Neither
HMG-14a
nor
HMG-17
gene function is required for growth of chicken DT40 cells or maintenance of
DNaseI-hypersensitive sites
Neither HMG-14a nor HMG-17 gene function is required for growth of chicken DT40 cells or maintenance of DNaseI-hypersensitive sites
Yi
Li
1
,
John R.
Strahler
2,3
and
Jerry B.
Dodgson
1,2,
*
1
Department of Microbiology,
2
Department of Biochemistry and
3
MSU-NIH Mass Spectrometry Facility, Michigan State University,
East Lansing
, MI 48824,
USA
Received November 1, 1996;
Accepted November 18, 1996
ABSTRACT
HMG-14 and HMG-17 form a family of ubiquitous non-histone chromosomal proteins and have been reported to bind
preferentially to regions of active chromatin structure. Our previous studies
demonstrated that the chicken
HMG-17
gene is dispensable for normal growth of the DT40 chicken lymphoid cell line.
Here it is shown that the major chicken
HMG-14
gene,
HMG-14a
, is also dispensable and, moreover, that DT40-derived cells lacking both HMG-17 and HMG-14a
proteins show no obvious change in phenotype with respect to the parental DT40
cells. Furthermore, no compensatory changes in HMG-14b or histone protein levels were observed in cells lacking both HMG-14a and HMG-17, nor were any alterations detected in such hallmarks of
chromatin structure as DNaseI-hypersensitive sites or micrococcal nuclease digestion patterns. It is
concluded that the HMG-14a and HMG-17 proteins are not required for normal growth of avian cell lines
in vitro
, nor for the maintenance of DNaseI-hypersensitive sites in chromatin.
INTRODUCTION
The high mobility group 17 (HMG-17) and 14 (HMG-14) non-histone chromosomal proteins are found in all higher
eucaryotic species. They bind to nucleosomes in chromatin (
1
-
4
), and this has been shown to stabilize core particles (
5
). The limited amounts of these proteins confines them to only a subset of
nucleosomes (
6
), and a number of experiments including nuclease digestion (
7
), protein-DNA cross-linking (
8
), and immunofractionation (
9
) have suggested that HMG-14 and HMG-17 are preferentially associated with nucleosomes in regions of
transcribed chromatin. HMG-17 and HMG-14 are expressed in virtually all avian and mammalian cell types
that have been examined. Unlike mammals, however, chickens express two rather
different types of HMG-14, HMG-14a and HMG-14b (
6
), with HMG-14a being the dominant form. HMG-17 and HMG-14 proteins appear to compete for the same binding sites
in vitro
(
4
), although the homology between the two proteins is <50% (
6
,
10
-
12
).
The abundance, wide distribution, and evolutionary stability of HMG-14 and HMG-17 suggest that these proteins play an important role in chromatin,
but their exact functions remain unknown. Previously, we reported that targeted
disruption of the
HMG-17
gene in the chicken cell line DT40 does not affect cell growth, and that no
major changes in phenotype and chromatin structure occurred in such cells (
13
). A possible explanation for this result could be that HMG-17 and HMG-14 are functionally redundant, although no increase in HMG-14 expression occurred in the cells lacking HMG-17. To test this possibility, both copies of the
HMG-14a
gene have been inactivated in both the DT40 cell line and in D108-1, which is a homozygous
HMG-17
null mutant. It is shown that cells lacking
HMG-14a
and cells lacking both
HMG-14a
and
HMG-17
gene function show no obvious phenotypic differences from the parental DT40
line.
MATERIALS AND METHODS
Plasmid constructs
Cassettes containing puromycin acetyltransferase (
puro
) and blasticidin S acetyltransferase (
bsr
) genes were kindly provided by Jean-Marie Buerstedde, Basel Institute of Immunology, Basel, Switzerland. Both
genes are under the control of the chicken [beta]-actin promoter and terminate in a SV40 poly(A) signal. pHM1.9HB and
pHM1.5HP are genomic clones from the
HMG-14a
locus (
14
). The 1.9 kb
Hin
dIII-
Bam
HI insert from pHM1.9HB was subcloned into the
Hin
dIII and
Bam
HI sites of the 3.9 kb pCRII vector (Invitrogen). The resulting construct was
digested with
Xho
I and
Xba
I and ligated to the 1.3 kb
Xho
I-
Xba
I insert from pHM1.5HP, yielding the plasmid pT14a. The
puro
gene cassette was cloned into pT14a using the
Bam
HI and
Xho
I sites, and the
bsr
cassette was cloned into pT14a using the
Eco
RI and
Xho
I sites. The resulting constructs, each containing 1.9 kb of the
HMG-14a
gene on the left and 1.4 kb on the right, were designated pT14apuro and
pT14absr, respectively (Fig.
1
). Targeting vectors were linearized by
Nsi
I or
Nsi
I plus
Spe
I digestion before electroporation. The
HMG-17
gene constructs, pBSH17neo and pBSH17his, have been described (
13
).
Electroporation and screening
Analysis of nucleosomes, nucleosomal proteins, and total cellular protein
Micrococcal nuclease digestion of isolated nuclei and gel electrophoresis on a
3.5% polyacrylamide-0.5% agarose gel containing 30% glycerol were performed as described
previously (
16
). Total histones and HMGs were extracted and analyzed by polyacrylamide gel
electrophoresis (PAGE) as described (
17
,
18
). Nuclei were isolated and digested with DNase I (
19
), followed by DNA isolation and digestion with either
Sal
I and
Xho
I or
Cla
I, gel electrophoresis, blotting and hybridization to either a 295 bp PCR-amplified fragment from the constant region of the chicken [lambda] light-chain gene (
13
) or a
Sal
I-
Cla
I fragment excised from a subcloned 1.8 kb
Sal
I-
Eco
RI fragment of pc-myc (
20
). For analysis of total cellular proteins, cells were washed with PBS and lysed
in 50 mM Tris-HCl, pH 6.8, 2% sodium dodecylsulfate (SDS), 5% 2-mercaptoethanol, 10% glycerol, and 0.01% bromophenol blue by
boiling for 5 min. A total of 74 [mu]g of protein per lane was analyzed by SDS-PAGE (15% acrylamide/0.1% SDS) followed by Western immunoblotting as
described previously (
13
). Two-dimensional protein gel electrophoresis was performed essentially as
described (
21
).
RESULTS AND DISCUSSION
Disruption of the
HMG-14a
gene
The general scheme used to prepare cell lines lacking functional
HMG-14a
genes involved disruption of one copy of this gene in DT40 cells by homologous
recombination with a
puro
-containing
HMG-14a
construct, followed by disruption of the second allele with a
bsr
-containing
HMG-14a
construct (Table
1
). The
puro
targeting vector, pT14apuro (Fig.
1
), was electroporated into DT40 cells to generate 34 resistant colonies. The
Southern hybridization patterns expected from integration by homologous
recombination at the
HMG-14a
gene for probes both 5' and 3' to the gene (Fig.
1
) are demonstrated in Figure
2
A (lane 6) and Figure
2
B (lane 6), respectively. By this assay, two colonies were determined to have
integration via homologous recombination, for a frequency of 11%. One colony,
DTpuro8, was chosen for a second round of transfection by the pT14absr
construct (Table
1
). A total of 38 blasticidin S-resistant colonies were obtained. The diagnostic pattern for homologous
integration is again shown in Figure
2
(lane 7 in A and B). By this assay, one colony, designated 8/bsr8, was
determined to have both copies disrupted by homologous recombination. Southern
hybridization of 8/bsr8 DNA with selectable marker sequences or the 1.8 kb
Hin
dIII-
Bam
HI genomic fragment deleted in making the targeting constructs gave the pattern
expected of a double disruption of the
HMG-14a
gene (not shown).
Isolation of cell lines lacking both
HMG-17
and
HMG-14a
genes
Two schemes were employed for the isolation of cell lines lacking both
HMG-17
and
HMG-14a
as illustrated in Table
1
. First, the D108-1 cell line which already lacks any functional
HMG-17
gene (
13
) was transfected with pT14absr to generate the triply disrupted cell line,
Bsr18, which was then transfected by pT14apuro to inactivate the second
HMG-14a
allele. Two colonies (Bp5 and Bp39) were determined to have both copies of
HMG-14a
disrupted as shown by Southern blotting analysis (Fig.
2
A and B). The converse strategy of disrupting the
HMG-17
gene in the
HMG-14a
null mutant cell line, 8/bsr8, was also successful (Table
1
). The
neo
targeting vector pBSH17neo (
13
) was used to disrupt one copy of
HMG-17
in 8/bsr8. Using the same assay for homologous recombination at the
HMG-17
gene as employed previously (
13
), 23 of the 44 resistant colonies were determined to have targeted integration
via homologous recombination for a frequency of 52%. One colony, 14N11 (lanes
3, Fig.
2
C and D), was chosen for a second round of transfection by the pBSH17his
construct (
13
). Of 14 L-histidinol-resistant clones, 11 colonies were determined to have both copies
disrupted by homologous recombination, yielding a targeting efficiency of 79%.
Two clones, Nh43 and Nh52 (lanes 1 and 2, Fig.
2
C and D), were chosen for further studies. The high frequencies of integration
by homologous recombination obtained in generating Nh43 and Nh52 argue strongly
against the possibility that cell lines were selected with mutations in other
genes that compensated for the loss of both HMG-14a and HMG-17 proteins.
Null mutants do not express
HMG-14a
RNA
Both
HMG-14a
targeting vectors were designed to replace four of the six
HMG-14a
exons with drug-selectable markers. Therefore, the
HMG-14a
doubly disrupted cell line 8/bsr8 and the quadruply disrupted
HMG-14a/HMG-17
mutant cell lines were not expected to make any
HMG-14a
message. Indeed, there was no transcript detected when total RNAs of these
cells were analyzed by northern hybridization (Fig.
3
A). As reported previously, disruption of both copies of the
HMG-17
gene resulted in low levels of
HMG-17
-hybridizing RNAs with a mobility marginally faster than that of wild type
(Fig.
3
B), which, however, do not encode the HMG-17 protein (
13
).
Quantitative analysis of the northern blots demonstrates that the heterozygous
null mutants DTpuro8 and Bsr18 make ~50% of the level of
HMG-14a
RNA observed in the parental DT40 cells and D108-1 (Fig.
3
, lanes 1, 2, and 5), suggesting that
HMG-14a
mRNA levels are proportional to gene copy number, as we have reported
previously for the
HMG-17
gene (
13
). This is also in agreement with the results of Pash
et al
. (
22
), who found that expression of human
HMG-14
mRNA in mouse cells did not affect the level of the endogenous mouse
HMG-14
message. Furthermore, the expression levels of any remaining chicken
HMG-17
and
HMG-14b
genes in both the
HMG-14a
doubly disrupted 8/bsr8 cells and the quadruply disrupted Bp5, Bp39, Nh43, and
Nh52 cells were not markedly altered (Fig.
3
B and C), indicating that the
HMG-14
and
HMG-17
genes are not coordinately regulated at the transcriptional level.
Quadruple disruptions do not make detectable HMG-14a or HMG-17 protein
HMG proteins extracted from cells were analyzed by Western immunoblotting using
antibodies elicited against a peptide that is common to both HMG-14 and HMG-17 (
11
,
23
). HMG-14a protein was not detected in the
HMG-14a
doubly disrupted cell line 8/bsr8 (Fig.
4
, lane 7) or in quadruply disrupted cells (Fig.
4
, lanes 1, 2, 9, 10). Because the epitope recognized by this antibody is in the
most conserved nucleosome-binding domain, common to both HMG-17 and HMG-14, any truncated form of HMG-14a that might retain normal function should be
detected, if present.
HMG-14a
gene expression was approximately linear with the functional gene copy number
(Fig.
4
, lanes 6 and 7) in accordance with our previous observations of the
HMG-17
gene (
13
). This gene copy number-protein level relationship is not affected by the presence or absence of
a functional
HMG-17
gene (Fig.
4
, lanes 3-6). HMG-17 expression is also unaffected by the presence or absence of HMG-14a (Fig.
4
, lanes 5-8), and the total absence of both
HMG-14a
and
HMG-17
did not affect the cellular level of the HMG-14b protein (Fig.
4
, lanes 1-10). Thus, expression of all three members of the HMG-14/HMG-17 family is not coordinately regulated.
a
Genotype of the cell line at the
HMG17
and
HMG14a
loci with + indicating wild type and - indicating null mutant.
b
The plasmid construct used to generate the desired homologous recombinant (see
Materials and Methods).
Lack of null-mutation effects on cellular phenotype
Analysis of bulk chromatin in mutant cell lines
The results discussed above (Figs
3
and
4
) indicated that no change in HMG-14b levels occurred in response to inactivation of both the
HMG-17
and
HMG-14a
genes. We also were unable to discern any alteration in the pattern of total
histone protein isoforms present in bulk chromatin of the quadruply disrupted
cells versus DT40, as detected by electrophoresis on a 15% Triton-acid-urea gel (not shown). This is not surprising, since only about 10%
of the nucleosomes would be expected to bind HMG-14 and HMG-17 proteins in normal cells. We also examined the structure of
mutant cell line chromatin at the level of individual nucleosomes. When
chromatin is digested by micrococcal nuclease, mononucleosomes of different
sizes are released depending upon the number of HMG-17 and -14 and histone H1 molecules bound. These different forms of
nucleoprotein complexes can be partially resolved in a polyacrylamide-agarose gel (
16
). We previously reported that in the region of the mammalian MII band, two
bands are discerned in DT40 cells, and the intensities of both bands
(especially the faster-migrating one) are decreased in
HMG-17
null mutants (
13
). When
HMG-14a
doubly disrupted 8/bsr8 and quadruply disrupted Bp5 and Nh43 cell chromatin was
examined, the lower MII band was absent in both Bp5 and Nh43 (not shown). Since
the MII particle normally contains one molecule of HMG-17 or HMG-14, it appears that the low levels of the remaining HMG-14b are insufficient to form detectable MII-type nucleosomes in quadruply disrupted cells.
Lack of null-mutation effects on DNaseI-hypersensitive sites
It has been reported that the increased sensitivity of active chromatin domains
to DNase I correlates with the presence of HMG-14 and/or HMG-17 (
7
,
27
). However, whether HMG-17 and HMG-14 are directly responsible for the increased DNase I-sensitivity is controversial (
28
-
32
). As an immature B-cell, DT40 expresses large amounts of immunoglobulin light-chain proteins from the rearranged copy of its [lambda] light-chain gene. It has been shown that DNase I-hypersensitivity exists in both the rearranged
and the germline loci with some sites specific for each type of locus (
33
). We have previously shown that the disruption of
HMG-17
in DT40 does not affect the preferential DNase I-sensitivity and the hypersensitive sites (
13
) in both the rearranged and the germline loci. To determine whether the
complete absence of both
HMG-14a
and
HMG-17
affects hypersensitive sites, nuclei from Nh43, 8/bsr8, and DT40 were digested
with limited amounts of DNase I. As shown in Figure
6
, the 7 kb and 5 kb [lambda] light-chain gene bands were about equally sensitive in all cell lines
tested, and no differences in number and intensity were seen for the smaller
bands produced due to cleavage at hypersensitive sites. This clearly suggests
that HMG-14a and HMG-17 are not required for the DNase I sensitivity and the
hypersensitive sites exhibited at the [lambda] light-chain locus. The c-
myc
locus was also examined for DNase I hypersensitivity (not shown), and again the
quadruply disrupted cells lines were just as sensitive to nuclease as the
HMG-14a
doubly disrupted cell line (8/bsr8) or the parental DT40 cells. Since HMG-14b exists in very limited amounts in these cells (Fig.
4
), it seems unlikely that it could be sufficient to induce the DNase I
sensitivity observed. Our results are consistent with a number of reports
suggesting that the DNase I hypersensitivity of active chromatin is not the
result of HMG proteins but rather is due to an altered higher order chromatin
configuration (
28
,
29
,
32
). However, our data do not argue against the evidence that HMG-17 and HMG-14 associate preferentially with active genes, and it remains
possible that these proteins are involved in the initial establishment of DNase
I-sensitive chromatin structures but are not required for their maintenance
during the generation of the mutant cell lines and their subsequent growth in
culture.
Cellular function of HMG-17 and HMG-14
In vitro
assays on isolated chromatin and more recent studies involving the over-expression of exogenous HMG-14 in cultured cells have suggested that these HMGs may act to
modulate gene expression and thereby influence cell proliferation and
differentiation through their regulated expression or activity. Our results
demonstrate that HMG-14a and HMG-17 are not required for cell growth in culture and, furthermore,
that no major phenotypic changes result from the complete absence of these
proteins in DT40 cells. One possible explanation for this is that the remaining
member of this family, HMG-14b, is functionally redundant with HMG-14a and HMG-17. We view this as unlikely since no compensatory increase in
HMG-14b protein expression was detected in quadruply disrupted lines, and HMG-14b is present at only ~10% of the amount of either HMG-14a or HMG-17 (Fig.
4
). The most straightforward interpretation of our results is that HMG-14a and HMG-17 are not required for the normal growth of, at least, chicken
lymphoid cells in culture. It is certainly possible that HMG-14a and HMG-17 are required for the growth and/or differentiation of other cell
types, even though they appear to be expressed ubiquitously in all cells. It is
also possible that the dispensable character of these proteins is peculiar to a
transformed cell line such as DT40. Perhaps the simplest interpretation of
these results is that HMG-14a and HMG-17 are not required for the growth or gross phenotypic properties of
any individual cell type, but rather function in some unknown way(s) in the
development of the animal. It is also possible that their function has only a
small effect in any individual cell, one which is significant enough to be
evolutionarily selected but which is negligible when examined in short term
culture in growth media.
ACKNOWLEDGMENTS
We thank Michael Bustin for the generous gift of the anti-HMG sera used, Jean-Marie Buerstedde for the histidinol, blasticidin S, and puromycin
resistant gene cassettes, Craig Thompson for the DT40 cell line, and Kathleen
Conklin for the c-
myc
probe used. We also thank Henry Hunt, Donald Salter and Louis King for advice
and technical assistance. This work was supported by grant GM41394 from the
National Institutes of Health and by USDA Grant 91-37204-6730.
