Bio 525/ Spring, 2010 Nuclear Architecture and Genomic Function Session 5 & 6: 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:

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?

MAINTAINING IN SITU FUNCTIONAL DOMAINS ON THE NUCLEAR MATRIX Chromosome Territories Splicing FactorsReplication Sites Transcript TracksTranscription Sites Extracting 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

Domains) The Functional Levels of Higher Order Chromatin Organization Are Associated With the Nuclear Matrix

2-D PAGE of 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

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,

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.

Time and temperature dependence of salt- resistant DNA binding to nuclear matrix, HAKES & BEREZNEY, JBC 1991, Figure 1

, HAKES & BEREZNEY, JBC 1991, Figure 3

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 total genomic DNA probe (nick translated) ss DNA probe Preference of the nuclear matrix for ss DNA over ds DNA or RNA

, HAKES & BEREZNEY, JBC 1991, Figure 5 Sequence specificity of DNA binding to the nuclear matrix

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 00C00C 37 0 C 37 0 C + salt Temperature dependence of salt- resistant DNA binding to nuclear matrix proteins

Hakes & Berezney, Fig 7 Labeled Genomic DNA Labeled Matrix DNA Excess DNA Excess total genomic DNA Excess RNA Nuclear matrix proteins show a preference for DNA over RNA and matrix DNA over total genomic DNA Fold Competitor 0x5x 10x 20x100x 1-D Southwestern Blots

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 N S MT N S T M 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, 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,

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 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 Intact Cell DNA – rich in situ Nuclear Matrix RNase A + 2.0M NaCl

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 corres- ponding 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 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,

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) AML Transcription Factors

MAJOR CONCLUSIONS OF ZENG et al., PNAS 1997 Conclusion 1 Transcriptionally active AML-1B binds to the nuclear matrix while inactive AML-1 does not (Figure 1). 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)

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/Δ ) 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 in situ immunofluorescence analysis

Zeng et al., PNAS 1997, Figure 3 contd… Delineation of the AML-1B NMTS by in situ immunofluorescence analysis AML-1B AML1-290/ AML-1B (NM) AML1-290/ (NM)

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 ] is necessary and sufficient to target the transcriptionally active AML-1B to the nuclear matrix.

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. MAJOR CONCLUSIONS OF ZENG et al., 1997

Zeng et al. Intranuclear Targeting of AML/CBFα Regulatory Factors to Nuclear Matrix- Associated Transcriptional Domains Proceedings of the National Academy of Sciences (1998) 95,

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 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, AML- 1B 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

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, Figures 2A and 2B

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., 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 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 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