Eukaryotic Genome

1. Eukaryotic Genome Organization and Regulation

2. Chromatin Organization Each human chromosome averages about 1.5 � 108 nucleotide pairs Each extended DNA molecule would be about 4 cm long � 1000X longer than the cell diameter (-) charged DNA wrapped around (+) charged histone proteins Extreme coiling shortens length of molecule by 100,000X

3. Histone Proteins Among eukaryotic organisms histone proteins are very similar What might this indicate? HINT: think about evolution DNA wrapped around histone proteins � nucleosome Two molecules each of four types of histone: H2A, H2B, H3, and H4 One molecule of H1 attaches to the DNA near the nucleosome The amino acid (N-terminus) of each histone protein (the histone tail) extends outward from the nucleosome. Histones leave the DNA briefly during DNA replication; but not during transcription

4. Why Causes the Coiling? Interactions between H1 + tails of nucleosome + linker DNA on either side cause formation of 30-nm chromatin fiber Fiber forms looped domains which attach protein scaffold Particular genes located in the same places on metaphase chromosomes in eukaryotic species � involved in coiling Interphase chromosomes: Highly condensed areas (heterochromatin) interspersed with less compact areas (euchromatin) Heterochromatin DNA cannot be transcribed The chromatin of each chromosome occupies a specific restricted area within the interphase nucleus � there is an order among the chaos! Looped domains attached to nuclear envelope in order to secure a place in nucleus

5. Cellular Differentiation Occurs shortly after blastocyst stage of embryo development Human cells express an average of 20% genes at any given time How many genes does this mean for the average cell? 1.5% of DNA in humans codes for protein A small fraction of the remainder codes for rRNA and tRNA The rest is currently thought to be noncoding HOWEVER researchers have found that much of the noncoding DNA is transcribed into RNAs of unknown function Gene expression = transcription Expression of specific genes is regulated at transcription - often in response to external signals Each stage in the process of gene expression can serve as a potential control point of gene expression Chromatin packing, transcription, RNA processing, translation, and various alterations to the protein product

7. Histone Modification Genes located in heterochromatin are usually not expressed A gene�s location in/near nucleosomes and scaffold attachment site can affect transcription Histone tails are a major site of modification Histone de/acetylation � removal or addition of an acetyl group (-COCH3) Acetylated histones grip DNA less tightly � easier for transcription proteins to bind Some acetylation enzymes are transcription factors Dual function � Modify chromatin structure Bind to and recruit components of the transcription machinery Histone Methylation condenses chromatin

8. DNA Modification DNA methylation Attachment of methyl groups (-CH3) to Cytosine after DNA synthesis inactivates genes Inactive DNA is highly methylated compared to DNA that is actively transcribed Ex. the inactivated mammalian X chromosome Genes are usually more heavily methylated in cells where they are not expressed � process can be reversed to turn genes back �on� DNA methylation enzymes recruit histone deacetylation enzymes and vice versa Genes usually stay methylated through successive cell divisions Methylation enzymes recognize sites on one strand that are already methylated and correctly methylate the daughter strand after each round of DNA replication

10. Genomic Imprinting Pre-programmed gene expression for each species Ex. Insulin-like Growth Factor 2 is only expressed from the paternal allele Discovery in mouse model Gynogenetic embryos - normal embryonic development; poor placental development Androgenetic embryos - poor embryonic development; normal placental development Each embryo received twice the normal level of maternal or paternal genes AND a complete lack of genes from the other parent What happens in meiosis? It is possible to erase and re-establish the imprint with each generation Alterations are epigenetic - modifications to the structure of the DNA rather than the sequence Methylation turns off either the maternal or paternal alleles of certain genes at the start of development Parental Conflict Hypothesis � each parent has a different interest for their child Father � wants to have strong offspring Mother � wants to have offspring that conserve resources and can therefore care for subsequent offspring

11. Transcription Factors Control elements - noncoding DNA segments that regulate transcription by binding certain proteins Ex. Promoter region (eukaryotic), control region (prokaryotic) Eukaryotic RNA polymerase requires the assistance of proteins called transcription factors General transcription factors bind TATA box Others are involved in protein-protein interactions binding each other Alone, these factors lead to a low rate of transcription Increased transcription rate of a particular gene depends on the interaction with specific transcription factors

12. Enhancers Proximal Control Elements � near the promoter Enhancers � distant control elements; thousands of nucleotides away from the promoter or possibly within an intron A given gene may have multiple enhancers

13. How Do Enhancers Work? Activator proteins - bind to enhancers; stimulate transcription of a gene Some recruit proteins that acetylate histones near the promoters of specific genes DNA Bending Protein - brings activators in contact with promoter This helps assemble and position the initiation complex on the promoter Mediator Protein � bind the activator proteins and the transcription factors Repressor proteins - inhibit expression of a gene. Block binding of activators to their control elements Block binding of transcription factors Turn off transcription even in the presence of activators Some recruit proteins that deacetylate histones -- reduces transcription

16. Like an Operon... How does a eukaryotic cell regulate the expression of genes that work together? Co-expressed genes located near each other on the same chromosome Each gene has its own promoter Chromatin modification makes the genes in a region available or unavailable Co-expressed genes on different chromsomes Often have the same control elements in the enhancer region Coordinated transcription due to presence of correct activator proteins Example: Steroid hormone enters a cell and binds to a specific receptor protein Hormone-receptor complex serves as a transcription activator Every gene whose transcription is stimulated by that steroid hormone has a control element recognized by that hormone-receptor complex

17. Post-Translational Modifications mRNA Life Span Prokaryotic mRNA degraded in minutes Eukaryotic mRNAs typically last for hours, days, or weeks Degraded by enzymatic shortening of the poly-A tail Triggers enzymatic removal of 5�cap Followed by rapid degradation of mRNA by nucleases

18. What regulates the degradation?? Nucleotide sequences in the untranslated region near 3� end Small single-stranded RNA molecules called microRNAs (miRNAs) bind complementary mRNA sequences miRNA binds to a protein -- complex then degrades the target mRNA OR blocks translation

19. RNA Interference Definition: Inhibition of gene expression by double-stranded RNA molecules dsRNA may be introduced by a researcher, a virus or created by an enzyme known as RNA-dependent-RNA-Polymerase (RDRP) When levels of RNA increase, RDRP turns ssRNA into dsRNA Dicer enzyme cuts the dsRNA into 21-25 base pair fragments called small interfering RNAs (siRNAs) Similar in size and function to miRNAs Bind to group of proteins creating RISC (RNA-induced silencing complex) siRNA is then unzipped and the RISC complex is activated RISC then recognizes target mRNA and mRNA is cleaved Why would scientists want to silence a gene???? RNAi Movie

21. Protein Processing and Degradation Many proteins must be short-lived to function appropriately � ex. cyclins Proteins intended for degradation are marked by the attachment of ubiquitin proteins Giant protein complexes called proteasomes recognize the ubiquitin and degrade the tagged protein When cell cycle proteins become impervious to proteasome degradation this can lead to cancer

22. What About Cancer? Involves genes that normally regulate cell growth and division Growth factors Growth factor receptors Intracellular molecules of signaling pathways Mutations altering any of these genes in somatic cells can lead to cancer Viruses can cause this alteration of genes Epstein-Barr Virus � Burkitt�s Lymphoma HPV � Cervical Cancer HIV � Kaposi�s Sarcoma Can also be caused by carcinogens � environmental factors that lead to an increased rate of gene mutation What are some known carcinogens? Oncogenes � tumor causing genes were identified in certain viruses Found to have counterparts in certain animals

23. Proto-Oncogenes and Tumor Suppressor Genes Proto-Oncogenes code for proteins involved in normal cell growth and division Can become a tumor causing gene (oncogene) following genetic mutation Translocations (movement of genetic information in a genome) are commonplace in cancer cells Genes may be moved near an active promoter Active promoter may be moved near a proto-oncogene, increasing its expression Point mutations In the promoter or enhancer of a proto-oncogene can increase expression In the coding sequence - protein that is more active or longer-lived Tumor-suppressor genes - normal protein products inhibit cell division Repair damaged DNA - prevent accumulation of mutations Control the adhesion of cells to each other - crucial for normal tissues and often absent in cancers Components of cell-signaling pathways that inhibit the cell cycle How might these genes play a role in tumor formation? Any decrease in the normal activity of a tumor-suppressor protein may contribute to cancer

25. ras Gene Mutations in the ras proto-oncogene are found in 30% of cancer cells Component of signal-transduction pathway that conveys external signals to the DNA ras protein is a G protein that relays a growth signal from a growth factor receptor Results in production of a protein that stimulates the cell cycle Many ras oncogenes have a point mutation that leads to a hyperactive version of the Ras protein Results in excessive cell division

26. p53 Gene Mutations in p53 occurs in 50% of human cancers Tumor suppressor protein p53 gene is activated when DNA is damaged The p53 protein is a transcription factor for several genes Can activate the p21 gene, which halts the cell cycle Can turn on genes involved in DNA repair. When DNA damage is irreparable it activates �suicide genes� which cause apoptosis A mutation that knocks out the p53 gene can lead to excessive cell growth and cancer