Barkha Bhatnagar 3 rd November, 2008

1. Barkha Bhatnagar 3rd November, 2008

2. Structure of chromosome

3. Interphase Nucleus

4. Chromatin Dynamics Gene poor chromosomes Gene rich chromosomes Spector et al, 2003 Schneider et al, 2007

5. Example of the model Branco et al, 2006

6. Drag image to reposition. Double click to magnify further.  Chromatin Dynamics Interchromosomal interaction Schneider et al, 2007 Branco et al, 2006

7. Present model – Interchromosomal network model Branco et al, 2006

8. Chromosome territories and nuclear bodies Schneider et al, 2007

9. Chromosome territories and nuclear bodies Kumaran et al, 2008

10. Main Objective The dynamic interactions between chromatin and nuclear bodies or their constituents and the impact of these interactions on nuclear structure and functions.

11. Outline • Genes on the move (Gene Positioning) • Nuclear Periphery • Loop out Chromosomal Territories - Intrachromosomal interaction • Interchromosomal interaction • 2. Estradiol-induced interchromosomal interaction • 3. Actin/motor-mediated interchromosomal interactions • 4. Gene interactions at nuclear speckles

12. 1. Nuclear Periphery Nuclear Pore Complex Active genes are associated with nuclear periphery – eg. - S. cerevisiae, D. melanogaster, and mammalian cells (β-globin locus during erythroid maturation)

13. Interaction of β-globin locus and Pol II in erythroid maturation Changing nuclear landscape with erythroid differentiation Ragoczy et al, 2006

14. 2. Loop out chromosome territories – short distance Example – mouse erythroid progenator cells Schneider et al, 2007 Fraser et al., 2007

15. 2. Loop out chromosome territories – long range (V/J interaction of Ig)

16. 2. Loop out chromosome territories – long range (T cell receptors - β and αδ)

17. 3. Interchromosomal interaction – loci present on different chromosomes Interaction of Th2 cytokine locus and the promotor of IFN-γ gene Schneider et al, 2007 LCR – locus control region

18. 3. Interchromosomal interaction – long distance (enhancer) • Olfactory genes – long distance chromatin interaction • 1,300 genes comprise of receptor family and only one olfactory receptor • is expressed in a particular neuron and expression is mono-allelic • Choice of specific olfactory receptor gene to be expressed in a given • neuron is mediated by interaction between the gene and the enhancer • element, called H element Enhancer – short region of DNA that can bound with proteins to enhance transcription

19. Cis and trans interactions of the H enhancer and olfactory-receptor genes Fraser et al., 2007

20. Fraser et al., 2007

21. 3. Interchromosomal interaction – genome inprinting Genome imprinting – genetic phenomenon by which certain genes are expressed in a parent-of-origin-specific manner CTCF – Insulator are cis acting DNA elements that prevent the enhancer and promotor interaction when present between them CTCF – chromatin insulator protein, possibly present during interphase ICR region – imprinting control region E – enhancer, Igf2 – insulin like growth factor 2, H19 – gene for non-coding RNA

22. Outline • Genes on the move (Gene Positioning) • Nuclear Periphery • Loop out Chromosomal Territories - Intrachromosomal interaction • Interchromosomal interaction • 2. Estradiol-induced interchromosomal interaction • 3. Actin/motor-mediated interchromosomal interactions • 4. Gene interactions at nuclear speckles

23. Article RETRACTED: Nuclear Receptor-Enhanced Transcription Requires Motor- and LSD1-Dependent Gene Networking in Interchromatin Granules Esperanza Nunez1, 2, 7, Young-Soo Kwon4, 7, Kasey R. Hutt1, 5, Qidong Hu1, Maria Dafne Cardamone1, 6, Kenneth A. Ohgi1, Ivan Garcia-Bassets1, David W. Rose3, Christopher K. Glass4, Michael G. Rosenfeld1, , and Xiang-Dong Fu3, , SUMMARY This article has been retracted at the request of the authors. Please see Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy).Reason: Shortly after publication of this paper, concerns were brought to the authors' attention regarding similarities in the plots quantifying the interchromosomal distances derived from FISH images. The authors undertook extensive investigation and confirmed a series of data duplications and transversions in the original dataset. Although they continue to believe the central conclusions of the paper based on confirmation and further studies by other scientists in their laboratories, given the extent of errors in the published plots, the authors feel compelled to retract the paper. They apologize to the scientific community for any confusion these errors may have caused and wish to sincerely thank the colleagues who communicated this problem to them in recent weeks.

24. Estradiol – estrogen (sex hormone), receptor ER-α • Purpose of experiment – to determine whether ligands for nuclear receptors induce interchromosomal interactions critical for specific programs of gene expression

25. TFF1 gene regulated by ER-α TFF1 – Trefoil factor 1, associated with breast cancer, located on chr 21 GREB1 – Gene regulated in breast cancer 1 protein, located on chr 2 SPECKLES – Interchromatin granule cluster, contain splicing factor

26. E2, steroid hormone in MCF7 and HMECs cell lines • 2 - 60 minute – interaction between TFF1 and GREB1 • 50% monoallelic and 50% diallelic • Non-homologous

27. Specific interchromosomal interactions in response to nuclear hormone • signaling • Question - How rapidly such signals are transduced resulting in the • coordinate movement and association of these genes • - How intranuclear signaling cascade is controlling the • movement and subsequent interaction of TFF1 and GREB1 • Hypothesize – Involvement of actin / motor elements • Use actin inhibitors, siRNA technique to block functioning of actin/myosin • – which abolished E2 induced interchromosomal interactions • Confirms – role for actin/myocin in ER-α-dependent interchromosomal interaction and gene movement

28. Where are interacting genes localized ? Gene interaction at nuclear speckles Speckles – rich in pre-RNA splicing factors, many other proteins involved in gene regulations Immuno-DNA FISH analysis by using 20 different probes to ER-α targeted genes and also marker for speckle protein (anti-SC35) - ER-α target genes colocalized with speckles upon E2 induction

30. Conclusion - 1- The whole issue of chromatin dynamics and Gene positioning - is a deep understanding of chromatin interaction through its element in the particular nuclear structure and its effect in determining the epigenetics of the Gene expression . 2-Nuclear Organization and Function are co-ordinated and intimately linked to each other- Long distance Interchromatin interaction and intrachromatin interaction are the result of a co-ordinated movement through motor protein to bring two long distance genes in the certain nuclear domain ( like ie speckle) to enhance their gene expression . 3-Nuclear bodies like speckle are active center for transient chromosomal interaction- Nuclear bodies provide a clear platform (a particular Nuclear domain) where the chromosomal interaction can perform its co-ordinated event like mRNA transcription and RNA processing much faster by using concentrated resources . 4- Nuclear periphery and nuclear domain interact with extra loop of chromatin , out of its chromosomal territory to bring out certain gene expression, to share the transcriptional machinery, to perform recombination to re-arrange the genes.

31. Thanks for your attention

32. http://en.wikipedia.org/wiki/Fluorescent_in_situ_hybridizationhttp://en.wikipedia.org/wiki/Fluorescent_in_situ_hybridization