From the Laboratory of Gene Regulation, The Wheeler Institute for
Biomedical Research, Huntington, New York 11743
The transcription factor, NF-Y, plays a critical
role in tissue-specific major histocompatibility complex class II gene
transcription. In this report the biochemical properties of the
heterotrimeric NF-Y complex have been characterized during
stage-specific B-cell development, and in several class
II
mutant B-cell lines, which represent distinct
bare lymphocyte syndrome class II genetic complementation groups. The
NF-Y complex derived from class II+ mature B-cells bound
with high affinity to anion exchangers, and eluted as an intact
trimeric complex, whereas, NF-Y derived from class II
plasma B-cells, and from bare lymphocyte syndrome group II cell lines,
RJ2.2.5 and RM3, dissociated into discrete NF-YA and NF-YB:C subunit
fractions. Recombination of the MPC11 plasma B-cell derived NF-Y A:B:C
complex with the low molecular mass protein fraction, NF-Y-associated factors (YAFs),
derived from mature A20 B-cell nuclei, conferred high affinity anion
exchange binding to NF-Y as an intact trimeric complex. Recombination
of the native NF-YA:B:C complex with the transcriptional cofactor, PC4,
likewise conferred high affinity NF-Y binding to anion exchangers, and
stabilized NF-Y interaction with CCAAT-box DNA motifs in
vitro. Interaction between PC4 and NF-Y was mapped to the
C-terminal region of PC4, and the subunit interaction subdomain of the
highly conserved DNA binding-subunit
interaction domain (DBD) of NF-YA. These results suggest
that in class II+ mature B-cells NF-Y is associated with
the protein cofactor, PC4, which may play an important role in
NF-Y-mediated transcriptional control of class II genes.
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INTRODUCTION |
The major histocompatibility complex
(MHC)1 class II genes encode
a set of highly polymorphic transmembrane glycoproteins that mediate
several critical immunological processes that include antigen
presentation to T helper cells, and the phenomenon of T-cell selection
in the thymus (reviewed in Refs. 1 and 2). MHC class II
and
subunits form a non-covalent heterodimer on the cell surface of a
restricted subset of mammalian cell types (e.g. mature
B-cells, activated T-cells, macrophages, and the thymic epithelium),
and their expression is modulated at the transcriptional and
post-transcriptional levels by a variety of cytokines, lymphokines, and
hormones (reviewed in Refs. 3-6). Additionally, in B lymphocytes MHC
class II proteins are regulated in a stage-specific manner, as the high
level of constitutive expression observed in mature B-cells is
completely extinguished during progression to the plasma B-cell stage
through repression at the transcriptional level (7, 8).
Three highly conserved MHC class II DNA elements, the S-, X-, and
Y-boxes, have been shown through extensive functional analyses to
represent, in large measure, the focal regulatory regions of class II
transcriptional control (9-12). The X-box contains two separable
functional elements: X1 and X2, where the X1 motif binds the regulatory
factor X (RFX) family of transcription factors (13, 14), and the X2
motif binds many members of the fos/jun and cyclic AMP
response element-binding protein (CREB)/activating transcription factor
families (15, 16). In contrast, a single activating transcription
factor, the heteromeric nuclear factor-Y (NF-Y), specifically interacts
with a CCAAT motif found in all MHC class II gene Y-boxes (10, 17, 18).
Proteins that interact with the X- and Y-box elements function in a
cooperative manner because alterations in their spacing lead to
significant reduction in in vivo promoter activity (19), and
disrupt stabilized complexes formed in vitro with their
corresponding proteins (20, 21).
NF-Y binding sites are frequently located within ~50-80 base pairs
of promoter transcriptional initiation sites, and also in more distal
enhancer control regions of eukaryotic genes (22). In these contexts,
the ubiquitous NF-Y factor is often juxtaposed with other general or
tissue-restricted transcription factors, which together mediate crucial
functional steps in constitutive and inducible gene regulation. For
example, cooperative protein interactions between NF-Y and the
CCAAT/enhancer-binding protein (C/EBP), and C/EBP-related proteins, DBP
and LAP, have been observed in the murine albumin promoter (23),
between NF-Y and the serum response factor, p67SRF, in the
human
-actin promoter (24), and between the NF-Y and the sterol
regulatory element-binding protein (SREBP) in the
3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) promoter (25). In the
last example, NF-Y:SREBP acts as a negative-feedback sensor to regulate
the transcriptional activity of HMG-CoA in response to serum
cholesterol levels.
Initial biochemical characterization of the HeLa cell CCAAT-box factor,
CP1 (26), and the rat liver CCAAT-box factor (CBF) (27), showed that
specific CCAAT-box DNA binding activity was lost following ion exchange
chromatography, but could be restored by recombining two separate
column fractions. The two HeLa column fractions, A and B, could also be
recombined with the separated Saccharomyces cerevisiae
subunit fractions, HAP2 and HAP3, to reconstitute specific CCAAT-box
binding activity (28). These results suggested a heterodimeric
structure for this CCAAT-box factor, which appeared to have been highly
conserved in eukaryotes (26-29). Isolation of cDNAs that encoded
the murine NF-YA/YB (18), and rat CBF-A(NF-YB), and CBF-B(NF-YA)
subunits (30-32) provided conclusive evidence of the equivalence of
CBF and NF-Y, and showed these subunits to be homologs of the yeast
HAP2/3 proteins (33-35). Cloning of an additional S. cerevisiae factor, HAP5 (36), and CBF-C(NF-YC) (37, 38), together
with biochemical characterization of the NF-Y complex, has now
confirmed the existence of a third subunit and a heterotrimeric
structure which is minimally required for specific CCAAT-box DNA
binding activity. Recently, the NF-Y complex has been shown to interact
functionally with the non-histone chromosomal high mobility group
protein, HMG-I(Y), and a protein-protein interaction site mapped to the
NF-YA DNA-subunit interaction domain (DBD) in NF-Y, and the AT-hook
motif in HMG-I(Y) (39).
Lack of appropriate MHC class II expression in humans results in a
severe autosomal recessive immunodeficient condition known as bare
lymphocyte syndrome (BLS) (6). The class II
mutant
B-cells in BLS subgroup type II synthesize normal levels of MHC class
I, and class II-associated invariant chain proteins, whereas expression
of all class II isotypes is universally extinguished. The
B-cell-specific gene product, class II transactivator (CIITA), represents the defective gene product in BLS group II class
II
cell lines, and is capable of fully restoring class II
gene transcription in this specific genetic complementation group (40).
CIITA has been shown to regulate both constitutive class II
transcription in mature B-cells (40), and mediate interferon-
(IFN-
)-induced expression of MHC class II genes during
monocyte/macrophage cell differentiation, and in several class
II
cell lines (41, 42). In addition, endogenous CIITA
mRNA expression has been shown to be completely suppressed in
plasma B-cells, whereas unregulated overexpression of CIITA in plasma
B-cells surprisingly restores MHC class II mRNA and protein
expression to the elevated levels observed in mature B-cells (43).
These results suggest active repression of CIITA expression accounts for the dominant suppressor plasma cell phenotype exhibited by mature
B:plasma B-cell somatic cell hybrids (7, 8), and further suggest CIITA
plays an obligatory role in the normal tissue-specific regulation of
MHC class II gene transcription.
Positive cofactor 4 (PC4), also referred to as p15 (44, 45), was
initially identified as an abundant nuclear protein in murine
plasmacytoma cells (46), and cloned independently on the basis of its
differential expression in rat embryo cells and B-cell tumors (47), and
the ability of this protein to bind polydeoxypyrimidines in
vitro (46). Subsequently, PC4/p15 was shown to function as a
general transcription accessory factor in the response of RNA
polymerase II to upstream activator proteins in in vitro
reconstituted systems (44, 45). Phosphorylated PC4, as modified by
casein kinase II (CKII), has been shown to be functionally inactive in
reconstituted cell-free in vitro transcription assays,
whereas both the purified native non-phosphorylated, and Escherichia coli derived forms of PC4, are potent
transcriptional activators in vitro (45, 48). In addition,
recent results suggest that PC4 regulation may occur while tethered to
specific promoter transcription factors, such as NF-Y (39).
In this report, the biochemical ion exchange properties of the NF-Y
complex were examined in stage-specific B lymphocytes, and compared
with a series of BLS mutant cell lines to determine if tissue-specific
changes in NF-Y structure occur during stages of active and inactive
MHC class II gene transcription. The NF-Y complex in class
II+ mature B lymphocytes was observed to bind as an intact
trimeric complex to anion exchangers. In contrast, the NF-Y complex
derived from class II
plasma B-cells dissociated into
discrete NF-YA and NF-YB:C subunit fractions following anion exchange
chromatography. A protein fraction derived from mature B-cell nuclei,
the NF-Y-associated factors (YAFs), in addition to recombinant PC4,
restored Q+ binding to the plasma B-cell NF-Y complex as an
intact trimeric complex. These results suggest that, in MHC class II
mature B-cells, PC4 functions to stabilize both NF-YA:B:C subunit
interactions, as well as NF-Y:CCAAT-box DNA interactions, and further
suggest PC4 plays important transactivator/transrepressor roles in MHC class II gene transcription during B-cell development.
 |
MATERIALS AND METHODS |
Recombinant Plasmids--
Human PC4 was cloned from pPC4 2T (39)
into the EcoRI site of pGEX2TK (Amersham Pharmacia Biotech)
using polymerase chain reaction. GST-PC4 mutants were derived from pPC4
2T using polymerase chain reaction, and cloned into the
BamHI-EcoRI sites of pGEX2TK. pPC4 (
C37)
contains the N-terminal 90 amino acids of PC4 (nucleotides 1-270 as
defined; Ref. 44), pPC4(
N64) contains the C-terminal 63 amino acids
(nucleotides 193-384), and pPC4(C37) contains the C-terminal 37 amino
acids of PC4 (nucleotides 271-384). pYA (DBD
C23) was prepared by
digesting pYA(DBD) (39) with StyI-EcoRI, followed by treatment with the large subunit of DNA polymerase I (Klenow) under
standard conditions to generate blunt-ends (49). The purified YA(DBD)
fragment was ligated and plasmids that lacked the C-terminal 23 amino
acids of YA(DBD) (nucleotides 1011-1081, as defined in Ref. 18) were
identified. Plasmids were prepared and verified using standard
techniques (49).
Expression and Purification of Recombinant Proteins--
The
cloning, expression, and purification of His-YB, His-YC, glutathione
S-transferase (GST), and GST fusion proteins (YA, YA(DBD),
YA(
DBD), PC4, Dr1, TFIIB, and HAP2(DBD)), and 32P
labeling of recombinant proteins using CKII, and heart muscle creatine
kinase (HMK) have been described previously (39). GST-PC4(
C37), GST-PC4(C37), GST-PC4(
N64), and GST-YA(DBD
C23) were expressed in
E. coli DH5
, and purified from the soluble fraction as
described previously (39). Thrombin cleaved recombinant proteins,
YA(DBD) and PC4, were further purified to >95% by centrifugation
through Centricon 30 filtration devices (Amicon) in BC-420 (50 mM Tris-HCl (pH 7.9), 0.42 M KCl, 20%
glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride), and stored at
80 °C
following buffer exchange to BC-100 (50 mM Tris-HCl (pH
7.9), 0.1 M KCl, 20% glycerol, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride) using Centricon 10 devices (Amicon).
Cell Culture and Nuclear Extract Preparation--
Cell lines
were kindly provided for these studies as follows: A20 (J. Durdik,
University of Arkansas, Fayetteville, AR), Raji (J. Jones, National
Jewish Center for Immunology and Respiratory Medicine, Denver, CO),
RJ2.2.5 (B. Mach, University of Geneva, Geneva, Switzerland), RM3 (M. Peterlin, University of California, San Francisco, CA), and 6.1.6 (D. Pious, University of Washington, Seattle, WA). MPC11 cells were
obtained from the ATCC, Rockille, MD. Lymphocyte cell lines were
maintained in suspension culture at ~106 cells/ml in RPMI
1640 (Life Technologies, Inc.) which contained 10% fetal bovine serum
(HyClone). Cell lines were routinely tested for cell surface MHC class
II protein using FITC-conjugated species-specific anti-class II
antibodies (PharMingen) and fluorescence-activated cell sorting
analysis. A20 and Raji cells express high levels of surface MHC class
II protein, whereas all other class II
cell lines tested
did not express measurable surface class II molecules.2
Nuclear extracts were prepared from these, and other cell lines,
according to the method of Dignam et al. (50), and as
described previously (39). Briefly, nuclear extracts were passed over DEAE-Sepharose (Sigma) in BC-420 to remove residual nucleic acids, and
the flow-through fraction was dialyzed for 5 h against BC-100 using a 10-12-kDa cut-off cellulose membrane (Life Technologies, Inc.). Extracts were centrifuged in an Eppendorf microcentrifuge (10,000 rpm for 10 min) at 4 °C following dialysis to remove
insoluble material, and aliquots of the supernatant frozen at
80 °C. Nuclear extract protein concentrations were determined
using the Bradford assay (Bio-Rad) with bovine serum albumin (BSA)
(Life Technologies, Inc.) as the protein standard (51), and ranged
between ~5-15 mg/ml.
Electrophoretic Mobility Shift Assay (EMSA)--
EMSA
assays, and the preparation and use of the E
, and S-collagen
CCAAT-box oligonucleotides in EMSA has been described previously (39).
The 32P-E
DNA oligomer was used in EMSA to normalize
nuclear extracts for relative NF-Y CCAAT DNA binding activities for use
in comparative chromatographic analyses. In EMSA, protein fractions
were mixed with ~2 µg of the nonspecific DNA competitor,
poly(dI-dC) (Amersham Pharmacia Biotech), prior to addition of ~0.2
ng of 32P-labeled DNA oligonucleotide probe in a 30-µl
binding reaction. Binding reactions were performed at 30 °C for 30 min, then loaded onto a 4% non-denaturing polyacrylamide gel (30:1
acrylamide/bisacrylamide ratio) containing 50 mM Tris, 50 mM boric acid, 1.0 mM EDTA (0.5 × TBE).
Gels were electrophoresed at ~150 V for 1.5 h following pre-running for 30 min at room temperature using 0.5 × TBE as the
running buffer. Dried gels were exposed to XAR-5 film (Eastman Kodak
Co.) with an intensifying screen (DuPont) at
80 °C.
Chromatographic Procedures--
Anion exchangers, Q+
Sepharose, DEAE-Sepharose, and the cation exchanger, S
Sepharose, were prepared according to the manufacturer's
specifications (Sigma), and equilibrated in BC-100. Nuclear extracts
were depleted of YAF proteins by first passing extracts over
DEAE-Sepharose in BC-420, followed by dialysis against several changes
of BC-420 over an 8-h period using a Spectra/Por 6 (25-kDa cut-off)
cellulose membrane (Spectrum). Subsequently, extracts were dialyzed
against BC-100 for 4 h and stored at
80 °C, following
centrifugation to remove insoluble material.
The NF-YA subunit was prepared from YAF-depleted nuclear extracts by
heating nuclear extracts to 65 °C for 5 min, placing extracts on ice
for 15 min, then centrifuging (10,000 rpm for 10 min) at 4 °C in an
Eppendorf microcentrifuge to remove insoluble material. NF-YB:C is
effectively inactivated following this heat treatment step (37). NF-YA
was further purified by passing this material over Q+
Sepharose equilibrated in BC-100, and collecting the flow-through fraction (27). NF-YA was concentrated using Centricon 10 filtration devices, stored in BC-100 at
80 °C, and used to complement NF-YB:C activity in recombination experiments.
NF-YB:C subunits were isolated following Q+ and
S
Sepharose chromatographic separations, as described
previously (27, 37), with the following modifications. A20 and MPC11
nuclear extracts, depleted of YAF proteins as described above, were
applied to a Q+ Sepharose column in BC- 100 (Tris-HCl
buffer). Following extensive washing with BC-100, NF-YB:C was
step-eluted using BC-600 (0.6 M KCl), and dialyzed against
BC-100 buffer where HEPES had been substituted for Tris- HCl. This
material was loaded onto a S
Sepharose column, and eluted
using BC-100 (HEPES). NF-YB:C elutes in the flow-through fraction,
whereas residual NF-YA activity binds tightly to S
Sepharose under these conditions (27). The NF-YB:C fractions in each
case were concentrated using a Centricon 10 filtration device, and the
buffer was exchanged to BC-100 (Tris-HCl). NF-YB:C fractions were
stored at
80 °C, and used to complement the NF-YA subunit in
recombination experiments. The elution position of separated NF-Y
subunits was identified in Q+ Sepharose column fractions by
incubating 5 µl of each column fraction with 10 µl of the
corresponding complementing partially purified YA or YB:C subunit
fraction in a 30-µl EMSA reaction.
In dialysis experiments, an aliquot of an A20 DEAE-Sepharose column
flow-through fraction (1.0 ml; ~10 mg of protein) in BC-420 was
dialyzed separately against BC-420 using a 10-12-kDa cut-off cellulose
membrane (Life Technologies, Inc.), against BC-420 using a 25-kDa
cut-off cellulose membrane (Spectrum), and also against BC-100 using a
25-kDa cut-off membrane. The dialysis buffers were changed in each case
after 3 h, and dialysis continued for an additional 3 h at
4 °C. After this period, all extracts were further dialyzed for
3 h against BC-100. Normalized NF-Y E
DNA binding activities
were determined, and assayed for binding to Q+
Sepharose.
To prepare NF-Y from nuclear extract fractions depleted of YAF
activity A20 or MPC11 nuclear extracts were first passed over DEAE-Sepharose in BC-420, dialyzed against BC-420 for 8 h using a
Spectra/Por 6 (25-kDa cut-off) membrane, then diluted 1:4 in BC-0
buffer. This material was applied to a heparin-agarose (Sigma) column
in BC-100, and the column was developed using a BC-KCl step gradient.
The NF-Y complex eluted in the 0.42 M KCl step was dialyzed
against BC-100, and aliquots stored at
80 °C A20 NF-Y derived from
the heparin-agarose fraction, and used in experiments with recombinant
PC4, was further purified using a CCAAT-box DNA affinity column as
described previously (39). Approximately 100 µg of the
affinity-purified NF-Y protein fraction was recombined with either
~50 µg of purified recombinant PC4, or purified BSA, as described
for YAF recombination experiments below.
In native YAF recombination experiments, A20 YAFs were prepared from
A20 nuclear extracts that had been passed over DEAE-Sepharose in
BC-420. The heparin-agarose A20 NF-YA:B:C and MPC11 NF-YA:B:C fractions
were compared with A20 nuclear extracts for NF-Y E
DNA binding
activity. A20 nuclear extracts that contained ~2-fold greater
equivalents of CCAAT-box DNA binding activity were used to prepare the
A20 YAF fraction by centrifuging the 0.42 M KCl DEAE-Sepharose fraction through a Centricon 30 filtering device (Amicon) in a DuPont Sorvall type SM-24 rotor (7000 rpm for ~3 h at
4 °C) as described previously (39). The individual heparin-agarose A20, or MPC11 NF-YA:B:C fraction, was adjusted to 0.42 M
KCl, recombined with the Centricon 30 filtrate, and incubated at room temperature for 5 min. This material was then dialyzed against BC-100
using a 10-12-kDa cut-off cellulose membrane for 3 h at 4 °C
to permit protein reassociation. NF-Y complex binding to Q+
Sepharose in these recombined fractions was determined following dialysis using EMSA.
The A20 YAF fraction, as prepared using Centricon 30 filtration
devices, was depleted of protein by incubation with StrataClean resin
according to the manufacturer (Stratagene). Silica beads were removed
by centrifugation, and the depletion procedure was repeated. This
depleted YAF fraction was recombined with A20 NF-YA:B:C, and the NF-Y
complex was tested for Q+ Sepharose binding using EMSA as
described above.
Normalized amounts of NF-Y E
DNA binding activity were loaded onto
ion exchange columns, and developed using a BC-KCl step gradient (0.1, 0.3, 0.6, and 1.0 M KCl). An aliquot of the column load
material and a 5-µl aliquot of each column fraction were then assayed
for CCAAT-box DNA binding activity to the E
oligomer probe using
EMSA. Equivalent film exposures were obtained for each chromatographic
analysis in order to directly compare the relative level of NF-Y
complex binding to ion exchange materials.
 |
RESULTS |
Anion Exchange Properties of the NF-Y Complex in Stage-specific B
Lymphocyte Cell Lines--
NF-Y is ubiquitously expressed in mammalian
tissues, and is known to play an essential functional role in the
transcriptional regulation of many tissue-specific (MHC class II,
collagen
2(I), lipoprotein lipase), cell-cycle regulated (cyclin A,
cdc2), and inducible (HMG-CoA, interleukin-4) eukaryotic genes;
however, the underlying transcriptional mechanisms that depend on NF-Y and its interaction with other combinations of general
proximal-promoter factors (e.g. Oct-1, Sp1, RFX), and
components of the basic RNA polymerase II machinery in these diverse
situations are largely unknown. The CCAAT-box DNA binding activity of
the HeLa CP1 complex in HeLa cells (26), the HAP2/3 complex in S. cerevisiae (28), and the CBF complex in rat liver and NIH3T3 cells
(27) were initially shown to be lost following ion exchange
chromatography, but could be reconstituted by recombining specific
column fractions. Subsequently, the CBF-A(NF-YB) fraction was shown to
contain an additional protein component, CBF-C(NF-YC), which associated
with CBF-A through strong hydrophobic interactions and coeluted as a
heterodimer through these ion exchange materials (37). Together with
cloning of the HAP5 subunit (36), these studies demonstrated that three
unique subunits were minimally required for specific CCAAT-box DNA
recognition.
In an effort to determine if the ubiquitous NF-Y complex exhibits
differential "tissue-specific" biochemical properties, the ion
exchange behavior of NF-Y was examined in a variety of stage-specific B
lymphocyte cell lines. The anion exchange properties of NF-Y derived
from the mature murine B-cell lymphoma, A20, were compared with the
terminally differentiated murine plasmacytoma B-cell line, MPC11 (Fig.
1). Analysis of the A20 NF-Y complex
revealed near quantitative recovery of E
CCAAT-box DNA binding
activity in the 0.6 M KCl step fractions (panel
A). In contrast, MPC11 NF-Y E
DNA binding activity was
virtually lost following Q+ Sepharose analysis (panel
B). A low level of residual MPC11 NF-Y was consistently observed
in the 0.6 M KCl step fraction (panel B,
fraction 10). The A20 and MPC11 E
DNA binding activities
identified in the 0.6 M KCl step fractions were shown to be
bona fide NF-Y complexes, respectively, using
-NF-YB
antibodies in EMSA upshift assays.2 These observations have
been extended to a number of additional murine and human mature B-cell
lines (e.g. CH27, Raji, and Daudi), to other murine
plasmacytoma B-cell lines (e.g. S107, MOPC 315, and
P3X63Ag8) using both Q+ Sepharose and DEAE-Sepharose. In
these cases, the mature B-cell NF-Y complex was observed to bind to
Q+ and DEAE-Sepharose with high affinity as an intact
multimeric complex, whereas the plasma B-cell NF-Y complex separated
into discrete subunit fractions. In addition, NF-Y derived from a
number of MHC class II
cell lines (e.g. HeLa,
3T3-L1, and TA1) exhibit identical Q+ Sepharose binding
behavior as observed in MPC11 cells.2 These results suggest
that the biochemical properties of NF-Y are altered during B-cell
development, and that high affinity NF-Y Q+ binding
correlates with activation of MHC class II genes in mature B-cells, and
loss of Q+ binding correlates with suppression of class II
gene transcription in terminally differentiated plasma B-cells.

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Fig. 1.
Anion exchange behavior of the NF-Y complex
derived from stage-specific B lymphocytes. Nuclear extracts
prepared from the murine mature B-cell lymphoma cell line, A20
(A), and the murine plasmacytoma cell line, MPC11
(B), were compared for NF-Y complex binding to the anion
exchanger, Q+ Sepharose. The columns were developed using a
KCl step gradient, and each fraction was assayed for E DNA binding
activity using EMSA. Positions of the KCl step-gradient are shown above
panel A, and the position of the NF-Y complex is denoted to
the left of each panel. Lane L, column
load material; lane P, free 32P-E DNA probe;
lanes 1-17, Q+ Sepharose column
fractions.
|
|
In order to locate and verify the identity of NF-Y subunits separated
during Q+ analysis in Fig. 1, individual complementing A20
and MPC 11 NF-Y subunits were prepared and used in recombination EMSA
assays. NF-YA and NF-YB:C fractions were added individually to A20 and MPC 11 Q+ fractions, and tested for E
DNA binding
activity (Figs. 2 and 3). Addition of exogenous A20 YB:C
activity to the A20 Q+ fractions identified a small
quantity of additional A20 YA activity in the early region (0.1 M KCl) of the step gradient (Fig. 2A). The
majority of YA subunit activity however, was observed in the 0.6 M KCl step fraction. Addition of exogenous A20 YA activity did not reveal any additional A20 YB:C activity in the A20
Q+ fractions (Fig. 2B). These analyses suggest
the majority of the A20 NF-YA and YB:C subunits elute together from
Q+ Sepharose as an intact multisubunit complex in the 0.6 M KCl fraction. Addition of exogenous MPC11 YB:C subunit
activity to the MPC11 Q+ fractions from Fig. 1 complemented
MPC11 YA activity, which had separated from the YB:C subunits during
application of the KCl gradient and was located in the low salt
gradient fractions (Fig. 3A). MPC11 YB:C activity was
identified following complementation with MPC11 YA activity only in the
0.6 M KCl step fraction of the gradient (Fig.
3B). These results clearly demonstrate that the loss of NF-Y
E
DNA binding activity in MPC11 nuclear extracts following
Q+ Sepharose chromatography (Fig. 1) is due to physical
separation of the YA and YB:C subunits. Furthermore, these comparisons
suggest that the biochemical properties of the NF-Y complex are altered in a stage-specific manner during the transition from a mature B-cell
to a plasma B-cell.

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Fig. 2.
Localization of additional A20 NF-YA subunit
activity following Q+ Sepharose analysis of the A20 NF-Y
complex. A20 NF-Y Q+ Sepharose fractions from Fig.
1A were assayed for additional NF-YA (A), and
NF-YB:C (B) activities through addition of exogeneous
purified complementing native A20 YB:C or YA subunit fractions,
respectively. NF-Y E DNA binding activity was determined using EMSA.
Complementing subunit activities are denoted above each panel.
Positions of the KCl step gradient are shown above A, and
the position of the NF-Y complex is denoted to the left of
each panel. In A, lane YB:C denotes the YB:C
fraction alone; lane C, control YA and YB:C subunits
recombined; lanes 1-17, Q+ fractions derived
from A20 Q+ analysis in Fig. 1A. In
B, lane A denotes YA subunit alone; lane
C, control YA and YB:C subunits recombined; lanes
1-17, Q+ fractions derived from A20 Q+
analysis in Fig. 1A.
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Fig. 3.
Identification of separated MPC11 NF-Y
subunits following Q+ Sepharose chromatography. MPC11
NF-Y Q+ Sepharose fractions from Fig. 1B were
assayed for YA (A), and YB:C (B) activities
through addition of exogeneous purified complementing native MPC11 YB:C
or YA subunit fractions, respectively. NF-Y E DNA binding activity
was determined using EMSA. Complementing subunit activities are denoted
above each panel. Positions of the KCl step gradient are
shown above A, and the position of the NF-Y complex is
denoted to the left of each panel. In
A, lane P denotes free 32P-E DNA
probe; lane YB:C denotes the YB:C fraction alone; lane
C, control YA and YB:C subunits recombined; lanes
1-17, Q+ fractions derived from MPC11 Q+
analysis in Fig. 1B. In B, lane A, YA subunit
alone; lane C, control YA and YB:C subunits recombined;
lanes 1-17, Q+ fractions derived from MPC11
Q+ analysis in Fig. 1B.
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Ethidium bromide (EtBr) is a compound that is known to intercalate
between duplex DNA strands, to prevent transcription factors from
binding to their sequence-specific elements, and to be useful in
assessing if associations observed between a protein and additional putative protein cofactors are specific, and not due to cofactor interaction with contaminating nuclear DNA (52-54). The results of
testing the effectiveness of EtBr in disrupting A20 NF-Y binding to
Q+ Sepharose are shown in Fig.
4. A20 nuclear extracts were treated in
control experiments with increasing EtBr concentrations, and tested for
E
DNA binding activity (panel A). EtBr concentrations of
50 or 100 µg/ml essentially inhibited A20 NF-Y E
DNA binding activity (lanes 3 and 4, respectively). A20
nuclear extracts were made 50 µg/ml in EtBr, applied to a
Q+ column equilibrated in the same EtBr concentration, and
KCl-step eluted (panel B). A20 Q+ column
fractions were assayed for DNA-binding by first reducing the [EtBr]
to 10 µg/ml. A20 NF-Y was quantitatively recovered in the 0.6 M KCl step fraction from this Q+ column. Peak
A20 NF-Y DNA binding fractions from the Q+ column
(fractions 9 and 10) were also shown to be sensitive to increasing
[EtBr], as an [EtBr] of 50 and 100 µg/ml inhibited E
DNA
binding activity (panel C, lanes 3 and
4, respectively). These results suggest that the A20 NF-Y
complex interacts with Q+ Sepharose anionic groups
directly, and not through nonspecific DNA, or DNA-protein interactions.
Further support for this conclusion was reached by treating A20 nuclear
extracts with high concentrations (0.1 unit/µl) of micrococcal
nuclease or DNase I prior to Q+ column analysis. Both
treatments had no effect on A20 NF-Y binding to Q+
Sepharose suggesting that contaminating DNA and/or nonspecific DNA-protein cofactor interactions are not responsible for A20 NF-Y
binding to anion exchange materials.2

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Fig. 4.
Analysis of A20 NF-Y binding to the E DNA
oligomer and to Q+ Sepharose in the presence of ethidium
bromide. In A, A20 nuclear extracts were tested for
NF-Y binding to the E DNA probe in the presence of increasing
concentrations of ethidium bromide. In B, A20 NF-Y binding
to Q+ Sepharose was analyzed in the presence of 50 µg/ml
ethidium bromide. The Q+ column fractionation and assay
conditions were as described in Fig. 1, and the BC-KCl buffers
contained 50 µg/ml ethidium bromide. EMSA of these Q+
fractions (B) was performed by diluting the ethidium bromide
concentration to 10 µg/ml. In C, A20 NF-Y peak
Q+ fractions (B; fractions 9 and
10) were tested for inhibition of E DNA binding by
ethidium bromide. In A and B, lane P,
free 32P-E probe. In A and C,
lane C, control NF-Y E binding activity. In A
and C, the ethidium bromide concentrations are as follows:
lane C, 0 µg/ml; lane 1, 10 µg/ml; lane
2, 25 µg/ml; lane 3, 50 µg/ml; lane 4,
100 µg/ml. In B, lanes 1-17, A20
Q+ Sepharose column fractions.
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High Affinity Binding of A20 NF-Y to Q+ Sepharose Is
Dependent on the Mature B-cell YAF Fraction--
To test the concept
that the NF-Y complex in mature B-cells is associated with one, or
more, small molecular mass polypeptides, in comparison to the known YA
(~42 kDa), YB (~36 kDa), and YC (~40 kDa) subunits, a series of
dialysis experiments were performed using cellulose membranes of
defined molecular mass cut-off and under specific KCl concentration
conditions (Fig. 5). A20 nuclear extracts
were first dialyzed against BC-420 using a 10-12-kDa cut-off cellulose
membrane (panel A), a 25-kDa cut-off cellulose membrane
(panel B), and against BC-100 using a 25-kDa cut-off cellulose membrane (panel C), then extracts were dialyzed
further against BC-100 in each case. The A20 NF-Y complex was tested
for its ability to retain or lose binding affinity for Q+
Sepharose following dialysis under these experimental conditions. These
analyses suggested that, under conditions of elevated [KCl] and using
a 25-kDa molecular mass cut-off membrane, the activity associated with
A20 NF-Y binding to Q+ Sepharose dissociated from the NF-Y
complex and was lost during dialysis (panel B). Control
experiments showed that the [KCl] and dialysis membrane cut-off size
were critical in separating this activity away from the NF-Y complex.
In particular, lower KCl concentrations of 0.1 M were
insufficient in facilitating dissociation of this activity using a
25-kDa cut-off membrane (panel C). These results suggest
that the activity associated with A20 NF-Y Q+ binding has a
nominal molecular mass between 10 and 25 kDa, and can be physically
separated from the NF-Y A:B:C subunit complex using 0.42 M
KCl. This operationally defined activity is referred to as the YAF
fraction. Subunit recombination assays using the Q+
fractions in panel B and exogenous complementing YA and YB:C subunits demonstrate that the A20 YA and YB:C activities derived from
dialyzed A20 extracts elute in a manner identical to MPC11 NF-Y (Fig.
1B).2

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Fig. 5.
Analysis of the A20 NF-Y complex anion
exchange binding properties following dialysis. A20 nuclear
extracts were dialyzed against BC-420 using a 10-12-kDa cut-off
cellulose membrane (A), a 25-kDa cut-off cellulose membrane
(B), and against BC-100 using a 25-kDa cut-off cellulose
membrane (C). Extracts were dialyzed further against BC-100
in each case, and tested for NF-Y binding to Q+ Sepharose
as described in Fig. 1. Positions of the KCl step-gradient are shown
above A, and the position of the NF-Y complex is denoted to
the left of each panel.
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|
Ultrafiltration has been used previously as a method for preparing
active YAF fractions, and dialysis using a 25-kDa cut-off membrane
under these same conditions has been used as a method to prepare
nuclear extracts depleted of YAF activity (39). In Fig.
6, an A20 NF-YA:B:C fraction depleted of
YAF activity failed to bind to Q+ Sepharose as an intact
heterotrimeric complex (panel A). Recombination of the A20
YA:B:C fraction with the separated A20 YAF fraction, however, fully
restored the ability of the A20 YA:B:C complex to bind to
Q+ Sepharose (panel B). In addition, prior
treatment of the A20 YAF fraction with DNase I or micrococcal nuclease
had no effect on its ability to confer Q+ binding to A20
NF-YA:B:C.2 Treatment of the A20 YAF fraction with protein
depleting silica beads (StrataClean resin) resulted in loss of its
ability to restore the Q+ binding phenotype (panel
C). The MPC11 NF-Y subunits were shown previously to separate
following Q+ Sepharose analysis (Figs. 1 and 3), suggesting
that NF-Y in a class II
plasmacytoma B-cell line differs
in biochemical properties from the NF-Y complex in mature B-cell class
II+ lymphocytes. The ability to transfer the A20
YAF-dependent Q+ phenotype to a class
II
NF-Y complex was tested by recombining MPC11 NF-Y
depleted of MPC 11 YAF proteins with the isolated A20 YAF fraction, and
assaying for high affinity NF-Y Q+ binding (panel
D). The ability to bind to Q+ Sepharose was
successfully transferred to MPC11 NF-YA:B:C by the A20 YAF fraction,
and the MPC 11 NF-Y elution profile was identical to A20 NF-Y
recombined with the A20 YAF fraction (compare panels B and
D). These results further suggest that the A20 YAF fraction
contains an activity capable of conferring high affinity Q+
binding to NF-Y. In addition, the partially purified A20 YA subunit derived from A20 nuclear extracts has been recombined with a partially purified HeLa YB:C subunit fraction derived from this class
II
cell line, and assayed for Q+ binding.
Recombination of this A20 YA fraction with HeLa YB:C restores E
DNA
binding activity, but this NF-Y complex fails to bind to Q+
Sepharose as an intact complex.2 These results suggest that
the ability to bind to Q+ Sepharose is not contained in the
A20 YA fraction, and furthermore suggests that A20 YA itself is not the
activity responsible for high affinity Q+ binding in the
A20 NF-Y complex. The ability to separate this activity from A20 NF-Y,
and to successfully recombine the YAF fraction with the depleted A20
NF-YA:B:C complex, which itself fails to bind to Q+
columns, represents a major step in efforts to characterize the functional properties of the NF-Y-associated cofactors in
vitro. Transfer of A20 YAF activity to the MPC 11 NF-Y complex
suggests that loss of YAF functional activity in the NF-Y complex of
terminally differentiated B-cells may represent a critical step in NF-Y
modulation of MHC class II genes during B-cell development.

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Fig. 6.
Recombination of the A20 YAF fraction with
depleted A20 and MPC 11 NF-YA:B:C nuclear extract fractions
reconstitutes high affinity NF-Y complex binding to Q+
Sepharose. A20 nuclear extracts were depleted of YAF proteins and
tested for Q+ Sepharose binding (A). The A20
NF-YA:B:C fraction, and MPC11 NF-YA:B:C fraction derived from depleted
nuclear extracts were recombined with A20 YAFs, then tested for binding
to Q+ Sepharose (B and D,
respectively). An A20 YAF fraction was depleted of protein using
StrataClean resin, recombined with the A20 NF-YA:B:C fraction, and the
complex tested for Q+ Sepharose binding (C).
Positions of the KCl step gradient are shown above A and
C. Lane P, free 32P-E DNA probe;
lane L, column load material. Column elution and EMSA assay
conditions were as described in Fig. 1.
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Physical Interaction between NF-YA(DBD) and PC4--
The
transcriptional cofactor, PC4, has been identified as a component in
isolated YAF fractions using Western blot assays,2 and was
shown to interact in vitro with NF-Y through the highly conserved DBD element contained in the YA subunit using Far Western assays in a previous study (39). To test the possibility that PC4 was
the YAF protein that alone conferred high affinity Q+
binding to NF-Y, affinity-purified A20 NF-YA:B:C derived from YAF-depleted nuclear extracts was recombined with purified recombinant PC4 and assayed for Q+ binding (Fig.
7). PC4 was observed to specifically
confer to NF-YA:B:C the ability to bind to Q+ Sepharose as
an intact trimeric complex with elution properties that were identical
to the NF-Y complex derived from unfractionated mature B-cell nuclear
extracts (Fig. 1A) and to NF-YA:B:C recombined with the
mature B-cell YAF fraction (Fig. 6, B and D).
Recombinant PC4 was also observed to specifically stabilize the
interaction of affinity-purified NF-Y binding to several known
CCAAT-box elements (Fig. 8), suggesting
that PC4 may stabilize NF-YA interactions with the YB:C heterodimer and
as a result may stabilize overall NF-Y interactions with CCAAT-box DNA
motifs.

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Fig. 7.
Recombinant PC4 restores Q+
Sepharose binding properties to the NF-YA:B:C complex.
Affinity-purified NF-Y derived from A20-depleted nuclear extracts was
recombined with recombinant PC4 (A) or BSA (B)
and assayed for binding to Q+ Sepharose. Positions of the
KCl step gradient are shown above A. Column elution and EMSA
assay conditions were as described in Fig. 1. Lane L, column
load material; lanes 1-17, Q+ Sepharose column
fractions.
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Fig. 8.
NF-Y binding to CCAAT-box DNA elements is
stabilized by recombinant PC4 in vitro. EMSA reactions were
performed using the affinity-purified native A20 NF-Y complex,
recombinant PC4, and the E (A) and S-collagen
(B) CCAAT-box DNA oligonucleotide probes. In A
and B, all lanes, except lane 1, received ~5
µg of NF-Y protein fraction. Lane 1, 32P-probe
alone; lane 2, no PC4 or BSA; lane 3, ~25 ng of
PC4; lane 4, ~50 ng of PC4; lane 5, ~25 ng of
BSA; lane 6, ~50 ng of BSA.
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To define the region in PC4 responsible for interaction with NF-Y,
32P-YA(DBD) was used to test several PC4 deletion mutants,
and control GST fusion proteins, in a glutathione-agarose bead
pull-down assay (Fig. 9A).
YA(DBD) was observed to stably interact with full-length PC4
(lane 3) and with a PC4 mutant that lacks both the
N-terminal serine-rich phosphorylation region and the region
responsible for binding to double-stranded DNA (lane 7).
YA(DBD) did not interact with a C-terminal PC4 mutant that lacked the
extreme 37 amino acids (lane 4), a GST fusion protein that
contained these 37 amino acids (lane 5), or the general
transcription repressor, Dr1 (lane 6). These results suggest
YA(DBD) interacts with the C-terminal region in PC4, and maps the
interaction site in PC4 near amino acid 90. To determine if
phosphorylated PC4 retains the ability to specifically interact with
NF-Y, PC4 was 32P-labeled with CKII and HMK and tested in
the glutathione-agarose pull-down assay using the NF-Y complex, several
NF-YA mutants, and other control GST fusion proteins (Fig.
9B). 32P-PC4 (CKII), as phosphorylated by CKII,
was observed to stably interact with the NF-Y complex (lane
3), full-length YA (lane 4), YA(DBD) (lane
6), TFIIB (lane 8), and the YA(DBD) homolog from
S. cerevisiae, HAP2(DBD) (lane 9).
GST-YA (
DBD), which lacked the DBD element, failed to interact with
32P-PC4 (CKII) (lane 5), as did GST-Dr1
(lane 7). 32P-PC4 (HMK), as phosphorylated by
HMK, was observed to stably interact with YA(DBD) (lane 12),
and a YA(DBD) mutant that lacked 23 amino acids in the C-terminal DBD
region, YA(DBD
C23) (lane 14), but failed to interact with
YA(
DBD) that lacked the highly conserved DBD region (lane
13). GST-YA (DBD
C23) fails to support CCAAT-box DNA binding
activity when recombined with full-length NF-YB:C.2 These
results suggest that internally and externally phosphorylated forms of
PC4, as modified by CKII and HMK respectively, do not impair or block
interaction with YA(DBD) in vitro, and that the PC4
interaction site maps to the subunit interaction subdomain in YA(DBD),
which is responsible for interaction with the YB:C heterodimer (32,
34).

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Fig. 9.
NF-YA(DBD) interacts with PC4 in
solution. GST-NF-YA(DBD) was expressed from pYA 2TK(DBD),
purified, then cleaved from GST with thrombin. Purified YA(DBD) was
32P-labeled in vitro with HMK, incubated with
GST, GST-PC4, a series of GST-PC4 deletion mutants, and GST-Dr1 bound
to glutathione-agarose beads in A. Retained
32P-YA(DBD) was assayed using SDS-polyacrylamide gel
electrophoresis. Lane 1, glutathione-agarose beads alone;
lane 2, GST; lane 3, GST-PC4; lane 4,
GST- PC4( C37); lane 5, GST-PC4(C37); lane 6,
GST-Dr1; lane 7, GST-PC4( N64). Molecular mass markers are
denoted at the left in kilodaltons. GST-PC4 was expressed
from pPC4 2TK, purified, cleaved from GST with thrombin, and purified.
PC4 was 32P-labeled in vitro using CK II
(lanes 1-9) or HMK (lanes 10-14), then
incubated with GST and a variety of GST fusion proteins bound to
glutathione-agarose beads in B. Retained 32P-PC4
was assayed using SDS-polyacrylamide gel electrophoresis. Lanes
1 and 10, glutathione-agarose beads alone; lanes
2 and 11, GST; lane 3, GST-YA/His-YB/His-YC;
lane 4, GST-YA; lanes 5 and 13,
GST-YA( DBD); lanes 6 and 12, GST-YA(DBD);
lane 7, GST-Dr1; lane 8, GST-TFIIB; lane
9, GST-HAP2(DBD); lane 14, GST-YA(DBD C23).
Molecular mass markers are denoted at the left in
kilodaltons.
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A group of MHC class II
cell lines have been
established from several human BLS patients' B-cells, and from several
class II+ mature human Burkitt's B-cell lymphoma cell
lines by
-irradiation or chemical mutagenesis, and immunoselected
for the loss of MHC class II expression (6). These BLS class
II
B-cell lines have been placed into four genetic
complementation groups, where each group represents a unique defect in
a gene product involved in MHC class II gene transcription. The genetic defect in BLS group II, CIITA, is thought to function as a
tissue-specific class II gene transcriptional cofactor (40), and the
genetic defect in BLS group IV, RFX5, has been identified as the large subunit of the X-box DNA-binding factor, RFX (54). The NF-Y complex
derived from the BLS group II cell lines, RJ2.2.5 and RM3, were
compared with their parental B-cell line, Raji, and a BLS group III
cell line, 6.1.6, for high affinity binding to Q+ Sepharose
(Fig. 10). The NF-Y complex derived
from both BLS Group II cell lines, RJ2.2.5 (panel B) and RM3
(panel C), dissociated following anion exchange
chromatography into NF-Y subunits fractions in a manner identical to
MPC11 NF-Y (Fig. 1B), and as shown using complementing YA
and YB:C subunit fractions in EMSA.2 In contrast, NF-Y
derived from the class II
, and CIITA+ BLS
group III cell line, 6.1.6 (panel D), and the class
II+ mature B- cell, Raji (panel A), eluted from
Q+ Sepharose as an intact multimeric complex in a manner
identical to the mature B-cell line, A20 (Fig. 1A). These
results suggest that inactivation of the B-cell-specific class II gene
factor, CIITA, in BLS group II cell lines is associated with
dissociation of the NF-Y subunit structure following anion exchange
analysis, whereas expression of functional CIITA in both "normal"
MHC class II+ mature B-cell lines (e.g. A20,
Raji), and in a BLS group III cell line, 6.1.6, results in an NF-Y
subunit structure that elutes as an intact multimeric complex from
anion exchangers. These results further suggest that CIITA may directly
or indirectly modulate the activity of PC4, in effect regulating its
ability to stabilize NF-Y subunit interactions.

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Fig. 10.
Comparison of NF-Y Q+ Sepharose
binding properties in MHC class II human mutant bare
lymphocyte syndrome B-cell lines. NF-Y derived from B-cell lines
representing the bare lymphocyte syndrome complementation group II:
RJ2.2.5 (B), and RM3 (C), and complementation
group III: 6.1.6 (D), were compared with the RJ2.2.5 and RM3
parental class II+ B-cell line, Raji (A). Column
elution and EMSA assay conditions were as described in Fig. 1.
Positions of the KCl step gradient are shown above A and
C. Lane P, free 32P-E DNA probe;
lane L, column load material; lanes 1-17,
Q+ column fractions.
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 |
DISCUSSION |
Differential expression of MHC class II genes during B lymphocyte
development represents an attractive system for studying the
transcriptional mechanisms underlying tissue-specific gene activation
and repression. Human MHC class II genes are activated at the mature
B-cell stage; however, progression to the plasma B-cell stage following
stimulation with foreign antigens, various mitogens, and T-cell factors
results in uniform repression of all class II gene expression at the
transcriptional level (7, 8). A number of ubiquitous transcription
factors, together with the B-cell-specific cofactors, CIITA (6) and the
Oct coactivator from B-cells (56), are believed to play critical
regulatory roles in stage-specific class II gene transcription;
however, the mechanistic steps involved in creating an active class II initiation complex and possible functional interactions between these
general activators, B-cell-specific cofactors, and basic components of
the RNA polymerase II machinery remain poorly understood.
In this study, the biochemical properties of the multimeric NF-Y
complex have been investigated and compared in stage-specific B-cells
in an effort to identify structural components in the NF-Y complex that
may be involved in regulating NF-Y function during states of active and
repressed class II transcription, and may contribute to the formation
of a functional class II initiation complex. The main observations from
this study are as follows. 1) The NF-Y complex derived from MHC class
II+ B-cells binds to anion exchangers with high affinity as
an intact trimeric species, whereas NF-Y derived from MHC class
II
plasma B-cells and BLS group II cells dissociates into
discrete YA and YB:C subunit fractions. 2) The NF-Y complex in mature
B-cells is associated with a low molecular mass protein fraction
(YAFs), which accounts for the NF-Y Q+ binding phenotype
(i.e. transfer of the YAF fraction to a YAF-depleted mature
B-cell NF-Y complex, or a NF-YA:B:C complex derived from class
II
cells fully restores NF-Y Q+ binding. 3)
Recombinant PC4 restores high affinity NF-YA:B:C binding to
Q+ Sepharose, and significantly stabilizes native
NF-Y:CCAAT-box DNA interactions in vitro.
Anion exchange analysis of the NF-Y complex in a variety of human
and murine MHC class II+ mature B-cell lines have shown
NF-Y to elute as an intact trimeric complex (Figs. 1 and 2). Western
blot analyses of the A20 Q+ fractions shown in Fig. 1,
using affinity-purified
-YA and
-YB antibodies, also confirmed
that the YA and YB subunits coelute in the 0.6 M KCl
fraction.2 In contrast, the NF-Y complex derived from a
variety of murine class II
plasma B-cell lines, several
other class II
cell types (e.g. P388D1,
3T3-L1, TA1, C3H10T1/2), and shown previously in HeLa (26), and NIH 3T3
(27) cell lines, dissociates into discrete YA and YB:C subunit
fractions following Q+ analysis. These observations
together with NF-Y subunit recombination experiments (Fig. 2) suggest
NF-YA:B:C subunit interactions are stabilized in class II+
mature B-cells, in comparison to a variety of class II
cell types. To test these conclusions further, the anion exchange properties of NF-Y were examined in the murine monocyte/macrophage cell
line, P388D1, during IFN-
-mediated differentiation. In the absence
of IFN-
, NF-Y dissociated into YA and YB:C fractions, whereas NF-Y
eluted as the trimeric complex following IFN-
-mediated induction of
class II transcription.2 These comparisons suggest that the
anion exchange properties of NF-Y are significantly altered during
periods of active, inactive, or repressed MHC class II
transcription.
In a previous report, physical interaction between NF-Y and the
transcriptional coactivator, PC4, was demonstrated in vitro using far Western assays, and mapped to the highly conserved DBD element in the NF-YA subunit (39). Biochemical characterization of the
YAF fraction from a variety of cell types and these initial studies
raised the possibility that PC4 was associated with NF-Y in
stage-specific B-cells, and responsible for the NF-Y Q+
binding properties. Recombination of affinity-purified NF-YA:B:C with
recombinant PC4 resulted in conversion of NF-Y into a complex that
bound Q+ Sepharose in a manner indistinguishable from
mature B-cell NF-Y, and NF-Y reconstituted with the mature B-cell YAF
fraction (Fig. 7). These results suggest that PC4 is the YAF component,
which itself is necessary and sufficient for conferring the Q+ binding phenotype to the NF-YA:B:C complex. PC4 is known to be phosphorylated in a group of N-terminal serine residues, primarily by CKII in vivo, and this modification of has been shown to prevent its
interaction with the viral activator, VP16, and its ability to function
as a coactivator in vitro (48). The PC4 recombination
results suggest that the non-phosphorylated form of PC4 is capable of
conferring the high affinity Q+ binding phenotype to NF-Y,
and further suggest that PC4 may be differentially regulated by
phosphorylation/dephosphorylation in association with NF-Y.
To determine if phosphorylated forms of PC4 interact with NF-Y, PC4 was
phosphorylated both with CKII and HMK and used to probe a series of GST
fusion proteins in a solution pull-down assay (Fig. 9). Both
32P-PC4 modified forms bound to YA(DBD), and not to the
N-terminal activation domain of YA, suggesting that phosphorylation did
not impair this interaction, and that the general nature of this
association was different from previous studies suggesting an
interaction between the N-terminal region of PC4 and the activation
domains of VP16 (48), and GAL4-AH (57). Of particular note was the observation that 32P-PC4 interacted with the subunit
interaction subdomain of YA(DBD). This region in YA(DBD) is known to be
important for the interaction of YA with the YB:C heterodimer in
creation of a unique structure, which then recognizes the CCAAT-box
motif (32). Deletion of amino acids in the DNA-binding subdomain of
YA(DBD), which prevent interaction with CCAAT-box motifs, did not
impair interaction with PC4. In addition, recombinant PC4 was observed
to specifically stabilize NF-Y:CCAAT- box DNA interactions in
vitro (Fig. 8). These results further suggest that the interaction
of PC4 with YA(DBD) may play a significant role in stabilizing YA
interaction with YB:C, and the interaction with CCAAT-box binding sites
following subunit trimerization. Further studies will be aimed at
determining the step(s) at which PC4 functions in these processes, and
in more precisely defining the region and amino acids in YA(DBD) that
support this interaction.
To more accurately define the region in PC4 that interacts with
YA(DBD), a series of GST-PC4 mutants were tested in vitro (Fig. 9). The N-terminal CKII phosphorylation region in PC4 was clearly
dispensable for this interaction, while the C terminus near amino acid
90 appeared to be an important interaction site. Phosphorylation of PC4
by CKII has been suggested to induce a conformational change that
prevents its interaction with activation regions and nullifies its
positive coactivator functions (45). Interaction of PC4 through its
C-terminal region with the highly conserved YA(DBD) region suggests PC4
may function in a unique manner when bound to NF-Y, since this
interaction was mapped to the region known at present only to be
responsible for nonspecific binding to single-strand DNA (57, 58). In
association with NF-Y, PC4 may act both as a potent activator through
its N-terminal region, and as a repressor when phosphorylated by CKII
since both forms interact with YA(DBD). In addition, PC4 may regulate
NF-Y CCAAT-box DNA binding by stabilizing overall subunit interactions, which in turn increase the rate of NF-Y association with its
DNA-binding site. An interaction between 32P-PC4(CKII) and
TFIIB was also observed (Fig. 9); however, the functional significance
of this observation is not known at present. These results contrast
with previous studies which suggest non-phosphorylated human PC4
interacts with TFIIA, and not TFIIB, in vitro (59), and with
Far Western assays, which suggest non-phosphorylated SUB1, the yeast
homolog of PC4, interacts with TFIIB, and acts as a clearance factor
in vivo by promoting the release of TFIIB from TBP (60).
SUB1 has been shown to activate a upstream activating sequence reporter
~4-fold in yeast which contains a CCAAT-box DNA-binding site for
HAP2/3/4/5, the yeast homolog of NF-Y (60). These results suggest PC4
may also play a functional role in mediating NF-Y transactivation
potential in vivo, and warrant further investigation into
the functional relationships between NF-Y, PC4, TFIIA, TFIIB, TBP, and
their concerted mechanism of action within NF-Y CCAAT-box containing
promoters.
Characterization of MHC class II gene expression during the mature to
plasma B-cell transition, and in somatic plasma:mature B-cell hybrids,
has suggested that the shift from active to inactive class II
transcription involves expression of a dominant plasma B-cell repressor
(7, 8). Overexpression of CIITA in plasma cells overrides this
repression and restores class II transcription (43), suggesting that
the hypothesized plasma B-cell repressor may act not on class II
promoters directly, but possibly on the CIITA promoter itself to
extinguish CIITA expression. These comparative analyses between
stage-specific B-cells demonstrate that CIITA plays a dominant role in
class II transcription; however, the relationships between CIITA
expression, class II promoter structure, and additional coactivator
proteins in relation to tissue-specific transcriptional initiation
remain unclear. CIITA functions as a critical nodal point in class II
gene activation, and transmission of its signal either directly, or
through downstream effector proteins, could result in specific
alterations in the structure and activity of the known class II
transcription factors during B-cell development. Several BLS group II
cell lines which possess defective CIITA genes, RJ2.2.5 and RM3, and
are class II
, as well all other class II
non-B-cell tested here, exhibit the class II
NF-Y
Q+ phenotype. In contrast, the parental cell line, Raji,
and all other mature B-cell lines that express CIITA, exhibit the class II+ NF-Y Q+ phenotype. The CIITA+
BLS group III cell line, 6.1.6, also exhibits the mature B-cell NF-Y
Q+ phenotype despite absence of class II transcription.
Collectively these comparative analyses from a diverse set of cell
types and cellular states provide support for the suggestion that PC4
activation is linked to CIITA expression. Transmission of the
biochemical signal initiated by CIITA may lead to conversion of PC4
from the phosphorylated to non-phosphorylated form, and stabilization
of NF-Y subunit interactions. The accumulated evidence presented in
this study supports a model of NF-Y structure based on a unique association with the abundant, ubiquitously expressed cofactor, PC4. In
this context, PC4 may be involved in mediating general NF-Y
transcription factor functions in class II
cells, and
undergo post-translational modification both during B-cell development
and during IFN-
-induced class II gene activation, which are critical
to NF-Y function in MHC class II gene transcription. Terminal
differentiation into the plasma B-cell stage may signal specific PC4
phosphorylation events, and be coupled to the overall process of
extinguishing class II transcription. Further in vitro analyses of the biochemical nature of the NF-Y complex in
stage-specific B lymphocytes and its in vivo function will
aid elucidation of the molecular mechanisms linking NF-Y function with
PC4 activity, and may provide insight into the signals initiated by
CIITA which regulate tissue-specific MHC class II gene
transcription.
I thank Drs. J. Durkik, J. Jones, B. Mach, M. Peterlin, and D. Pious for providing cell lines; H. Ge and R. Roeder,
D. McNabb and L. Guarente, I. Verma, D. Reinberg, and their colleagues
for providing various plasmids; and Poornima Bhat-Nakshatri and Raichal Melathe for technical assistance.