Download Dosage Compensation: Transcription-Level Regulation of X

AMER. ZOOL., 17:685-693 (1977).
Dosage Compensation: Transcription-Level Regulation of X-Linked Genes in
Drosophila
JOHN CHARLES LUCCHESI
Department of Zoology and Genetics Curriculum, University of North Carolina,
Chapel Hill, North Carolina 27514
SYNOPSIS In Drosophila melanogaster, the level of X-linked gene products is found to be
equivalent in normal males and females (dosage compensation) and in metafemales
(3X;2A); it is also equivalent in triploid females, intersexes (2X;3A) and metamales
(XY;3A). In all instances, the expression of X-linked genes is regulated in such a fashion
that it is concordant with the level of autosomal gene activity. This means that at least five
different transcriptional levels exist for X-linked structural genes; the lowest in
metafemales, the highest in metamales, and three different intermediate levels, in females
(diploid or triploid), intersexes and males. Two models have been proposed for the
regulatory mechanism. These models are discussed and current experimental approaches
are reviewed.
INTRODUCTION
THE PHENOMENON
With the probable exception of genes
for sex determination, structural genes on Dosage compensation in diploid males and
the X chromosome of Drosophila are ex-females
pressed equally in both sexes, in spite of
The characteristic relationships involved
the fact that they are present in two doses are represented in Figure 1. If compensain somatic cells of females and in a single tion is operative, normal males and
dose in males. This phenomenon was females have an equivalent level of prodnamed "dosage compensation" by Muller uct for a given X-linked gene g. Note that
(Muller et al., 1931). It represents an excel- a single dose of g in a female leads to half
lent model system with which to study of the product found in a normal male,
coordinate regulation of gene activity in a while two doses of g in a male lead to twice
eukaryotic organism since it appears to the product found in a normal female. In
involve modulation of the transcriptional fact, it is evident that, while dosage comlevel of a sizable fraction of the genome. In pensation exists between the normal sexes,
this paper, I propose to review the various "dosage dependence" prevails within one
characteristics of dosage compensation, cit- sex (viz., the levels of gene product exhibing references only as illustrations of the ited by females with one, two or three
work reported. I shall also discuss two doses of g, or by males with one or two
models of the regulatory mechanism and doses of this gene).
the working hypothesis motivating our
The relationships just discussed have
current experimental attack. All observabeen
established at three levels of gene
tions and analyses described herein have
been performed on Drosophila melanogaster activity. They were uncovered using measurements of terminal phenotypic pro(Meigen).
ducts: the gene g of Figure 1 could be
replaced by wa (white apricot), a hypomorI am indebted to Dr. Gustavo P. Maroni for his phic mutant allele of a gene involved in the
valuable comments and suggestions during the preparation of this manuscript. Research in my labora- synthesis and/or deposition of eye pigments (Bridges, 1922; Muller et al., 1931).
tory was supported by NIH Grant GM-15691.
685
686
JOHN CHARLES LUCCHESI
Low
Gene Product
I
High
2
FIG. 1. Diagrammatic statement of dosage compensation and its corollaries. Normal males and females
have equivalent levels of total gene product for a
given X-linked gene g, arbitrarily set at 2. In comparison, females heterozygous for a deficiency encompass-
ing the locus of g exhibit less gene product, while
females and males heterozygous for a duplication of g
exhibit 50 per cent and 100 per cent more product,
respectively. Note the gene dosage dependence of
product within a sex.
Dosage compensation was also found to
occur at the level of RNA synthesis measured, in autoradiographs, as a function of
the incorporation of tritiated uridine along
the giant polytene chromosomes of larval
salivary glands. Total chromosomal RNA
synthesis by both X's in females is equivalent to that synthesized by the single X
chromosome in males (Mukherjee and
Beermann, 1965). Dosage dependence
within a given sex can also be demonstrated by measurements of RNA synthesis
(Korge, 1970; Holmquist, 1972). Finally,
dosage compensation and dosage dependence were established for activity levels of
enzymes whose structural genes are located on the X chromosome (Komma,
1966; Seecof et al., 1969). For example, the
gene g of Figure 1 could represent the
structural gene for 6-phosphogluconate
dehydrogenase; in this case, gene product
would be the level of 6-PGD activity in
crude extracts.
The concordance of the measurements
made at the level of terminal phenotypic
product, enzyme activity and chromosomal RNA synthesis strongly suggests that the
regulatory mechanism affects the level of
X chromosome transcription, a contention
sustained by the recent work of Korge
(1976). This investigator reported equivalent amounts of a given protein in the
salivary gland secretion of male and
female larvae. The structural gene of this
protein is located on the X chromosome at
(or near) the site of a puff whose presence
seems to be correlated with the presence of
the protein in the secretion. In addition,
Korge was able to demonstrate dosage
dependence of the secretory protein
within each sex.
Both X chromosomes are active in females
Equalization of X-linked gene products
in males and females is not achieved by
inactivating one of the two X's in female
somatic cells as is the case in mammals. In
Drosophila, there is no Barr body equivalent. Furthermore, two lines of evidence
indicate that both X chromosomes are
simultaneously active in cells of the female
soma. The first consists of the fact that
females heterozygous for recessive mutants such as y (yellow hairs, bristles and
body color), w (white eyes),/ (forked hairs
and bristles) are uniformly wild type in
phenotype. If the X bearing the wild type
allele were inactivated in some cells at
some time during development, patches of
DOSAGE COMPENSATION IN DROSOPHILA
687
mutant tissue would ensue, leasing to a
mosaic phenotype.
The second line of evidence, provided
by the study of allozymes, is illustrated in
Figure 2. In this example, two purebred
lines are shown to exhibit a slow and a fast
electrophoretic variant of 6-phosphogluconate dehydrogenase. A cross between
the two lines yields F t females which bear
the two parental forms of the enzyme plus
an intermediate form. This observation is
consistent with a dimeric structure for
6-PGD and the simultaneous synthesis of
the fast and slow subunits within the same
cells.
Drosophila, it is not surprising that gene
activity of X and autosome segments is
autonomous in translocations: X-linked
genes are subjected to dosage compensation even when they are relocated
elsewhere in the genome; autosomal genes
transposed to the X do not become dosage
compensated. An example of the evidence
upon which the former contention is based
is provided in Figure 3A. The gene g is the
structural gene for the enzyme tryptophan
pyrrolase. Although it has been translocated to an autosome, this gene exhibits
dosage compensation in that males with
one dose and females with two doses of the
transposed segment have equal levels of
Autonomous behavior of chromosome segments tryptophan pyrrolase activity.
in X-autosome translocations
That an autosomal gene relocated to the
X is not ipso facto dosage compensated has
A corollary of Barr body formation in been recently established by Dr. R. L.
female mammals is the potential inactiva- Roehrdanz and J. M. Kitchens in our
tion of an autosomal segment translocated laboratory (unpublished data). One of the
to the X; in the reciprocal occurrence, a pertinent comparisons which they persegment of the X translocated to the auto- formed is presented in Figure 3B. The
somes may be refractory to inactivation translocated autosomal segment bears the
and heterochromatization. Since there is structural gene for aldehyde oxidase; if it
no somatic X chromosome inactivation in were subjected to dosage compensation, it
should prove twice as active in males as in
I
females. This is not the case since the
males and females in Figure 3B have equivalent levels of enzyme activity.
77Z
Dosage compensation in heteroploids
The work to be discussed in this section
deals with X-linked gene activity in
genotypes which differ from the basic male
and female constitutions by the presence
of additional whole X chromosomes or
whole sets of autosomes.
X-linked gene activity has been determined in triploid females (3X;3A) and
intersexes (2X;3A), as a function of enzyme levels (Lucchesi and Rawls, 1973)
B
A
Pgd
Pgd
and chromosomal RNA synthesis (Maroni
and Plaut, 1973a). Similar measurements
Pgd B
PgdA
were performed on metafemales (3X;2A)
FIG. 2. Polyacrylamide gel electrophoresis of by Lucchesi et al., (1974). I should men6-phosphogluconate dehydrogenase allozymes. Gels tion that, while our results regarding
1 and 2 represent extracts of slow and fast variant- X chromosome activity in metafemales
bearing strains, respectively; gel 3 is a mixture of are at odds with those of a group of
these two extracts; gel 4 represents enzyme activity
zones in an extract from females heterozygous for the Russian investigators (Faizullin and
Gvozdev, 1973; Ananiev et al., 1974),
two variants (after Kazazian et al., 1965.)
77T,
688
JOHN CHARLES LUCCHESI
V///////////.
'////////A
Y//////////A
'////////A
A
Y///////////. V///////A
Y//////A
B
X/////////////////A
|
FIG. 3. Autonomous behavior of X and autosomal
fragments in translocations. X chromosome material
is represented by thick, solid lines; the Y chromosome
is a thin, solid, J-shaped symbol; autosomes are
cross-hatched. 3A. A section of the X chromosome,
bearing the structural gene for tryptophan pyrrolase,
relocated to an autosome, remains dosage compensated as evidenced by the equivalence in enzyme
activity of males with one and females with two doses
of the translocation (after Tobler et al., 1971). 3B.
Autosomal genes translocated to the X fail to become
dosage compensated as evidenced by the observation
that males and females heterozygous for a relocated
structural gene for aldehyde oxidase exhibit similar
levels of enzyme activity (Roehrdanz et al. unpublished results).
they were recently confirmed by Stewart
and Merriam (1975). Finally, in collaboration with Dr. G. Maroni and J. Belote, we
have recently succeeded in measuring
X-linked gene activity in metamales
(XY;3A). These preliminary data indicate
that metamales are almost equivalent to
triploid females and, thereby, exhibit substantial, if not complete, dosage compensation.
The conclusions which can be drawn
from the work with heteroploids are rep-
DOSAGE COMPENSATION IN DROSOPHILA
689
resented in Figure 4. Total product per basically triploid forms: females, intercell for X-linked genes is the same in sexes and, presumably, metamales. Here,
males, females and metafemales, with one, total gene product per cell is fifty per cent
two and three X chromosomes, respec- greater than in diploids and is concordant
tively. One may well ask why this is the with a similar increment in autosomal gene
case. It seems likely to me that X-linked expression mediated by the presence of an
genes are regulated in such a fashion that extra set of autosomes.
their activity is in harmony with that of
In order to maintain a balance between
autosomal genes. The latter is obviously X and autosomal activity, metasexes and
equivalent in all three genotypes since they intersexes make use of dosage compensahave similar autosomal complements. The tion, the mechanism evolved to equalize X
same logical argument can be extended to chromosome output in normal males and
2.5
(/)
O
Q
CD
\
2.0
Y//////////A
1.5
>
<
.0
Y//////////1
0.66
Total Product/Cell
FIG. 4. Summary of measurements of X-linked
gene activity in normal and heteroploid genotypes.
Metamales (XY;3A) are shown as being substantially,
if not fully, compensated. See text for the various
references upon which this figure is based.
690
JOHN CHARLES LLCCHESI
females. We have measured five levels at
which X-linked structural genes can operate (ordinate in Figure 4). Note that the
range of gene activity is probably not discontinuous and that any level within the
limits of the range represented can occur.
In fact, I believe that some of the published results of Stewart and Merriam
(1975) show this to be the case, although
they chose to interpret these data differently. In any event, this constitutes a
unique model system with which to study
transcriptional regulation since it appears
that one can set the activity of a large
group of structural genes (perhaps all of
the genes on the X, i.e. 20 per cent of the
structural genome of D. melanogaster) at a
given level by synthesizing genotypes with
the appropriate combination of X's and
autosomes.
THE MODELS
Two basic models have been proposed
to explain dosage compensation and all of
its corollaries.
The first model, originally formulated
by Muller (1950), states that dosage compensation can be achieved by a decrease in
gene expression such that two doses of an
X-linked gene in females are reduced to a
level of activity comparable to that of a
single dose in males. The assumption is
made that there are on the X chromosome
certain regulatory genes called "compensators" whose products have an overall
inhibitory effect, commensurate with their
concentration in the nucleus, on other
X-linked genes. This model is, therefore,
conveniently labelled a "mass action
model." The rate of transcription (Tr) for
a given structural gene would be maximal,
were it not for the presence of specific
inhibitors (I) in the nucleus:
Tr(max)
[I]
A necessary aspect of the model is that the
compensators themselves are not compensated. If [I] were, in fact, the concentration
of inhibitor molecules present in a male, in
a female there would be twice as much [I],
since there are two doses of compensators
present; this would halve the value of Tr.
In metafemales, there would be three
times as much [I] and Tr would be onethird of its value in males. Total gene
product per cell is obtained by multiplying
the rate of transcription per gene for a
given karyotype by the number of doses of
the gene in that karyotype. This yields
equivalent levels in males, females and
metafemales. X activity in the basically
triploid genotypes (metamales, intersexes
and females) is explained by this formulation if one considers that, since the cells
and their nuclei are proportionately
larger, the concentration of the inhibitor
in these forms is two-thirds of its value in
diploids.
One extreme version of this model
would have separate regulatory elements
for each structural gene on the X chromosome. Each of these elements may have
either a stimulating or inhibitory action
while their overall effect would be one of
repression of transcription. Needless to
say, such compensation would be difficult
to demonstrate and even more difficult to
characterize. The other extreme version of
the model calls for a single, major compensator regulating the activity of most or all
X-linked structural genes. Stewart and
Merriam (1975) have searched for such a
major locus on the X. They did this by
systematically duplicating the entire X
chromosome, one small segment at a time.
If it included the major compensator, such
a duplication in a male would result in
twice the concentration of inhibitor normally present. This would reduce the activity of all structural genes on the X by one
half. No such region was uncovered. I
would like to point out, once again, that
such a reduction in the expression of
X-linked genes should cause such imbalance between X and autosomal products
that viability should be grossly impaired.
The above considerations are illustrated in
Figure 5. Stewart and Merriam's data also
failed to demonstrate the existence of
major compensators specific for the two
X-linked structural genes monitored. In
conclusion, if they exist, negative compen-
DOSAGE COMPENSATION
691
WDROSOPMLA
MASS ACTION MODEL
X/A Gene Products
Balanced,viable
Lethal?
FIG. 5. Upper portion: effect of a single major
compensator in a male. Lower portion: two doses of
the compensator reduce the activity of X-linked structural genes to one-half of its normal value. The Y
chromosome is omitted for the sake of simplicity.
Negative signs are used to emphasize the repressive
effect of the regulatory factor in the mass action
model. The X chromosome is solid, the autosomes
are cross-hatched.
sators must be numerous and have small
individual effects.
The second, or "competition model,"
first published by Maroni and Plaut
(I973a,b) and, independently, by Schwartz
(1973) suggests that equalization of gene
products in males and females is achieved
by enhancing gene activity. The assumption is made that there are autosomal
genes whose products are necessary to
mediate or enhance the transcription of
X-linked structural genes. The level of
enhancer molecules in the nucleus (E) is
proportional to the sets of autosomes in
the genome. If the number of molecules is
relatively low and the number of X-linked
genes (n) competing for them is relatively
high, an increase in doses of X chromosomes will reduce the amount of enhancer
available per gene, i.e.., the rate of its
transcription:
doses of the gene in the genome. When
this is done, the levels of total gene product observed in the euploids and heteroploids of Figures 1 and 4 are, in fact,
expected. Here again, one extreme version
of this model would have different autosomal regulatory genes for small groups
of structural genes on the X chromosome.
The other extreme is a situation where a
single autosomal compensator regulates
the activity of most or all structural genes.
„
, E
no. A
Tr = k — =
—
n
no. X
As in the previous model, the total product
per cell for a given X-linked structural
gene is obtained by multiplying the rate of
transcription per gene by the number of
A WORKING HYPOTHESIS
It occurred to me that if there were a
major autosomal compensator, a duplication including its locus, introduced in a
basically diploid male or female, should
lead to a fifty per cent increase in X-linked
gene activity without any concomitant increase in autosomal expression. This
should result in lethality. Similarly, a single
dose of the regulatory site (in individuals
heterozygous for a small deficiency encompassing its locus) should halve X activity, again leading to genie imbalance and
lethality. These considerations are illustrated in Figure 6. So I asked myself if
there were regions of the genome which
are triplo-lethal and haplo-insufficient.
692
JOHN CHARLES LUCCHESI
COMPETITION MODEL
X/A Gene Products
s///////////////\
+
1
-^^^
'///////////////*
+v
////////////////A
+
%
\ ^ -
Balanced, viable
^
Lethal ?
Lethal?
^
FIG. 6. Upper portion: effect of a major autosomal
compensator in a male. Middle and lower portions: a
single and a triple dose of the compensator leading to
a deficiency or excess of X-linked gene products in
relation to autosomal genes. The Y chromosome is
omitted for simplicity. Positive signs are used to
underscore the enhancing effect of regulatory
molecules in the competition model. The X chromosome is solid, the autosomes are cross-hatched.
The answer appeared to be affirmative: gene function or compensation. The meiLindsley and Sandier et al. (1972) reported otic mutant may represent a defective regthe occurrence of a region located at ulatory function which would allow proper
83D-E on the cytological map of D. compensation to occur (since the flies are
melanogaster which seems to be the only perfectly viable) but would interfere in
region of the genome to cause lethality some indirect fashion with the preparation
when present in a single dose or in three of the X for the process of crossing-over.
The involvement of 83D-E (and of mei-1,
doses in otherwise diploid flies.
An additional fact, concerning the kindly made available by Dr. J. Valentin) in
83D-E region, was recently brought to my X chromosome activity is currently being
attention by Dr. Bruce Baker. There exists investigated by Dr. R. Roehrdanz in our
a meiotic mutant, mei-1, isolated by Valen- laboratory, in collaboration with Dr. R. E.
tin (1973), which is unique among all other Denell of Kansas State University, who has
meiotic mutants tested to date in that its synthesized some very useful chromoeffect (reduction of recombination) is re- somes for this purpose.
stricted to the X chromosome. This unBefore ending this presentation I should
usual mutant happens to map at or near briefly mention two additional experi83D-E. This amazing coincidence en- mental approaches which we are currently
hances the hope that the region contains a undertaking to study the regulatory
regulatory site responsible for X-linked mechanism responsible for dosage com-
DOSAGE COMPENSATION IN DROSOPHILA
693
pensation. The first is a genetic study of Lindsley, D. L., L. Sandier, B. S. Baker, A. T. C.
Carpenter, R. E. Denell, J. C. Hall, P. A.Jacobs, G.
regulatory sites, adjacent to X-linked strucL. Gabor Miklos, B. K. Davis, R. C. Gethmann, R.
tural genes, which mediate the latter's reW. Hardy, A. Hessler, S. M. Miller, H. Nozawam,
sponse to the regulatory signal. To this
D. M. Parry, and M. Gould-Somero. 1972. Segmental aneuploidy and the genetic gross structure of
- end we are screening for mutants asthe Drosophila genome. Genetics 71:157-184.
sociated with several X-linked structural
genes coding for specific enzymes; the Lucchesi, J. C. and J. M. Rawls. 1973. Regulation of
gene function: a comparison of X-linked enzyme
desired mutant phenotype would be a lack
activity levels in normal and intersexual triploids of
of dosage compensation. The second apDrosophila melanogaster. Genetics 73:459-464.
proach, a major effort to isolate X-binding, Lucchesi, J. C.,J. M. Rawls, Jr., and G. Maroni. 1974.
non-histone chromosomal proteins is
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