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Bio 525/ Spring, 2010 Nuclear Architecture and Genomic Function

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?.

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Bio 525/ Spring, 2010 Nuclear Architecture and Genomic Function

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  1. Bio 525/ Spring, 2010Nuclear 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

  2. 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.

  3. Isolation of the Nuclear Matrix Berezney & Coffey (1974) Biochem Biophys Res Commun 60: 1410-1417

  4. Nuclear Matrix Isolation from Liver Tissue

  5. Functional Properties Associated with Nuclear Matrix

  6. Functional Properties Associated with Nuclear Matrix (contd.)

  7. How are multiple genomic processes organized and coordinated in space and time in the cell nucleus?

  8. Chromosome Territories Splicing Factors Replication Sites Nuclear Matrix Extracting Transcript Tracks Transcription Sites MAINTAINING IN SITU FUNCTIONAL DOMAINS ON THE NUCLEAR MATRIX

  9. 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

  10. Domains) Domains) The Functional Levels of HigherOrder Chromatin Organization Are Associated With the Nuclear Matrix

  11. 2-D PAGE of Nuclear Matrix Proteins

  12. 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)

  13. 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 domainspecific for hyperphosphorylated CTD of RNA pol II LS (Mol. Cell Biol. 18, 1998, 2406)

  14. 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.

  15. HAKES D & BEREZNEY RDNA Binding Properties of the Nuclear Matrix and Individual Matrix ProteinsJournal of Biological Chemistry (1991) 266, 11131-11140

  16. 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.

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

  18. ,HAKES & BEREZNEY, JBC 1991, Figure 3

  19. 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).

  20. ,HAKES & BEREZNEY, JBC 1991, Figure 4 total genomic DNA probe (nick translated) Preference of the nuclear matrix for ss DNA over ds DNA or RNA ss DNA probe

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

  22. 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).

  23. ,HAKES & BEREZNEY, JBC 1991, Figure 6 00C Temperature dependence of salt- resistant DNA binding to nuclear matrix proteins 37 0C 37 0C + salt

  24. Fold Competitor 0x 5x 10x 20x 100x Hakes & Berezney, Fig 7 1-D Southwestern Blots Labeled Genomic DNA Excess DNA Nuclear matrix proteins show a preference for DNA over RNA and matrix DNA over total genomic DNA LabeledGenomic DNA Excess RNA Excess total genomic DNA LabeledMatrix DNA

  25. 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).

  26. ,HAKES & BEREZNEY, JBC 1991, Figure 8 S N M N T M S T The nuclear matrix DNA binding proteins represent an enrichment of a subset of nuclear DNA binding proteins

  27. ,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

  28. 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). MAJOR CONCLUSIONS OF HAKES & BEREZNEY, 1991

  29. MA H, SIEGEL, AJ & BEREZNEY RAssociation of chromosome territories with the nuclear matrix: Disruption of human chromosome territories correlates with the release of a subset of nuclear matrix proteinsJournal of Cell Biology (1999) 146, 531-541

  30. 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).

  31. 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

  32. 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).

  33. MA et al., JCB, 1999, Figure 2 Relationship of nuclear matrix structure to chromosome territory disruption in NHF-1cells

  34. 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).

  35. MA et al., JCB, 1999, Figure 4 Protocol for releasing nuclear matrix associated proteins that correlates with disruption of chromosome territories

  36. 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).

  37. MA et al., JCB, 1999, Figure 5 Two-dimensional PAGE analysis of nuclear matrix proteins released during disruption of chromosome territories

  38. 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)

  39. Chromosome Territory Anchoring Proteins (CTAPs)

  40. 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).

  41. 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.

  42. 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.

  43. 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

  44. 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)

  45. 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).

  46. Zeng et al., PNAS 1997, Figure 1 AML-1B is associated with the nuclear matrix

  47. 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

  48. Zeng et al., PNAS 1997, Figure 2 Nuclear matrix association of AML-1B is independent of DNA binding and CBF-β interaction

  49. Zeng et al., PNAS 1997, Figure 3 Delineation of the AML-1B NMTS by in situ immunofluorescence analysis

  50. Zeng et al., PNAS 1997, Figure 3 contd… Delineation of the AML-1B NMTS by in situ immunofluorescence analysis

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