(Received for publication, May 10, 1995; and in revised form, June 12, 1995)
From the
We compared the nucleosomal organization, histone H1 subtypes,
and histone H1 phosphorylated isoforms of ras-transformed and
parental 10T mouse fibroblasts. In agreement with previous studies, we
found that ras-transformed mouse fibroblasts have a less
condensed chromatin structure than normal fibroblasts. ras-transformed and parental 10T cells had similar amounts of
H1 subtypes, proteins that have a key role in the compaction of
chromatin. However, labeling studies with P and Western
blot experiments with an antiphosphorylated H1 antibody show that
interphase ras-transformed cells have higher levels of
phosphorylated H1 isoforms than parental cells. G
/S
phase-arrested ras-transformed cells had higher amounts of
phosphorylated H1 than G
/S phase-arrested parental cells.
Mouse fibroblasts transformed with fes, mos, raf, myc, or constitutively active mitogen-activated
protein (MAP) kinase kinase had increased levels of phosphorylated H1.
These observations suggest that increased phosphorylation of H1 is one
of the consequences of the persistent activation of the
mitogen-activated protein kinase signal transduction pathway. Indirect
immunofluorescent studies show that phosphorylated H1b is localized in
centers of RNA splicing in the nucleus, suggesting that this modified
H1 subtype is complexed to transcriptionally active chromatin.
Due to their role in chromatin compaction, H1 histones are considered as general repressors of transcription (Weintraub, 1984). However, H1 histones are associated with transcriptionally active chromatin (Ericsson et al., 1990; Kamakaka and Thomas, 1990). Both nucleosomes and H1 are present in the coding regions of active genes, but both are absent in the promoter (Nacheva et al., 1989; Postnikov et al., 1991; Belikov et al., 1993). It is currently thought that the H1 interacts differently with active and repressed chromatin (Garrard, 1991; van Holde et al., 1992).
The H1 histones are a heterogeneous group of several subtypes
that differ in amino acid sequence (Cole, 1987; Lennox and Cohen,
1988a; Parseghian et al., 1994a). The relative amounts of H1
subtypes vary with cell type within a particular species, as well as
among various species. For example, mouse tissues contain various
levels of H1 subtypes, H1a, H1b, H1c, H1d, H1e, and H1 (Lennox and Cohen, 1983). Since H1 subtypes differ in their
abilities to condense DNA and chromatin fragments, it has been proposed
that the differential distribution of the H1 subtypes with chromatin
domains may generate chromatin regions with different degrees of
compaction (Cole, 1987; Lennox and Cohen, 1988a). Indirect
immunofluorescence studies with H1 subtype-specific antibodies have
shown that the nuclear location of specific H1 subtypes is non-uniform.
Rodent histone H1
colocalized with nucleoli, human H1-3 is
found primarily in the nuclear periphery, and human H1-1 is
distributed in parallel to the DNA concentration (Gorka et
al., 1993; Breneman et al., 1993; Parseghian et
al., 1993; Parseghian et al., 1994b). Antibodies to human
H1-2 and H1-4 generated a punctate staining pattern, reminiscent of the
speckled staining patterns described when the nuclear sites of splicing
factors, small nuclear RNAs, and RNA synthesis were localized
(Parseghian et al., 1994b; Huang and Spector, 1992; Jackson et al., 1993; Lu et al., 1994).
Further
heterogeneity in the H1 population arises from post-translational
modification. The H1 histones are phosphorylated at serine and
threonine residues located in the N- and C-terminal domains of the
protein (van Holde, 1988). Immunochemical and biochemical data show
that H1 phosphorylation increases dramatically as cells progress
through the cell cycle (Lu et al., 1994; Hohmann, 1983).
Phosphorylation of H1 begins in G, continues at an
increasing rate and extent throughout S and G
, and reaches
a maximum in mitosis. The H1 subtypes, however, differ in their extent
of phosphorylation and the scheduling of some of their phosphorylation
during the cell cycle (Hohmann, 1983).
Phosphorylation of the H1
subtypes is likely to influence their interaction with DNA and, in
turn, modulate chromatin structure (Hill et al., 1990; Roth
and Allis, 1992). It has been proposed that H1 phosphorylation drives
chromosome condensation during mitosis (Bradbury, 1992). However,
recent studies indicate that chromosome condensation can occur in the
absence of H1 (Ohsumi et al., 1993), and that other non-H1
chromosome-associated polypeptides play an important role in this
process (Hirano and Mitchison, 1994). Reconstitution studies of
chromatin with rat thymus H1 or phosphorylated H1, which was
phosphorylated in vitro by p34 kinase
to an average of 5.3 phosphates per molecule, showed that
phosphorylation of H1 did not induce a condensation of chromatin
structure. Instead, H1 phosphorylation caused a destabilization of
chromatin structure at low ionic strength (Kaplan et al.,
1984). Another example of where H1 phosphorylation appears to have a
function in the decondensation, rather than the condensation, of
chromatin is with the Tetrahymena H1. H1 of Tetrahymena macronuclei, which are transcriptionally active, is highly
phosphorylated. Tetrahymena H1 is completely dephosphorylated
during conjugation when transcription ceases and chromatin becomes
condensed (Lin et al., 1991). Based upon these (and other)
examples, it has been proposed that phosphorylation of H1 acts as a
first step mechanism for inducing chromatin decondensation enabling
access of factors for gene activation or replication as well as
chromosome condensation (Roth and Allis, 1992).
Laitinen et
al.(1990) observed that ras-transformed mouse (NIH-3T3)
fibroblasts had a more decondensed nucleosomal structure than normal
fibroblasts. Such an alteration in chromatin structure could be a
result of alterations in H1 subtype levels or increased amounts of
phosphorylated H1. In this report, we show that ras-transformed mouse fibroblasts have higher levels of
phosphorylated isoforms of H1 subtypes b and c than the parental cells.
Furthermore, mouse fibroblasts that were transformed with oncogenes (raf, fes, mos, or myc), most of
which impact on the mitogen-activated protein (MAP) ()kinase
signal transduction pathway, had higher levels of phosphorylated H1
than the parental cell line (Davis, 1993; Blumer and Johnson, 1994;
Hunter and Pines, 1994). Kinases involved in this pathway are MAP
kinase kinase and MAP kinase. We show that NIH-3T3 cells transformed by
a constitutively activated MAP kinase kinase have a greater level of
phosphorylated H1 than the parental cells. These data suggest that the
persistent activation of the MAP kinase pathway, which is thought to
have a role in oncogenesis, leads to elevated amounts of the
phosphorylated H1. In indirect immunofluorescence studies with an
antibody to phosphorylated H1b, we observed a punctate pattern of
nuclear staining in parental cells. This pattern of staining was also
found in the ras-transformed cells. Furthermore, in both
parental and ras-transformed cells, phosphorylated H1b
co-localized to centers for RNA splicing.
Cell lines were grown in plastic tissue
culture plates in a humidified atmosphere containing 7% CO in medium supplemented with penicillin G (100 units/ml) and
streptomycin sulfate (100 µg/ml). The ras-transformed cell
lines Ciras-2 and Ciras-3 and NIH-3T3 derived cell lines were grown in
-minimal essential medium plus 10% fetal bovine serum (Intergen,
Purchase, NY). The proportion of cells in the different cell cycle
phases was determined by fluorescent-activated cell sorting (Blosmanis et al., 1987).
Figure 1:
Nucleosomal organization and histone
composition of chromatin of ras-transformed and parental mouse
fibroblasts. Mouse fibroblast 10T (A) or Ciras-3 (B)
nuclei at 0.27 or 0.34 mg of DNA/ml, respectively, were digested with
micrococcal nuclease for various times (minutes, indicated at the bottom of the figure). DNA fragments (10T, 15 µg;
Ciras-3, 20 µg) were resolved on 1% agarose gels which were stained
with ethidium bromide. The cell cycle distribution of the cells used in
this analysis was as follows: G
, 48.2 (47.1); S, 22.8
(23.6); G
/M, 29.0 (29.3) for 10T (Ciras-3). C and D, histones (10 µg) isolated from nuclei of 10T (C) and Ciras-2 (D) were electrophoretically resolved
on AUT-15% polyacrylamide gels. The Coomassie Blue-stained gels are
shown.
Changes in the content of H1 subtypes and/or modified histone H1
isoforms could account for the observed alterations in chromatin
compaction (Laitinen et al., 1995). Thus, we next investigated
the level of H1 subtypes and H1-modified forms in nuclei from cells. H1
was isolated by perchloric acid extraction from the nuclei of mouse 10T
fibroblasts and transformed derivatives of these cells (Ciras-2,
Ciras-3) and resolved by two-dimensional polyacrylamide gel
electrophoresis (SDS by AUT), which separates H1 subtypes and their
phosphorylated isoforms. Using the nomenclature of Lennox et
al.(1982), Fig. 2, A, B, and C,
shows that H1 subtypes H1b, -d, -e, -c, and H1 were present
in both ras-transformed and parental 10T1/2 mouse fibroblast
nuclei; subtype H1a was absent. Densitometric scans of the H1 subtypes
resolved on SDS gels demonstrated that the relative levels of the H1
subtypes were similar in ras-transformed and parental 10T
fibroblasts (data not shown). For example, the relative amount of
H1
in the H1 population in the cell lines 10T
,
Ciras-2, and Ciras-3 was comparable.
Figure 2:
H1 of ras-transformed and parental mouse fibroblasts. H1 was
isolated from 10T (lane 1), Ciras-2 (lane 2),
and Ciras-3 (lane 3) mouse fibroblasts. H1 (2 µg) was
electrophoretically resolved on SDS (A) (10T
, lane
1; Ciras-2, lane 2; Ciras-3, lane 3), AUT (B) (10T
, lane 1; Ciras-2, lane 2),
or two-dimensional gels (AUT into SDS) (10T
, C;
Ciras-2, D). E and F, H1 (2 µg)
extracted from 10T (lane 1) and Ciras-2 (lane 2)
cells metabolically labeled with
[
P]orthophosphate were electrophoretically
separated on SDS-15% polyacrylamide gels. On this gel, c-pb and c-pc
were not resolved from b and c, respectively. E shows the Coomassie Blue-stained gel, and F shows the
accompanying autoradiogram. pb is the phosphorylated isoform
of H1b. c-pb and c-pc are the c-phosphorylated
isoforms of H1b and H1c, respectively.
Lennox and Cohen (1988b) have shown that some of mouse H1 subtypes (H1b and H1c) undergo two types of phosphorylation. One phosphorylated isoform has an altered mobility in SDS gels. This type of phosphorylation is called c-phosphorylation because of its effect on protein conformation. The other phosphorylated isoform retains the same mobility as the parent band. The c-phosphorylated isoforms of H1 subtypes H1b and H1c migrate slower than the unmodified H1 subtype on SDS gels. Fig. 2A shows that H1 from ras-transformed cell nuclei had greater amounts of an H1 isoform (c-pb) running slower than H1b and a isoform (c-pc) migrating between H1c and H1d or -e on SDS gels. Consistent with these bands corresponding to the c-phosphorylated isoforms, treatment of H1 with alkaline phosphatase resulted in reduction or disappearance of these bands (Fig. 3C). The amounts of the c-phosphorylated H1b and H1c were approximately 2- to 3-fold greater in the ras-transformed cells (Fig. 2A and Table 1). These observations provided evidence that the level of the phosphorylated H1 was elevated in ras-transformed cells.
Figure 3: Phosphorylated H1 subtypes of ras-transformed and parental mouse fibroblasts. H1 (2 µg) isolated from 10T (lane 1), Ciras-2 (lane 2), or Ciras-3 (lane 3) was resolved on SDS-15% polyacrylamide gels. A and C show the India ink-stained patterns of H1 transferred to the membranes. The membranes shown in B and D were immunochemically stained with the antiphosphorylated H1 antibody as described under ``Materials and Methods.'' C and D, the Ciras-2 H1 were treated with alkaline phosphatase (lane 2AP). pb, c-pb, and c-pc are the phosphorylated isoforms of H1b and c-phosphorylated isoforms of H1b and H1c, respectively.
To gain further evidence that H1
of ras-transformed and parental mouse fibroblasts were
differentially phosphorylated, cells were metabolically labeled with
[P]orthophosphate, and H1 was
electrophoretically resolved on SDS-polyacrylamide gels. H1 of parental
10T cells was labeled to lower levels than the H1 isolated from ras-transformed mouse fibroblasts (Ciras-2) (Fig. 2, E and F). Densitometric scanning of autoradiograms of
labeled H1 resolved on SDS gels indicated that labeling of H1 from the
highly metastatic Ciras-2 cell line was approximately 5-fold higher
than that of the H1 from the normal parental 10T cell line.
Previous
studies have shown that the level of phosphorylation of H1 varies
considerably during the cell cycle. Possibly, a greater percentage of
the ras-transformed mouse fibroblasts were in S and
G/M phases of the cell cycle than that of the parental
cells. The proportion of cells in the different phases of the cell
cycle was determined by flow cytometry of cells stained with ethidium
bromide (Table 1). The ras-transformed cells had less
cells in the G
/M phase of the cell cycle, where H1
phosphorylation is at its peak, than did the parental cells. Further,
the Ciras-3 cells had a greater percentage of the cell population in
G
than did the parental cell line. It is clear, therefore,
that the increased level of H1 phosphorylation in the ras-transformed cells was not due to a change in the
proportion of these cells in S and G
/M.
The level of the phosphorylated H1b isoform in ras-transformed and parental 10T cell lines was determined in Western blot experiments with the antiphosphorylated H1 antibody. Fig. 3B shows the abundance of phosphorylated H1b was greater in the ras-transformed cells (Ciras-2 and Ciras-3) than in the parental 10T cells. The ras-transformed cells had an approximate 4-fold increase in the amount of phosphorylated H1b (Table 1). Similar results were obtained when H1 was acid-extracted directly from the cells.
ras-transformed Ciras-2 cells treated with colcemid, which arrests cells at mitosis (Fig. 4), had high levels of hyperphosphorylated histones (data not shown, see Lu et al. (1994)). Although phosphorylation of H1 subtypes should be maximal in mitotically arrested Ciras-2 cells, the antiphosphorylated H1 antibody still detected only the phosphorylated H1b isoform (Fig. 5). Thus, it appears that the antiphosphorylated H1 antibodies are remarkably specific for the H1b isoform.
Figure 4: Effect of hydroxyurea or colcemid on cell cycle progression of ras-transformed and parental mouse fibroblasts. Parental 10T cells and Ciras-2 cells were either untreated (top panels) or treated with 2 mM hydroxyurea for 24 h (middle panels) or with 0.06 µg/ml colcemid for 16 h (bottom panels) as described under ``Materials and Methods.'' DNA content in the ethidium bromide-stained cells was determined by flow cytometry. The number of cells is represented on the y axis, and the amount of DNA (fluorescence intensity) is shown on the x axis.
Figure 5:
Immunoblot analysis of phosphorylated H1
isoforms of normal and ras-transformed mouse fibroblasts
treated with hydroxyurea or colcemid. Cells (10T, lane
1; Ciras-2, lane 2; R2, lane 3) were treated
with hydroxyurea (A-D) or colcemid (E and F) as described in the legend to Fig. 4. H1 was
resolved on SDS-15% polyacrylamide gels and then transferred to
membranes which were immunochemically stained for phosphorylated H1.
The amount of protein loaded in each lane was as follows: A from left to right: lane 1,
10T
, 1.5 µg; lane 1 (second),
10T
, 2.0 µg; lane 2, Ciras-2, 2.0 µg; lane
3, R-2, 1.5 µg. C: lane 1, 10T
, 2
µg; lane 2, Ciras-2, 2.0 µg. E, H1 histones
(2 µg) were isolated from Ciras-2 cells that were not treated (lane 1) or treated (lane 2) with colcemid. A, C, and E show membranes stained with
India ink, and B, D, and F show the
corresponding immunochemically stained membranes. pb is
phosphorylated H1b.
Figure 6: Phosphorylated H1 isoforms of parental and oncogene-transformed NIH-3T3 mouse fibroblasts. H1 (2 µg) isolated from NIH-3T3 cells (lane 1) or NIH-3T3 cells transformed with v-fes (lane 2), v-mos (lane 3), c-myc (lane 4), or A-raf (lane 5). H1 was resolved on SDS-15% polyacrylamide gels, transferred to membranes which were stained with India ink (A) and then immunochemically stained with antiphosphorylated H1 antibody (B). pb is phosphorylated H1b. c-pb and c-pc are the c-phosphorylated isoforms of H1b and H1c, respectively.
Recently, Mansour et
al.(1994) demonstrated that constitutively active MAP kinase
kinase transformed NIH-3T3 mouse fibroblasts. Fig. 7shows the
content of H1 subtypes and their phosphorylated isoforms in the
parental NIH-3T3, K97M (cells transfected with catalytically inactive
MAP kinase kinase), and N3S222D (cells transfected with
constitutively active MAP kinase kinase). Densitometric scanning of the
Coomassie Blue-stained SDS gels indicated that the
N3S222D cells
had slightly higher amounts of c-phosphorylated H1b than the parental
or K97M cell lines. Differences in the levels of the H1 subtypes
including H1
were not observed. In Western blot analysis of
the level of phosphorylated H1b in the H1 preparations, the content of
this phosphorylated H1 isoform was found to be highest in the H1 from
N3S222D cells (Fig. 7B and Table 3). For the
Western blot shown in Fig. 7, the antiphosphorylated H1 antibody
detected two bands. However, in two repeats of this Western blot
experiment, only one band was immunochemically stained.
Figure 7:
Phosphorylated H1 of NIH-3T3 cells
transformed with constitutively active MAP kinase kinase. H1 isolated
from NIH-3T3 cells (lane 1), K97M (NIH-3T3 cells transfected
with inactive MAP kinase kinase, lane 2), N3S222D
(NIH-3T3 cells transfected and transformed with constitutively active
MAP kinase kinase, lane 3). H1 (2 µg) was resolved on
SDS-15% polyacrylamide gels, transferred to membranes which were
stained with India ink (A) and then immunochemically stained
with antiphosphorylated H1 antibody (B). pb is
phosphorylated H1b. c-pb and c-pc are the
c-phosphorylated isoforms of H1b and H1c,
respectively.
Before staining ras-transformed and parental
10T cells with the antiphosphorylated H1 antibody, we
ascertained the specificity of the antiphosphorylated H1 antibody using
total cellular protein from Ciras-2 cells. Western blot analysis
indicated that the antibody detected only the phosphorylated H1b
isoform in total protein. (
)Cells growing on coverslips were
fixed, and the phosphorylated H1b isoform was localized by indirect
immunofluorescence using the antiphosphorylated H1 antibody. The
majority of the interphase 10T and ras-transformed (Ciras-2)
cells had a weak nuclear fluorescence and punctate fluorescent dots
dispersed throughout the nucleoplasm ( Fig. 8and 9). The
staining of the Ciras-2 nuclei was more diffuse than the 10T nuclei.
This was probably due to cell and nuclei shape. The 10T cells were flat
and their nuclei large, while the Ciras-2 cells were round and their
nuclei appeared smaller. The number of speckles observed for ras-transformed and parental cells were equivalent.
Occasionally, we observed cells that were in M phase (see Fig. 8B). As might be expected, the mitotic
chromosomes stained intensely for phosphorylated H1.
Figure 8:
Phosphorylated H1b colocalizes with the
B1C8 nuclear matrix antigen in 10T mouse fibroblast nuclei. Cells were
fixed on coverslip and double-stained by indirect immunofluorescence
with antiphosphorylated H1 antibody and B1C8 monoclonal antibody and
then goat anti-rabbit antibody conjugated to fluorescein isothiocyanate (A and B) and goat anti-mouse antibody conjugated to
Texas red (C and D). The immunochemically stained 10T
cells were photographed at 60 magnification. B and D show a cell in M phase of the cell
cycle.
The
punctate/speckled pattern of nuclear staining observed with this
antibody (also see Lu et al., 1994) is reminiscent of the
finding that nuclear sites of splicing factors, small nuclear RNAs, and
RNA synthesis were colocalized (Huang and Spector, 1991, 1992; Carter et al., 1991; Bassim-Hassan et al., 1994; Jackson et al., 1993). To ascertain whether the phosphorylated H1b was
colocalizing with the sites of splicing factors and ``transcript
domains'' (Xing et al., 1993), the monoclonal antibody
B1C8 was also used in indirect immunolocalization studies. The B1C8
monoclonal antibody recognizes a 180-kDa human nuclear matrix protein
and co-immunoprecipitates exon-containing RNA from in vitro splicing reactions (Wan et al., 1994; Blencowe et
al., 1994). Western blot experiments with the B1C8 monoclonal
antibody and total protein isolated from 10T mouse fibroblasts showed
that the monoclonal antibody detected a 180-kDa mouse protein. The relative nuclear positioning of phosphorylated H1b and the
B1C8 antigens was done by double staining with antiphosphorylated H1b
rabbit antibody and B1C8 mouse monoclonal antibody. Fig. 8and Fig. 9show that the majority of the speckles in the nuclei of
10T and Ciras-2 cells observed with the antiphosphorylated H1 antibody
were detected with the B1C8 monoclonal antibody. Of the 40 to 50
speckles illuminated by the antibodies, approximately 80% overlapped.
Thus, phosphorylated H1b appeared to be localized to sites of RNA
processing and gene transcription in the majority of the interphase 10T
and ras-transformed cells. However, for cells in mitosis, the
B1C8 staining did not colocalize with the staining for phosphorylated
H1b, where intense staining of B1C8 was found at the spindle poles (see Fig. 8D). This pattern of redistribution of the B1C8
antigen during mitosis in mouse fibroblasts is similar to that observed
in human CaSki cells (Wan et al., 1994).
Figure 9:
Phosphorylated H1b colocalizes with the
B1C8 nuclear matrix antigen in Ciras-2 mouse fibroblast nuclei. Ciras-2
cells were fixed on the surface of a coverslip and stained by indirect
immunofluorescence with antiphosphorylated H1 antibody and B1C8
monoclonal antibody and then goat anti-rabbit antibody conjugated to
fluorescein isothiocyanate (A, B, and C) and
goat anti-mouse antibody conjugated to Texas red (D, E, and F). The immunochemically stained Ciras-2 cells
were photographed at 60
magnification.
ras- and c-myc-transformed mouse
fibroblasts (NIH-3T3 and 10T) have a less condensed chromatin
structure than the parental cell line (this report; Laitinen et
al. (1990, 1995)). Since H1 stabilizes higher order chromatin
structure, it was reasonable to suggest that changes in H1 would be a
factor in chromatin decondensation. Alterations in H1 subtypes have
been observed in transformed cells (Tan et al., 1982; Davie
and Delcuve, 1991; Nagaraja et al., 1995). A reduction in the
content of H1
in ras-transformed NIH-3T3 mouse
fibroblasts may contribute to the decondensation of the chromatin in
the ras-, raf-, fes-, mos-, and myc-transformed cells (Laitinen et al.(1995) and
references therein). However, the lower level of histone H1
in oncogene-transformed mouse fibroblasts was not a general
observation. ras-transformed 10T mouse fibroblasts and
constitutively active MAP kinase kinase-transformed NIH-3T3 cells had a
content of H1 subtypes (including H1
) that was similar to
that of parental cells.
The one consistent alteration we observed in the oncogene-transformed 10T and NIH-3T3 mouse fibroblasts was an increase in the level of phosphorylated H1 subtypes. The content of phosphorylated H1b and H1c that have a reduced mobility on SDS gels was elevated in the oncogene-transformed cells. Furthermore, in Western blot experiments with an antibody that is highly selective for the phosphorylated form of H1b in these cells, we found that the level of this phosphorylated subtype was increased in all oncogene-transformed cells used in this study. Similar results were obtained when the levels of phosphorylated H1b and the c-phosphorylated isoforms of H1c and H1b were analyzed from cells transformed with combinations of ras, myc, and mutant p53 (data not shown).
Studies with native and reconstituted chromatin show that phosphorylated H1 destabilize chromatin structure (Kaplan et al., 1984; Hill et al., 1991). Further, in studies with avian fibroblasts transfected with H5 (an H1 variant), H5 was shown to inhibit proliferation in normal fibroblasts but not in transformed cells, in which H5 was phosphorylated. Aubert et al.(1991) proposed that phosphorylated H5 lacked the ability to condense chromatin. Thus, these and other studies (see Lu et al. (1994) and references therein) provide support for the idea that an increase in the phosphorylation of H1 leads to destabilization of the chromatin in oncogene-transformed cells.
Mouse fibroblasts transformed with raf, fes, or mos had elevated levels of phosphorylated H1 subtypes. These oncogenes code for serine/threonine or tyrosine kinases and act upon the MAP kinase signal transduction pathway (Hunter and Pines, 1994). NIH-3T3 cells transformed with constitutively active MAP kinase kinase also had an increased content of phosphorylated H1b and, to a lesser extent, c-phosphorylated H1b. These observations suggest that persistent activation of the MAP kinase pathway has a role in the intensified phosphorylation of H1 in the transformed cells. One of the downstream targets of the MAP kinase pathway is the c-Myc protooncogene product. Phosphorylation of c-Myc increases its ability to transactivate genes (Davis, 1993). Myc and activated Ras enhance the expression of cyclins D1, E, and A (Jansen-Durr et al., 1993; Daksis et al., 1994; Filmus et al., 1994). Overexpression of cyclin E in cells significantly enhances cyclin E-associated H1 kinase activity (Resnitzky et al., 1994). It is possible that activation of the MAP kinase signal transduction pathway leads to the phosphorylation of c-Myc which in turn increases the expression of cyclins E and A and activity of a cyclin E- or cyclin A-associated H1 kinase (Jansen-Durr et al., 1993; Hunter and Pines, 1994).
The high selectivity of the antiphosphorylated H1 antibody allowed us to ascertain the cellular location of the phosphorylated isoform of H1b. A punctate pattern of nuclear staining of ras-transformed and parental 10T cells was observed. Clearly, the results show that the phosphorylated isoform of H1b is non-uniform in the nuclei of parental and ras-transformed mouse fibroblasts. In M-phase cells, the punctate pattern of staining is lost, and an intense staining of the mitotic chromosomes is observed. A similar staining was observed for non-S phase HeLa nuclei (Lu et al., 1994).
The speckled pattern observed with the antiphosphorylated H1 antibody closely matched the pattern found with the B1C8 monoclonal antibody. B1C8 colocalizes with other RNA splicing components (SC35, snRNPs) (Blencowe et al., 1994). Several transcribed genes (e.g. c-fos) have been located near sites containing the RNA splicing components (Huang and Spector, 1991; Carter et al., 1993; Xing et al., 1993). Furthermore, the punctate pattern of splicing factors is sensitive to inhibitors of transcription (Huang et al., 1994). Recently, Durfee et al.(1994) found that the nuclear matrix protein p84, which is present in human and mouse cells and binds to the N-terminal domain of dephosphorylated Rb, colocalizes with the B1C8 antigen. Dephosphorylated Rb binds to the transcription factor E2F, which is involved in the activation of several early response genes, including c-myc. Although inactive when bound to dephosphorylated Rb, E2F still has the capacity to bind its target DNA (Nevins, 1992; Weintraub et al., 1992). Thus, the nuclear matrix-bound p84, which is near sites containing splicing factors, through its association with dephosphorylated Rb-E2F complex, may attract early response genes to sites of RNA processing. Following the release of E2F from Rb, E2F forms a complex with cyclin E (and later with A), p107, and CDK2 (Hunter and Pines, 1994). It is an attractive hypothesis that the transcription factor E2F directs the H1 kinase activity of CDK2 to transcriptionally active chromatin regions (Devoto et al., 1992).