From the Fondazione "Istituto Pasteur-Fondazione
Cenci-Bolognetti", c/o Dipartimento di Genetica e Biologia Molecolare
Università "La Sapienza", P. le A. Moro 5, 00185-Roma Italy
and
Centro Acidi Nucleici Consiglio Nazionale delle Ricerche,
Roma Italy
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ABSTRACT |
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The chromatin organization of eukaryotic telomeres is essential for telomeric function and is currently receiving great attention. In yeast, the structural organization of telomeres involves a complex interplay of telomeric proteins that results in the formation of heterochromatin. This telomeric heterochromatin involves homotypic and heterotypic protein interactions that have been summarized in a general model.
Recent analyses have focused on the study of the structural complexity
at yeast telomeres to the level of specific nucleosomes and of the
distribution of protein complexes in a natural telomeric region (LIII).
In this report, we further analyze the structural complexity of LIII
and the implication of this structure on telomeric silencing. It is
shown that the establishment of repressive heterochromatin structures
at LIII requires the recruitment of Sir3p through interaction with the
N terminus of histone H4. The establishment of such structures does not
require acetylation of any of four lysines located in the H4 N terminus
(lysines 5, 8, 12, and 16).
Telomeres stabilize chromosomes, facilitate replication of
chromosomal termini, and are functionally linked to aging and
tumorigenesis (1, 2). The chromatin organization of telomeres is
believed to be essential for their function and is currently receiving great attention. In particular, the chromatin structure of yeast telomeres is probably the best known among all eukaryotes because of
their heterochromatic nature, required for transcriptional silencing in
both artificial (3) and natural (4) contexts.
In Saccharomyces cerevisiae, the telomeric sequence is
composed of about 350 base pairs containing the
(C1-3A)n repeats. This element is followed by two
main subtelomeric sequences: the X and the Y' elements (5). X elements
are present at all telomeres and can exist in two main forms, a
complete form containing the X core and the STR-A, B, C, D elements or
a short form containing the X core or part of it (5-8). Y' elements
are highly conserved and are present in about 70% of the telomeres
(5-7). Thus, yeast telomeres contain tandem repetitions of the
(C1-3A)n motif flanked by subtelomeric, middle
repetitive sequences (reviewed in Ref. 9), internally followed by
unique sequences.
As for the primary sequence, the chromatin structure of yeast telomeres
is also organized in different domains. The terminal (C1-3A)n repeats are organized into a
nuclease-resistant structure called the telosome (10, 11) that does not
contain nucleosomes and is firmly associated with the protein Rap1
(10-12). This protein binds to the repetitive
(C1-3A)n sequences (13, 14) and interacts with
other proteins including Rif1p, Rif2p, Sir3p, and Sir4p
(15-17). In addition, Sir3p and Sir4p interact among them and with
nucleosomes located in subtelomeric regions through the N termini of
histones H3 and H4. The nature of the available evidence obtained so
far for Sir3p-histone H4 interactions is genetic and biochemical
(reviewed in Ref. 18).
The crystal structure of the nucleosome core particle has been recently
resolved at 2.8 Å of resolution (19). This structure shows that the
highly basic N-terminal tail of histone H4 (amino acids 1-25)
potentially binds as an extended chain to a region of extreme acidity
on the exposed face of the H2A-H2B dimer of a neighboring particle. The
H4 residues involved in the interparticle contact are the amino acids
16-24, which are also involved in the binding to the silencing protein
Sir3p. These data predict that Sir3p interacts with histone H4 at
telomeres and alters nucleosomes interactions while participating in
the formation of telomeric heterochromatin.
The complex interplay of telomeric proteins has been summarized in a
model for telomeric heterochromatin (18). This general model applies to
all the 32 telomeres of S. cerevisiae and takes into account
homotypic and heterotypic protein interactions. In summary, in a
telomeric region, a telosome is internally followed by subtelomeric
heterochromatin, then by "normal" chromatin. Comprehensive reviews
of structural, genetic and functional yeast telomere organization are
available (18, 20).
In addition to the growing body of general information described above,
the chromatin structure of a natural telomeric region from S. cerevisiae has been recently described. This region corresponds to
the terminal 1.4 kilobases of the left arm of chromosome III (LIII)
(21). In LIII, the terminal (C1-3A)n repetitive sequences are followed by a complete X element and by the single copy
Ty5-1 retrotransposon (see map in Fig. 1). Both the telosome and the X
element are characterized by a tight chromatin structure organization.
The X element contains protein complexes and irregularly distributed
but well localized nucleosomes, whereas a regular array of phased
nucleosomes is associated with the promoter region of Ty5-1. The lack
of Sir3p causes a nucleosome rearrangement within the promoter region
of Ty5-1 and releases Ty5-1 silencing. In contrast, Sir3p is not
required to keep the overall tight organization of the X element (21).
This previous analysis of LIII has focused the study of the structural
complexity at telomeres to the level of specific nucleosomes and of the
distribution of protein complexes. In addition, it has provided
in vivo evidence linking the modification of heterochromatin
structure with the loss of transcriptional telomeric silencing (4).
We describe in this report that the deletion of residues 4-28 of
histone H4 disrupts the heterochromatin structure of LIII. This
disruption mimics the rearrangement of heterochromatin caused by the
absence of Sir3p (21). A previous report has shown that the 4-28
N-terminal domain of histone H4 contains residues that are required for
Sir3p-H4 in vitro interaction (22). In addition, H4 residues
4-28 are required for the recruitment of Sir3p to telomeres and for
telomeric silencing (4, 22). Thus, we conclude that the establishment
of repressive heterochromatin structures at LIII requires the
recruitment of Sir3p through interaction with the N terminus of histone
H4. The establishment of such structures does not require acetylation
of any of the four lysines located in the H4 N terminus (lysines 5, 8, 12, and 16).
Yeast Strains and Culture Conditions
All strains were provided by M. Grunstein.
SIR3 Studies--
The following strains were used: wild type
LJY153 and mutant LJY155 (isogenic to LJY153 and lacking Sir3p,
indicated as sir3). LJY153: MATa ade2-101,
his3- H4 Studies--
The following strains were used: wt PKY501 and
mutants PKY813 and LDY722. PKY501: MATa,
ade2-101, his3-
Strains LJY153 and LJY155 were grown at 28 °C on SC medium lacking
tryptophan containing glucose to an A600 of
about 0.5/ml. Strains PKY501, PKY813, and LDY722 were grown similarly
on SC medium lacking uracil.
Nuclease Sensitivity Analyses
Cells from 50-ml cultures were collected by centrifugation,
treated with zymolyase, and digested with micrococcal nuclease (MNase)1 as described
previously (27). After digestions of chromatin or naked DNA, the
sensitivity to the enzyme was analyzed by the indirect end-labeling
technique (28). The DNA samples were purified, digested with
BamHI, resolved in agarose gels, and transferred to
nitrocellulose membranes. The cutting profile generated by MNase was
visualized after hybridization with the probe indicated in the map in
Fig. 1. This probe abuts the BamHI site selected for the
analyses and extends in an outer direction from position 1495 to 1725 of S. cerevisiae chromosome III.
Nucleosomal spacing analyses were performed with MNase. After digestion
of chromatin with MNase, the purified DNA samples were directly
resolved in agarose gels, generating a nucleosome ladder that could be
visualized by staining with ethidium bromide. The DNA samples were then
transferred to nitrocellulose membranes and hybridized with the probe
indicated in the legend to Fig. 1.
Deletion of Residues 4-28 of Histone H4 Disrupts Telomeric
Heterochromatin Structure--
The chromatin structure of S. cerevisiae left telomeric region of chromosome 3 (LIII) has been
analyzed in different wild type and mutant strains. Permeabilized cells
were digested with increasing concentrations of MNase, and the
sensitivity to the enzyme was monitored by indirect end-labeling using
the probe indicated in Fig. 1. This
figure shows the MNase digestion profiles of chromatin from the
parental strain (PKY501) and from two derived H4 mutants (PKY813 and
LDY722). PKY813 is isogenic to PKY501 and bears a deletion of residues
4-28 of histone H4 (termed
In PKY813 the length of the band detected in the absence of MNase
digestion is higher (lane 7) than in the wild type strain (lane 3). This is because of the fact that the subpopulation
of cells from PKY813 used in this study bears a 5.5 kilobase Y'-short subtelomeric element inserted into the (C1-3A)n repeat at ~150 base pairs from the X element border (to be detailed elsewhere; see the map on the right side of Fig. 1). This insertion is
favored by the
The digestion profiles in lanes 7-10 show that the Y'-short
element inserted into the (C1-3A)n repeat of
PKY813 has a complex MNase digestion pattern, too distant from the
probe to allow any detailed analysis. Two separate
(C1-3A)n tracts are present in LIII in these
cells: one terminal and one internal, between the Y' and X elements.
The terminal (C1-3A)n repeat is organized into the
so-called telosomic structure and is expected to be MNase-inaccessible,
as actually observed. In addition, also the internal 150-base pairs
long (C1-3A)n tract is MNase-inaccessible, thus
suggesting a closed heterochromatic and telosome-like organization.
Moving inward from the internal (C1-3A)n repeat,
the X element of the
The digestion pattern generated by MNase inside the X element is in
general more smeared in the
To analyze whether the insertion of Y'S element had any effect on the
chromatin structure of LIII, the MNase digestion profiles of
Y'-short-bearing and Y'-less subpopulations of the PKY813 strain were
analyzed. The analysis was made possible by the existence of
subpopulations bearing or devoid of Y' elements that are stable for a
sufficient number of generations. The digestion profiles in both the X
elements and in the Ty5-1 sequences were essentially indistinguishable
in the various subpopulations analyzed, pointing to the independence of
the heterochromatin organization of these sequences upon proximity of
the telosome structure that characterizes the C1-3A
tracts. The digestion profile of these tracts is always the same,
independently on the presence (see the profile shown in Fig. 1,
lanes 7-10, and in Fig. 2, lanes 7-9) or
absence of Y' elements in distal position.
Our restriction analyses of LIII (Refs. 4 and 21, this paper, and
unpublished results2) have revealed no differences between
the sequence of the parental and of the mutant strains analyzed in this
work for the region encompassed between the
(C1-3A)n repeat/X element border and the
BamHI site.
K to R Mutations of the Four Acetylatable Residues in the H4 N
Terminus (5, 8, 12, 16) Do Not Alter Heterochromatin
Structures--
The last three lanes in Fig. 1 show the
MNase digestion profiles of the Ac The Lack of Histone H4 N-terminal Tail and the Absence of Sir3p
Cause Similar Changes of Heterochromatin Structure--
Fig.
2 compares the MNase digestion profiles
of LIII in two different mutant strains: LJYl55 (lacking Sir3p) and
PKY813 (
Interestingly, the MNase digestion pattern of the STR A-D and X core
regions remains unaffected by either mutation. This pattern (extending
from the STR-A sequences to the ABF1 and the ACS binding sites, a
silenced origin of replication (8)) is characterized by an overall
lower accessibility.
The indirect end-labeling analyses shown in Fig. 2 do not absolutely
demonstrate that the change of MNase digestion pattern observed for the
Chromatin from these strains was digested with increasing
concentrations of MNase. The purified DNA was resolved on an agarose gel, blotted and hybridized with the probe indicated in Fig. 1. The
nucleosomal ladder generated on whole-genome chromatin (that is,
stained with ethidium bromide) is shown for comparison (Fig. 3,
panel B). This procedure reveals whether the DNA used as a probe is associated with nucleosomal particles or with complexes of
different nature. By its very nature, nucleosomal spacing analysis complements the indirect end-labeling data. In Fig. 3, probe 1 displays
a normal nucleosomal array in all the strains, thus corroborating that
the shift of MNase cutting detected around the 5' long terminal repeat
of Ty5-1 in the Repressive Heterochromatin Structures Require Recruitment of Sir3p
through Histone H4 Interaction--
The model derived from the 2.8-Å
crystal structure of the nucleosome suggests that an H4-based
internucleosome connection in the higher order structure is replaced in
heterochromatin by an interaction with Sir3p (19, 30). The region of
histone H4 that binds to the silencing factor localizes in its N
terminus and is also required for telomeric silencing. Consequently, it has been suggested (30) that the binding of Sir3p to telomeric areas
and the establishment of telomeric silencing would require the
disruption of nucleosome-nucleosome interactions. These considerations highlight the interest of analyzing the direct in vivo
interaction between Sir3p and the H4 N-terminal tail and its structural consequences.
Studies on natural, nonengineered telomeres often yield
population-averaged data because they rely on probes for repetitive sequences; hence, the difficulty of establishing precise boundaries among the various structural compartments of the chromosomal
extremities and precise mapping of nucleosomes and other proteins.
We compare in this report the in vivo structural
consequences of mutations that affect two different heterochromatin
components (histone H4 and Sir3p). The deletion of residues 4-28 of
histone H4 cause modifications of LIII heterochromatin that are similar to those caused by the absence of Sir3p. Interestingly, both kinds of
mutations release Ty5-1 silencing and silencing at the HM
loci (4, 22). In addition, the 4-28 N-terminal domain of histone H4 is required for the recruitment of Sir3p to the telomeres and contains residues required for Sir3p-H4 in vitro interaction
(22, 23). Thus, the present data show that the establishment of
repressive heterochromatin structures at LIII requires the recruitment
of Sir3p through interaction with the N terminus of histone H4.
The most interesting attribute of the structural alterations caused by
the lack of Sir3p and by the deletion of histone H4 N terminus is that
they are undistinguishable. The major changes observed in both mutant
strains is an alternate localization of nucleosomes on the Ty5-1
sequences. To our knowledge, this is an unprecedented observation.
Nucleosomes are known to undergo in vivo destabilization or
loss, but not displacement. Their structural modification can reach
various degrees of severity and occurs in different genetic processes,
the most thoroughly analyzed being the activation of transcription
(31-33). A limited ability of translational displacement has been
described in vitro, in artificial conditions (34). Multiple
alternate positions on the same rotational phase (reviewed in Ref. 35)
can be occupied in vivo, ranging usually from 1 to 5 different positions (an extreme case of 15 different positions occupied
being provided by the 5 S rDNA gene (36). Thus, the occupation of
different positions is documented both as a static possibility in
vivo by alternate locations along the same rotational frame and as
a temperature-driven dynamic process in vitro in the
"mobile nucleosomes" experiments (34), not as an actual process
in vivo. Interestingly, two among the mutations known to
relieve transcriptional silencing at telomeres (4, 37-39) cause the
differential localization of in vivo nucleosomes (Figs. 1-3).
A possible explanation for the shift of nucleosome positioning caused
by the lack of Sir3p or the deletion of histone H4 N terminus is that,
in the absence of a staple-like function provided by the Sir3p or by
its H4 amino-tail counterpart, the tight structure of heterochromatin
(in which nucleosomes are constrained in certain positions) does not
occur. Under these unconstrained conditions, a more accessible
unrepressed configuration (with differently located nucleosomes) is
organized that allows transcription to start. In addition, the
recruitment of the transcriptional preinitiation complex to the Ty5-1
promoter region could also favor the shift of nucleosome positioning in
the absence of Sir3p-H4 interaction.
One of the features defined by the 2.8-Å resolution model of the
nucleosome structure is that one H4 N-terminal tail segment has the
potential to make a strong interparticle connection, perhaps relevant
to the higher-order structure of chromatin (19). Because this H4 domain
is also required for the binding of Sir3p, the similarity of the
effects caused by the lack of Sir3p or of its reacting counterpart in
histone H4 is coherent, with the possibility that binding of Sir3p
locks down nucleosomes by changing the normal nucleosome-nucleosome interactions.
It was suggested that the R1 domain of histone H4 is involved in
stabilizing the bend caused on DNA by the interaction with the histone
octamer (25) at the site defined as dyad. The R1 domain is functionally
defined as a basic region spanning residues 16-19 (23, 40), very
sensitive to mutations that alter charge and lead to derepression at
HML and at telomeres (37-39). R1 was also shown to be
necessary for nucleosome positioning by the
In conclusion, the similarity of the modifications caused by the lack
of Sir3p and by the deletion of H4 N-terminal tail supports the
in vivo interaction of these components at LIII, which
agrees with the nucleosome high resolution model. This interaction is required to keep the repressed heterochromatin configuration of the
Ty5-1 retrotransposon (4).
The Role of H4 N-terminal Acetylation in Heterochromatin--
As
mentioned above, the establishment of heterochromatic repressive
structures at LIII requires the N terminus of histone H4. In contrast,
the simultaneous mutation of the four acetylatable lysines (5, 8, 12,
16) of histone H4 to arginines does not significantly affect it.
Previous in vitro binding analyses suggest that the K to R
mutation of the four H4 N-terminal lysines does not impair
Sir3p-histone H4 interaction (22). In addition, studies on the HM
loci have demonstrated that, as in other eukaryotes,
heterochromatin in yeast is hypoacetylated at the H4 lysines 5, 8, and
16 but not at lysine 12 (41). However, lysine 12 is not required for
the interaction with Sir3p or for the establishment of functional heterochromatin (22, 23).
We have shown that the K to R mutation of the four acetylatable lysines
does not affect Ty5-1 silencing. In addition, although these mutations
moderately inhibit mating (42), they do not lead to derepression of the
HML
It is interesting to note that the two mutations of histone H4 analyzed
in this report (
INTRODUCTION
Top
Abstract
Introduction
References
EXPERIMENTAL PROCEDURES
200, leu2-3, leu2-112, lys2-801, Trp1-
901, ura 3-52,
hhf1[HIS3], with plasmid
pRS424 (TRP1, 2 µm). Additional details are provided in Refs.
22-24.
200, leu2-3, -112, lys2-801,
trp1-
901, ura 3-52, thr-,
arg4-1, hhf1::HIS3, hhf2::LEU2, with pPK301
(CEN3, ARS1, URA3, HHF2). PKY813: isogenic to PKY501, with plasmid
pPK613 (hhf2 del(4-28)). For further descriptions of
these strains, see Refs. 23 and 25. LDY722 is isogenic to PKY501 with
plasmid pLD722 (CEN4, ARS1, URA3, HHF2-K5, 8, 12,16R)
(26).
RESULTS
NtH4). LDY722 (termed
Ac
NtH4) has the lysine residues 5, 8, 12, and 16 changed
into arginine, thus preventing acetylation in these positions. The
chromatin organization of LIII in the wild type strain has been
previously reported (21). The digestion profile of this strain is shown in lanes 3-6. One fuzzy telomeric band is observed in the
lane containing undigested chromatin from PKY501. Fuzziness
is expected from the fact that the yeast population is
length-heterogeneous for the (C1-3A)n repeat
(29). The X element shows low accessibility to MNase, reflecting a
closed overall chromatin structure organization. Inside the X element,
this overall protection extends from the STRA sequences to the ACS
binding site, a silenced origin of replication. Protein complexes and
irregular, but well positioned nucleosomes coexist inside the X
element. In contrast, the 5' region of the Ty5-1 retrotransposon
accommodates a regular array of nucleosomes that are associated with
specific DNA sequences. The map at the sides of the gel images
schematically depicts such organization both in the wild type
(filled half-circles) and in the
NtH4 mutant (empty
half-circles). Lanes 7-10 and 11-14 show the MNase digestion profiles of the two histone H4 mutants (PKY813 and
LDY722, respectively).
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Fig. 1.
Chromatin structure of LIII in histone H4 N
terminus mutants. The bottom map is a
representation of LIII. The position of the
(C1-3A)n repeats (filled bar), the X
element, and the Ty5-1 retrotransposon are indicated as well as the
TBF1 (gray oval), ABF1 (filled oval), and ACS
(empty oval) binding sites (determined in Ref. 21). The STR
A-D sequence elements are localized in the left part of the X element.
The 5' and 3' long terminal repeats (LTR) of Ty5-1 are also
shown. The three arrowed rectangles with decreasingly
gray shades represent the open reading frames previously
defined by sequencing (44): YCL76w, 75w, and 74w (from left to right).
The positions of the probe (black bar) and the
BamHI restriction site used for indirect end-labeling are
also indicated. S. cerevisiae wild type (wt)
strain PKY501 (lanes 3-6) and mutants PKY813 (lacking the
N-terminal tail: NtH4) (lanes 7-10) and LDY722
(substituted in the N-terminal tail acetylatable Lys residues:
Ac
NtH4) (lanes 11-14) were grown in 50-ml
cultures, collected, treated with zymolyase, and digested with MNase as
described previously (27). Indirect end-labeling analysis is shown.
Samples 1 and 2 show purified PKY501 DNA (10 µg) digested with 1.2 and 2.5 units/ml MNase, respectively. Samples 3, 7, and 11; 4, 8, and
12; 5, 9, and 13; and 6, 10, and 14 show chromatin digested with 0, 4, 12, and 40 units/ml, respectively. The size marker lane
(M) contains a 123-base pair ladder from Life Technologies,
Inc. A scaled representation of LIII is shown both on the right and on
the left-sides. The representation on the left side depicts a wt LIII,
and the right side representation shows the same LIII when bearing a
5.5-kilobase Y' element inserted into the (C1-3A)n
repeat at about 0.15 kilobases from the X element border. This
insertion is stably present in a subpopulation of the
NtH4 mutant.
Half-circles along these maps represent the
translationally phased nucleosomes on the Ty5-1 sequences
(filled, PKY501 wt; empty, PKY813 mutant). The
lower intensity of the digestion patterns shown in lanes
11-14 is because of an occasional lower yield of the LDY722
samples.
NtH4
mutation,2 is stable (being
constantly observed for at least 100 generations), and is not present
in all the subpopulations derived from the original mutated clone (see below).
NtH4 mutant (lanes 7-10) shows, as
in the wild type strain, overall protection against MNase. Thus, the
4-28 residues from the N terminus of histone H4 are not essential to
keep the closed overall organization of the X element. In addition, the
digestion pattern of the X element is quite similar in the wild type
and the
NtH4 strains (see also next sections), suggesting that the
lack of H4 N terminus does not cause major changes in the specific
chromatin structure of the X element.
NtH4 mutant than in the wild type
strain. This can be explained assuming that most nucleosomes and
protein complexes within the X element are in a more relaxed conformation in the mutant. The digestion pattern generated by MNase in
the promoter region of Ty5-1 is altered in the
NtH4 mutant,
suggesting a change of nucleosome distribution.
NtH4 mutant. They are
similar to the wild type digestion profiles, indicating that the
heterochromatin organization of LIII in this mutant does not undergo
major changes. This result prompted us to check the level of Ty5-1
expression in the Ac
NtH4 mutant. As in the wild type
strain, the expression of Ty5-1 was completely repressed in this
mutant (data not shown), whereas it was derepressed in the
NtH4
mutant strain, as previously reported (4). Thus, the deletion of the
4-28 N-terminal residues of histone H4 causes derepression of Ty5-1
and disruption of LIII heterochromatin structures. On the contrary,
mutations that mimic the hypoacetylated state of histone H4 N terminus
do not significantly alter LIII chromatin structure nor Ty5-1 silencing.
NtH4). The digestion profiles of the respective parental
strains (LJY153 and PKY5O1) are also shown as a control. The results
indicate that the chromatin organization of LIII is similar in the two mutant strains and that they differ from the wild-type parental organization. The major changes in the mutants consist of a shift of
MNase cutting in the region that contains the 5' long terminal repeat
of Ty5-1 (where the promoter of the retrotransposon is located). In
the strain lacking Sir3p, this shift corresponds to a change of the
nucleosome positioning previously reported (21). Thus, these results
prompt us to propose that a similar change of nucleosome distribution
occurs in the
NtH4 mutant (see vertical schematic maps on both sides
of Fig. 2).
View larger version (64K):
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Fig. 2.
The absence of Sir3p and of the histone H4
N-terminal tail cause similar alterations of LIII heterochromatin.
Indirect end-labeling analysis (MNase digestion patterns, obtained as
described in Fig. 1) of strains LJY153 (wt, lanes
1-3), LJY155 (sir3) (lanes 4-6), PKY813 ( NtH4)
(lanes 7-9), and PKY501 (wt, lanes
10-12). Lane D, naked DNA digestion. The similarity of
the alterations produced by the sir3 and
NtH4 mutations is shown by
the comparison of lanes 4-6 versus lanes
7-9. M, size markers; LTR, long terminal
repeat.
NtH4 mutants is because of a shift of nucleosomes positions. Such
conclusions can be further sustained by a "nucleosomal spacing"
analysis in vivo, as already reported for the wt LJY153 (21), to show that nucleosomes are actually present on the Ty5-1 promoter region of the
NtH4 mutant. Fig.
3 shows the results obtained performing
the nucleosomal spacing analyses on all the strains studied in the
present report.
View larger version (71K):
[in a new window]
Fig. 3.
Nucleosomes are present on the Ty5-1
sequences of histone H4 mutants. Nucleosomal spacing analysis.
After digestion of chromatin with increasing concentrations of MNase
(0, 20, and 40 units/ml for the left-to-right samples in each group),
the purified DNA samples were resolved in agarose gels, visualized
after staining with ethidium bromide (panel B), transferred,
and hybridized with probe 1 (panel A). Lanes
1-3, LJY153 (wt); lanes 4-6, LJY155
(sir3); lanes 7-9, PKY501 (wt); lanes
10-12, PKY813 ( NtH4); lanes 13-15, LDY722
(Ac
NtH4).
NtH4 mutant actually corresponds to a shift of
nucleosome positioning.
DISCUSSION
2 repressor (15). Thus,
it is likely that the lack of these residues weakens the DNA-H4 tail
interactions, allowing a more relaxed and accessible heterochromatin configuration.
locus, as analyzed by Northern hybridization (data
not shown), which suggests that they exert a minimal effect of HM
repression. Taken together, these data prompt us to propose that
the recruitment of Sir3p to telomeres and the consequent establishment
of heterochromatic-epressive structures do not require acetylation of
any of the H4 N-terminal lysines.
NtH4 and Ac
NtH4) exert a negative
effect on the activation of regular non-telomeric polymerase II genes
(26). Such gene activation failing has been correlated with the
establishment of a closed chromatin structure (43). In addition, the N
terminus of histone H4 has been shown to be required for
2 to
position nucleosomes and repress gene expression (15). In summary, the
N terminus of histone H4 plays an essential role in gene activation and
silencing through the establishment of specific chromatin structure
organizations. The role of the acetylatable lysines differs depending
on the specific process and probably on the proteins with which they interact.
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ACKNOWLEDGEMENTS |
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The support of G. P. Ateneo, La Sapienza, of MURST project "Protein-nucleic acid interactions" and of CNR Target Project on Biotechnology is acknowledged.
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FOOTNOTES |
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* This work was supported by European Community Human Capital Mobility Grant CHRX CT94-0047.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ These authors contributed equally to this work.
¶ Present address: Instituto de Bioquímica Vegetal y Fotosíntesis (Universidad de Sevilla-CSIC), Centro de Investigaciones Isla de la Cartuja, c/Américo Vespucio s/n, 41092 Sevilla, Spain.
** To whom correspondence should be addressed: Dipartimento di Genetica e Biologia Molecolare, Università La Sapienza, P. le Aldo Moro, 5 00185 Roma, Italy. Tel.: +39.6.4991.2880; Fax +39.6.4440.812; E-mail: Dimauro{at}axrma.uniroma1.it.
The abbreviations used are: MNase, micrococcal nuclease; wt, wild type.
2 S. Venditti, M. A. Vega-Palas, and E. Di Mauro, unpublished results.
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REFERENCES |
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