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Lectures 3 & 4

Lectures 3 & 4. Transcription / Splicing/ Replication in the Nucleus. Major Genomic Functions of Eukaryotic Cells. ( b) Transcription (selective regions of genome many times) DNA  RNA. DNA. RNA transcripts. ( c) Genes which code for proteins (RNA polymerase II)

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Lectures 3 & 4

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  1. Lectures 3 & 4 Transcription / Splicing/ Replication in the Nucleus

  2. Major Genomic Functions of Eukaryotic Cells (b)Transcription (selective regions of genome many times) DNA RNA DNA RNA transcripts (c) Genes which code for proteins (RNA polymerase II) DNA mRNAprotein synthesis (a) DNA replication (whole genome 1X): DNA  DNA

  3. Transcription • Transcription is initiated at specific sequences at the 5’ end of the gene called thepromoterwhich typicallycontains aTATA boxconsensus sequence. • Transcriptional machinery is assembled at the promoter viaTFIID[TBP (TATA binding prot) & TAFs (TBP binding proteins)] • Key components of thepre- initiation complexareTFIIA and TFIIBwhich lead to the recruitment ofTFIIF/pol II and other TF’s. • Initiation/Elongation requires phosphorylation of theCTD (C-Terminal Domain)of theRNA polymerase II large subunit Initiation elongation PTEFb CTD: hepapeptide (52x) Y1,S2,P3,T4,S5,P6,S7 Initiation: S2P (TFIIH) Elongation: S5P (PTEFb) SII and ELL (processivity) Elongating RNAP II ~ 50 components (>3 mill Dal) initiation complex

  4. Ribosomal (r), Transfer (t) & Small Nuclear (sn) RNAs • Nucleolus is the site of synthesis and maturation of the rRNA as well as the site for packaging with the preribosomal proteins. • rRNA is synthesized in the nucleolus by RNA polymerase I as a 45 S precursor rRNA which is then processed into 28, 18 and 5.8 S rRNA • The RNA and the proteins form pre rRNP ( ribosomal ribonucleoproteins) which mature into ribosomal subunits and exit from the nucleus into the cytoplasm for protein synthesis. • The 5S rRNA is synthesized in the extranucleoloar regions of the nucleus by RNA pol III. • snRNA [small nuclear RNAs]: RNA pol II, involved in RNA processing (splicing) • t RNA’s: RNA pol III

  5. Sucrose Gradient Separation of Nucleolar RNA Cells were labeled for 10 min with [14C]- methionine and chased as indicated

  6. RNA Splicing and Other Processing • RNA splicing involves a series of steps for intron removal and exon splicing to form a functional mRNA. • Splicesome complexes composed of many splicing factors and snRNPs are involved in the splicing process. • Intermediates of splicing are characterized by lariat structures. • Other processing of RNA include addition of methylated cap at the 5’ end of mRNA ( capping) and a polyadenosine tail at the 3’end. E3 E1 I1 E2 I2 Splicing mRNA

  7. Functional Genomics of the Cell Nucleus: The New Frontier

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

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

  10. Replication Timing and the Spatial Organization of RS Early S-Replication of Actively Transcribed Genes Mid and Late S- Replication ofNon-Active Genes and Other Non-Transcribed DNA Sequences (e.g., Satellite/Repetitive DNA)

  11. Visualizing Replication Sites in the Cell Nucleus Cells grown on coverslips Label replication sites with fluorescent probes Examine by fluorescence microscopy Computer image analysis

  12. Replication Timing and the Spatial Organization of DNA Replication Sites Replication Sites (RS) are Arranged into Distinctly Different Patterns As S-Phase Progresses Early SMid S Late S

  13. Research Journal Article Ma H, SAMARABANDU J, DEVDHAR RS, ACHARYA R, CHENG P-C, MENG C & BEREZNEY R Spatial and Temporal Dynamics of DNA Replication Sites in Mammalian Cells Journal of Cell Biology (1998) 143, 1415-1425.

  14. MAJOR CONCLUSIONS OF MA et al. ,1998 • (1)There is an average of ~1,000 replication sites (RS) active at any moment in early S phase (Fig 1 & 2). • (2) The average life-time of an early S RS is about 45 minutes and contains ~ 1 mbp of DNA (Fig 3 & 4). • (3) RS persist in the cell cycle and future generations as higher order chromatin domains (Fig 5, 6 & 7).

  15. MAJOR CONCLUSIONS OF MA et al. ,1998 • (4) In replication timing specific 1 mbp chromatin domains are recruited as RS at specific times in S. • (5) The same population of RS that replicate in early S of one cell generation, replicate at the same time in early S of the next cell generation as presumably identical higher order chromatin domains (Fig 8).

  16. MAJOR CONCLUSIONS OF MA et al. , 1998 Conclusion 1 There are an average of ~1,000 replication sites (RS) active at any moment in early S phase (Fig 1 & 2).

  17. Visualizing DNA Replication Sites In Mammalian Cells BrdU BrdU Daughter DNA Strand Parental DNA Strand DNA Replication Occurs at Discrete Sites

  18. ANALYZING DNA REPLICATION SITES (RS) IN THE CELL NUCLEUS BY 3-D MICROSCOPY & COMPUTER IMAGING Single halogenated nucleoside labeling experiment 1. Mammalian cells are grown on cover slips and synchronized in early S-phase. 2. Pulse with halogenated nucleoside e.g., 5 min, bromodeoxyuridine (BrdU). 3. Fix cells and label with anti BrdU, and a 20 Ab with FITC (green). 4. Collect optical sections by confocal microscopy. 5. Do computer imaging contour analysis of the individual RS and 3-D reconstruction of the optical sections. 6. Determine the average number of RS in early S phase at any moment of time and the x,y,z coordinates and volumes of all the individual sites.

  19. Quantitative Image Analysis CONTOUR ANALYSIS OF DNA REPLICATION SITES 10 9 11 3 5 3-D organization 1 4 2 Optical section 8 ~1000 sites per nucleus 6 7 Number, XY / XYZ Coordinates & Quantitative co-localization

  20. MA et al. , JCB 1998, Figure 1 Computer analysis of individual replication sites Original Image In-house software Commercial software

  21. MA et al., JCB 1998, Figure 2 Quantitative image analysis of replication sites

  22. MAJOR CONCLUSIONS OF MA et al. , 1998 contd.. Conclusion 2 The average life-time of an early S RS is about 45 minutes and contains ~ 1 mbp of DNA (Fig 3 & 4)

  23. DNA Double Labeling IdU CldU Daughter DNA Strand Parental DNA Strand Earlier Replication Later Replication IdU: Later Replication CldU: Earlier Replication Overlay

  24. DETERMINING THE AVERAGE LIFETIME OF A REPLICATION SITE (RS) IN EARLY S PHASE [Double halogenated nucleoside labeling experiment] • Mammalian cells grown on cover slips are synchronized in early S-phase. • 2. A pulse-chase-pulse experimentis then performed with CldUto label one temporal population of RS and after a chase from 0 minutes to 2 hoursIdU is used to label a second temporal population of RS. • 3.The anti-CldU, anti-IdU and appropriate 20 Abs are used to stain theCldU incorporatedRS greenand theIdU sites in red. • 4.If replication is still continuing during IdU incorporation at sites previously labeled with CldU those sites will appear yellow.Green sitesare sites that have finished replication before or at the IdU pulse.Red sitesare RS that have initiated after the CldU pulse is completed.

  25. Application of Halogenated Nucleotides for Double Labeling of DNA Replication Sites Pulse-Chase-Pulse Experiment Earlier S Sites 5 min pulse CldU Chase 0-36 hours Later S Sites5 min pulse IdU Anti CldU FITC Anti IdU Texas Red Earlier Replication SitesGreen Later Replication Sites Red Earlier and Later Replication at the Same SitesYellow

  26. MA et al. , JCB 1998, Figure 3 CldU-IdUdouble labeling of DNA replication sites

  27. DETERMINING THE AVERAGE LIFETIME OF A REPLICATION SITE (RS) IN EARLY S PHASE Double halogen nucleoside labeling experiment(CONTINUED) 5. Measure the number of yellowand green sites for each time point 6. The total of green sites plus yellow sites for each chase time equals the total number of original sites before the chase as either finished sites (green) or still replicating sites (yellow). 7. Determine the proportion of yellow sites as the percent of total green plus yellowsites for each chase time and plot the percent yellowsites (overlaps) versus chase time. This shows the experimental kinetics of decay for the yellowsites. 8. To determine the average lifetime, fit the experimental curve to a model lifetime curve that best fits the data :This is a 45 minute theoretical lifetime decay curve in this case.

  28. MA et al. , JCB 1998, Figure 4 Average time to replicate an RS = 45 min

  29. ESTIMATION OF AVERAGE AMOUNT OF DNA AND NUMBER OF REPLICONS PER REPLICATION SITE Determine average number of RS (1,000) Determine the average lifetime of RS (45 minutes) 3. Determine average amount of DNA per nucleus (biochem) 4. From 1, 2 and 3 can estimate the ~ amount of DNA per RS 5. From 4 and average bidirectional fork rate (3.5 Kbp/min) estimate the average minimal number of replicons/per RS

  30. 1.0 Mb 0.16 Mb Minimal Avg. # of Replicons Per Site = =6.25 ESTIMATION OF AVERAGE AMOUNT OF DNA AND NUMBER OF REPLICONS PER REPLICATION SITE Total DNA per Nucleus Total Number of RS per Nucleus 10,000 Mb 10,000 sites Estimated DNA Per Site = = = 1.0Mbp If each site contained one huge replicon it would take ~300 minutes to complete (fork rate of 3.5 Kbp/min). Since only ~160 Kbp of DNA can be replicated in 45 minutes, the average 1 Mbp RS is composed of at least six 160 Kbp replicons which replicate in a relatively synchronous manner.

  31. MAJOR CONCLUSIONS OF MA et al. , 1998 contd.. Conclusion 3 RS persist in the cell cycle and future generations as higher order chromatin domains (Fig 5, 6 & 7) .

  32. Replication Timing and the Spatial Organization of DNA Replication Sites Replication Sites (RS) are Arranged into Distinctly Different Patterns As S-Phase Progresses Early SMid S Late S

  33. Ma et. al., Figures 5 & 6:RS are Maintained Throughthe Cell Cycle as Higher Order Chromatin Domains CldUchaseIdU early/late early/G2 early/mid l propidium iodide 12 hr chase

  34. G1 Non-Replicating Chromatin Domain S Replicating ChromatinDomain G2 Non-ReplicatingChromatin Domain Higher Order Chromatin Domains (1 Mbp) as Units of DNA Replication (RS) Sites ~1 mbp domain Replication machinery

  35. 3-D Model of a 1 mbp Multi-Loop Chromatin Domain

  36. MAJOR CONCLUSIONS OF MA et al. , 1998 contd.. Conclusion 4: A Proposal In replication timing specific 1 mbp chromatin domains are recruited as RS at specific times in S. Thus basic unit of control for replication timing is not the individual gene but higher order chromatin domains that contain multiple genes.

  37. FUNCTION GENOMICS OF DNA REPLICATION SITES (RS) IN THE CELL NUCLEUS Is There A Precise Timing In The Replication Of The Thousands Of Genes During The First Hour Of S-Phase?

  38. MAJOR CONCLUSIONS OF MA et al. , 1998 contd.. Conclusion 5 The same population of RS that replicate in early S of one cell generation, replicate at the same time in early S of the next cell generation as presumably identical higher order chromatin domains (Fig 8).

  39. MA et al., JCB 1998, Figure 8 Demonstration of a high degree of replication timing among 1000’s of genes in early S phase

  40. Figure 8 (continued): Demonstration of a High Degree of Replication Timing Among 1000’s of Genes in Early S Phase

  41. MAJOR CONCLUSIONS OF MA et al. , 1998 (1)There is an average of ~1,000 replication sites (RS) active at any moment in early S phase (Fig 1 & 2). (2) The average life-time of an early S RS is about 45 minutes and contains ~ 1 mbp of DNA (Fig 3 & 4). (3) RS persist in the cell cycle and future generations as higher order chromatin domains (Fig 5, 6 & 7). (4) In replication timing specific 1 mbp chromatin domains are recruited as RS at specific times in S. (5) The same population of RS that replicate in early S of one cell generation, replicate at the same time in early S of the next cell generation as presumably identical higher order chromatin domains (Fig 8).

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