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 QuantStudio12K 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
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,