Neither Hmg-14a nor Hmg-17 Gene Function Is Required for Growth of Chicken Dt40 Cells or Maintenance of Dnasei-hypersensitive Sites

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)(2)(3)(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)(11)(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.

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 β-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 HindIII-BamHI insert from pHM1.9HB was subcloned into the HindIII and BamHI sites of the 3.9 kb pCRII vector (Invitrogen).The resulting construct was digested with XhoI and XbaI and ligated to the 1.3 kb XhoI-XbaI insert from pHM1.5HP, yielding the plasmid pT14a.The puro gene cassette was cloned into pT14a using the BamHI and XhoI sites, and the bsr cassette was cloned into pT14a using the EcoRI and XhoI 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 NsiI or NsiI plus SpeI digestion before electroporation.The HMG-17 gene constructs, pBSH17neo and pBSH17his, have been described (13).

Electroporation and screening
The DT40 cell line (15) was obtained from Craig Thompson, University of Chicago, Chicago, IL.Cell culture conditions, cell cycle analysis, and electroporation were performed as described previously (13).Selection was by one of the following drugs: blasticidin S (ICN, 30 µg per ml), puromycin (Sigma, 0.5 µg per ml), G418 (Gibco-BRL, 2 mg per ml), and L-histidinol (Sigma, 1.5 mg per ml).Resistant colonies were expanded in drug-free media.DNA extraction and Southern blotting analysis were as described previously (13).The diagnostic probes used were a 1.4 kb BamHI-HindIII fragment (5′ to the 1.9 kb fragment in pHM1.9HB,Fig. 1) and a 1.8 kb HindIII-AccI fragment (3′ to the HMG-14a gene, Fig. 1).Colonies whose DNA yielded the expected bands were further screened by hybridization with selectable marker gene plasmids and the 1.8 kb HindIII-BamHI fragment deleted in making the HMG-14a targeting constructs.RNA isolation, RNA blotting, and RNA hybridization probes were as described previously (13).

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 SalI and XhoI or ClaI, gel electrophoresis, blotting and hybridization to either a 295 bp PCR-amplified fragment from the constant region of the chicken λ light-chain gene (13) or a SalI-ClaI fragment excised from a subcloned 1.8 kb SalI-EcoRI 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 µg 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).

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 2A (lane 6) and Figure 2B (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 HindIII-BamHI 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. 2A 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. 2C 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. 2C 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. 3A).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. 3B), 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. 3B 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.

Lack of null-mutation effects on cellular phenotype
All four quadruply disrupted cell lines divided normally in standard growth media, without any observable difference in phenotype from DT40.No change in morphology was found among any of these cell lines when they were examined by phase contrast microscopy.Growth rates of the mutant lines and DT40 were determined following plating each line at 10 3 cells/ml in 24-well plates (Fig. 5).It is clear that the quadruply disrupted cells grow equally as well as the DT40 parental cell line.There was also no significant difference in growth and/or survival of these lines in reduced fetal calf serum (2, 4, 6, and 8%, as opposed to 10% in Fig. 5), nor in the percentage of cells in G1, S and G2 phases of the cell cycle when examined by flow cytometry (not shown).
Normal cell proliferation and differentiation have been suggested to require the regulated expression of HMG-14 and HMG-17.Their expression is down-regulated during differentiation of osteoblasts and promyelocytic leukemia cells (24), and over-expression of HMG-14 inhibits differentiation of mouse myoblast cells in vitro (22).To assess whether the inactivation of the HMG-17 and HMG-14a genes affected the existing level of differentiation of the parental DT40 cells, we examined the targeted cell lines with Giemsa staining, light-scattering profile analysis, and IgM fluorescence staining.Giemsa staining and light-scattering analysis with flow cytometry detect the complexity of the cytoplasmic structure.A more complex cytoplasmic structure would be expected if DT40 cells proceeded toward the later stages of their normal differentiation, which would be to become mature B cells and then plasma cells.Both Giemsa and light-scattering analysis did not detect any significant difference between mutant cell lines and the DT40 parental cells.Surface IgM is a differentiation marker of B-cells, present on immature and mature B-cells but not on plasma cells.When DT40 cells and the quadruply disrupted cells (Bp5, Bp39, Nh43, and Nh52) were examined for surface IgM expression by flow cytometry, no difference was detected (results not shown).Similar analysis of total λ light chain expression using an anti-chicken light chain antibody detected no difference in the profiles of DT40 and the quadruply disrupted mutant cells, indirectly suggesting that IgD expression was also unchanged.
To further investigate the potential regulatory role of HMG-14 and HMG-17 proteins on cell growth and function, we examined the protein profiles of mutant lines and the parental DT40 line by 2-D gel electrophoresis.Takami et al. (25) and Seguchi et al. (26) noted a few changes in 2-D gel protein profile due to disruption of the chicken histone H2B-V and 01H1 genes, respectively.The 2-D gel protein profiles of all the quadruply disrupted cell lines and DT40 are very similar (results not shown), suggesting that HMG-14a and HMG-17 are probably not involved in the regulation of the expression of predominant cell proteins in DT40.Careful examination of the gels revealed three spots that were consistently different between quadruply disrupted lines and DT40.One of these spots appears to be due to the expression of the histidinol dehydrogenase selectable marker gene since it is present in all his1-transfected cells but not in the other cell lines, regardless of their HMG genotype, and it is about 50 kDa in size, in agreement with that predicted.In addition, two small proteins (∼10 kDa each) showed some decrease in expression in the quadruply disrupted cells with respect to DT40.The identity and function, if any, of these proteins remains unclear, but the effect of the disruptions is only a limited one.

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)(29)(30)(31)(32).As an immature B-cell, DT40 expresses large amounts of immunoglobulin light-chain proteins from the rearranged copy of its λ 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 λ 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 λ 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.

Figure 1 .
Figure 1.Schematic diagram of the targeting vectors and endogenous and disrupted HMG-14a alleles.Solid boxes represent the exons of the HMG-14 gene.Hatched boxes represent the homologous sequences present in the targeting vectors.Shaded boxes represent the cassettes for drug-selectable markers.Open boxes represent the pCRII vector.E, B, H, X, P, N, and S represent the cleavage sites for EcoRI, BamHI, HindIII, XhoI, PstI, NsiI, and SpeI, respectively.(A) Diagram of pT14absr.(B) Diagram of pT14apuro.(C) Map of the endogenous HMG-14a gene in DT40.Black bars below the map indicate the location of the three hybridization probes for the 5′ flanking region, replaced sequences, and 3′ flanking region, respectively, from left to right.(D) Map of the HMG-14a disrupted mutant gene generated by homologous recombination with pT14apuro.

Figure 2 .
Figure 2. Southern blotting analysis of representative cell lines.Genomic DNAs from the indicated cell lines were extracted, digested with EcoRI (A and C) or BamHI (B and C), fractionated on agarose gels, blotted, and hybridized to 32 P-labeled 5′-flanking (A) or 3′-flanking (B) HMG-14a probes (Fig. 1) or to the 0.2 kb (C and D) HMG-17 diagnostic probe (13).The molecular sizes in kb are indicated.The notations 8.8 kb (A), 6.2 kb (B), 11.2 kb (C), and 5.7 kb (D) indicate endogenous bands found in the DT40 cell line, 4.35 kb (A) and 5.15 kb (B) indicate those expected after homologous recombination mediated by pT14apuro, 3.3 kb (A) and 5.5 kb (B) are those expected after homologous recombination mediated by pT14absr, 12.9 kb (C) and 7.2 kb (D) indicate those expected after homologous recombination mediated by pBSH17neo (15), and 7.3 kb (C) and 8.2 kb (D) indicate those expected after homologous recombination mediated by pBSH17his (13).

Figure 3 .
Figure 3. Northern blotting analysis of RNA from representative cell lines.(A) Total RNA (15 µg per lane) extracted from the indicated cells lines was fractionated on a 1.2% agarose-formaldehyde gel, blotted to a membrane, and probed with 32 P-labelled HMG-14a cDNA.The membrane was then stripped and reprobed sequentially with HMG-17 (B), HMG-14b (C), and GAPDH (D) hybridization probes (13).

Figure 4 .
Figure 4 .Western blotting analysis of representative cell lines.Total cell lysates (74 µg per lane) were separated by SDS-PAGE (15% polyacrylamide) and blotted to a polyvinylidene difluoride (Gelman) membrane.The membrane was probed with antibody against the DNA-binding domain of HMG-14/17 (23).Enzyme chemiluminescence (DuPont-NEN) was used to locate the bands after incubation in horseradish peroxidase conjugate(13).Cell lines tested are as designated at the top of each lane, and the locations of the HMG proteins are indicated by arrows on the left.

Figure 5 .
Figure 5. Growth curves of representative cell lines.Cell lines as indicated in the insert were plated out as 10 3 cells per ml and counted at 12 h intervals.Triplicate samples were counted at each time point, and the standard deviation values were used to plot the error bars.

Figure 6 .
Figure 6.DNase I-sensitivity analysis of representative cell lines.Nuclei from the indicated cell lines were prepared and incubated in the presence of DNase I at the indicated concentrations for 20 min at room temperature.The partially digested DNAs were extracted and digested with SalI and XhoI, gel electrophoresed, and blotted as described previously(13).The blots were hybridized to a 295 bp fragment amplified from the constant region of the chicken λ light-chain gene(13).Triangles and arrows at right designate parental and hypersensitive bands, respectively.

Table 1 .
Outline of gene disruption strategies a Genotype of the cell line at the HMG17 and HMG14a loci with + indicating wild type and -indicating null mutant.bTheplasmid construct used to generate the desired homologous recombinant (see Materials and Methods).