Structure of the nucleus

1. Structure of the nucleus • Nucleoli: rDNA, rRNA synthesis, ribosome assembly • Chromatin: Genomic DNA - protein complexes, transcription • Nuclear envelope • Two membranes (10-50nm separation)

2. Structure of the nucleus • Nucleoli: rDNA, rRNA synthesis, ribosome assembly • Chromatin: Genomic DNA - protein complexes, transcription • Nuclear envelope • Two membranes (10-50nm separation)

3. Structure of the nucleus • Nucleoli: rDNA, rRNA synthesis, ribosome assembly • Chromatin: Genomic DNA - protein complexes, transcription • Nuclear envelope • Two membranes (10-50nm separation) • Continuous with endoplasmic reticulum (ER) • Supported on nuclear side by nuclear lamina • Meshwork of proteins on inner surface for mechanical support • Lamins are related to intermediate filaments of cytoskeleton • Mutations in Lamin A linked to disease • Molecular phenotype is misshapen nuclei

4. Structure of the nucleus • Nucleoli: rDNA, rRNA synthesis, ribosome assembly • Chromatin: Genomic DNA - protein complexes, transcription • Nuclear envelope • Two membranes (10-50nm separation) • Continuous with endoplasmic reticulum (ER) • Supported on nuclear side by nuclear lamina • Meshwork of proteins on inner surface for mechanical support • Lamins are related to intermediate filaments of cytoskeleton • Mutations in Lamin A linked to disease • Molecular phenotype is misshapen nuclei

5. Structure of the nucleus • Nucleoli: rDNA, rRNA synthesis, ribosome assembly • Chromatin: Genomic DNA - protein complexes, transcription • Nuclear envelope • Two membranes (10-50nm separation) • Continuous with endoplasmic reticulum (ER) • Supported on nuclear side by nuclear lamina • Meshwork of proteins on inner surface for mechanical support • Lamins are related to intermediate filaments of cytoskeleton • Mutations in Lamin A linked to disease (HGPS) • Molecular phenotype is misshapen nuclei

6. Structure of the nucleus • Nuclear envelope • Gated by nuclear pore complex (NPC) • 15-30 times larger than a ribosome • Composed of ~30 nucleoporin proteins • Exhibits 8-fold symmetry • Small molecules (< 40 kD) diffuse freely

7. Regulation of nuclear import/export • Proteins contain nuclear import and/or nuclear export signal sequences • Import: Nuclear Localization Signal (NLS): n-PKKKRKV-c • Importin beta/alpha binds to the NLS of the “cargo” protein in the cytoplasm • The beta-alpha-”cargo” complex binds the cytoplasmic filaments of the NPC • The docked complex translocates through the NPC to the nucleoplasm

8. Regulation of nuclear import/export • Proteins contain nuclear import and/or nuclear export signal sequences • Import: Nuclear Localization Signal (NLS): n-PKKKRKV-c • On nuclear side, Ran-GTP binds and disrupts the beta-alpha-”cargo” complex • Cargo is released in nucleus • Importin-beta is bound to Ran-GTP

9. Regulation of nuclear import/export • Proteins contain nuclear import and/or nuclear export signal sequences • Ran-GTP bound to Importin-beta travels down its concentration gradient • Cytoplasmic Ran-GTP hydrolyzes its bound GTP • Ran-GDP releases Importin-beta in cytoplasm • Export: Nuclear Export Signal (NES) • Exportin carries alpha back to cytoplasm (bind beta) GTP GDP Ran-GDP Ran-GTP GNEF GAP Ran-GDP Ran-GTP (release beta) Pi

10. Chromosomes and chromatin • Chromatin = DNA + associated proteins • Histone octamer • ( H2A, H2B, H3, H4 ) x2 • Nucleosome = histone octamer + 146 bp DNA

11. Chromosomes and chromatin • Chromatin = DNA + associated proteins • H1 linker protein connects adjacent nucleosomes • 10nm “beads-on-a-string” compacts to a 30nm fiber • Packaged DNA is protected from damaging agents • Octamer tails also contribute to higher-order compaction

13. Euchromatin & heterochromatin • Euchromatin • Dispersed, not compacted • Readily accessed by TXN factors and RNAp • Transcriptionally active • Histone modifications • Histone Acetyltransferase enzymes (HATs) • Acetylation of Lysine residues in H3 and H4 DNA (-) <--> Histones (+) • Neutralize (+) on histones, reducing DNA - histone tail interaction • Create binding sites for additional factors Lysine Acetyl-lysine

14. Euchromatin & heterochromatin • Heterochromatin • Highly compacted • Not readily accessed by TXN factors or RNAp • Transcriptionally inactive • Histone modifications • Histone Methylransferase enzymes (HMTs) • Methylation of Lysine residues in H3 and H4 • Create binding sites for additional factors • Constitutive: always compacted • Facultative: conditionally compacted (e.g. cell type specific) • X-inactivation in females + Lysine trimethyl-lysine

15. Euchromatin & heterochromatin • X-inactivation • Males have only 1 X Chromosome • Females have 2 X Chromosomes • Gene dosage in females is regulated by only using 1 of the 2 available X chromosomes • Cats have a pigment gene on X • Black allele vs orange allele • Female calico cats have random patches of black vs orange fur • Cloning of a calico cat confirmed the random nature of X-inactivation

16. Euchromatin & heterochromatin • X-inactivation • Convert one X chromosome to facultative heterochromatin • Random event early in development • Stably maintained through subsequent cell divisions Before inactivation After Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm Xp Xm

17. Heterochromatin & euchromatin • X-inactivation • Actively transcribed chromosomes stain strongly for acetylated histones • Inactivated X chromosome does not • Histones of inactivated X are instead methylated by a HMT enzyme • HP1 binds methylated sites and facilitates chromatin condensation

18. Heterochromatin & euchromatin • Examples of factors that specifically bind various chromatin modifications

19. Heterochromatin & euchromatin • The Nucleus as an Organized Organelle • Chromosome ordering is directed by the nuclear envelope proteins. • In the nucleus, mRNAs are synthesized at discrete sites. • Estrogen Receptor txn of: • GREB1 gene on Chr 2 • TRFF1 gene on Chr 21 - Estrogen (E2) = separate + Estrogen (E2) = together

20. Heterochromatin & euchromatin • The Nucleus as an Organized Organelle • Transcription occurs in distinct locations, “transcription factories” • Genes from different chromosomal locations are brought together • DNA sequences that participate in a common biological response but reside on different chromosomes interact within the nucleus. • Nuclear Matrix • May function as scaffold for organization

21. Regulation of Gene Expression • Inducible gene expression • kinetics of β-galactosidase enzyme induction • Add inducer • start transcription = mRNA accumulation • mRNA translation = protein accumulation • Remove inducer • Stop txn • mRNA and protein levels slowly return to original level

22. Gene expression: bacteria • Inducible gene expression • Example: Sugar catabolism • In the absence of lactose, no need to have enzymes that metabolize it • In the presence of lactose, cell should make enzymes for metabolizing it • Repressible gene expression • Example: Amino acid anabolism • In the absence of tryptophan, cell must synthesize tryptophan • In the presence of tryptophan, cell does not need to make it • Both systems make use of a TXN repressor protein • DNA binding protein that interferes with TXN • Acts as an ON/OFF switch for gene expression • Binds a DNA sequence called the “Operator” • Steric blockade to promoter binding • Binds relevant metabolite that allosterically affects DNA binding

23. Gene expression: bacteria • Inducible gene expression • Example: Sugar catabolism • In the absence of lactose, no need to have enzymes that metabolize it - Lactose, Lac Repressor binds Operator and blocks TXN

24. Gene expression: bacteria • Inducible gene expression • Example: Sugar catabolism • In the presence of lactose, cell should make enzymes for metabolizing it + Lactose, Lac Repressor can’t bind Operator Allosteric effector inactivates Lac Repressor DNA binding

26. Gene expression: bacteria • Inducible gene expression • Lac operon can only be induced when glucose level is low • Low glucose = high cAMP level inside cell • CRP protein binds and activates TXN in presence of cAMP • cAMP-CRP complex binds DNA • Helps RNAp bind to promoter region • Look for -35 (TTGACA) and -10 (TATAAT) elements!

27. Gene expression: bacteria • Repressible gene expression • Example: Amino acid anabolism • In the absence of tryptophan, cell must synthesize tryptophan • Trp Repressor can only bind to Operator sequence when tryptophan is present • Tryptophan, Trp Repressor can’t bind Operator Allosteric effector needed for effective DNA binding

28. Gene expression: bacteria • Repressible gene expression • Example: Amino acid anabolism • In the presence of tryptophan, cell does not need to make it • Trp Repressor can only bind to Operator sequence when tryptophan is present + Tryptophan, Trp Repressor binds Operator tightly, blocks TXN Allosteric effector needed for effective DNA binding

30. Gene expression: eukaryotes

31. Gene expression: eukaryotes • TXN-level control • TXN factors bind specific DNA sequence “elements” • Activators • DNA binding domain + activation domain • Repressors • DNA binding domain + repression domain

32. Gene expression: eukaryotes • How do we identify promoter “elements” important for gene expression? • Deletion Mapping • DNA footprinting • Using DNase I digestion • Genome-wide location analysis • Using chromatin immunoprecipitation

33. Gene expression: eukaryotes • TXN-level control • Promoter structure • TATA Box (core element) • Response elements < 1 kb away • Can be isolated sites for individual factors or clustered together • Enhancer elements > 1 kb away • 200 bp size containing many binding sites • Insulator elements separate one transcription unit from an adjacent unit INSULATE ENHANCE PEPCK gene

34. Gene expression: eukaryotes • TXN-level control • Mechanism • Co-activation • Co-operative binding between Activator and GTFs • Histone modification: recruit HAT enzymes

36. Gene expression: eukaryotes • TXN-level control • Mechanism • Co-repression • Antagonistic binding: block GTFs • Histone modification • recruit Histone deacetylase (HDAC) enzymes • Recruit HMTs

37. Gene expression: eukaryotes • TXN-level control • Mechanism • Co-repression • DNA methylation: recruit DNA methyltransferases (DNMTs) • Methylated DNA serves as binding sites for proteins (MeCP2) • Recruit… HDACs, HMTs

38. Gene expression: eukaryotes • Processing-level control • Alternative splicing • Exonic Splicing Enhancers • ESE binding proteins • Cell-type specific • Fibroblast vs Hepatocyte • (RNA editing too) Fn

39. Gene expression: eukaryotes • TLN-level control • mRNA localization • Bicoid @ anterior • Oskar @ posterior • Beta-actin mRNA at leading edge of a migrating fibroblast

40. Gene expression: eukaryotes • TLN-level control • mRNA translation • Masking by specific proteins that bind to 5’-/3’-UTR sequences • IRE is an RNA sequence • IRP binds to IRE - Iron = bind and inhibit + Iron = no bind

41. Gene expression: eukaryotes • TLN-level control • mRNA stability • polyA tail length 200nt --> 30nt (destroyed) • Specific sequence effects • 5’-CCUCC-3’ stabilizing (factors bind to mediate this) • 5’-AUUUA-3’ destabilizing (factors bind to mediate this) • Just one of these can reduce ½-life from 10hrs to 90 minutes

42. Gene expression: eukaryotes • TLN-level control • mRNA stability • polyA tail length 200nt --> 30nt (destroyed) • Decapping enzyme • 5’  3’ exonuclease