bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2024,
Номер
unknown
Опубликована: Март 16, 2024
Abstract
Immunomodulatory
imide
drugs
(IMiDs)
including
thalidomide,
lenalidomide,
and
pomalidomide,
can
be
used
to
induce
degradation
of
a
protein
interest
that
is
fused
short
zinc
finger
(ZF)
degron
motif.
These
IMiDs,
however,
also
endogenous
neosubstrates,
IKZF1
IKZF3.
To
improve
selectivity,
we
took
bump-and-hole
approach
design
screen
bumped
IMiD
analogs
against
8380
ZF
mutants.
This
yielded
analog
induces
efficient
mutant
degron,
while
not
affecting
other
cellular
proteins,
In
proof-of-concept
studies,
this
system
was
applied
TRIM28,
disease-relevant
with
no
known
small
molecule
binders.
We
anticipate
will
make
valuable
addition
the
current
arsenal
systems
for
use
in
target
validation.
One-Sentence
Summary
Engineered
zinc-finger-based
degrons
enable
targeted
induced
by
selective
molecular
glues.
Disease Models & Mechanisms,
Год журнала:
2024,
Номер
17(4)
Опубликована: Апрель 1, 2024
Degron
tagging
allows
proteins
of
interest
to
be
rapidly
degraded,
in
a
reversible
and
tuneable
manner,
response
chemical
stimulus.
This
provides
numerous
opportunities
for
understanding
disease
mechanisms,
modelling
therapeutic
interventions
constructing
synthetic
gene
networks.
In
recent
years,
many
laboratories
have
applied
degron
successfully
cultured
mammalian
cells,
spurred
by
rapid
advances
the
fields
genome
editing
targeted
protein
degradation.
this
At
Glance
article,
we
focus
on
efforts
apply
mouse
models,
discussing
distinct
set
challenges
posed
vivo
environment.
Cell Reports,
Год журнала:
2022,
Номер
41(13), С. 111865 - 111865
Опубликована: Дек. 1, 2022
How
transcription
is
regulated
as
development
commences
fundamental
to
understand
how
the
transcriptionally
silent
mature
gametes
are
reprogrammed.
The
embryonic
genome
activated
for
first
time
during
zygotic
activation
(ZGA).
RNA
polymerase
II
(Pol
II)
and
productive
elongation
this
process
remains
elusive.
Here,
we
generate
genome-wide
maps
of
Serine
5
2-phosphorylated
Pol
after
ZGA
in
mouse
embryos.
We
find
that
both
phosphorylated
forms
display
similar
distributions
across
genes
ZGA,
with
typical
enrichment
emerging
ZGA.
occurs
at
prior
their
activation,
suggesting
2
phosphorylation
may
prime
gene
expression.
Functional
perturbations
demonstrate
CDK9
SPT5
major
regulators
prevents
precocious
some
genes.
Overall,
our
work
sheds
molecular
insights
into
transcriptional
regulation
beginning
mammalian
development.
Genes & Development,
Год журнала:
2022,
Номер
36(13-14), С. 770 - 789
Опубликована: Июль 1, 2022
Promoter-proximal
RNA
Pol
II
pausing
is
a
critical
step
in
transcriptional
control.
has
been
predominantly
studied
tissue
culture
systems.
While
shown
to
be
required
for
mammalian
development,
the
phenotypic
and
mechanistic
details
of
this
requirement
are
unknown.
Here,
we
found
that
loss
stalls
pluripotent
state
transitions
within
epiblast
early
mouse
embryo.
Using
Nelfb
−/−
mice
NELFB
degron
stem
cell
model,
show
embryonic
cells
(ESCs)
representing
naïve
pluripotency
successfully
initiate
transition
program
but
fail
balance
levels
induced
repressed
genes
enhancers
absence
NELF.
We
an
increase
chromatin-associated
NELF
during
from
later
states.
Overall,
our
work
defines
acute
long-term
molecular
consequences
reveals
role
continuum
as
modulator
transitions.
It
was
previously
considered
that
protein
knockdown
using
a
combination
of
zinc
finger
degron
tag
and
thalidomide
analog
is
impossible
in
mouse
cells,
however,
EGFP
expression
as
an
indicator,
we
found
possible
super-degron
(SD)
with
iberdomide
or
mezigdomide
cells.
Despite
the
efficient
degradation
SD-tagged
wild-type
PD-1
could
not
be
degraded
but
human
Jurkat
cells
human-type
CEREBLON
(CRBNI391V).
In
mice
CRBNI391V,
endogenous
tagged
SD
efficiently
suppressed
T
both
cultured
whole
body.
addition,
comparison
treatment
anti-PD-1
antibody
suggested
transient
downregulation
might
prevent
cell
exhaustion
compared
to
treatment.
brain
also
reduced
by
pomalidomide
crosses
brain-blood
barrier.
These
results
suggest
analogs
are
effective
for
vitro
vivo
knockdown,
although
some
conditional
settings
required.
Studying
essential
genes
required
for
dynamic
processes
in
live
mice
is
challenging
as
genetic
perturbations
are
irreversible
and
limited
by
slow
protein
depletion
kinetics.
The
auxin-inducible
degron
(AID)
system
a
powerful
tool
analyzing
inducible
loss
vitro,
but
it
toxic
to
mice.
Here,
we
use
an
optimized
second-generation
AID
achieve
the
conditional
reversible
of
centrosomal
CEP192
We
show
that
auxin
derivative
5-phenyl-indole-3-acetic
acid
well
tolerated
over
2
weeks
drives
near-complete
degradation
less
than
1
hour
vivo.
did
not
affect
centriole
duplication
decreased
γ-tubulin
recruitment
centrosomes
impairing
mitotic
spindle
assembly.
Sustained
vivo
led
cell
division
failure
death
proliferative
tissues.
Thus,
suited
rapid
and/or
sustained
study
functions
bioRxiv (Cold Spring Harbor Laboratory),
Год журнала:
2025,
Номер
unknown
Опубликована: Апрель 18, 2025
Abstract
Loss
of
function
studies
are
a
central
approach
to
understanding
gene/protein
function.
In
mice,
this
often
relies
upon
heritable
recombination
at
the
DNA
level.
This
is
slow
and
non-reversible,
which
limits
both
spatial
temporal
resolution
analysis.
Recently,
degron
techniques
that
directly
target
proteins
for
degradation
have
been
successfully
used
quickly
reversibly
knockdown
proteins.
Currently,
these
systems
limited
by
lack
tissue/cell
type
specificity.
Here,
we
generated
mice
allow
control
GFP-tagged
protein
degradation.
Degron
GFP
line
leads
in
different
cellular
compartments
distinct
cell
types.
Further,
it
rapid
reversible.
We
probe
glucocorticoid
receptor
epidermis
demonstrate
has
functions
proliferative
differentiated
cells
–
an
analysis
would
not
possible
with
traditional
approaches.
propose
ability
use
knock-in
lines
loss
will
provide
additional
motivation
generation
useful
tools.
Toxicological Sciences,
Год журнала:
2023,
Номер
194(1), С. 53 - 69
Опубликована: Май 25, 2023
Abstract
The
degradation
tag
(dTAG)
system
for
target
protein
can
remove
proteins
from
biological
systems
without
the
drawbacks
of
some
genetic
methods,
such
as
slow
kinetics,
lack
reversibility,
low
specificity,
and
inability
to
titrate
dosage.
These
make
it
difficult
compare
toxicity
resulting
pharmacological
interventions,
especially
in
vivo.
Because
dTAG
has
not
been
studied
extensively
vivo,
we
explored
use
this
study
physiological
sequalae
CDK2
or
CDK5
adult
mice.
Mice
with
homozygous
knock-in
sequence
onto
were
born
at
Mendelian
ratios
despite
decreased
levels
comparison
wild-type
In
bone
marrow
cells
duodenum
organoids
derived
these
mice,
treatment
degrader
dTAG-13
resulted
rapid
robust
but
caused
no
appreciable
change
viability
transcriptome.
Repeated
delivery
vivo
studies
proved
challenging;
multiple
formulations
an
effort
maximize
while
minimizing
formulation-related
toxicity.
Degradation
all
organs
except
brain,
where
likely
did
cross
blood
brain
barrier,
only
microscopic
changes
testis
CDK2dTAG
findings
corroborated
conditional
knockout
Our
results
suggest
that
provide
loss
mice
causes
previously
unknown
phenotypes.
Life Science Alliance,
Год журнала:
2023,
Номер
6(11), С. e202302225 - e202302225
Опубликована: Сен. 4, 2023
Numerous
efforts
to
understand
pluripotency
in
mammals,
using
pluripotent
stem
cells
culture,
have
enabled
the
generation
of
artificially
induced
cells,
which
serve
as
a
valuable
source
for
regenerative
medicine
and
creation
disease
models.
In
contrast
these
tremendous
successes
field
past
few
decades,
our
understanding
totipotency,
is
highlighted
by
its
broader
plasticity
than
pluripotency,
still
limited.
This
largely
attributable
scarcity
available
materials
lack
vitro
However,
recent
technological
advances
unveiled
molecular
features
that
characterize
totipotent
cells.
Single-cell
or
low-input
sequencing
technologies
allow
dissection
pre-
post-fertilization
developmental
processes
at
level
with
high
resolution.
this
review,
we
describe
some
key
findings
totipotency
discuss
how
acquired
beginning
life.