Download Nuclear Matrix Proteins and Nuclear Targeting

Bio 405/505 Advanced Cell &
Developmental Biology II
The Cell Nucleus Lectures
Dr. Berezney
Lecture 4 : Nuclear Matrix Proteins and
Nuclear Targeting; Background & Figures for
Hakes & Berezney, 1991; Ma et al., 1999;
Zeng et al., 1997 & 1998
What is the nuclear matrix?
The nuclear matrix is the proteinaceous
nuclear structure remaining after nuclease,
salt and detergent treatments of isolated
nuclei which maintains features of the
overall nuclear architecture including the
nuclear lamina-pore complex, the nucleolus
and the nonchromatin fibrogranular matrix.
Isolation of the Nuclear Matrix
Berezney & Coffey (1974) Biochem Biophys Res Commun 60: 1410-1417
Nuclear Matrix Isolation from Liver Tissue
Functional Properties Associated with Nuclear Matrix
Functional Properties Associated with Nuclear Matrix
(contd.)
How are multiple genomic processes organized and
coordinated in space and time in the cell nucleus?
Chromosome Territories
Splicing Factors
Replication Sites
Nuclear
Transcript Tracks
Transcription Sites
MAINTAINING IN SITU FUNCTIONAL DOMAINS
ON THE NUCLEAR MATRIX
3-D Model of a 1 mbp Multi-Loop Chromatin Domain
The chromatin loops in each domain are attached to
nuclear matrix protein complexes (“Loop Base Spring”)
which form a dynamic network underlying the chromatin
domains
The Functional Levels of Higher Order Chromatin
Organization Are Associated With the Nuclear Matrix
2-D PAGE of Nuclear Matrix Proteins
Nuclear Matrix Proteins
1. The nuclear matrix is composed of a major group of ~dozen highly
conserved proteins (termed nuclear matrins: PNAS 88, 1991: 10,312)
and many other (100’s) less abundant ones including those with cell
type, tissue, species, developmental and human tumor (bladder,
breast, uterine, cervical, prostate, colon and kidney cancer) specificity.
2. Many of the nuclear matrins are pre-mRNP, RNA splicing or
transcriptional factors but many have yet to be identified.
3. Matrin 3 is an ~ 96 kDa protein that contains 2 Zn finger motifs, RNA
Recognition Motifs (RRM’s) and an acidic rich domain at the C-T
common among transcriptional activators (J Biol Chem 266, 1991: 9893)
Nuclear Matrix Proteins (cont.)
4. Matrin Cyp (cyclophilin) a ~88 kDa protein that contains
the complete cyclophilin protein sequence at the N-T and SR
repeats - characteristic of splicing factors – within the
carboxyl half. The protein has peptidylprolyl cis-trans
isomerase activity and co-localizes with splicing factor-rich
nuclear speckles (J. Biol Chem. 273, 1998: 8183)
5. Matrin SRm 160 (~160 kDa protein) is an exon junction
splicing factor (Mol. Cell. Biol. 22, 2002: 148).
6. Matrin 250 (~250 kDa) is the hyperphosphorylated form of
RNA pol II LS (PNAS 93, 1996: 8253).
7. Matrin SCAF 8 (140 kDa) contains SR-rich motifs and a
binding domain specific for hyperphosphorylated CTD of
RNA pol II LS (Mol. Cell Biol. 18, 1998, 2406)
Nuclear Matrix Proteins (cont.)
DNA (Chromatin) Loop Anchoring Proteins
or MAR/SAR Binding Proteins ????
• DNA Topoisomerase
• SAF-A and SAF-B (Bind both DNA and RNA)
• SAT B1 – MAR protein specific for lymphocytes
• What else ???? More Research is needed at the
levels of DNA (chromatin loops), multi-loop
chromatin domains ( ~1 mbp domains) and
whole chromosome territories.
HAKES D & BEREZNEY R
DNA Binding Properties of the
Nuclear Matrix and Individual Matrix
Proteins
Journal of Biological Chemistry (1991) 266,
11131-11140
MAJOR CONCLUSIONS OF HAKES & BEREZNEY, JBC 1991
Conclusion 1
Salt resistant binding of DNA to isolated nuclear matrix was
saturable and temperature dependent (Figures 1 & 3) with an
estimate of 150,000 binding sites per nuclear matrix
structure.
,HAKES
& BEREZNEY, JBC 1991, Figure 1
Time and temperature dependence of salt- resistant DNA binding to
nuclear matrix
,HAKES
& BEREZNEY, JBC 1991, Figure 3
SS DNA
Saturation binding of
single and double
stranded rat liver
genomic DNA to nuclear
matrices
ds DNA (nick translated)
ds DNA (S1 treated)
Total
Salt resistant
MAJOR CONCLUSIONS OF HAKES & BEREZNEY, JBC 1991
Conclusion 2
Single stranded regions of DNA were preferentially bound
with RNA poorly competing for the DNA binding sites
(Figures 3 & 4) and a preference for matrix DNA and poly
(dA).(dT) over total genomic DNA (Figure 5).
,HAKES
& BEREZNEY, JBC 1991, Figure 4
Competitors
Total ds DNA (n.t)
ss DNA
X RNA
Total ss DNA
ds DNA probe
(nick translated)
Preference of the nuclear
matix for ss DNA over ds
DNA or RNA
ds DNA (S1
treated)
X RNA
ss DNA probe
,HAKES
& BEREZNEY, JBC 1991, Figure 5
genomic DNA
Sequence specificity of DNA binding
to the nuclear matrix
X E.coli DNA
Matrix DNA
poly (dA), poly (dT)
MAR
MAJOR CONCLUSIONS OF HAKES & BEREZNEY, JBC 1991
Conclusion 3
The properties of temperature and salt resistant binding of
DNA and preference for DNA binding over RNA and matrix
DNA over total genomic DNA was observed for DNA binding
to individual proteins on Southwesterns (Fig 6 & 7).
,HAKES
& BEREZNEY, JBC 1991, Figure 6
0.05 0.1
0.2 0.5 1.0 2.0
M NaCl
00 C
37 0C
37 0C + salt
Temperature
dependence of
salt- resistant DNA
binding to nuclear
matrix proteins
Fold Competitor
Labeled
Genomic
DNA
Labeled
Genomic
DNA
Labeled
Matrix
DNA
0x
5x 10x 20x 100x
Hakes & Berezney, Fig 7
1-D Southwestern Blots
Excess
DNA
Nuclear matrix proteins
show a preference for DNA
over RNA and matrix DNA
over total genomic DNA
Excess
RNA
Excess total
genomic DNA
MAJOR CONCLUSIONS OF HAKES & BEREZNEY, JBC 1991
Conclusion 4
The nuclear matrix is enriched in the higher molecular weight
DNA binding proteins in the cell nucleus (50,000 - >150,000)
and seven of the twelve major proteins of the nuclear matrix
were shown to bind DNA (lamins A, C, matrins D,E,F,G and 4)
(Figures 8-10).
,HAKES
& BEREZNEY, JBC 1991, Figure 8
The nuclear matrix DNA binding proteins represent an
enrichment of a subset of nuclear DNA binding proteins
,HAKES
& BEREZNEY, JBC 1991, Figures 9 & 10
Two- dimensional analysis of
individual nuclear matrix DNA binding
polypeptides (2-D Southwesterns)
Verification of individual nuclear
matrix DNA binding polypeptides
MAJOR CONCLUSIONS OF HAKES & BEREZNEY, 1991
1. Salt resistant binding of DNA to isolated nuclear matrix was saturable and
temperature dependent (Figures 1 & 3) with an estimate of 150,000 binding sites
per nuclear matrix structure.
2. Single stranded regions of DNA were preferentially bound with RNA poorly
competing for the DNA binding sites (Figures 3 & 4) and a preference for matrix
DNA and poly (dA).(dT) over total genomic DNA (Figure 5).
3. The properties of temperature and salt resistant binding of DNA and preference
for DNA binding over RNA and matrix DNA over total genomic DNA was observed
for the individual DNA binding of the proteins on Southwesterns (Figures 6 & 7).
4. The nuclear matrix is enriched in the higher molecular weight DNA binding
proteins in the cell nucleus (50,000 - >150,000) and seven of the twelve major
proteins of the nuclear matrix were shown to bind DNA (lamins A, C, matrins
D,E,F,G and 4) (Figures 8-10).
MA H, SIEGEL, AJ & BEREZNEY R
Association of chromosome territories
with the nuclear matrix: Disruption of
human chromosome territories correlates
with the release of a subset of nuclear
matrix proteins
Journal of Cell Biology (1999) 146, 531-541
MAJOR CONCLUSIONS OF MA et al., JCB, 1999
Conclusion 1
Chromosome territory organization is maintained after in
situ extraction of cells with 2M NaCl for nuclear matrix
preparation (Fig 1).
MA et al., JCB, 1999, Figure 1
Intact Cell
DNA – rich in situ Nuclear Matrix
RNase A + 2.0M NaCl
Chromosome territories are maintained after extraction of WI-38 cells
for DNA- rich nuclear matrix, but are disrupted when RNase A digestion
precedes 2.0 M NaCl extraction
MAJOR CONCLUSIONS OF MA et al., JCB, 1999
Conclusion 2
Disruption of nuclear matrix organization by pre-treatment
with RNase A before 2M NaCl extraction leads to a corresponding disruption of territorial organization (Fig 1 & 2).
MA et al., JCB, 1999, Figure 2
Relationship of nuclear matrix structure to chromosome
territory disruption in NHF-1 cells
MAJOR CONCLUSIONS OF MA et al., JCB, 1999
Conclusion 3
The finding that extraction with ammonium sulfate at similar
ionic strength (0.65 M) as 2M NaCl following RNase does
not lead to territorial disruption (Fig 2) has led to a
procedure to isolate proteins that are released in
association with disruption of territories (Fig 4).
MA et al., JCB, 1999, Figure 4
Protocol for releasing nuclear matrix associated proteins that correlates
with disruption of chromosome territories
MAJOR CONCLUSIONS OF MA et al., JCB, 1999
Conclusion 4
These released proteins comprise a distinct subset of
proteins in nuclear matrix preparations (Fig 5) and are
termed CTAPs (Chromosome Territory Anchoring Proteins).
MA et al., JCB, 1999, Figure 5
Two-dimensional PAGE analysis of nuclear matrix proteins
released during disruption of chromosome territories
MAJOR CONCLUSIONS OF MA et al., 1999
• Chromosome territory organization is maintained after in situ
extraction of cells with 2M NaCl for nuclear matrix preparation
(Fig 1)
• Disruption of nuclear matrix organization by pre-treatment with
RNase A before 2M NaCl extraction leads to a corresponding
disruption of territorial organization (Fig 1 & 2)
• The finding that extraction with ammonium sulfate at similar
ionic strength (0.65 M) as 2M NaCl following RNase does not
lead to territorial disruption (Fig 2) has led to a procedure to
isolate proteins that are released in association with disruption
of territories (Fig 4)
• These released proteins comprise a distinct subset of proteins
in nuclear matrix preparations (Fig 5) and are termed CTAPs
(Chromosome Territory Anchoring Proteins)
Chromosome Territory Anchoring Proteins (CTAPs)
Nuclear Targeting [Leonhardt et al. Cell 71 (1992) 865]
• Aside from NLS’s and NES’s
there is growing evidence that
many nuclear proteins contain
an Nuclear Targeting Sequence
(NTS’s) that target individual
proteins to the sites of genomic
function/organization.
• A classic example is the DNA
methyl transferase (MTase)
which is an enzyme associated
with replication sites in cells and
is responsible for maintaining
the methylation patterns of the
DNA from cell generation to
generation.
• This is important for regulation
of transcription ( highly
methylated genes are generally
not transcribed).
Co-localization of MTase (red)
with BrdU labeled (green) DNA
replication sites (RS).
Nuclear Targeting contd…
Question: How is MTase targeted to RS? Is there a
specific region of the MTase protein that is
responsible for targeting the MTase to RS??
•
Construct a series of deletion mutants of MTase
• Transfect mammalian cells with MTase constructs fused to the
beta-galactosidase (β-gal) gene.
• Use anti-β-gal antibodies to detect localization of the fusion
protein in the nucleus and with RS labeled with BrdU method.
Nuclear Targeting contd…
Results: A region of the N-terminal MTase is necessary
and sufficient to target β-gal to RS. The targeting
sequence is a 248 aa track from aa 207-455 of the 1,502
aa sequence of the whole protein.
Zeng et al
Identification of a nuclear matrix targeting
signal in the leukemia and bone-related
AML/CBF-α transcriptional factors
Proceedings of the National Academy of Sciences
(1997) 94, 6746-6751
AML Transcription Factors
• AML genes code for a class of transcriptional factors
(activators) that mediate tissue specific gene expression
in cells of lymphoid, myeloid and osteoblast lineages.
• The AML protein family is a series of alternatively
spliced (which define tissue specificity) and
chromosome translocation forms of the AML gene.
• The chromosomal translocations forms of AML are
characteristic of the childhood disease AML (acute
myeloid leukemia)
MAJOR CONCLUSIONS OF ZENG et al., PNAS 1997
AML-1B – active – nuclear matrix associated (480 aa)
AML-2 – active – nuclear matrix associated
AML-3- active – nuclear matrix associated
AML-1 –inactive – not nuclear matrix associated (250
aa; truncated at C-terminal missing 230 aa)
Conclusion 1
Transcriptionally active AML-1B binds to the nuclear
matrix while inactive AML-1 does not (Figure 1).
Zeng et al., PNAS 1997, Figure 1
AML-1B is associated with the nuclear matrix
MAJOR CONCLUSIONS OF ZENG et al., PNAS 1997
Conclusion 2
Association of AML-1B with the nuclear matrix is
independent of DNA binding (Figure 2), but requires a 31 a.a.
sequence near the C-terminus termed the Nuclear Matrix
Targeting Sequence (NMTS; Figure 3).
Test AML-1B substitution mutants in rhd region
(contains motifs for DNA binding and CBF-β binding)
or a deletion mutant (AML/Δ155-258) that lacks distal
portion of rhd for nuclear matrix association
Zeng et al., PNAS 1997, Figure 2
Nuclear matrix association of AML-1B is independent of
DNA binding and CBF-β interaction
Zeng et al., PNAS 1997, Figure 3
Delineation of the AML-1B NMTS by in situ
immunofluorescence analysis
Zeng et al., PNAS 1997, Figure 3 contd…
Delineation of the AML-1B NMTS by insitu
immunofluorescence analysis
Zeng et al., PNAS 1997, Figure 3 contd…
AML-1B
AML-1B
(NM)
AML1-290/351-381
AML1-290/351381 (NM)
Delineation of the AML-1B NMTS by in situ
immunofluorescence analysis
MAJOR CONCLUSIONS OF ZENG et al., PNAS 1997
Conclusion 3
Fusion of the AML-1B NMTS to the Gal 4 protein directs GAL
4 to the nuclear matrix (Figure 5A).
Zeng et al., PNAS 1997, Figure 5(A)
The NMTS is sufficient to direct heterologous nuclear protein
to the nuclear matrix
MAJOR CONCLUSIONS OF ZENG et al., PNAS 1997
Conclusion 4
Thus the NMTS [aa 351-381] is necessary
and sufficient to target the transcriptionally
active AML-1B to the nuclear matrix.
MAJOR CONCLUSIONS OF ZENG et al., 1997
1. Transcriptionally active AML-1B binds to the nuclear matrix while
inactive AML-1 does not (Figure 1).
2. Association of AML-1B with the nuclear matrix is independent of DNA
binding (Figure 2), but requires a 31 a.a. sequence near the C-terminus
termed the Nuclear Matrix Targeting Sequence (NMTS; Figure 3).
3. Fusion of the AML-1B NMTS to the Gal 4 protein directs GAL 4 to the
nuclear matrix (Figure 5A).
4. Thus the NMTS is necessary and sufficient to target the transcriptionally
active AML-1B to the nuclear matrix.
Zeng et al.
Intranuclear Targeting of AML/CBFα
Regulatory Factors to Nuclear MatrixAssociated Transcriptional Domains
Proceedings of the National Academy of Sciences
(1998) 95, 1585-1589
MAJOR CONCLUSIONS OF ZENG et al., PNAS 1998
Conclusion 1
The NMTS Sequence of AML-1B Efficiently Transactivates
Transcription of a Heterologous Reporter Gene (Figure 1).
Thus the NMTS is Potentially is Involved in Both Binding to
Nuclear Matrix Sites of Transcription and Activation of
Transcription at Certain Promoter Sites
Zeng et al., PNAS, 1998, Figure 1`
The NMTS of AML-1B Transactivates Heterologous
Reporter Gene Expression
MAJOR CONCLUSIONS OF ZENG et al., PNAS 1998
Conclusion 2
Consistent with a Role in Transcriptional Regulation, AML1B Co-Localizes With a Limited Number of RNAP II Sites
(Figure 2) which Mark Active Sites of Transcription in the
Nucleus (Figure 3). Moreover, a Mutation in the DNA Binding
Domain of AML1B That Abrogates Binding to Potential Gene
Promoter Sites, Results in a Complete Lack of Colocalization
with RNAP II Sites in the Nucleus
Zeng et al., PNAS, 1998, Figures 2A and 2B
Immunolocalization of AML-IB with RNAP II
AML-1B
RNAP II
Merged
Zeng et al., PNAS, 1998, Figures 2A and 2B
Merged image of mutant AML-1B (green) and RNAP II (red).
L148D mutant contains a single point mutant in the “runt”
homology domain and thus lacks DNA binding activity and
is incapable of directly binding to gene promoter sites
Zeng et al., PNAS, 1998, Figure 3
Co-localization of transcription sites (BrUTP) and RNAP II
MAJOR CONCLUSIONS OF ZENG et al., PNAS 1998
Conclusion 3
AML-1B Does not Colocalize with Splicing Factors Domains
(Nuclear Speckles) (Figure 4).
Zeng et al., PNAS, 1998, Figure 4
AML-1B Does Not Colocalize With
SC-35 RNA Splicing Domains
MAJOR CONCLUSIONS OF ZENG et al., 1998
1. The NMTS Sequence of AML-1B Efficiently Transactivates
Transcription of a Heterologous Reporter Gene (Figure 1). Thus the
NMTS is Potentially Involved in Both Binding to Nuclear Matrix Sites
of Transcription and Activation of Transcription at Certain Promoter
Sites
2. Consistent with a Role in Transcriptional Regulation, AML-1B CoLocalizes With a Limited Number of RNAP II Sites (Figure 2) which
Mark Active Sites of Transcription in the Nucleus (Figure 3).
Moreover, a Mutation in the DNA Binding Domain of AML1B That
Abbrogates Binding to Potential Gene Promoter Sites, Results in a
Complete Lack of Colocalization with RNAP II Sites in the Nucleus
3. AML-1B Does not Colocalize with Splicing Factors Domains (Nuclear
Speckles) (Figure 4).
Nuclear Matrix Proteins
• The most abundant proteins are highly
conserved in mammals
• Species, Cell type and tissue specific
• Developmental specific
• Cell growth and proliferation specific
• Human Cancer Specific: Bladder,
Breast, Uterine, Cervical, Prostate,
Colon and Kidney