Nuclear Organization and Gene Expression Dr. David L. Spector

Transcription

1 NUCLEAR ORGANIZATION AND GENE EXPRESSION David L. Spector, Ph.D. Cold Spring Harbor Laboratory One Bungtown Road Cold Spring Harbor, New York Visit our website at 1 Orphanides and Reinberg (2002) Cell 108, Spector, D.L. (2001) J. Cell Sci. 114, The screen versions of these slides have full details of copyright and acknowledgements 1

2 Chromosomes are arranged in territories in the interphase nucleus 4 Bolzer et al. (2005) PLoS Biology 3, Visualizing gene expression in living cells 5 Characterization of the U2OS stable cell line Southern Northern Western 3.4 kb 2.8 kb 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 The locus is stably integrated in a euchromatic region of chromosome 1p hours post-transfection (-) Dox CFP-lac repressor MS2 binding protein-yfp hours post-transfection hours after the addition of Dox MS2 binding protein-yfp 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 RNA is present at the focal concentration of MS2 binding protein-yfp CFP-lac repressor MS2 binding protein-yfp Intron probe 10 MS2 binding protein-yfp msec exposure every 2.5 min for 4.5 hrs YFP-MS2 0 min (-) dox 5 min (+) dox 7.5 min 17.5 min 130 min 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 3-D organization of gene expression 2 hours after initiation 13 Nuclear mrnp dynamics is consistent with a diffusion based mechanism msec exposure for 30 sec Shav-Tal et al. (2004) Science 304, mrnp particle tracking Simple diffusion Corralled diffusion 60% 40% Average diffusion coefficient 0.04 µm 2 /sec 15 Shav-Tal et al. (2004) Science 304, The screen versions of these slides have full details of copyright and acknowledgements 5

6 Tet-ON-VP16-YFP is present at the locus 5 min after the addition of Dox Tet-ON-VP16-YFP rtetr VP16 YFP 16 Tet-ON-VP16-YFP is increasingly recruited to the locus over time (15 min after the addition of Dox) Tet-ON-VP16-YFP rtetr VP16 YFP 17 The RNA polymerase II large subunit is recruited to the active locus YFP-RNA pol II 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 The pre-mrna splicing factor, SF2/ASF, is recruited to the active locus YFP-SF2/ASF 19 CstF-64 is recruited to the active locus YFP-lac repressor CstF Active Chromatin Repressed Chromatin Adapted from: Kouzarides, 21 T. The screen versions of these slides have full details of copyright and acknowledgements 7

8 HP1α, β and γ are enriched at the inactive locus YFP-HP1α YFP-HP1β YFP-HP1γ 22 Histone H3 tri-methylated at Lysine 9 is enriched at the inactive locus 0 min (-) Dox 2.5 hrs (+) Dox YFP-lac repressor Histone H3 trimethyl-k9 23 The Suv39h1 and G9a-L histone methyltransferases localize at the inactive locus YFP-Suv39h1 YFP-G9a-L 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 HP1α clears from the locus upon transcriptional induction 0 min (-) Dox 2.5 hr (+) Dox YFP-HP1α 25 YFP-HP1α 100 msec exposure every 2.5 min for 3 hrs 26 0 min 17.5 min 30 min 50 min 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 Histone H3 and the H3.3 variant Deposition Replication coupled Replication coupled and independent S31 Histone Fold Domain A31 S87 V89 M90 A87 I89 G90 Ahmad and Henikoff (2002) Mol. Cell 9, ; PNAS 99, pseudo-colored red H3.3-YFP Exposure every 2.5 min for 5 hrs 16 min 29 H3.3-YFP 0 min (-) dox 7.5 min (+) dox 40 min 75 min 180 min 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 Summary (1) The cell nucleus is an organized cellular compartment containing chromosome territories and numerous nuclear compartments which may play a role in sequestering and releasing factors as well as function in their modification and assembly. Using an inducible system we have been able to directly visualize a genetic locus as well as its RNA and protein products in living mammalian cells. Nascent RNA can be detected within 5-8 min after induction, while dramatic changes in chromatin structure are seen by min. Factors from various nucleoplasmic regions are recruited to the locus in a transcription-dependent manner. 31 Summary (2) mrnp nuclear dynamics is consistent with a diffusion based mechanism. HP1α, β and γ, histone H3 tri-methyl K9, and several HMTases are enriched at the inactive locus. HP1α clears from the locus as it decondenses and is not detected at the locus ~ 50 min after transcriptional activation. Histone H3.3 is enriched at the transcriptionally active locus. 32 Acknowledgements Cold Spring Harbor Susan Janicki Prasanth Kannanganattu Himeji Inst. Tech. Toshiro Tsukamoto NIH/NCI Thomas Ried Fred Hutchinson Kami Ahmad Steven Henikoff University of Illinois Andrew Belmont Albert Einstein College of Medicine Yaron Shav-Tal Robert Singer CNRS - Montpellier Edouard Bertrand Visit our web site at 33 The screen versions of these slides have full details of copyright and acknowledgements 11