
additional 2 h. Beads were washed four times with 1 ml of ChIP-RIPA
wash buffer (50 mM HEPES-KOH [pH 7.6], 500 mM LiCl, 1 mM EDTA,
1.0% IGEPAL-CA630, 0.7% sodium deoxycholate) and once with TE
containing 50 mM NaCl.
Following the final wash, DNA was recovered as described by Nelson
et al. (29). Briefly, Dynabeads and precipitated input chromatin were
resuspended in 100
l of 10% Chelex resin (Bio-Rad) and incubated for
10 min at 100°C. Samples were cooled to room temperature and then
digested with proteinase K (0.2 mg/ml) for 30 min at 55°C. Samples
were again boiled for 10 min to inactivate proteinase K and centrifuged
at 20,000 gfor 3 min to pellet the Chelex-Dynabead mixture. Super-
natants (80
l) containing the immunoprecipitated DNA were trans-
ferred to clean 1.5-ml tubes, and the Chelex-Dynabead resins were
resuspended in an additional 120
l of water, vortexed, and centri-
fuged as before. Supernatants were combined, yielding 200
lofim-
munoprecipitated DNA.
Determination of relative enrichment was performed by qPCR using
an ABI PRISM 7900HT 384-well real-time PCR machine with SYBR green
PCR master mix (Applied Biosystems) and primers designed as described
below. Threshold cycle (C
T
) values of ChIP-enriched DNA were exponen-
tiated and expressed as percent recovery relative to the input DNA ana-
lyzed in parallel.
Primer pairs for qPCR were designed using Primer3Plus against hu-
man genome sequence (NCBI36/hg18) retrieved using the UCSC Ge-
nome Browser (19). The primer sequences and relative positions of the
ChIP amplicons are provided in the supplemental material. The antibod-
ies (amounts used and sources [antibody names]) used for ChIP were
against THAP11 (5
l, custom affinity purification), HCF-1 (1
g, Bethyl
Laboratories [A301-399A]), RNAPII (4
g, Santa Cruz [SC-899X]),
RNAPII-S5P (2
g, Bethyl Laboratories [A300-655A]), RNAPII-S2P (2
g, Bethyl Laboratories [A300-654A]), histone H3 (4
g, Abcam
[ab1791]), histone H3 acetyl K27 (2
g, Abcam [ab4729]), histone H3
trimethyl K4 (4
g, Abcam [ab8580]), histone H3 acetyl K9 (4
g, Abcam
[ab4441]), and FLAG-M2 (4
g, Sigma [F1804]).
Sequential ChIP. For sequential ChIP, first-round ChIPs were per-
formed as described above except that following the final wash, beads were
resuspended in 10 mM Tris-HCl (pH 7.6)–1 mM EDTA–2% SDS–20 mM
DTT and the precipitated complexes were eluted by incubation at 37°C for
30 min. The 50-
l eluate was divided into two 20-
l aliquots with 10
l
reserved for qPCR analysis. Eluates were diluted 20-fold with 10 mM
Tris-HCl (pH 7.6)–100 mM NaCl–1 mM EDTA–1% Triton X-100 and
adjusted to 1
g/
l BSA. THAP11 ChIP eluates were subjected to ChIP
again (reChIP) with 1
g of IgG or HCF-1, and HCF-1 ChIP eluates were
subjected to reChIP with 1
g of IgG or 5
l of THAP11 antibodies
overnight at 4°C. The resulting reChIP products were collected using pro-
tein G Dynabeads, washed, and eluted as described above for conventional
ChIP. Enrichment was determined by qPCR and expressed as percent
recovery relative to the input of the first-round ChIP.
alamarBlue cell proliferation assay. Proliferation of SW620 knock-
down cells was determined using the alamarBlue cell viability reagent
according to the manufacturer’s instructions (Invitrogen). Puromycin-
selected SW620 or control THAP11 knockdown cells were seeded at 5,000
per well (eight wells per condition) in black-wall 96-well tissue culture
plates in 100
l of DMEM–1% FBS. Five identical 96-well plates were
seeded for determination of cell viability on 5 consecutive days. Twenty-
four hours after seeding, 10
l of alamarBlue reagent was added per well to
one 96-well plate and this plate was incubated for2hat37°C in a cell
culture incubator. Fluorescence measurements were then performed with
a BioTek Synergy HT multidetection microplate reader equipped with
540-nm excitation and 590-nm emission filters. Average background flu-
orescence was calculated from wells (n16) containing medium alone
and subtracted from the fluorescence of wells containing cells. This pro-
cess was repeated every 24 h for 5 consecutive days using one replicate
96-well plate per day.
Crystal violet cell proliferation assay. SW620 knockdown cells were
plated at 3 10
5
per well in six-well tissue culture plates and grown in
DMEM–1% FBS. At the indicated time points, cells were fixed with 4%
paraformaldehyde for 15 min at room temperature, rinsed with PBS, and
stained with 0.2% crystal violet. Cells were washed extensively with water
and air dried. Bound crystal violet was eluted with 10% acetic acid and
measured by absorbance at 595 nm in a BioTek Synergy HT multidetec-
tion microplate reader.
RESULTS
THAP11 expression in colon cancer. THAP domain proteins re-
main poorly characterized, and their role in human diseases, in-
cluding cancer, is largely unknown. In an effort to further charac-
terize the physiological role of THAP domain-containing
proteins, we performed a systematic survey of publicly available
gene expression data sets to identify conditions of differential
THAP protein expression. Using this approach, we identified in-
creased THAP11 mRNA expression in a microarray data set orig-
inally designed to elucidate gene expression differences in the
Transcriptional Regulation by THAP11–HCF-1 Complex
May 2012 Volume 32 Number 9 mcb.asm.org 1657
SW480/SW620 cell culture model of colon cancer progression
(16, 31).
To corroborate the microarray finding, we prepared total RNA
and whole-cell extracts from SW480 and SW620 cells and deter-
mined the relative amounts of THAP11 mRNA and protein by
quantitative RT-PCR and immunoblotting, respectively. Both cell
types express THAP11 mRNA, but metastasis-derived SW620
cells were found to express approximately 4 times as much
THAP11 mRNA as isogenic, primary-tumor-derived SW480 cells
(Fig. 1A). SW620 cells also expressed more THAP11 protein than
SW480 cells (Fig. 1B, compare lanes 1 and 2), but SW480 cells
contain greater amounts of THAP11 than primary-tumor-de-
rived colon cancer cell lines HCT-116 and HT29 or the metastasis-
derived LoVo cell line (Fig. 1B, compare lanes 1 and 4 to 7). The
neuroendocrine colon cancer cell line Colo320-HSR contains
nearly as much THAP11 as SW620 cells, suggesting that elevated
THAP11 expression is not restricted to the SW480/SW620 model
system (Fig. 1B, compare lanes 2 and 3). Immunofluorescence
analysis revealed endogenous THAP11 to be located almost exclu-
sively within the nuclei of SW620 (Fig. 1C) and SW480 cells (data
not shown), consistent with a putative function in chromatin-
dependent processes, including transcriptional regulation. These
results suggest that gain of THAP11 expression may play a role in
colon cancer cell function.
To further explore this possibility, we next examined a large
cohort of human colon cancer samples to determine if THAP11 is
overexpressed in colon cancer and determine a potential link be-
tween THAP11 expression and disease progression. Immunohis-
tochemical analysis of THAP11 expression in human colon cancer
TMAs revealed that colon cancer has significantly higher THAP11
expression than normal colonic mucosa (Fig. 1D). The majority of
samples from normal colonic epithelium (n33) and benign
adenomas (n7) stained both weakly and infrequently for
THAP11 (Fig. 1D, part N), while increased THAP11 staining fre-
quency and intensity were observed in primary colon adenocarci-
nomas (n133) (Fig. 1D, compare parts gI, gII, and gIII with part
N), as well as both liver (n3) and lymph node metastases (n
37) (Fig. 1D, parts LM and LNM, respectively). A quantitative
assessment of THAP11 immunoreactivity scored as a combina-
tion of the intensity and the percentage of staining revealed signif-
FIG 1 THAP11 expression in human colon cancer cell lines and tumors. (A) THAP11 mRNA levels in SW480 and SW620 cells determined by quantitative
RT-PCR and expressed relative to the level in SW480 cells. Values represent the mean standard deviation of triplicate quantitative RT-PCRs from a
representative experiment performed at least three times. (B) Immunoblot assays of whole-cell extracts from the indicated colon cancer cell lines. (C) Immu-
nofluorescence localization of endogenous THAP11 in SW620 cells. Nuclei were identified by counterstaining with DAPI, and cells were visualized by differential
interference contrast (DIC) microscopy. MERGE is an overlay of the THAP11, DAPI, and DIC images. (D) Representative images of THAP11 immunohisto-
chemical staining in normal colon epithelium (N); grade I (gI), grade II (gII), and grade III (gIII) adenocarcinomas; liver metastasis (LM); and lymph node
metastasis (LNM). (E) Quantitative analysis of THAP11 expression in TMA samples. THAP11 immunoreactivity was scored as described in Materials and
Methods, and samples were placed into either the high/strong (3) or the low/weak (3) THAP11 expression group. An asterisk denotes a statistically significant
difference from normal/benign adenomas, as measured by chi-square analysis (P0.001).
Parker et al.
1658 mcb.asm.org Molecular and Cellular Biology
icantly higher THAP11 expression in primary colon tumors and
metastases than in normal tissues/benign adenomas (P0.001)
(Fig. 1E). Taken together, these data demonstrate that increased
THAP11 expression occurs in multiple stages of colon cancer and
suggest that SW620 cells may represent a tractable model to eval-
uate the molecular function of THAP11 in colon cancer.
Profiling of gene expression in SW620-THAP11 knockdown
cells. Previous work in our laboratory and others has suggested
that THAP proteins can both activate and repress transcription (9,
24–26). Initial experiments performed using a Gal4 DNA-binding
domain fusion of THAP11 in luciferase reporter assays suggested
that THAP11 functions as a transcriptional repressor (data not
shown). However, considering the artificial nature of the Gal4
fusion-based luciferase reporter assay and to independently eval-
uate whether endogenous THAP11 has transcriptional regulatory
properties, we performed profiling of gene expression in SW620
cells depleted of THAP11 via retrovirally expressed shRNA.
THAP11-specific shRNA constructs shT11A and shT11C signifi-
cantly diminished THAP11 mRNA and protein, as determined by
quantitative RT-PCR and immunoblotting (see Fig. S1 in the sup-
plemental material). Two independent pools of SW620 cells ex-
pressing either control (shNS, shNS2) or THAP11 (shT11A,
shT11C) shRNAs were analyzed for global gene expression
changes using oligonucleotide microarrays. Genes displaying a
1.5-fold change and a Pvalue of 0.01 (Student’s ttest) between
control (shNS, shNS2) and THAP11 (shT11A, shT11C) shRNA-
expressing cells were defined as differentially expressed. This gene
set was further processed to remove predicted genes which have
been subsequently “discontinued” by the NCBI and additionally
lack supporting mRNA or expressed sequence tag sequences. This
stringent analysis identified only 80 transcripts (excluding
THAP11) as differentially expressed between THAP11 and con-
trol knockdown groups (Fig. 2A). Of these differentially expressed
RNAs, 70% (56/80) showed increased expression with THAP11
knockdown while 30% of the genes were downregulated, suggest-
ing that THAP11 possesses transcriptional regulatory activity. In-
dependent verification of microarray-determined gene expression
changes by quantitative RT-PCR of activated and repressed genes
recapitulated the majority of these findings (Fig. 2B; data not
shown), suggesting that the data set as a whole likely represents a
THAP11-dependent gene expression program.
Gene expression measurements by oligonucleotide microarray
and quantitative RT-PCR reflect steady-state mRNA levels and by
themselves are incapable of assessing whether regulation occurs at
the transcriptional or posttranscriptional level. To determine if
the gene expression changes observed in THAP11 knockdown
cells were attributable to increased transcription, we performed a
modified nuclear run-on assay with SW620 cells expressing either
control or THAP11 shRNAs (8). Run-on transcription from iso-
lated nuclei was performed using BrU to label nascent RNA tran-
scripts in the presence of 0.5% Sarkosyl. Inclusion of Sarkosyl in
run-on reactions prevents reinitiation of transcription, thus al-
lowing only the completion of transcripts actively engaged by
RNA polymerase II (RNAPII) at cell lysis (8). BrU-labeled nascent
transcripts were then immunoprecipitated, and transcript levels
were determined by quantitative RT-PCR. In THAP11 knock-
down cells, we found nascent transcript levels for LSMD1,
NCRNA00095,AA862256, and ANXA1 (annexin A1) upregulated
in a manner qualitatively similar to that of their steady-state
mRNAs (Fig. 2C; data not shown). Importantly, THAP11 steady-
state but not nascent transcript levels were depleted in SW620 cells
expressing THAP11 shRNAs (Fig. 2C). This expected discrepancy
is in agreement with the proposed mechanism of RNA interfer-
ence as a posttranscriptional gene silencing event and provides an
important verification of the specificity of the nuclear run-on as-
say to detect nascent rather than mature transcripts (27). Taking
these findings together, we conclude that the increased RNA levels
observed in THAP11 knockdown cells for LSMD1,NCRNA00095,
AA862256, and ANXA1; decreased levels of ZNF32 and OPHN1
genes; and perhaps the microarray data set as a whole likely reflect
THAP11-mediated changes in the transcription of these target
genes. However, we cannot exclude the possibility that posttran-
scriptional regulatory mechanisms also contribute to the gene ex-
pression profile of THAP11 knockdown cells.
To determine if THAP11 knockdown similarly changes gene
expression in other colon cancer cell lines, we expressed THAP11
shRNAs in Colo320-HSR cells, which also have elevated THAP11
expression (Fig. 1B). Knockdown of THAP11 in Colo320-HSR
cells derepressed the putative THAP11 target genes ALG14,
LSMD1,AA862256,NCRNA00095, and AK021933, similar to its
knockdown in SW620 cells (Fig. 2D; data not shown). Interest-
ingly, annexin A1 was not derepressed in Colo320-HSR cells upon
THAP11 knockdown (data not shown), suggesting that some gene
targets may also be cell type specific.
Transcripts induced by THAP11 knockdown may represent an
authentic cellular response to diminished THAP11 or, alterna-
tively, may arise from nonspecific shRNA events despite our use of
multiple control and THAP11-targeted shRNAs. To discriminate
between these possibilities, we performed rescue experiments us-
ing a THAP11 expression construct rendered nonsilenceable by
mutation of three consecutive codons in each of the shRNA tar-
geting sequences. SW620 cells were first transduced with either
control or THAP11 shRNA and then with either control (empty)
or THAP11-Rescue-3xFLAG retroviruses. Cells expressing both
constructs were selected by puromycin resistance (shRNA) and
FACS (THAP11 rescue). Immunoblotting with THAP11 antibody
revealed robust expression of rescue but not endogenous THAP11
in cells transduced with THAP11-Rescue-3xFLAG but not control
(empty) virus (Fig. 2E). The identity of the THAP11 bands in
rescue-expressing cells was further validated by immunoblotting
with monoclonal anti-FLAG antibody (Fig. 2E).
If the gene expression profile observed in THAP11 knockdown
cells is attributable to specific depletion of THAP11, then restora-
tion of THAP11 status in THAP11-Rescue-3xFLAG cells should
reverse this effect. Indeed, quantitative RT-PCR analysis of puta-
tive THAP11 gene targets revealed that expression of nonsilence-
able THAP11 prevented the differential gene expression previ-
ously observed in THAP11 knockdown cells (Fig. 2F; see Table S1
in the supplemental material). Importantly, this rescue effect was
functional irrespective of the magnitude of target gene induction.
Modestly (1.2- to 1.6-fold) induced genes such as SMARCA1 and
ATG4A and robustly (3- to 8-fold) induced genes, including
LSMD1 and AA862256, were rescued by nonsilenceable THAP11.
The expression of THAP11-Rescue-3xFLAG rerepressed putative
THAP11 gene targets below the level observed in cells expressing
endogenous amounts of THAP11 (shNS and empty), likely due to
rescue construct overexpression. Next, we asked whether a gain in
THAP11 expression is sufficient to alter transcription at these tar-
get genes in colon cancer cells that express small amounts of en-
dogenous THAP11. Retrovirus-mediated overexpression of
Transcriptional Regulation by THAP11–HCF-1 Complex
May 2012 Volume 32 Number 9 mcb.asm.org 1659
FIG 2 THAP11 regulates transcription in colon cancer cells. (A) Heat map depicting microarray-based profiling of gene expression in SW620 cells expressing
either control (NS and NS2) or THAP11 (T11A and T11C) shRNAs. (B) Validation of microarray-determined gene expression changes by quantitative RT-PCR.
Parker et al.
1660 mcb.asm.org Molecular and Cellular Biology
THAP11 in HCT-116 cells also resulted in repression (ALG14,
C1orf83,LSMD1,NCRNA00095,AA862256,ZSCAN20,PRAF2)
or activation (OPHN1,ZNF32) of genes previously shown to be
derepressed or repressed by THAP11 knockdown, respectively
(Fig. 2G). Taken together, these results indicate that human
THAP11 functions as a transcriptional regulator of these genes in
colon cancer cells.
Identification of direct THAP11 gene targets in SW620 cells.
THAP11-dependent changes in gene expression may reflect a di-
rect role for THAP11 in transcriptional regulation. To identify
direct THAP11 gene targets, we performed ChIP with normal
SW620 cells using our custom anti-THAP11 antibody and moni-
tored the enrichment of immunoprecipitated chromatin by qPCR
using amplicons spaced approximately 300 to 600 bp apart and
spanning at least 1 kb on either side of the transcriptional start
sites of both repressed (Fig. 3A; see Fig. S2 in the supplemental
material) and activated (Fig. 3B) putative THAP11 target genes,
including PRAF2,LSMD1,AA862256,ALG14,NCRNA00095,
C1orf83,ZSCAN20,ZNF32, and OPHN1. This ChIP scanning ap-
proach identified endogenous THAP11 binding within 500 bp of
the annotated transcriptional start site at each putative THAP11
target gene examined. Similar patterns of enrichment were ob-
served when ChIP was performed using anti-FLAG monoclonal
antibody with SW620 cells depleted of endogenous THAP11 but
expressing THAP11-Rescue-3xFLAG (data not shown). Together,
these results show that THAP11 directly binds to both activated
and repressed genes.
To further confirm the specificity of the THAP11 ChIP assay,
we repeated the experiment with THAP11 knockdown SW620
cells. As expected, cells expressing THAP11 shRNA exhibited re-
duced, albeit detectable, levels of chromatin-bound THAP11. Re-
sidual binding in THAP11 knockdown cells ranged from 18% at
LSMD1 to 45% at C1orf83 relative to that observed with control
knockdown cells (Fig. 3C), despite undetectable levels of THAP11
in whole-cell extracts (Fig. 3D), suggesting that knockdown was
substantial but incomplete. Because each of the aforementioned
genes was effectively regulated by and contained chromatin-
bound THAP11 near the transcriptional start site, we conclude
that these genes are likely direct targets of THAP11-mediated
transcriptional regulation.
Decreased RNAPII occupancy at THAP11-repressed genes.
Since 70% of differentially expressed genes were derepressed in
THAP11 knockdown cells, we focused our attention on further
characterizing the mechanism of THAP11-mediated transcrip-
tional repression. We examined RNAPII occupancy at THAP11
target genes by ChIP using an antibody that recognizes the N-ter-
minal domain of RNAPII and found increased total RNAPII bind-
ing near the transcriptional start sites of genes induced by
THAP11 knockdown (Fig. 4A; data not shown). The increase in
total RNAPII was paralleled by increases in C-terminal domain
(CTD)-phosphorylated and transcriptionally active RNAPII, as
revealed by ChIP using S2P and S5P CTD-specific antibodies (Fig.
4B; data not shown). The elevated RNAPII occupancy observed at
LSMD1 in THAP11 knockdown cells was reversed in SW620 res-
cue cells that simultaneously express nonsilenceable THAP11,
thus confirming that increased RNAPII occupancy reflects dimin-
ished THAP11 levels (Fig. 4C). Importantly, no change in RNAPII
occupancy was observed at the
-actin promoter upon either
THAP11 knockdown or nonsilenceable THAP11 rescue expres-
sion, demonstrating the specificity of the assay for THAP11 target
genes (Fig. 4D). These results suggest that THAP11 may repress
transcription by limiting or destabilizing RNAPII at THAP11 tar-
get genes.
Analysis of histone modifications at THAP11-repressed
genes. To determine whether THAP11 occupancy modulates his-
tone acetylation and methylation patterns associated with tran-
scriptional activation, we analyzed levels of histone H3 lysine 4
trimethylation (H3K4me3), lysine 9 acetylation (H3K9ac), and
lysine 27 acetylation (H3K27ac) in SW620 THAP11 knockdown
cells using ChIP assays (Fig. 5). Our results show that while the
level of total histone H3 and H3K4me3 did not change at any of
the target genes analyzed, H3K9ac was upregulated at the LSMD1
(Fig. 5A), ZSCAN20 (Fig. 5B), and C1orf83 (Fig. 5C) genes upon
THAP11 knockdown. Interestingly, however, while enhanced
acetylation of lysine 27 was observed for the ZSCAN20 and
C1orf83 genes, no such changes were noticeable in LSMD1 under
similar conditions. These results suggest that histone H3 hyper-
acetylation of target genes may contribute to their upregulation in
THAP11-depleted cells.
HCF-1 associates with THAP11 and regulates THAP11 tar-
get gene expression. THAP11 and most other THAP proteins
have recently been shown to contain a functional HBM, a four-
amino-acid signature (E/DHxY) that mediates the interaction of
DNA- and chromatin-associated proteins with the N-terminal
Kelch domain of the transcriptional coregulator HCF-1 (10, 26).
These observations inspired us to ask whether THAP11 associates
with HCF-1 in colon cancer cells and if this interaction contrib-
utes to THAP11-mediated transcriptional regulation. To address
this question, we immunoprecipitated HCF-1 from SW620 nu-
clear extract and probed the immunoprecipitate for endogenous
THAP11. As shown in Fig. 6A, a significant fraction of THAP11
was found to specifically coprecipitate with HCF-1. The reciprocal
experiment revealed that a small but detectable fraction of HCF-1
also coprecipitated with THAP11 (data not shown). This observa-
tion was also extended to additional colon cancer cell lines where
THAP11 was found to coprecipitate with HCF-1 from nuclear
extracts prepared from Colo320-HSR, SW480, and HCT-116 cells
(see Fig. S3 in the supplemental material). Since THAP11 associ-
Values represent the mean standard deviation of four independent experiments. Double asterisks denote a statistically significant difference (P0.01) from
control (NS) shRNA, as determined by Dunnett’s posttest following analysis of variance. (C) Steady-state and nascent mRNA levels of putative THAP11 gene
targets in control (NS) or THAP11 knockdown (T11C) SW620 cells. (D) Gene expression determined by quantitative RT-PCR in Colo320-HSR cells expressing
control (NS) or THAP11 (T11A or T11C) shRNA. (E, top) THAP11 immunoblotting with SW620 cells expressing either control (Empty) or THAP11-Rescue-
3xFLAG (Rescue) and the indicated shRNA. Endogenous THAP11 is indicated by the red arrow. The black arrow indicates THAP11-Rescue-3xFLAG. (E,
bottom) Blots were stripped and reprobed with anti-FLAG and anti-
-actin antibodies. (F) Quantitative RT-PCR of THAP11 and LSMD1 expression in SW620
cells expressing either control (Empty) or THAP11-Rescue-3xFLAG (Rescue) and the indicated shRNA. RNA levels are normalized to
-actin and expressed as
n-fold changes relative to SW620 cells expressing control shRNA (NS) and rescue (Empty) constructs. (G) Quantitative RT-PCR analysis of THAP11 target genes
in HCT-116 cells transduced with either control (Empty) or THAP11-overexpressing retrovirus. In panels C, D, F, and G, values represent means standard
deviations of triplicate quantitative RT-PCRs of a representative experiment performed at least three times.
Transcriptional Regulation by THAP11–HCF-1 Complex
May 2012 Volume 32 Number 9 mcb.asm.org 1661
ated with HCF-1 in cells, we next analyzed the effect of HCF-1
knockdown on THAP11 target gene expression. As shown in Fig.
6B, knockdown of HCF-1 alone was sufficient to regulate tran-
scription at THAP11 target genes in a manner qualitatively similar
to that of THAP11 knockdown.
Because endogenous THAP11 and HCF-1 physically associate
and coordinately regulate gene expression, we next wished to de-
termine if HCF-1 is recruited to promoter regions of THAP11
target genes. To address this question, we used ChIP to determine
the chromatin occupancy profile of HCF-1 in SW620 cells at
THAP11-repressed target genes LSMD1,ALG14,NCRNA00095,
AA862256,ZSCAN20,PRAF2, and C1orf83 (Fig. 7A; see Fig. S3B
in the supplemental material) and THAP11-activated target genes
ZNF32 and OPHN1 (Fig. 7B). Compared with the chromatin oc-
cupancy profile previously determined for THAP11 (Fig. 3), we
found that the distribution of chromatin-bound HCF-1 was strik-
ingly similar in each genomic region analyzed (Fig. 7A and B; see
Fig. S3B in the supplemental material), suggesting that HCF-1 is
recruited to THAP11-bound promoters. Sequential ChIP, or
ChIP-reChIP, experiments subsequently confirmed that THAP11
and HCF-1 simultaneously co-occupy promoters of THAP11-
regulated genes. Chromatin that was immunoprecipitated with
anti-HCF-1 antibody was effectively subjected to reChIP with
anti-THAP11 antibody but only at THAP11-bound target genes
and not the control ACTB (
-actin) promoter or the THAP11–
HCF-1-regulated RRM1 promoter (Fig. 7C, left half) (26). Similar
results were observed when the order of antibodies was reversed,
i.e., ChIP with anti-THAP11 and reChIP with anti-HCF-1 (Fig.
7C right half). Interestingly, we also find that THAP11 co-occu-
pies previously identified HCF-1- and E2F-responsive promoters
CDC25A and RBL1(p107) (Fig. 7C), suggesting that THAP11, in
addition to E2F transcription factors, may contribute to HCF-1
binding at these genes (39).
HCF-1 is not known to possess intrinsic DNA-binding activity,
and its association with chromatin is thought to require interac-
tion with DNA-bound factors (43). Accordingly, sequence-spe-
cific binding by THAP11 in a targeted genomic region may result
in HCF-1 recruitment by virtue of their physical interaction. To
test this directly, we performed ChIP assays for HCF-1 and
THAP11 in SW620 cells depleted of either THAP11 or HCF-1 by
shRNA. THAP11 knockdown results in a marked reduction in
HCF-1 occupancy in each THAP11-bound genomic region exam-
ined (Fig. 8A, left half) but not the THAP1 target RRM1 (Fig. 8A,
right half), suggesting that THAP11 specifically recruits HCF-1 to
its target promoters. Importantly, HCF-1 protein levels were un-
altered in THAP11 knockdown cells (Fig. 8B), indicating that the
differential recruitment of HCF-1 observed at THAP11 target
promoters does not result from a reduction in total HCF-1 pro-
tein. The reciprocal experiment, ChIP assay for THAP11 in
HCF-1 knockdown cells (Fig. 8C), unexpectedly revealed that
THAP11 binding to chromatin was HCF-1 dependent, suggesting
that a functional THAP11–HCF-1 complex is necessary for chro-
matin association of both factors. However, we note that in mul-
tiple repeat experiments, HCF-1 knockdown resulted in a modest
but reproducible decrease in total THAP11 protein expression
(Fig. 8B; data not shown). To demonstrate that physical associa-
tion with HCF-1 is indeed necessary for THAP11 targeting to
chromatin, we generated THAP11 HBM mutants and determined
their abilities to bind chromatin and repress transcription in
HCT-116 cells, which express small amounts of endogenous
THAP11. The FLAG-tagged wild-type and THAP11
H243A
and
THAP11
Y245A
HBM mutant forms were stably expressed in HCT-
116 cells at similar levels, but the HBM mutant forms showed
strongly diminished HCF-1 interaction, as determined by coim-
munoprecipitation (Fig. 8D). A ChIP assay performed with anti-
FLAG monoclonal antibody to detect only ectopic THAP11 re-
FIG 3 ChIP analysis of endogenous THAP11. THAP11 ChIP at THAP11-
repressed genes (A) and THAP11-activated genes (B) in SW620 cells using
anti-THAP11 antibody or control IgG. (C) Control (shNS) or THAP11
(shT11) knockdown SW620 cells were analyzed by ChIP as described for panel
A. Values represent the mean standard deviation of duplicate qPCRs from a
representative experiment performed three times with similar results. (D) Im-
munoblotting of SW620 cells from panel C expressing control (shNS) or
THAP11 (shT11) shRNAs.
Parker et al.
1662 mcb.asm.org Molecular and Cellular Biology
vealed a similarly strong reduction in chromatin binding of HBM
mutant but not wild-type THAP11 (Fig. 8E). Consistent with their
reduced chromatin association, we found that overexpressed
THAP11
H243A
and THAP11
Y245A
not only failed to repress tran-
scription but instead stimulated gene expression relative to that in
empty-vector-expressing cells (Fig. 8F), suggesting that HBM mu-
tants may contain weak dominant negative activity. From these
data, we conclude that THAP11–HCF-1 complex formation is
necessary for the targeting of both factors to THAP11 target pro-
moters and subsequent transcriptional regulation.
Cell growth suppression in THAP11 knockdown SW620
cells. We have shown that THAP11 expression is upregulated in
primary and metastatic colon cancer tumors and cell lines (Fig. 1),
suggesting that increased THAP11 expression may confer a
growth and/or survival advantage on expressing cells. To explore
the possibility that THAP11 knockdown affects cell proliferation,
we performed an alamarBlue cell enumeration assay, which de-
tects the metabolic conversion of nonfluorescent resazurin to flu-
orescent resorufin in viable cells. SW620 cells were transduced
with control (shNS) or THAP11 shRNA (shT11A, shT11C, or
shT11E), at 2 days postransduction, selected with puromycin for
an additional 2 days, and then seeded into 96-well plates for the
alamarBlue assay. As shown in Fig. 9A, THAP11 knockdown re-
sulted in a significant decrease in the number of viable cells over
time with each THAP11-specific shRNA examined. Knockdown
with shRNA T11E was found to be substantially more effective at
reducing cell proliferation than shRNAs T11A or T11C, and this
difference correlates with the extent of knockdown, as determined
by immunoblotting of nuclear extracts (Fig. 9A, right half). Sim-
ilar findings were obtained when the alamarBlue assay was per-
formed with Colo320-HSR cells (Fig. 9B) or when cell prolif-
eration was measured by crystal violet staining of SW620 cells
(Fig. 9C).
DISCUSSION
In this work, we investigated the role of THAP11 in regulating the
transcription and proliferation of colon cancer cells. We identified
a novel set of genes that have previously not been linked to colon
cancer cell function. Our work also demonstrates that HCF-1 is an
obligatory partner for stable chromatin association and target
gene regulation by THAP11. Based on these and additional data
presented here, we propose that THAP11 plays a role in colon
cancer cell function, at least in part, by regulating a subset of cod-
ing and noncoding RNAs (ncRNAs).
Several lines of evidence presented here suggest that THAP11,
in complex with HCF-1, functions as a transcriptional regulator in
colon cancer cells. Profiling of gene expression in THAP11 knock-
down cells in conjunction with ChIP assays revealed direct
THAP11-mediated transcriptional regulation. The majority of
differentially expressed genes were derepressed upon THAP11
knockdown, suggesting that the THAP11–HCF-1 complex may
function predominantly as a transcriptional repressor. Nonethe-
less, we find that 30% of the genes are repressed upon depletion of
endogenous THAP11 and several are indeed direct THAP11–
HCF-1 target genes. This finding indicates that THAP11–HCF-1
can both activate and repress transcription in colon cancer cells
FIG 4 THAP11 regulates RNAPII occupancy at repressed genes. Total RNAPII (A) and RNAPII-S5P (B) occupancy as determined by ChIP assay of SW620 cells
expressing the indicated shRNAs. (C) ChIP analysis of RNAPII occupancy at LSMD1 in SW620 cells expressing either control (shNS) or THAP11 (shT11) shRNA
and either the control (Empty) or THAP11-Rescue-3xFLAG (Rescue). (D) ChIP analysis of RNAPII occupancy at the ACTB (
-actin) promoter from the
indicated SW620 cells as in panel C. Values represent the mean standard deviation of duplicate qPCRs from a representative experiment performed three times.
Transcriptional Regulation by THAP11–HCF-1 Complex
May 2012 Volume 32 Number 9 mcb.asm.org 1663
and is in good agreement with a recent genome-wide analysis of
THAP11-dependent transcription in mouse ES cells which has
identified both directly activated and repressed genes (10). While
our work extensively characterizes the role of THAP11–HCF-1 in
transcriptional regulation, the precise molecular mechanism by
which THAP11–HCF-1-mediated activation and repression oc-
cur remains to be determined. As a transcriptional coregulator,
HCF-1 has been alternately linked to both activation and repres-
sion of transcription (39, 42). In the context of transcriptional
repression, HCF-1 is known to associate with the SIN3/HDAC
histone deacetylase complex, O-linked glycosyltransferase, and
protein phosphatase 1 (1, 42). Consistent with the above observa-
tions, we find that THAP11 depletion at repressed genes increases
histone acetylation concomitant with increased RNAPII binding.
FIG 5 THAP11 regulates histone acetylation at repressed genes. Histone H3 modifications in SW620 cells expressing either control (shNS) or THAP11 (shT11)
shRNAs as determined by ChIP using histone H3, H3K4me3, H3K9ac, and H3K27ac antibodies. Values represent the mean standard deviation of duplicate
qPCRs from a representative experiment performed three times.
Parker et al.
1664 mcb.asm.org Molecular and Cellular Biology
The identity of HCF-1-associated activities that may contribute to
THAP11-mediated repression and/or activation is an unanswered
but pertinent question. Our studies, together with previous work,
suggest that the newly discovered THAP family of proteins func-
tions in part by regulating the transcription of their target genes
(6, 10, 24–26).
THAP11 expression was found to positively correlate with dis-
ease progression in human primary tumor specimens. The in-
crease in THAP11 expression in colon cancer tumors and cell lines
suggests that THAP11-dependent transcriptional regulation may
contribute to the pathogenesis of colon cancer. Consistent with
this hypothesis, we find that knockdown of THAP11 in SW620
colon cancer cells results in a significant decrease in cell prolifer-
ation. Interestingly, however, the THAP11-dependent gene ex-
pression profile is largely devoid of genes with previously known
functions in tumorigenesis or cell proliferation. Exceptions to this
generalization are annexin A1, a member of the annexin family of
calcium-dependent phospholipid-binding proteins, and PRAF2,
a novel proapoptotic Bcl-xL/Bcl2-interacting protein (40). The
role of annexin A1 in cancer cell function is complex, but several
reports have suggested that annexin A1 can function in an anti-
proliferative capacity (15, 17, 20). We speculate that increased
PRAF2 and/or annexin A1 gene expression resulting from
THAP11 knockdown may contribute to the cell proliferation de-
fect observed in these cells. Alternatively, the proliferation defect
resulting from THAP11 knockdown may arise from the cumula-
FIG 6 HCF-1 associates with THAP11 and coregulates transcription. (A) SW620 nuclear extract was immunoprecipitated with the indicated antibody, and the
immunoprecipitates (IP) were immunoblotted for HCF-1 and THAP11. The input corresponds to 10% (50
g) of the starting material. The HCF-1 precursor
and the HCF-1
C
subunit polypeptides are indicated by the arrow and the bracket, respectively. (B) Gene expression in SW620 cells expressing the indicated
shRNAs. Values represent the mean standard deviation of four independent experiments. The dashed line indicates the relative expression of control
shRNA-expressing cells.
Transcriptional Regulation by THAP11–HCF-1 Complex
May 2012 Volume 32 Number 9 mcb.asm.org 1665
tive effect of multiple dysregulated genes. In this context, we also
note that, in addition to protein coding genes, several annotated
or putative long ncRNAs were also identified as direct targets
of THAP11-mediated transcriptional repression, including
NCRNA00095 and AA862256. The fortuitous discovery of these
ncRNAs as THAP11-regulated transcripts by microarray-based
gene expression profiling likely reflects their previous but errone-
ous annotation as protein coding genes with subsequent inclusion
in the microarray design. Since many long ncRNAs have been
discovered to be regulated by the same transcriptional control
mechanisms utilized by protein coding mRNAs (14), we speculate
that additional long ncRNAs are likely regulated by THAP11. It is
now well established that long ncRNAs contribute to a diverse
array of biological functions, including transcriptional regulation,
cell growth, and apoptosis, but the vast majority of long ncRNAs
remain uncharacterized (37). We were surprised to observe such a
small THAP11 target gene set in our microarray analysis. One
possible explanation for this is that THAP11 target genes are in-
dividually sensitive to a given level of THAP11 in cells, while
knockdown by THAP11 A and C constructs decreases the level of
THAP11 to a point where only the gene set identified in this
microarray are uncovered. The use of a more robust knockdown
construct or THAP11 knockout cells may uncover additional and
novel THAP11 targets. Additional studies are required to identify
FIG 7 THAP11 and HCF-1 co-occupy chromatin in colon cancer cells. ChIP assay at THAP11-repressed (A) and -activated (B) genes in SW620 cells. THAP11
ChIP data from Fig. 3 are replotted here for comparison. (C) Sequential ChIP assays of SW620 cells at THAP11 and previously identified HCF-1 target genes. (Left
half) HCF-1 ChIP followed by control IgG or THAP11 reChIP. (Right half) THAP11 ChIP followed by control IgG or HCF-1 reChIP. Enrichment was analyzed
by qPCR and expressed as percent recovery relative to the input from the first ChIP. Values are means standard deviations of duplicate PCRs from a single
experiment performed at least three times with similar results.
Parker et al.
1666 mcb.asm.org Molecular and Cellular Biology
the full complement of THAP11-regulated transcripts, as well as
the protein coding or noncoding gene(s) downstream of THAP11
that contributes to cell proliferation.
THAP11 associates with and recruits HCF-1 to promoters, and
all of the THAP11 target genes analyzed here contain one or more
putative THAP11-binding sites near their respective transcription
start sites. The stable association of the THAP11–HCF-1 complex
on chromatin was unexpectedly found to require both proteins;
FIG 8 Interaction with HCF-1 is necessary for THAP11 chromatin association. (A) HCF-1 ChIP with SW620 cells expressing the indicated shRNAs. (B)
Immunoblotting of THAP11 and HCF-1 in SW620 cells from panels A and C. (C) THAP11 ChIP with SW620 cells expressing the indicated shRNAs. (D)
Coimmunoprecipitation of THAP11 by HCF-1 in HCT-116 cells expressing FLAG-tagged wild-type (WT) THAP11 or HCF-1 binding domain H243A and
Y245A mutant proteins. Coprecipitating THAP11 was detected by FLAG immunoblotting. IP, immunoprecipitate. (E) ChIP assay of HCT-116 cells from panel
D using anti-FLAG antibody. (F) Quantitative RT-PCR of THAP11 target genes in HCT-116 cells from panel D. The dashed line indicates the relative expression
of empty-vector-expressing cells.
Transcriptional Regulation by THAP11–HCF-1 Complex
May 2012 Volume 32 Number 9 mcb.asm.org 1667
depletion of either protein was sufficient to disrupt the binding of
both factors at THAP11 target genes. This mutual interdepen-
dence of HCF-1 and a sequence-specific transcription factor has
not been described previously and may represent a unique feature
of THAP11. Recent structural analyses of prototypical THAP do-
mains bound to their respective DNA elements have revealed that
THAP proteins bind DNA in a bipartite manner, making simul-
taneous major- and minor-groove DNA contacts (5, 35). Nonspe-
cific contacts with the sugar-phosphate backbone of the minor
groove are critical for THAP domain binding to DNA and are
mediated primarily by basic amino acids present in a flexible loop
structure positioned between the zinc-coordinating residues and
the conserved AVPTIF box. However, the length of this loop
structure is dramatically shortened in THAP11 and has been sug-
gested to result in reduced minor-groove contacts and a dimin-
ished affinity for DNA (35). Since HCF-1 does not bind DNA
directly, it appears unlikely that HCF-1 is functioning as a true
DNA-binding heterodimeric partner of THAP11. Alternatively,
HCF-1 may function as a non-DNA-binding heterodimeric
partner or instead as a bridge between THAP11 and another tran-
scription factor. Indeed, sequence-based analysis of the THAP11-
bound promoters described here has identified several high-prob-
ability Sp1-binding sites close to THAP11-bound regions. Sp1 has
been previously shown to associate with HCF-1 but in a region
separate from the THAP11-interacting Kelch domain (13, 42),
suggesting that the binding of Sp1 and that of THAP11 to HCF-1
are not likely to be mutually exclusive. We hypothesize that
THAP11 guides HCF-1 to promoters harboring a THAP11 re-
sponse element but that stable association of the THAP11–HCF-1
complex requires additional interaction between HCF-1 and fac-
tors like Sp1.
The role of HCF-1 in cell proliferation and cell cycle progres-
sion has been attributed largely to HCF-1-dependent transactiva-
tion of E2F target genes during G
1
/S-phase progression (18, 39,
43). Our results suggest that THAP11-dependent gene regulation
is a novel and previously unknown component of HCF-1-depen-
dent cell proliferation. Consistent with this interpretation, we
note that other non-E2F factors have also recently been linked to
HCF-1-dependent cell proliferation. HCF-1 has been shown to
link the transcriptional coactivator and deubiquitinating enzyme
BAP1 with YY1 to regulate cell proliferation and growth control
genes (44), while Mazars et al. have shown that THAP1, rather
than E2F proteins, recruits HCF-1 to activate the cell cycle-regu-
lated gene RRM1 in proliferating endothelial cells (26). Interest-
ingly, we find that, in addition to the novel gene set described here,
THAP11 and HCF-1 are also corecruited at the cell cycle- and
E2F-regulated genes CDC25A and RBL1. This finding raises the
intriguing possibility that THAP11 complements or cooperates
with E2F proteins for the recruitment of HCF-1 at some cell cycle-
regulated genes. These findings further underscore the notion that
THAP proteins represent a large but poorly characterized family
of HCF-1-associated transcription factors with potential roles in
cell proliferation, development, and apoptosis.
Our findings contrast with a previous report that suggests that
THAP11 is downregulated in several human cancers and func-
tions as a cell growth suppressor through direct transcriptional
repression of MYC (45). These conflicting results may represent a
tissue-specific role for THAP11 in human colon cancer versus
other types of cancer. However, we note that the assertion by Zhu
et al. that THAP11 is largely repressed in human cancers is based
solely on mRNA expression data and may not accurately reflect
THAP11 protein status. Nonetheless, close inspection of the
FIG 9 Knockdown of THAP11 decreases cell proliferation. Shown are results of alamarBlue cell proliferation assays of SW620 cells (A, left half) or Colo320-HSR
cells (B, left half) expressing the indicated shRNAs. Values represent the mean standard deviation (n8 wells) from a representative experiment performed
at least three times with similar results. Also shown are immunoblot assays of extracts from SW620 cells (A, right half) or Colo320-HSR cells (B, right half)
expressing the indicated shRNAs. NS, control shRNA. (C) Crystal violet cell proliferation assay with SW620 cells expressing either control or THAP11 shRNAs.
shNS, control shRNA.
Parker et al.
1668 mcb.asm.org Molecular and Cellular Biology
THAP11 expression data from their multiple-tissue Northern blot
array (Fig. 1 in reference 45) suggests elevated THAP11 mRNA in
several tumor versus normal colon tissues, consistent with our
immunohistochemistry results. We were unable to detect endog-
enous THAP11 or HCF-1 at the MYC promoter in SW620 cells
(data not shown), and knockdown of either factor similarly failed
to increase MYC gene expression (data not shown), suggesting
that MYC is not a THAP11 target gene in colon cancer cells and
providing a potential explanation for the contrasting results re-
garding THAP11-dependent cell proliferation.
THAP11 has recently been identified as a critical factor in
the maintenance of ES cell pluripotency and proliferation (9).
Interestingly, small-interfering-RNA-mediated knockdown of
THAP11 was reported not to affect ES cell proliferation signif-
icantly, despite an approximately 85% reduction in THAP11 ex-
pression (9). These findings are in good agreement with our re-
sults indicating that the significant but incomplete knockdown
observed with shRNAs T11A and T11C results in a modest cell
proliferation defect while nearly complete depletion of THAP11
with shRNA T11E yields a markedly enhanced reduction of cell
proliferation.
In summary, our studies show for the first time that THAP11
expression increases dramatically in colon cancer and plays an
important role in colon cancer cell proliferation. We show that
THAP11 functions as a transcriptional regulator that requires
HCF-1 as an obligatory partner in DNA binding and target gene
regulation. Finally our study identifies a novel set of genes that
previously have not been linked to cancer cell function. Future
studies will investigate the roles of these novel protein coding and
ncRNA target genes, identify the full complement of THAP11 and
THAP11–HCF-1 target genes, and determine whether THAP11
has a role in other human cancers.
ACKNOWLEDGMENTS
This work was supported by NIH/NCI grant R01 CA133755 (D.C.). J.B.P.
is supported by NIH/NCI Institutional NRSA Training Program in Signal
Transduction and Cancer grant T32 CA070085. We gratefully acknowl-
edge the Northwestern University Cell Imaging Facility, Flow Cytometry
Facility, and Genomics Core Facility, which are supported by Cancer Cen-
ter Support grant NCI CA060553.
We have no conflict of interest to declare.
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    • "In addition to the known interactors described above, ChromNet also revealed previously uncharacterized, high-scoring interactions, including the transcriptional regulator Host Cell Factor C1 (HCFC1) (Additional file 1: Table S3). HCFC1 binds largely to active promoters [39] and is involved in biological processes, such as cell cycle progression [46, 50] and oncogenesis [12, 45, 48] . This further supports its possible role as an interactor of MYC in regulating these activities. "
    [Show abstract] [Hide abstract] ABSTRACT: A cell’s epigenome arises from interactions among regulatory factors—transcription factors and histone modifications—co-localized at particular genomic regions. We developed a novel statistical method, ChromNet, to infer a network of these interactions, the chromatin network, by inferring conditional-dependence relationships among a large number of ChIP-seq data sets. We applied ChromNet to all available 1451 ChIP-seq data sets from the ENCODE Project, and showed that ChromNet revealed previously known physical interactions better than alternative approaches. We experimentally validated one of the previously unreported interactions, MYC–HCFC1. An interactive visualization tool is available at http://chromnet.cs.washington.edu. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0925-0) contains supplementary material, which is available to authorized users.
    Full-text · Article · Dec 2016
    • "Genome-wide analysis of THAP11 binding to DNA revealed a novel consensus sequence that is distinct from motifs recognized by other potent transcriptional regulators, and that is present in the promoters of about 800 human genes (Dejosez et al., 2010; Ngondo-Mbongo et al., 2013; Worsley Hunt and Wasserman, 2014 ). Indeed, a stringent microarray analysis performed in colon cancer cells confirmed that THAP11 either activates or represses expression of at least 80 genes (Parker et al., 2012). Importantly, THAP11 does not possess an activation domain and therefore it has to achieve its functions by recruiting different effector proteins via its C-terminal domain (Dejosez et al., 2008Dejosez et al., , 2010 Mazars et al., 2010). "
    [Show abstract] [Hide abstract] ABSTRACT: Thanatos associated protein 11 (THAP11) is a cell cycle and cell growth regulator differentially expressed in cancer cells. THAP11 belongs to a distinct family of transcription factors recognizing specific DNA sequences via an atypical zinc finger motif and regulating diverse cellular processes. Outside the extensively characterized DNA-binding domain, THAP proteins vary in size and predicted domains, for which structural data are still lacking. We report here the crystal structure of the C-terminal region of human THAP11 protein, providing the first 3D structure of a coiled-coil motif from a THAP family member. We further investigate the stability, dynamics and oligomeric properties of the determined structure combining molecular dynamics simulations and biophysical experiments. Our results show that the C-ter region of THAP11 forms a left-handed parallel homo-dimeric coiled-coil structure possessing several unusual features.
    Full-text · Article · Mar 2016
    • "After processing, the HCF-1 N and HCF-1 C subunits remain tightly, but noncovalently associated131415 and regulate distinct phases of the cell-division cycle [16]. HCF-1 N promotes G1-to-S-phase transition [16] via association with E2F transcription factors [17, 18] and Thap11 [19], and HCF-1 C promotes proper M-phase progression [16] . OGTmediated HCF-1 proteolytic processing ensures proper cell-cycle progression through activation of HCF-1 C -subunit M-phase functions [9]. "
    [Show abstract] [Hide abstract] ABSTRACT: Human HCF-1 (also referred to as HCFC-1) is a transcriptional co-regulator that undergoes a complex maturation process involving extensive O-GlcNAcylation and site-specific proteolysis. HCF-1 proteolysis results in two active, noncovalently associated HCF-1N and HCF-1C subunits that regulate distinct phases of the cell-division cycle. HCF-1 O-GlcNAcylation and site-specific proteolysis are both catalyzed by O-GlcNAc transferase (OGT), which thus displays an unusual dual enzymatic activity. OGT cleaves HCF-1 at six highly conserved 26 amino acid repeat sequences called HCF-1PRO repeats. Here we characterize the substrate requirements for OGT cleavage of HCF-1. We show that the HCF-1PRO-repeat cleavage signal possesses particular OGT-binding properties. The glutamate residue at the cleavage site that is intimately involved in the cleavage reaction specifically inhibits association with OGT and its bound cofactor UDP-GlcNAc. Further, we identify a novel OGT-binding sequence nearby the first HCF-1PRO-repeat cleavage signal that enhances cleavage. These results demonstrate that distinct OGT-binding sites in HCF-1 promote proteolysis, and provide novel insights into the mechanism of this unusual protease activity.
    Full-text · Article · Aug 2015
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