Transcript Lect_03_312014x

Mechanisms?
How does actomyosin contractility influences gene
transcription?
Actin and myosin in
transcription
a) Pol II
- Pre-initiation complex assembly
together with WASP and Arp2/3.
- Actin enhances activity of chromatinremodeling complexes and elongation
machinery.
- Splicing machinery (MVa)
- Backward motor (MVI) counteracting
motor functions (NMI).
b) Pol I
- Transcription initiation of rRNA
- Chromatin remodeling
- rRNA elongation and maturation
de Lanerolle & Serebryannyy (2011) Nat Cell Biol 13: 1282-1288
Actin in the nucleus
de Lanerolle & Serebryannyy (2011) Nat Cell Biol 13: 1282-1288
Role of actin in the nucleus
Actin/cofilin enter the nucleus via nuclear pore (NPC), while actin/profilin
is exported via exportin 6.
Nuclear actin can interact with all three polymerases and engage in
transcription.
Nuclear actin can form uncoventional polymers (rods) that are shorter
than cytoplasmic F-actin).
Nuclear actin undergo post-translational modifications, such as
SUMOylation (small ubiquitin-like modifier).
Steroid receptors (co-activator of gene transcription) that translocate to
the nucleus upon ligand binding also interact with actin, actin binding
proteins and myosins.
Some transcription factors (PREP2, YY1, MAL) bind to cytoplasmic actin.
Changes in actin polymerization can sequester or release these factors so
that they can enter the nucleus to regulate transcription.
Myosin in the nucleus
de Lanerolle & Serebryannyy (2011) Nat Cell Biol 13: 1282-1288
Role of myosins in the nucleus
Gene transcription: NMI, MVI and actin required for transcription by RNA pol,
through structural roles, motor functions or a combination of the two.
Co-activators: steroid receptors (co-activator of gene transcription) translocate to
the nucleus upon ligand binding also interact with actin, actin binding proteins and
myosins.
Transcription factors?: Heavy chain of MII and MXVIIIb have been directly
implicated in the activation of genes required for differentiation.
Scafolding complexes : myosins (NMI, MII) together with actin and intermediate
filament proteins (lamins and emerin) form complexes that provide a link between
the nucleoskeleton and chromatin.
Cell cycle: MXVIb interacts with cyclin A and proliferating cell nuclear antigen
(PCNA). Binding to protein phosphatase 1 (PP1) may control its activity and
transport through NPC.
RNA processing: MVa found in speckles and thought to be involved in RNA
processing
Many questions remain…
• Form of actin in the nucleus and its regulation?
monomer? filament? non-canonical-oligomer?
Bellin et at (2013) Mol Biol Cell 24:982-04:
monomer: speckles RNA-processing factors
filament: submicron lengths, excluded from chromatin rich
regions and part of viscoelastic mesh
scaffold for organizing nuclear complexes?
• How actin and myosins associate with each other
and with other nuclear components?
Lamin and emerin are proteins of the
nuclear envelope
Inner nuclear membrane proteins
Lamins
•
IF proteins divided in Type A (Lamin A/C)
and Type B (Lamin B)
•
bind to DNA directly or indirectly via other
nuclear proteins (emerin, LINC complex)
•
mutations in Type A lamins are found in
human laminopathies including: EmeryDreifuss muscular dystrophy (EDMD),
dilated cardiomyopathy (DCM) and
Hutchinson-Gilford progeria syndrome.
Emerin
•
•
http://circres.ahajournals.org/content/103/1/16/F2.large.jpg
•
•
actin capping protein, promotes actin
polymerization
form a complex with nuclear spectrin and
4.1 proteins
binds to LINC complex (nesprin/SUN)
mutated in X-linked EDMD
Nuclear abnormalities caused by lamin A/C
deficiency
• Lmna-/- cells have increase nuclear deformation, defective
mechanotransduction and impaired viability under mechanical stress.
• NFkB-mediated transcription in response to mechanical or cytokine
stimulation (anti-apoptotic signal) is attenuated.
• Tissue-specific effects of lamin A/C deficiency may arise from impaired
nuclear mechanics and transcriptional activation.
Increased nuclear deformation
Lmna+/+
Lmna-/-
No strain
22% strain
No strain
19% strain
Compromised survival
Lammerding (2004) J Clin Inv 113:370-378
Increased nuclear fragility
Attenuated transcription
Background
• MKL1 (aka MAL or MRTF-A) is a mechanosensitive transcription
factor.
• Intracellualr localization is regulated by changes in actin
polymerization:
- cytoplasmic: binds G-actin and has constitutive nuclear export signal.
- nuclear: mitogenic/mechanical stimulation triggers RhoA-mediated actin
polymerization; releases MKL1 from G-actin and exposes NLS within
actin-binding domain.
•
MKL1 in the nucleus co-activates serum-response factor (SRF) and activates
genes involved in cell motiliy/contractility: vinculin, actin and SRF itself.
•
Does loss/mutations of lamin A/C affect MKL1-SFR signaling?
Nuclear translocation of MKL1 is impaired in lamindeficient and mutant cells
Serum stimulation
Time-lapse
Cardiac muscle
Expression of MKL1-SRF target genes* is
downregulated in lamin-deficient and mutant cells
Fibroblasts
*SRF and vinculin
Cardiac muscle
Reduced nuclear import and increased nuclear
export in lamin-deficient and mutants cells
IMPORT
IMPORT
LMB: leptomycin B inhibitor
of nuclear export
EXPORT
Impaired nuclear accumulation of MKL1 is caused
by altered actin dynamics
RPEL: actin-binding motif; SAP: DNA binding domain;
TAD: transcription activation domain
Altered actin dynamics in lamin-deficient and
mutants cells
Emerin expression rescues actin dynamics and
restores MKL1 translocation
Lamin mutants
Emerin mutant (Emd-/Y)
Emerin rescue
Rescue of MKL1 translocation requires emerinbinding to actin
Actin motility (FRAP)
M151, M164 and Q133H: emerin mutants
that do no binding to actin
Cytoplasm
Nucleus
Summary
• Lamin-deficient and mutant cells have impaired nuclear translocation and
downstream signalling of MKL1.
• Abnormal nuclear translocation of MKL1 was caused by altered actin dynamics.
• Ectopic expression of emerin, which is mislocalized in lamin mutant cells,
restored MKL1 nuclear translocation and rescued actin dynamics.
• The findings provide insight into the etiology of laminopathies, whereby lamin
A/C and emerin might regulate gene expression through modulation of nuclear
and cytoskeletal actin polymerization.
Why is this experiment important?
Starved
Stimulated
NLS-GFP-NES: GFP fused to nuclear localization signal/nuclear export signal