Bio 405/505 Advanced Cell & Developmental Biology II The Cell Nucleus Lectures Dr. Berezney Lecture 3 : Background & Figures; Huang & Spector, 1996; Wei et al., 1999; Wei et al., 1998; Osborne et al., 2004;

Conventional View of Eukaryotic Transcription and RNA Splicing as two Separate Processes 1.DNA  Transcription  pre-mRNA 2. pre-mRNA  RNA Splicing  mRNA Current View of Eukaryotic Transcription and RNA Splicing as Being Functionally and Spatially Linked as a Highly Coordinated Process DNA  Transcription/RNA Splicing  mRNA

HUANG S & SPECTOR D Intron-dependent recruitment of pre- mRNA splicing factors to sites of transcription Journal of Cell Biology (1996) 133,

Huang & Spector, JCB 1996, Figure 1 Constructs used in transient transfections

Major Conclusions of Huang & Spector, 1996 Association of nascent RNA transcripts with splicing factors is intron dependent during transient or stable expression (Figs 2-5). Conclusion 1

RNA SC-35 merged Huang & Spector, Fig 2 Intronless RNA’s or RNA’s with a partial intron are not spatially associated with splicing factors in the cell nucleus β-galactosidase β-globin (cDNA) CMVTAT (partial intron) AD VA RNA

RNA SC-35 merge Huang & Spector, Fig 3 Transiently expressed RNA transcripts containing introns are spatially associated with splicing factors β-globin (genomic DNA) CGTAT β- tropomyosin β-tropo/U2 snRNP

Huang & Spector, JCB 1996, Figure 4 Association of transcripts and splicing factors

Huang & Spector, Fig 5 Intron dependent association of RNA transcripts and splicing factors in stably transfected cell lines RNA SC-35merged β-globin minus β-globin plus

Major Conclusions of Huang & Spector, 1996 The foci of expressed RNA are also the sites of transcription (Fig 6). Conclusion 2

Huang & Spector, JCB 1996, Figure 6 Transiently expressed RNAs co-localize with sites of transcription β-globin minus β-tropo plus RNABrUTPmerged

Major Conclusions of Huang & Spector, 1996 contd… The foci of intron-containing RNA are sites of pre-mRNA splicing and co- localize with mature mRNA (Fig 7 & 8). Conclusion 3

Huang & Spector, JCB 1996, Figure 7 Diagram illustrating the design of hybridization probes for the specific detection of β- tropomysin pre-mRNA and mature RNA. Introns 6 & 7 were used as pre-mRNA specific probes Splice junction constructs 12 Nt from 3’ and 5’ ends, respectively, between exons 5 & 6 or 5 & 8 were used as mRNA specific probes

Huang & Spector, Fig 8 Sites of transiently expressed intron-containing pre-mRNA & mRNA co-localize with each other and with sites of pre-mRNA splicing β-tropo mRNA (a) & pre-mRNA (b) β-tropo pre-mRNA (d) & SC-35 (e) β-tropo mRNA (g) & SC-35 (h) after 5 hr in α-amanitin

Major Conclusions of Huang & Spector, 1996 Association of nascent RNA transcripts with splicing factors is intron dependent during transient or stable expression (Figs 2-5) The foci of expressed RNA are also the sites of transcription (Fig 6) The foci of intron-containing RNA are sites of pre-mRNA splicing and co-localize with mature mRNA (Fig 7 & 8) Taken together these findings support a model for coordinate transcription and RNA splicing at discrete assemblies arranged within the nuclear architecture.

A Model for Transcription-Coupled Pre-mRNA Splicing

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, Review paper and powerpoint lecture 4 (Section II) from Bio 402/502 website

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

Functional Model of ~1 Mbp Chromatin Domains G1G1 Non-Replicating Chromatin Domain S Replicating Chromatin Domain S or G 2 Non-Replicating Chromatin Domains Replication factory

DO THE HIGHER ORDER CHROMATIN DOMAINS (1 Mbp) FUNCTION AS GENE TRANSCRIPTION FACTORIES AND IF SO, WHAT IS THEIR RELATIONSHIP TO RNA SPLICING SITES (NUCLEAR SPECKLES)?

Wei et al. Three-dimensional visualization of transcription sites and their association with splicing factor-rich nuclear speckles Journal of Cell Biology (1999) 146,

Major Conclusions of Wei et al., 1999 Transcription Sites (TS) labeled with BrdUTP in permeabilized 3T3 mouse fibroblast cells are characterized by inhibitor and enzyme digestions studies as pol I- and II- RNAP mediated TS (Figure 1). Conclusion 1

Figure 1 (Wei et al., 1999): Properties of Transcription Sites in 3T3 Mouse Fibroblast Cells

Major Conclusions of Wei et al., 1999 Approx. 2,000 sites (94% pol II-mediated TS) are detected following segmentation and are arranged in clusters and 3-D network arrays (Figures 2 & 3). Conclusion 2

Figure 2 (Wei et al., 1999) Segmentation of Transcription Sites

Figure 3 (Wei et al., 1999) 3-D Visualization of TS and Individual Sites

Major Conclusions of Wei et al., 1999 The number of TS, their 3-D organization and arrangement into higher order functional zones are maintained after extraction for nuclear matrix (Figures 4, 5 & 6). Conclusion 3

Figure 4 (Wei et al., 1999) TS After Nuclear Matrix Extraction and Synthesis on Nuclear Matrix

Figure 5 (Wei et al., 1999) 3-D Visualization of TS and Individual Sites After Nuclear Matrix Extraction

Figure 6 (Wei et al., 1999) Replication and Transcription Sites Higher Order Zones After Nuclear Matrix Extraction

Major Conclusions of Wei et al., 1999 Significant levels (43%) of pol II TS are associated with splicing factor-rich nuclear speckles (Figure 8, Table II) and are found along the periphery as well as inside individual speckles that are optically sectioned (Figure 9). Conclusion 4

Figure 8 (Wei et al., 1999) Association of TS With Nuclear Speckles

Table II (Wei et al., 1999) Quantitation of TS Associated with Nuclear Speckles

Figure 9 (Wei et al., 1999) Evaluation of TS Inside Speckles By Optical Sectioning

Major Conclusions of Wei et al., 1999 A similar distribution of TS along nuclear speckles was detected following in vivo labeling of TS with BrdU (Figure 10). Conclusion 5

Figure 10 (Wei et al., 1999) In vivo Labeling of TS and Association With Nuclear Speckles

Major Conclusions of Wei et al., 1999 Taken together these findings support a model in which coordinate transcription/RNA splicing of pre-mRNA occurs in close association with the nuclear speckles as functional transcription/splicing factories that are associated with the nuclear matrix. Overall Conclusion

Major Conclusions of Wei et al., 1999 Transcription Sites (TS) labeled with BrdUTP in permeabilized 3T3 mouse fibroblast cells are characterized by inhibitor and enzyme digestions studies as pol I- and II- RNAP mediated TS (Figure 1). Approx. 2,000 sites (94% pol II-mediated TS) are detected following segmentation and are arranged in clusters and 3-D network arrays (Figures 2 & 3). The number of TS, their 3-D organization and arrangement into higher order functional zones are maintained after extraction for nuclear matrix (Figures 4,5 & 6). Significant levels (43%) of pol II TS are associated with splicing factor- rich nuclear speckles (Figure 8, Table II) and are found along the periphery as well as inside individual speckles that are optically sectioned (Figure 9). A similar distribution of TS along nuclear speckles was detected following in vivo labeling of TS with BrdU (Figure 10). Taken together these findings support a model in which coordinate transcription/RNA splicing of pre-mRNA occurs in close association with the nuclear speckles as functional transcription/splicing factories.

This raises the key question: When are the Genes in These Early S Replicated Chromatin Domains Transcribed? Replication Timing Experiments in Mammalian Cells Have Demonstrate that Actively Transcribed Genes are Preferentially Replicated in Early S-Phase

WEI X, SAMARABANDU J, DEVDHAR RS, SIEGEL AJ, ACHARYA R & BEREZNEY R Segregation of transcription and replication sites into higher order domains. Science (1998) 281,

Simultaneous Replication and Transcription at Chromatin Domains during S-Phase RS TS RS/TS

Major Conclusions of Wei et al., 1998 The ~ 1,000 RS & 2,000 TS at any moment in early S are spatially separate chromatin domains (Fig 1). Conclusion 1

Wei et al., Science 1998, Figure 1 Spatial seperation of replication and transcription sites throughout S phase

These Findings Are Consistent With Model 2

Major Conclusions of Wei et al., 1998 contd.. Individual RS & TS are arranged into separate nuclear zones of replication or transcription (Fig 3). Conclusion 2

Wei et al., Science 1998, Figure 3 Cluster distribution of replication and transcription sites extending into several optical sections

These Findings Support A Novel Model 3

These segregated zones are arranged into spatially separate 3-D networks that extend throughout the nuclear interior (Fig 4). Conclusion 3 Major Conclusions of Wei et al., 1998 contd..

Wei et al., Science 1998, Figure 4 Replication and transcription sites in early S phase separate into distinct higher order domains in 3D

Wei et al., Science 1998, Figure 4 contd…. Replication and transcription sites in early S phase separate into distinct higher order domains in 3D

Replication timing and transcription of newly replicated genes likely involves a corresponding dynamic networking of replication & transcription processes in which nuclear zones of replication and transcription undergo coordinate rezoning. These networks of functional replication & transcription zones are the organizational basis for the regulation of replicational and transcriptional activity. Overall Conclusions Major Conclusions of Wei et al., 1998 contd..

Major Conclusions of Wei et al, 1998 The ~ 1,000 RS & 2,000 TS at any moment in early S are spatially separate chromatin domains (Fig 1). Individual RS & TS are arranged into separate nuclear zones of replication or transcription (Fig 3). These segregated zones are arranged into spatially separate 3-D networks that extend throughout the nuclear interior (Fig 4). Replication timing and transcription of newly replicated genes likely involves a corresponding dynamic networking of replication & transcription processes in which nuclear zones of replication and transcription undergo coordinate rezoning. These networks of functional replication & transcription zones are the organizational basis for the regulation of replicational and transcriptional activity in the cell.

Transcription Factories Gene Regulation By Higher Order Arrangement of Chromatin Loops and Multi-Loop Domains Based On Transcription

Osborne et al., Nature Genetics 36, (2004) Active Genes Dynamically Colocalize to Shared Sites of Ongoing Transcription The intranuclear position of many genes has been correlated with their activity state, suggesting that migration to functional subcompartments may influence gene expression. Indeed, nascent RNA production and RNA polymerase II seem to be localized into discrete foci or 'transcription factories'. Current estimates from cultured cells indicate that multiple genes could occupy the same factory, although this has not yet been observed. Here we show that, during transcription in vivo, distal genes colocalize [close proximity?] to the same transcription factory at high frequencies. Active genes are dynamically organized into shared nuclear subcompartments, and movement into or out of these factories results in activation or abatement of transcription. Thus, rather than recruiting and assembling transcription complexes, active genes [may] migrate to preassembled transcription sites. More generally, these findings indicate a dynamic interplay between multiple actively transcribed genes and the transcriptional machinery to form highly regulated transcription factories.

Gene Probes From a ~40 Kbp Region on Mouse Chromosome 7 Hbb-b1 (β-like globin, highly transcribed in erythroid cells) Eraf (essential globin-stabilizing protein) Uros (heme biosynthetic enzyme essential for globin) Igf2 (insulin-like growth factor, unrelated to globin) Kcnq1ot1 (long QT intronic transcript, unrelated to globin) Hba (α-globin gene on chromosome 11)

Major Conclusions of Osborne et al., 2004 Actively transcribed genes show significant levels of co-localization (proximity) (Fig 1 and 2) Conclusion 1

(b) RNA FISH on adult anemic spleen erythroid cells with intron probes for Uros. Note cell nuclei with zero, one or two signals (green). DAPI staining is blue. Scale bar, 5 µm. (c) The percentage of alleles with a gene transcription signal by RNA FISH for Hbb-b1, Hba, Eraf, Uros, Igf2 and Kcnq1ot1 in erythroid cells. (d) Relative primary transcript levels for Hbb-b1, Eraf, Uros and Igf2, measured by quantitative RT- PCR of intron sequences. Average values are shown with Eraf set to 1. Fig 1. Transcription frequencies of genes on mouse chr 7

Fig 2. Colocalization of transcribed genes with Hbb-b1 (red) on mouse chr 7 using RNA FISH Hba Kcnq1ot1 Uros IgF2 Eraf % of gene signals that overlap with Hbb-b1 gene signals

Major Conclusions of Osborne et al., 2004 Co-localization (proximity) of genes correlates with an active transcriptional state (Fig 3) Conclusion 2

Fig 3. Colocalization of genes is transcription- dependent- 3-D measurement of distances between Hbb-b1(red) and Eraf (green)

Major Conclusions of Osborne et al., 2004 Actively transcribed genes show significant levels of co-localization (proximity) with RNA pol II (Fig 4) Conclusion 3

Fig 4. Actively transcribed genes associate with RNAP II foci BLACK (RNA FISH % alleles)-GRAY (% foci overlap with pol II)

Major Conclusions of Osborne et al., 2004 Co-localization (proximity) of actively transcribed genes and RNA pol II (Fig 5) Conclusion 4

Fig 5. Actively transcribed genes colocalize to shared transcription factories DNA FISH: (Hbb (green), Eraf (red), pol II (blue) RNA FISH: (Hbb (red), Eraf (green), pol II (blue) Distances Hbb  Eraf

Major Conclusions of Osborne et al., 2004 Multiple actively transcribed genes are dynamically associating with individual transcription factories. It is conceivable that that there is a dynamic and coordinate assembly of chromatin loops (actively transcribed genes) and transcription factories. (Fig 8) Conclusion 5 (Overall)

Fig 8. Model of dynamic associations of genes with transcription factories Schematic representation of chromatin loops (black) extruding from a chromosome territory (gray). Transcribed genes (white) in RNAP II factories (black circles). Potentiated genes (free loops) that are not associated with RNAP II factories are temporarily not transcribed. Potentiated genes can migrate to a limited number of preassembled RNAP II factories to be transcribed (dotted arrows). We propose that both cis and trans associations are possible.

Transcription Factories Gene Regulation By Higher Order Arrangement of Chromatin Loops and Loop Domains