ISSN 0006-2979, Biochemistry (Moscow), 2024, Vol. 89, No. 4, pp. 663-673 © Pleiades Publishing, Ltd., 2024.
663
Functional Role of C-terminal Domains
in the MSL2 Protein of Drosophila melanogaster
Evgeniya A. Tikhonova
1
, Pavel G. Georgiev
1
, and Oksana G. Maksimenko
1,a
*
1
Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia
a
e-mail: maksog@mail.ru
Received September 30, 2023
Revised December 11, 2023
Accepted December 12, 2023
AbstractDosage compensation complex (DCC), which consists of five proteins and two non-coding RNAs roX,
specifically binds to the X chromosome in males, providing a higher level of gene expression necessary to compen-
sate for the monosomy of the sex chromosome in male Drosophila compared to the two X chromosomes in females.
The MSL2 protein contains the N-terminal RING domain, which acts as an E3 ligase in ubiquitination of proteins
and is the only subunit of the complex expressed only in males. Functional role of the two C-terminal domains
of the MSL2 protein, enriched with proline (P-domain) and basic amino acids (B-domain), was investigated. As a
result, it was shown that the B-domain destabilizes the MSL2 protein, which is associated with the presence of
two lysines ubiquitination of which is under control of the RING domain of MSL2. The unstructured proline-rich
domain stimulates transcription of the roX2 gene, which is necessary for effective formation of the dosage com-
pensation complex.
DOI: 10.1134/S0006297924040060
Keywords: dosage compensation, long non-coding RNAs, MSL1, roX, MSL complex, ubiquitination
Abbreviations: CES,chromatin entry sites; DCC,dosage compensation complex.
* To whom correspondence should be addressed.
INTRODUCTION
Dosage compensation is the phenomenon of equal-
izing gene expression levels in organisms with differ-
ent numbers of sex chromosomes. The mechanisms of
dosage compensation in insects have been studied us-
ing the model organism Drosophila melanogaster [1-4].
Dosage compensation in Drosophila is based on forma-
tion of the RNA–protein complex, which is recruited
to the male X chromosome and enhances gene expres-
sion by approximately two-fold. The dosage compensa-
tion complex (DCC) in Drosophila comprises five pro-
teins (MSL1, MSL2, MSL3, MOF, and MLE) and two long
non-coding RNAs (roX1 and roX2). The subunits of DCC
are highly conserved among animals, and the complex
consisting of MSL1, MSL2, MSL3, and MOF proteins
plays an important role in transcription regulation but
not in dosage compensation in humans [5,6].
Protein MSL2 is expressed exclusively in males
and is considered as a key component of the dos-
age compensation complex [1, 2]. The MSL2 protein
(Fig. 1a) consisting of 773 amino acids contains two
highly conserved domains: N-terminal RING-domain
and CXC-domain [4,7]. The RING-domain is a conserved
domain in MSL2 proteins of humans and Drosophila,
which functions as a ubiquitin E3 ligase mediating
ubiquitination of the specific substrates including core
subunits of the dosage compensation complex [8, 9].
At the same time, the RING-domain is involved in in-
teraction of MSL2 with the N-terminal coiled-coil do-
main of MSL1, which forms a homodimer [10-13].
MSL1 and MSL2 form core of the complex, which
can specifically bind to certain male X-chromosome
sites independent on other components of the dosage
compensation complex [10]. MSL3 protein and acetyl
transferase MOF interact with the C-terminal domain
of MSL1 [14, 15]. The MLE helicase, belonging to the
ATP-dependent RNA/DNA helicase family, specifical-
ly remodels secondary structure of the roX RNAs to
increase their efficiency in formation of the dosage
TIKHONOVA et al.664
BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024
compensation complex [16-18]. The second conserved
domain of MSL2, CXC-domain (Zn3Cys9), is the only
DNA binding domain found in the Drosophila dosage
compensation complex proteins [19]. Structural analy-
sis showed that the two CXC-domains can specifically
bind to GA-repeats [20]. The incomplete dosage com-
pensation complex comprising the core part of the
MSL1–MSL2 complex binds to approximately 200 sites
on the X chromosome, called chromatin entry sites
(CES) [21] or high affinity sites (HAS) [22]. DNA ele-
ments rich in GA-repeats have been found in the CES
region, which can bind the CXC-domain of the MSL2
protein [23]. The transcription factor CLAMP with its
N-terminal zinc finger C2H2-type domain interacting
with the unstructured region of amino acids 618-655 of
the MSL2 protein [25-27] also binds to CES [24]. CLAMP
has an N-terminal homodimerizing domain [28] and
is involved in organizing distant contacts between the
DCC binding sites [29]. It has been shown that the CXC
and CLAMP-interacting domains of MSL2 jointly par-
ticipate in the binding of MSL complex to the X chro-
mosome in males [25].
The unstructured C-terminus of the MSL2 protein
contains two regions: proline-rich (P-domain) and ba-
sic amino acid-rich (B-domain). The B-domain contains
one of the numerous sites where self-ubiquitination of
the MSL2 protein occurs in vitro [8]. It is hypothesized
that the C-terminus specifically binds to roX RNA, pro-
viding efficient assembly of the MSL complex and in-
clusion of the MLE protein [30]. Some experimental
data suggest that the C-terminus is involved in specific
recognition of the GA-rich regions on the X chromo-
some by the CXC-domain in the male mammals [23].
Aim of this study was to elucidate functional role of
the C-terminal regions in the MSL2 protein.
MATERIALS AND METHODS
Plasmid construction. For expression of the
3xFLAG-tagged full-length MSL2, wild-type and dele-
tion variants corresponding to the P- and B-domains
of the protein were fused with 3xFLAG at the C-termi-
nus and cloned into an expression vector. The vector
contains an attB site for φC31-dependent integration,
a strong Ubi-p63E gene promoter with its 5′-UTR, last
intron of the dctcf gene with its 3′-UTR, and SV40 polya-
denylation signal. The intronless yellow gene was used
as a reporter to screen for transformants. Details of
the cloning procedures, primer sequences, and plas-
mids are available upon request.
Fly crosses and transgenic lines. Drosophila
melanogaster lines were maintained at 25°C on stan-
dard yeast medium. Transgenic constructs were in-
jected into preblastodermal embryos. Integration of
constructs into the genome was achieved through the
φC31-mediated site-specific integration at the 86F8 lo-
cus in the corresponding line with an attP site [31].
Flies obtained after injection were crossed with the
y
1
w
1118
laboratory flies, and transgenic offspring were
identified by cuticular structure pigmentation. Homo-
zygous lines were obtained through a series of crosses
via balancer chromosomes. Lines that were lethal in
the homozygous state were maintained on balancer
chromosomes. Details of crosses are available upon
request.
Antibodies. Antibodies against MSL1 [423-1030],
MSL2 [421-540], CLAMP [222-350] were raised in rab-
bits and purified from serum using ammonium sulfate
fractionation followed by affinity purification on a CNBr-
activated sepharose (GE Healthcare, USA) or Amino-
link Resin (Thermo Fisher Scientific, USA) according to
the standard protocols. Mouse monoclonal antibodies
against FLAG epitope (clone M2) were obtained from
Sigma (USA).
Fly extract preparation. Twenty adult flies were
homogenized with a pestle in 200 µl of 1×PBS contain-
ing 1% β-mercaptoethanol, 10 mM PMSF, and 1 : 100
Calbiochem Complete Protease Inhibitor Cocktail VII.
The suspension was sonicated 3 times for 5 s at 5 W.
Next, 200 µl of 4×SDS-PAGE buffer was added, and the
mixture was incubated for 10 min at 100°C and centri-
fuged at 16,000g for 10 min.
Immunostaining of polytene chromosomes.
Drosophila 3rd instar larvae were raised at 18°C un-
der standard conditions. Immunostaining of polytene
chromosomes was performed as described previously
[32]. The following primary antibodies were used: rab-
bit anti-MSl1 at dilution of 1 : 100, rabbit anti-MSL2 at
dilution of 1 : 100, and mouse monoclonal anti-FLAG at
dilution of 1 : 100. Secondary antibodies were goat an-
ti-mouse conjugated with Alexa Fluor 488 used at di-
lution of 1 : 2000 and goat anti-rabbit conjugated with
Alexa Fluor 555 used at dilution of 1 : 2000 (Invitrogen,
USA). Polytene chromosomes were also stained with
DAPI (AppliChem, USA). Images were captured using
a Nikon Elclipse Ti fluorescent microscope equipped
with a Nikon DS-Qi2 digital camera and processed us-
ing ImageJ 1.50c4 and Fiji bundle 2.0.0-rc-46 software.
Three to four independent stainings were performed,
and 4-5 samples of polytene chromosomes were ob-
tained for each transgenic line expressing MSL2.
Chromatin immunoprecipitation. Chromatin
preparation was performed according to the described
protocols [33, 34] with some modifications. Samples of
500 mg each of adult flies were ground in a mortar in
liquid nitrogen and resuspended in 10 ml of a buffer A
(15 mM HEPES-KOH, pH 7.6, 60 mM KCl, 15 mM NaCl,
13 mM EDTA, 0.1 mM EGTA, 0.15 mM spermine, 0.5 mM
spermidine, 0.5% NP-40, 0.5 mM DTT, supplemented with
0.5 mM PMSF and Calbiochem Complete Protease In-
hibitor Cocktail V). The suspension was then homoge-
FUNCTIONS OF C-TERMINAL DOMAINS IN THE MSL2 PROTEIN 665
BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024
nized in a Dounce homogenizer with tight pestle and
filtered through a 70-µm Nylon Cell Strainer (BD Bio-
sciences, USA). The nuclei were pelleted by centrifuga-
tion at 4000g, 4°C, for 5 min in a buffer supplemented
with sucrose, resuspended in a wash buffer (15 mM
HEPES-KOH, pH 7.6, 60 mM KCl, 15 mM NaCl, 1 mM
EDTA, 0.1 mM EGTA, 0.1% NP-40, Calbiochem Complete
Protease Inhibitor Cocktail V), and cross-linked using
1% formaldehyde for 15 min at room temperature.
Cross-linking was stopped by adding glycine to a final
concentration of 125 mM. The nuclei were washed with
three 10-ml portions of wash buffer and resuspend-
ed in 1.5 ml of a nuclear lysis buffer (15 mM HEPES,
pH 7.6, 140 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 1% Tri-
ton X-100, 0.5  mM DTT, 0.1%  sodium deoxycholate,
0.1% SDS, Calbiochem Complete Protease Inhibitor
Cocktail V). The suspension was sonicated (20×30  s
with 60 s intervals, on ice at 50% output), and 50-µl
aliquots were used to test the extent of sonication and
to measure DNA concentration. Debris was removed by
centrifugation at 14,000g, 4°C, for 10  min, and chroma-
tin was pre-cleared with a Protein A agarose (Pierce,
USA), blocked with BSA and salmon sperm DNA; 50-µl
aliquots of such pre-cleared chromatin samples were
stored as input material. Samples containing 10-20 µg
of DNA equivalent in 1 ml of nuclear lysis buffer
were incubated overnight at 4°C with rabbit antibod-
ies against MSL1 (1 :  500), MSL2 (1  : 200), and CLAMP
(1 :  200), or with nonspecific IgG purified from rabbit
preimmune sera (control). Chromatin–antibody com-
plexes were collected using blocked Protein A agarose
at 4°C over 5 h.
After three rounds of washing with lysis buffer
(as such and with 500 mM  NaCl) and a single wash
with TE buffer (10 mM  Tris-HCl, pH 8; 1 mM  EDTA),
the DNA was eluted with an elution buffer (50 mM
Tris-HCl, pH 8.0; 1  mM  EDTA, 1% SDS) at 65°C, proteins
and RNA were removed by adding proteinase K and
RNase A. DNA was purified using phenol-chloroform
extraction followed by reprecipitation. Enrichment of
specific DNA fragments was analyzed by real-time PCR
using a QuantStudio12K Flex Cycler (Applied Biosys-
tems, USA).
At least three independent biological replicates
were made for each chromatin sample. The results of
chromatin immunoprecipitation are presented as per-
centage of input genomic DNA normalized to a positive
control genomic site (a genomic site outside the CES to
which the protein of interest binds). The tubulin-γ37C
coding region (devoid of binding sites for the tested
proteins) was used as a negative control; autosomal
MSL1-binding region 26E3, MSL2-binding region 25A3,
and CLAMP-binding region 39A1 were used as positive
genomic controls.
RNA isolation and quantitative analysis. Total
RNA was isolated from 2- to 3-day-old adult males and
females using a TRI reagent (Molecular Research Cen-
ter, USA) according to the manufacturers instructions.
RNA was treated with two units of Turbo DNase I (Am-
bion, USA) for 30 min at 37°C to eliminate genomic
DNA. Synthesis of cDNA was performed using 2 μg of
RNA, 50 U of ArrayScript reverse transcriptase (Ambi-
on), and 1 μM of oligo(dT) as a primer. The amounts
of specific cDNA fragments corresponding to roX1 and
roX2 were quantified by real-time PCR with Taqman
probes. At least three independent measurements
were made for each RNA sample. Relative levels of
mRNA expression were calculated in the linear am-
plification range by calibration using a standard ge-
nomic DNA curve to account for differences in primer
efficiencies. Individual expression values were nor-
malized to RpL32 mRNA as a reference.
RESULTS
Study of functional role of the B- and P-domains
of the MSL2 protein. The unstructured C-terminus
of MSL2 (Fig. 1a) contains a proline-rich region (Pro-
line-rich, P-domain 685-713 aa) and a region rich in
basic amino acids (Basic-rich, B-domain, 715-728 aa).
Both regions have a moderate level of conservation
among different Drosophila species (Fig. 1b). However,
several studies [30, 35-37] have provided experimental
evidence that the C-terminal region of MSL2 interacts
with roX RNA. Moreover, interaction of MSL2 with roX
is important for the specific recruitment of DCCs to the
male X chromosome [36, 37]. It was previously shown
that deletion of the region 743-773 aa does not affect
functions of the MSL2 protein in vivo [30]. Therefore,
in this work, we investigated functional role of the ad-
jacent P- and B-domains of the MSL2 protein.
For this purpose, MSL2 cDNA variants with dele-
tions of sequences encoding regions 685-713 aa (MSL2
ΔP
)
or 715-728 aa (MSL2
ΔB
) were obtained. To express the
tested proteins, cDNA was inserted into an expression
vector (Fig. 2a) under control of the strong promoter
of the Ubiquitin-p63E (U) gene. cDNA for the MSL2 pro-
tein did not contain noncoding parts of the msl-2 gene
mRNA, which have binding motifs for the translation
repressor Sxl in females [38]. As a result, the U:msl-2
WT
transgene is expressed at the same level in males and
females.
The cDNAs to be cloned were fused in a single frame
with the sequence encoding 3 copies of the FLAG epi-
tope. The resulting transgenes (U:msl-2
ΔP
and U:msl-2
ΔB
)
were integrated into the 86Fb region on chromosome
3 using recombination system based on the φC31 inte-
grase [31]. As a control, we used the previously obtained
U:msl-2WT (86Fb) line expressing wild-type MSL2
protein, MSL2
WT
-FLAG [25]. To determine the level of
expression of MSL2 mutants relative to the control,
TIKHONOVA et al.666
BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024
Fig. 1. Structural organization of the MSL2 protein. a) Scheme of the MSL2 protein. Main domains are shown: RING-, CXC-,
CLAMP-binding, P- and B-domains. b) Clustal Omega sequence alignment of the C-terminal part of the MSL2 protein in the
well-studied Drosophilidae species. P-domain is highlighted with an orange frame, and B-domain is highlighted with a green
frame.
the amount of protein was determined using immuno-
blot analysis of the extracts obtained from the adult
flies (Fig. 2b). It turned out that the MSL2
ΔP
-FLAG
protein is expressed at the level comparable to the
MSL2
WT
-FLAG, while at the same time expression of
the MSL2
ΔB
-FLAG increased 2-3-fold in comparison
with the MSL2
WT
-FLAG.
In the region 715-728 aa there are two sequen-
tially located lysines (K715K716) ubiquitination of
which invitro is catalyzed by the RING-domain of the
MSL2 protein [8]. The remaining lysines ubiquitinated
invitro by the RING-domain were localized in the re-
gion of aa 420-510.[8]. To determine contribution of the
K715K716 lysines to stability of the MSL2 protein, con-
structs under the control of the Ubiquitin-63E promot-
er were obtained for transient expression in the S2
cell culture (Fig. 2c): MSL2
WT
-FLAG (control), MSL2
ΔRING
-
FLAG (deletion of the RING-domain in the MSL2 pro-
tein), and MSL2
ΔB
-FLAG. Expression levels of the MSL2
variants were detected using immunoblot analysis.
The MSL2
ΔB
-FLAG and MSL2
ΔRING
-FLAG proteins were
expressed at approximately the same level, several
times higher than expression of the MSL2
WT
-FLAG.
Thus, it can be assumed that the amino acids K715K716
are the main targets for self-ubiquitination reducing
stability of the MSL2 protein.
To clarify functional role of the P- and B-domains
in the dosage compensation, ability of the mutant vari-
ants of the protein to restore survival of the males
homozygous for the msl2
γ227
null mutation (2nd chro-
mosome), which leads to complete inactivation of the
msl-2 gene, was investigated [7]. The msl2
γ227
mutation
causes death of 100% of the males, predominantly at
the embryonic and early larval stages and does not af-
fect survival of the females. For the study, transgenic
lines msl2
γ227
/CyO were obtained; U:msl-2*/TM6,Tb, in
which msl2
γ227
and U:msl-2* transgenes were bred re-
spectively onto the CyO (2nd chromosome) and TM6,Tb
(3rd chromosome) balancers. Expression of the MSL2
variants was examined only in the males that have
one copy of the transgene (U:msl-2*/TM6,Tb). At the
same time, comparison was made of the survival rate
of males homozygous for the null mutation (msl2
γ227
/
msl2
γ227
) relative to the msl2
γ227
/CyO (control) males
with normal survival. As a result, it was shown that
survival rate of the males expressing MSL2
ΔP
and
FUNCTIONS OF C-TERMINAL DOMAINS IN THE MSL2 PROTEIN 667
BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024
Fig. 2. Obtaining transgenic lines expressing mutant MSL2 proteins. a) Scheme of the used expression vector. Promoter and
5′-UTR of the Ubiquitin-63E gene, last intron and 3′-UTR of the dctcf gene, and polyadenylation signal from the SV40 virus are
shown. MSL2 variants are presented below; dashed lines indicate locations of the introduced deletions. b) Immunoblot analy-
sis of the protein extracts obtained from the adult flies expressing various MSL2 variants tagged with the 3×FLAG epitope (WT,
ΔP,ΔB). Immunoblot analysis was performed using antibodies that specifically recognize FLAG and GAF (internal loading con-
trol). c) Comparison of the expression of MSL2
WT
-FLAG, MSL2
ΔRING
-FLAG, and MSL2
ΔB
-FLAG proteins in S2 cells. Immunoblot
analysis was performed using antibodies that specifically recognize FLAG and lamin (internal loading control). d) Comparison
ofviability (in relative percentage terms) of adult males msl2
γ227
/msl2
γ227
, in which the MSL2-3×FLAG variants were expressed
(WT, ΔP, ΔB). Number of msl2
γ227
/CyO males expressing MSL2 variants was used as an internal control with normal viability.
Ratio of adult males of line y
1
w
1118
; +/+ to males y
1
w
1118
; +/CyO was used as an indicator of survival of the wild-type line. Histogram
shows the means with standard deviations obtained from three independent experiments. *p< 0.05. e) Viability (in relative per-
centage terms) of females homozygous for the transgene relative to the adult males expressing MSL2 variants. Histogram shows
themeans with standard deviations obtained from three independent experiments. **p< 0.01.
MSL2
WT
at the background of null mutation in the
homozygote is slightly lower than that of the control
males, while survival of the MSL2
ΔB
is comparable to
the control males (Fig. 2d, Table 1). Thus, deletion of
the P-domain does not have a visible effect on the ac-
tivity of MSL2 in dosage compensation, while, at the
same time, MSL2
ΔB
functions more efficiently than
MSL2
WT
, which is probably due to the greater stability
of this MSL2 variant.
A sensitive model system for studying dosage
compensation has previously been described, which
is based on ectopic expression of MSL2 in females,
TIKHONOVA et al.668
BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024
Table 1. Male survival study (positive function of
dosage compensation)
Genotype
Msl2
γ227
/
msl2
γ227
msl2
γ227
/CyO
U:MSL2
WT
/TM6 138±3.2 163±1.1
U:MSL2
ΔP
/TM6 112±5.2 145±3.1
U:MSL2
ΔB
/TM6 90±1.2 86±2.3
Note. Analysis of the ratio of males with the msl2
γ227
/msl2
γ227
;
U:MSL2*/TM6 genotype to males with the msl2
γ227
/CyO;
U:MSL2*/TM6 genotype, which were obtained by crossing
the (F0) males with the msl2
γ227
/CyO; U:MSL2
WT
/TM6 genotype
with the msl2
γ227
/msl2
γ227
; U:MSL2
WT
/TM6 females.
Table 2. Female survival study (negative function
ofdosage compensation)
Genotype
MSL2/MSL2
males
MSL2/MSL2
females
U:MSL2
WT
/TM6 141±2.2 35±0.6
U:MSL2
ΔP
/U:MSL2
ΔP
162±5.2 159±3.1
U:MSL2
ΔB
/U:MSL2
ΔB
114±2.8 8±2
Note. Analysis of the ratio of the U:MSL2*/U:MSL2* females
toU:MSL2*/U:MSL2* or U:MSL2*/TM6 males.
resulting in the assembly of a functional DCC [39,40].
The more efficiently DCC assembles on the X chromo-
some, the more gene transcription increases, which di-
rectly correlates with the decrease in female viability
as a result of imbalance in the gene expression profile.
As expected (Fig. 2e, Table 2), the females carrying the
homozygous U:msl-2
WT
transgene are characterized by
the reduced viability (about 25% relative to the males).
In the females homozygous for the U:msl-2
ΔB
trans-
gene, there is a further decrease in survival. Sur-
prisingly, the females homozygous for the U:msl-2
ΔP
transgene have close to normal survival rates. Thus,
deletion of the P-domain in the MSL2 protein leads to
the partial disruption of dosage compensation only in
the more sensitive model system.
Comparison of MSL1 and MSL2 binding in males
and females expressing MSL2 variants. To study ef-
ficiency of the DCC binding to the X chromosome of
males, immunostaining of polytene chromosomes iso-
lated from the salivary glands of Drosophila larvae is
most often used, which makes it possible to visual-
ize proteins on the interphase chromatin [10, 41-43].
Inthe line msl2
γ227
;U:MSL2
WT
proteins MSL1 and MSL2
efficiently bind only to the X chromosome (Fig. 3a).
Similar results were obtained on the polytene chro-
mosomes of the males of the line expressing MSL2
ΔP
.
Thus, the results of binding of the MSL1 and MSL2 pro-
teins to polytene chromosomes fully confirm the re-
sults of the functional test (Fig. 2d), according to which
there are no disturbances in the process of formation
of the dosage compensation complex in the males ex-
pressing MSL2
ΔP
.
A similar study was carried out on the polytene
chromosomes from the salivary glands of the female
larvae (Fig. 3, b, c) expressing variants of the MSL2 pro-
tein. In the larvae expressing MSL2
WT
, the MSL1 and
MSL2 proteins cover the entire X chromosome ex-
cept for a few small regions. However, binding of the
MSL proteins to the X chromosome is less intense in
the females compared to the males. This is due to the
fact that in the females expression of the MSL1 pro-
tein and roX RNA is much weaker. Binding of the MSL
proteins is visually enhanced on the X chromosome of
the larvae expressing MSL2
ΔB
, which can be explained
by the significant increase in stability of the mutant
protein. The results are consistent with the function-
al test, according to which survival rate of the females
expressing MSL2
ΔB
is significantly lower compared to
the MSL2
WT
females (Fig. 2e). Binding of MSL2
ΔP
and
MSL1 to the X chromosome of the U:MSL2
ΔP
females is
significantly reduced. Intense staining with antibod-
ies against MSL1 and FLAG (MSL2) is observed only in
certain regions of the chromosome, which, apparently,
coincide with the strongest CES. Thus, MSL2
ΔP
disrupts
effective binding of DCC to the X chromosome of the
females.
Previous studies [10, 21, 22, 41, 44] showed that in-
activation of MSL3, or MLE, or roX RNA resulted in
the DCC recruitment to only small part of the regions
corresponding to the main CES including the regions
of the genes encoding roX1 (3F) and roX2 (10C). Thus,
the MSL2
ΔP
variant similarly leads to the decrease in
efficiency of the DCC formation, which is visualized
by preservation of the binding of the MSL1 and MSL2
proteins to the strongest CES on the X chromosome and
decrease in the binding to the secondary sites of DCC
recruitment.
To confirm this assumption, we compared binding
of the MSL1, MSL2, and CLAMP proteins with the most
well-studied CES on the X chromosome of the 2-3-day old
males using chromatin immunoprecipitation (Fig. 3d).
To compare DCC binding in the lines expressing
MSL2
WT
and MSL2
ΔP
, previously characterized repre-
sentative CES of the complex were selected: PionX sites
[23], HAS/CES sites [21, 22]. As a result, it was found
that MSL2
ΔP
and MSL2
WT
bind to all sites with approx-
imately the same efficiency. At the same time, at some
sites there is an excessive accumulation of the MSL2
ΔP
protein, which can be explained by partial redistri-
bution of the complex in the line expressing MSL2
ΔP
.
Similar results were obtained for MSL1. The CLAMP
protein binds to the tested CES with equal efficiency