Article

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

, Pavel G. Georgiev

, and Oksana G. Maksimenko

1,a

Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia

e-mail: maksog@mail.ru

Received September 30, 2023

Revised December 11, 2023

Accepted December 12, 2023

Abstract— Dosage 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

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 manufacturer’s 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

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

-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

-FLAG, while at the same time expression of

the MSL2

ΔB

-FLAG increased 2-3-fold in comparison

with the MSL2

-FLAG.

In the region 715-728 aa there are two sequen-

tially located lysines (K715K716) ubiquitination of

which in vitro is catalyzed by the RING-domain of the

MSL2 protein [8]. The remaining lysines ubiquitinated

in vitro 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

-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

-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

-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

1118

; +/+ to males y

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

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

, 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

/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

/TM6 genotype

with the msl2

γ227

/msl2

γ227

; U:MSL2

/TM6 females.

Table 2. Female survival study (negative function

ofdosage compensation)

Genotype

MSL2/MSL2

males

MSL2/MSL2

females

U:MSL2

/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

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

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

, 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

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

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

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

FUNCTIONS OF C-TERMINAL DOMAINS IN THE MSL2 PROTEIN 669

BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

Fig. 3. a) Comparison of the MSL1 and MSL2 binding to the polytene chromosomes of the msl2

γ227

male larvae expressing dif-

ferent variants of MSL2 (MSL2

, MSL2

ΔP

). b) Comparison of the MSL1 and MSL2 binding to the polytene chromosomes of the

female larvae heterozygous for the transgene expressing one of the MSL2 variants (MSL2

, MSL2

ΔP

, MSL2

ΔB

). The photographs

show immunostaining with mouse anti-FLAG antibodies (MSL2, green) and rabbit anti-MSL1 antibodies (red). DNA staining–

DAPI (blue). c) Comparison of distribution of the MSL2 protein along the polytene X chromosome in the females heterozygous

for the transgene expressing one of the MSL2 variants (MSL2

, MSL2

ΔP

). Two independent stainings are shown. MSL2 staining–

mouse anti-FLAG antibodies (red), DNA– DAPI (blue). d)Comparison of the MSL1, MSL2, and CLAMP proteins binding to CES

in the males expressing MSL2 variants (WT and ΔP) at the msl2

γ227

background. Red letters indicate regions to which MSL2 is

able to bind directly according to [23]. Results are presented as percent enrichment of DNA after immunoprecipitation to input

DNA (% input), normalized relative to the corresponding positive control MSL1 (26E3), MSL2 (25A3), and CLAMP (39A1) binding

sites on autosomes. The histograms show comparison of the level of MSL2

ΔP

protein binding with the level of MSL2

binding

(scaledto “1”). Whiskers show standard deviations of three independent experiments. *p < 0.05.

in all lines. Thus, the results obtained confirm that

MSL2

ΔP

maintains its effective binding to CES on the

Xchromosome.

P-domain determines ability of MSL2 to acti-

vate transcription. To study in more detail function-

al role of the P-domain in the process of dosage com-

pensation, we examined expression of the roX RNAs,

which are necessary for dosage compensation. In the

wild-type females, roX RNAs are not expressed due to

the absence of the MSL-containing complex that acti-

vates their transcription [45]. Expression of the wild-

type MSL2 in females results in significant activation

of the roX2 RNA and, to a lesser extent, of the roX1

RNA [46]. CES located near the roX genes are necessary

TIKHONOVA et al.670

BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

Fig. 4. Comparison of roX1 and roX2 in the females expressing MSL2

, MSL2

ΔP

, MSL2

ΔB

. a) Comparison of binding of MSL1, MSL2,

CLAMP proteins in the lines expressing MSL2

, MSL2

ΔB

, MSL2

ΔP

in the regions of the roX1 and roX2 genes. *p < 0.05; **p < 0.01.

b) Levels of roX1 and roX2 RNA expression in the larvae of male and female flies of the y

1118

line (wild type) and flies express-

ing MSL2

, MSL2

ΔP

, MSL2

ΔB

at the background of the msl2

γ227

null mutation. Histograms show changes in the mRNA levels of the

tested roX genes in the lines expressing MSL2

, MSL2

ΔP

, MSL2

ΔB

, compared to the expression level in the males of the y

1118

line

(corresponding to the “1” mark on the scale). Whiskers show standard deviations for three independent measurements.

to stimulate their transcription in males and to repress

them in females [47, 48]. Chromatin immunoprecip-

itation showed that MSL2 and MSL1 bind efficiently

to these sites in the female larvae expressing MSL2

MSL2

ΔB

, and MSL2

ΔP

proteins (Fig. 4a). Moreover, in

the female larvae of the MSL2

ΔB

line, the MSL1 protein

binds approximately 1.5 times stronger to the roX1

CES and 1.2 times stronger to the roX2 CES compared

to the control line MSL2

. A more pronounced in-

crease in the binding to the studied CES was detected

for the MSL2 protein (1.8 times stronger for the roX1

CES and 1.6 times stronger for the roX2 CES compared

to the control line MSL2

). In the case of females of

the MSL2

ΔP

line, the MSL1 protein binds to the roX

CES approximately 1.8-2 times weaker compared to

the control line MSL2

, and the MSL2 protein also

binds 2 times weaker with the roX1 CES, and approx-

imately 1.2 times weaker with the roX2 CES. Howev-

er, binding of the CLAMP protein in all lines remains

the same. In the males of the MSL2

ΔP

line, it was also

not possible to detect statistically significant changes

in the strength of binding of the MSL1, MSL2, CLAMP

proteins to the roX CES compared to the control

line MSL2

In the last part of the work, we confirmed that

the MSL2 expression in females induces roX RNA tran-

scription (Fig. 4b). However, the level of roX expres-

sion in such females is reproducibly lower compared

to the males of the same lines. Expression of MSL2

ΔB

the females results in an approximately 2-fold increase

in the roX RNA expression compared to the control

MSL2

line. Since increased binding of the MSL1 and

MSL2 proteins with the roX CES in females of this line

was observed, we can conclude that there is a direct

correlation between the efficiency of MSL1/MSL2 bind-

ing to the CES and activation of transcription of the

roX genes. This result is consistent with the data for

roX1 obtained for the MSL2

ΔP

line: approximately two-

fold decrease in the binding of MSL1/MSL2 proteins is

accompanied by the proportional decrease in the level

of roX1 expression. However, in the case of roX2 in the

MSL2

ΔP

line, a slightly different picture is observed:

the level of roX2 expression decreases 3.7-fold with

slight decrease in the level of MSL2 binding (1.2-fold)

compared to the control line MSL2

. The obtained

difference can be explained by the relative accuracy

of the qChIP method in measuring the amount of pro-

tein on chromatin. However, the significant difference

obtained between the amount of MSL2 associated with

CES and the level of roX2 expression suggests that the

P-domain in the MSL2 protein is involved in transcrip-

tion activation of the roX2 gene.

FUNCTIONS OF C-TERMINAL DOMAINS IN THE MSL2 PROTEIN 671

BIOCHEMISTRY (Moscow) Vol. 89 No. 4 2024

DISCUSSION

In this work, we investigated functional role of

two domains (rich in prolines and basic amino acids)

at the C-terminus of the MSL2 protein. As a result, it

was demonstrated that these domains do not have

a visible effect on the activity of DCC functioning in

males. According to the currently dominant ideas

[36, 37], C-terminus of the MSL2 protein interacts with

the roX RNA, which is critical for the DCC assembly.

Itcan be assumed that the C-terminal regions adjacent

to the P- and B-domains are responsible for specific in-

teraction of MSL2 with roX RNA, which requires fur-

ther study.

The results obtained in this work suggest that the

B-domain contains two lysines, which are the main

residues in MSL2 subjected to autoubiquitination lead-

ing to the significant decrease of the protein stabili-

ty. It can be assumed that upon interaction with the

coiled-coil domain of the MSL1 protein catalytic ac-

tivity of the RING-domain decreases, and, as a result,

the MSL2 protein, which is in complex with MSL1, is

stabilized. Thus, efficiency of the complex formation

increases, and, at the same time, concentration of the

free MSL2 decreases.

The MSL2 protein is capable of stimulating tran-

scription within DCC and also has an independent

function in activating transcription of a group of au-

tosomal promoters [49]. In mammals, the MSL2 ortho-

logue has been shown to ubiquitinate histone H3 at

lysine 24 [5]. Histones are enriched with this mark in

the areas of intense transcription. We have shown that

the proline-rich region of MSL2 may be involved in ac-

tivation of the roX2 gene transcription by the dosage

compensation complex. It is known that the unstruc-

tured proline-rich regions are capable of stimulating

transcription by attracting and stabilizing transcrip-

tion complexes at promoters. Interestingly, MSL1 also

stimulates transcription independently on the dosage

compensation complex by stabilizing binding to the

promoters of the cyclin-dependent kinase CDK7, which

accelerates release of the RNA polymerase II from the

pausing state [50].

In conclusion, our data indicate that the reduced

levels of roX RNA in the females expressing MSL2

ΔP

negatively affect efficiency of DCC formation. This is

manifested as a decrease in the amount of MSL1/MSL2

proteins associated with the roX CES and provides ad-

ditional confirmation of the key role of the roX RNA in

organizing dosage compensation.

Acknowledgments. Equipment of the Center for

High Precision Editing and Genetic Technologies for

Biomedicine of the Institute of Gene Biology of the

Russian Academy of Sciences was used in this work,

as well as equipment purchased with the financial

support of the Ministry of Science and Higher Edu-

cation of the Russian Federation under agreement

no. 075-15-2021-668.

Contributions. E.A.T. planned and performed ex-

periments, discussed the results; P.G.G. supervised the

study, discussed the results, and edited the manuscript;

O.G.M. planned and performed the experiments, dis-

cussed the results, supervised the study, and prepared

the manuscript.

Funding. This work was financially supported by

the Russian Science Foundation (grant no.21-14-00211).

Ethics declarations. This work does not con-

tain any studies involving human and animal sub-

jects. Theauthors of this work declare that they have

noconflicts of interest.

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