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

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

3. Transcription Transcription is initiated at specific sequences at the 5’ end of the gene called the promoter which typically contains a TATA box consensus sequence. Transcriptional machinery is assembled at the promoter via TFIID [TBP (TATA binding prot) & TAFs (TBP binding proteins)] Key components of the pre- initiation complex are TFIIA and TFIIB which lead to the recruitment of TFIIF/pol II and other TF’s. Initiation/Elongation requires phosphorylation of the CTD (C-Terminal Domain) of the RNA polymerase II large subunit CTD: hepapeptide (52x) Y1,S2,P3,T4,S5,P6,S7Y1,S2,P3,T4,S5,P6,S7 Initiation: S 2 P (TFIIH) Elongation: S 5 P (PTEFb) SII and ELL (processivity) Elongating RNAP II ~ 50 components (>3 mill Dal) PTEFb initiation complex Initiation elongation

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. E1E1 I1I1 E2E2 I2I2 E3E3 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. MAINTAINING IN SITU FUNCTIONAL DOMAINS ON THE NUCLEAR MATRIX Chromosome Domains Splicing FactorsReplication Sites Transcript TracksTranscription Sites Extracting Nuclear Matrix

10. Replication Timing and the Spatial Organization of RS Early S - Replication of Actively Transcribed Genes Mid and Late S - Replication of Non- 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. Early S Mid S Late S Replication Timing and the Spatial Organization of DNA Replication Sites Replication Sites (RS) are Arranged into Distinctly Different Patterns As S-Phase Progresses

13. 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. Research Journal Article

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

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

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 2 0 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 1 2 3 4 5 911 10 8 7 6 Optical section Number, XY / XYZ Coordinates & Quantitative co-localization ~1000 sites per nucleus 3-D organization

20. MA et al., JCB 1998, Figure 1 Computer analysis of individual replication sites Original ImageIn-house softwareCommercial 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 Earlier ReplicationLater Replication Parental DNA Strand Daughter DNA Strand CldU: Earlier Replication IdU: Later Replication Overlay CldU

24. DETERMINING THE AVERAGE LIFETIME OF A REPLICATION SITE (RS) IN EARLY S PHASE [Double halogenated nucleoside labeling experiment ] 1.Mammalian cells grown on cover slips are synchronized in early S-phase. 2. A pulse-chase-pulse experiment is then performed with CldU to label one temporal population of RS and after a chase from 0 minutes to 2 hours IdU is used to label a second temporal population of RS. 3. The anti-CldU, anti-IdU and appropriate 2 0 Abs are used to stain the CldU incorporated RS green and the IdU sites in red. 4. If replication is still continuing during IdU incorporation at sites previously labeled with CldU those sites will appear yellow. Green sites are sites that have finished replication before or at the IdU pulse. Red sites are 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 Replication Sites Green Later Replication SitesRed Earlier and Later Replication at the Same SitesYellow Earlier S Sites 5 min pulse CldU Chase 0-36 hours Later S Sites5 min pulse IdU Anti CldU FITC Anti IdU Texas Red

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

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 yellow and 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 yellow sites for each chase time and plot the percent yellow sites (overlaps) versus chase time. This shows the experimental kinetics of decay for the yellow sites. 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. Average time to replicate an RS = 45 min MA et al., JCB 1998, Figure 4

29. ESTIMATION OF AVERAGE AMOUNT OF DNA AND NUMBER OF REPLICONS PER REPLICATION SITE 1.Determine average number of RS (1,000) 2.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. ESTIMATION OF AVERAGE AMOUNT OF DNA AND NUMBER OF REPLICONS PER REPLICATION SITE 10,000 Mb 10,000 sites = 1.0Mbp Minimal Avg. # of Replicons Per Site = 1.0 Mb 0.16 Mb = 6.25 Estimated DNA Per Site = 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. Total DNA per Nucleus Total Number of RS per Nucleus =

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

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

33. Ma et. al., Figures 5 & 6: RS are Maintained Through the Cell Cycle as Higher Order Chromatin Domains CldU chase IdU early/mid early/lateearly/G2 l 12 hr chase propidium iodide

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

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

36. 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. Conclusion 4: A Proposal MAJOR CONCLUSIONS OF MA et al., 1998 contd..

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

38. 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). Conclusion 5 MAJOR CONCLUSIONS OF MA et al., 1998 contd..

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

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).