Centromere- Lecture notes 1-7 PDF

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Nuclear transport The nuclear envelope (NE) is a double lipid bilayer, outer membrane is continuous with the ER whereas the inner nuclear membrane is associated with the nuclear lamina. The NE provides a much stronger physical barrier than the plasma membrane. The NE is studded with nuclear pore com...

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Nuclear transport The nuclear envelope (NE) is a double lipid bilayer, outer membrane is continuous with the ER whereas the inner nuclear membrane is associated with the nuclear lamina. The NE provides a much stronger physical barrier than the plasma membrane. The NE is studded with nuclear pore complexes (NPC) which form a diffusion barrier. There are 2,000-5,000 NPC complexes per somatic nucleus. The NPC is a huge protein complex that fuses the internal and external nuclear membrane to form an aqueous cylindrical channel. It is composed of over 400 protein subunits (integral proteins and remaining non-membrane nuclear Pore proteins) generating ring-core that spans both membranes with 8-fold symmetry. There are 8 filaments on cytoplasmic side, 8 in the nuclear side forming ring “basket”. Crystallography and structural analysis identifies different nucleoporins that make up NPC • transmembrane group • central scaffold group • FG repeat group. FG repeat proteins line the pore. They are highly unstructured hydrophobic molecules. “Virtual gating” by Mobility of unstructured domains that result in highly dynamic pore and is filled quickly. By creating barrier to proteins that have low affinity, high specificity interactions with transport receptors that escort cargo proteins through the nuclear pore by diffusion. Several theories on assembly, one possibility is that as a prepore generated by joining of several Nup complexes through transportation via existing NPCs, it binds to the chromatin. It is then inserted into the double membrane close to the chromatin. This leads to the fusing of double membrane. During mitosis the NPC appears to disassemble in order. Peripheral nucleoporins disassociate from the NPC. The rest scaffold proteins remain stable as cylindrical ring complexes within the nuclear envelope. This disassembly is phosphate driven but the enzyme involved in the phosphorlyation is unknown. The entry and exit of large molecules from the cell nucleus is tightly controlled by the nuclear pore complexes (NPCs). Although small molecules can enter the nucleus without regulation, macromolecules such as RNA and proteins require association with transport factors like karyopherin called importins to enter the nucleus and exportins to exit. The ability of both importins and exportins to transport their cargo is regulated by the small Ras related GTPase, Ran. GTPases are enzymes that bind to a molecule called guanosine triphosphate (GTP) which they hydrolyse to create guanosine diphosphate (GDP) and release energy. GTPase is active when its GTP bound and inactive when its GDP bound. Its function requires GAP – GTPase activator protein that stimulates GTPase activity as well as GEF – GTP/GDP exchange factor (stimulating the release of GDP to allow binding of GTP). The dominant nucleotide binding state of Ran depends on whether it is in the nucleus (RAN-GEF-RanGTP) or the cytoplasm (Ran-GAP=RANGDP). Protein that must be imported to the nucleus from the cytoplasm carry nuclear localization signals (NLS) and NLSs are bound by importins. A NLS is a sequence of amino acids that acts as a tag. They are diverse most commonly hydrophilic. NLS receptors: importins/ karyopherins are multifunctional binding proteins. Importin α and β also function in mitotic spindle regulation. Importin β and α are major workhorses that are regulated by multigene in nuclear transport. They bind cargo via NLS and different proteins interact with different classes of NLS even they are related. They bind nucleoporins for localization within

NPC. The binding of Ran for regulation. As Importin proteins beta/alpha bind their cargo with high affinity in the cytoplasm they can form the import complex. It interacts with the cytoplasmic filaments in nuclear pore complex and slide through a network of FG repeat nucleoporin proteins, which produce a series of weak interactions that concentrate the import complex within the nuclear pore. Once inside the nucleus, interaction of the importin β of the complex with Ran-GTP causes a conformational change in the importin that causes the dissociation of cargo. Cargo is “trapped” in nucleus unless it has an NES (export signal). The resulting complex of importin and Ran-GTP then diffuse to the cytoplasm. Cargo is now free to function. If cargo is bound to importin α it is then exchanged for CAS, a nuclear export receptor. CAS carries the importin α/Ran-GTP complex (low affinity for cargo) to the cytoplasm. In the cytoplasm, a protein called Ran Binding Protein separate Ran-GTP from importin. Separation allows access to a GTPase activating protein (GAP) that binds Ran-GTP and induces the hydrolysis of GTP to GDP. The Ran-GDP produced from this process now binds the nuclear transport factor NUTF2 which returns it to the nucleoplasm. Now in the nucleus, the Ran-GDP interacts with a guanine nucleotide exchange factor (GEF) which replaces the GDP with GTP, resulting again in Ran-GTP, and beginning the cycle again. Ran-dependent nuclear export is organized in a very similar fashion, except that transport receptors bind cargo in the presence of Ran- GTP and discharge in the presence of Ran-GDP. Proteins, transfer RNA, and assembled ribosomal subunits are exported from the nucleus due to association with exportins, which bind signalling sequences called nuclear export signals (NES). The exportin binds the cargo with Ran-GTP whereas importins depend on RanGTP to dissociate from their cargo. The complex diffuses through the pore to the cytoplasm and dissociates. Ran-GTP binds GAP and hydrolyses GTP, and the resulting Ran-GDP complex is restored to the nucleus where it exchanges its bound ligand for GTP. Exporting 1 Xpo1 is a major export protein which is homology with importin β, but cargo is bound in presence of Ran-GTP. It binds to NES (short, leucine rich sequences) on proteins. Snurportin cycle involves the nuclear import of U snRNAs. For snRNA import snurportin binds importin β without interference with RNA cap binding domain. For recycling snurportin back to cytoplasm XPO1 binds/blocks the cap binding domain ensuring no snRNA binding during export. In RNA export, there is a Ran-dependent mechanism. Xpo1/Crm1 functions as transport adaptor for several types of mRNA transport, mediated by different adaptor proteins. Or Xpot and Xpo5 bind tRNAs and miRNAs respectively in a Ran dependent pathway. CRM1 does not bind RNA directly but via NES-containing adaptor proteins that bind to RNA. Mutations can affect RNA's structure which inhibit the binding to exportin-t, and consequentially, to be exported, providing the cell with another quality control step. There is also a Ranindependent mechanism, NXF1 is a major mRNP transporter that does not bind Ran. NXF1 complex mediates binding to NPC and transport cargo. The transport complex disassembles in the cytoplasm. It is notable that all viral RNAs and cellular RNAs except mRNA are dependent on RanGTP. Conserved mRNA export factors are necessary for mRNA nuclear export. In higher eukaryotes, mRNA export is thought to be dependent on splicing which in turn recruits a protein complex, TREX, to spliced messages.

Regulated access to nucleus is a key mechanism of gene regulation especially in Embryonic development and Signal-dependent gene regulation. The nucleo-cytoplasmic transport processes are regulated by cellular signaling systems via cargo protein modification and the transport machinery, including the transport receptors, the NPC and the Ran system. Regulation occurs via Phosphorylation in which Positive: P activates transport and Negative: P inhibits transport. Or via Complex formation such as NF-kappaB transport inhibitor and beta-catenin and Wnt signalling. Example: NF-AT is a transcription factor that mediates T-cell activation, possessing both NLS and NES. At rest, two NLSs of NF-AT are phosphorylated and located in the cytoplasm. Ca++ entry activates calcineurin, protein phosphatase which binds NES. Dephosphorylation unmasks NLS resulting in nuclear import. Low Ca++ in the nucleus causes dissociation of calcineurin revealing NES. Nuclear kinase activity inactivates NLS resulting in export.PLC and growth factor. The deregulation of the nuclear import and export machinery is a key marker of various diseases. Many tumor suppressor transcription factors show cytoplasmic sequestration ablating their nuclear functions allowing uncontrolled cell division. Examples p53, BRCA1, APC. Some stomatitis virus interact with Nups to inhibit the export of host mRNAs that encode anti-viral factors and make the translation machinery available for expression of viral mRNAs.

Histone assembly and transmission Epigenetic features of the chromosome are A branch of biology which studies the causal interactions between genes and their products which brings the phenotype into being. It is also the study of changes in gene function that are mitotically and/or meiotically heritable that do not involve a change in DNA sequence. Chromosome replication is simple in prokaryotes in which dsDNA could be unwound, and each strand could template a new partner strand during its replication in S phase assisted by the action of polymerase (leading and lagging strand). In eukaryotes, it is a complicated programme as chromatin is the form DNA takes in vivo and chromatin also replicate in addition to DNA. In general, there are three levels of chromatin organization: 1. DNA wraps around histone proteins forming nucleosomes (euchromatin). 2. Multiple histones wrap into a fibre consisting of nucleosome arrays in their most compact form (heterochromatin, transcriptionally silent genes). 3. Higher-level DNA packaging of the 30 nm fibre into the metaphase chromosome (during mitosis and meiosis). Chromatin is actively assembled and dissembled according to nuclear processes including DNA replication in S phase, DNA repair, and transcription. Histone synthesis in the cytoplasm is upregulated in S-phase. Newly synthesized histones are associated with chaperones to promote their ordered assemble: Hsp90, small Nuclear Antigen Specific Protein (sNASP), and Histone acetyltransferase complex. Chaperones show histone dimers to sites of assembly: H3:H4 tetramers (replicative chaperons Asf1) H2A:H2B 2 dimers. Importin then takes over Histone complexed for nuclear translocation. During the first steps of assembly, incorporation of differentially modified (e.g. methylated) DNA and histones (e.g. acetylation, phosphorylation, methylation) can produce differences in chromatin

structure and activity. Modifications signal is specific at chromatin states, such as the acetylation/deacetylation cycle of H3-H4 during replication at S phase while ATP establish a regular spacing. The incorporation of histone variants such as CENP-A are associated with silent centromeric regions. Chromatin assembly factor 1 (CAF-1) catalyses deposition of new histone H3:H4 tetramers, it interacts with Asf1 to transfer histone dimers to DNA as a tetramer. It assembles nucleosomes onto newly synthesized DNA during replication and repair. NAP1 completes nucleosome assembly by catalysing the assembly of H2A:H2B dimers to new H3:H4 tetramers. In the maturation step, incorporation of linker histones and other specific DNA-binding factors helps to space and fold the nucleosomal array. Further folding and Histone modifications can operate at this late stage to organise chromatin domain in the nucleus. Histone H3 phosphorylation in chromosome condensation and segregation, and methylation on Lys9 of histone H3 mediates chromatin silencing. The final group of stimulatory factors is targeting proteins e.g. PCNA, which may help to bring specialized proteins to specific domains in the nucleus. Or by exhibiting tissue specificity (e.g. neuronspecific NAP-1 which associate with all core histones and NAP-2 interacts with both linker and core histones). CAF1 mechanism accounts for deposition of new histones, but not the parental histones. Parental histones contain epigenetic marks and are maintained during replication. Parental histones are long lived proteins as t1/2 of histones in non-cycling mammalian cells is measured in months. Pre-existing H3:H4 tetramers are probably transmitted intact which are supported by density labelling experiments that indicate H3:H4 stay together over generations. Cross-linking experiments also show that chemically stabilized H3:H4 tetramers can be transmitted for multiple generations. During DNA replication, parental histones are retained at their original sites, +/- about 2 nucleosomes. They are re-used thus, providing a potential basis for transmission of histone-dependent epigenetic information. A method for swapping epitope tags to measure the disposition of ancestral histone H3 across the yeast genome over six generations. Ancestral H3 is preferentially retained at the 5' ends of most genes, with strongest retention at long, poorly transcribed genes. Most maternal histones are reincorporated within their pre-replication locus during replication. Replication-independent replacement and transcription-related retrograde nucleosome movement shape the distributions of ancestral histones. There is a key role for Topoisomerase I in retrograde histone movement during transcription. Specific loci are enriched for histone proteins synthesized several generations beforehand, and that maternal histones re-associate close to their original locations on daughter genomes after replication. Accumulation of ancestral histones could play a role in shaping histone modification patterns. FACT (facilitates chromatin transcription) is a heterodimeric protein complex that affects eukaryotic RNA polymerase II (Pol II) transcription elongation both in vitro and in vivo. It is a histone remodelling factor that associates with the helicase complex at the fork and may pass H3:H4 tetramers “around the fork”. It enables chromatin replication in vitro. FACT consists of 140 and 80 kilodalton (kDa) subunits. The 140 kDa subunit is encoded in human gene like yeast gene Spt16 and the 80 kDa subunit is human SSRP1. Spt16 - binds H2A/H2B mononucleosomes and SSRP1 binds H3/H4 tetramers. Purified human FACT binds Co-

immunoprecipitation specifically to mononucleosomes and the histone H2A/H2B dimer, but not to the H3/H4 tetramer. It destabilises nucleosomes during polymerase passage and supports leading and lagging strand synthesis from a functional origin of replication. Nucleosomes substantially inhibited replication and FACT is sufficient to restore DNA replication. FACT’s precise role remains to be worked out may promote nucleosome displacement at fork? Or Nucleosome assembly/reincorporation after fork? Histone modifications are all considered “epigenetic” but how is this information passed on? Histone H3 methylation (K9, K27) is associated with gene silencing. H3K9me3 polycomb regulates developmental gene expression, maintains gene repression stably in cell lineages. The regulation is established by DNA binding components (genetic) that directs a histone modifying complex, PRC2, to chromatin, mediating silencing through H3K27 methylation. PRC2 contains both histone methyltransferase activity and methyl-histone binding activity. Binding of PRC2 to parental methylated histone H3 promotes methylation of newly deposited histones in a positive feedback loop that regenerates H3K27 methylation in a locus-specific fashion. In drosophila, a suppressor gene, SuVar3-9 was shown to be a histone methyltransferase of H3K9-me3. There are two domains, SET domain – catalytic and chromo domain which is a K9me3 binding domain. SuVar3-9 binds its product, a methylated nucleosome, it is localised to methylate nearby nucleosomes. This spreads the binding site for SuVar3-9. H3K9 methylation can spread in cis which accounts for the variegation phenotype as H3K9me3 associated with transcriptional silencing and the spreading leads to random clonal silencing of gene in cell lines. Moreover, it accounts for stability of heterochromatin, H3K9me3 inherited from parental chromosomes can direct replenishment via SuVar3-9 (a positive feedback loop is established through enzyme binding to its product)....