MCB 252 Topic 10 Chromosome Organization and Gene Regulation II Prof David Rivier MCB 252 Spring 2015 Reading For topics 10 and 11 Lodish sections 7 2 7 5 7 7 Outline Localization of Heterochromatin in Nucleus Nuclear Lamina Mis localization and Progeria Basic Structure of Chromatin Histone Code How chromatin is structured and organized within the nucleus Heterochromatin Euchromatin Nucleolus How chromatin is structured and organized within the nucleus RER Ribosome Outer nuclear membrane NPC Inner nuclear membrane Nuclear Lamina Heterochromatin How chromatin is structured and organized within the nucleus EM image of the nuclear lamina in the frog oocyte How chromatin is structured and organized within the nucleus Nuclear lamins type V intermediate filaments N ter head domain Coiled coil nuclear lamins rod domain Dimerization and higher structure assembly C ter tail domain Nuclear and INM targeting domains A type Lamins LMNA gene codes for Lamin A and Lamin C B type Lamins LMNB1 and LMNB2 genes coding for Lamin B1 and Lamin B2 respectively How chromatin is structured and organized within the nucleus A type lamins Lamin A and Lamin C 5 1 2 3 1 Alternative splicing 2 4 3 5 4 6 5 6 7 7 8 8 9 9 10 10 11 d 12 11 12 3 Lamin A 664 aa NLS CaaX Head domain 1 Head domain Coiled coil domain 2 3 4 5 Coiled coil domain Tail domain 6 7 8 9 Tail domain 10 Lamin C 572 aa NLS but no CaaX How chromatin is structured and organized within the nucleus Dimer Polymer Fiber How chromatin is structured and organized within the nucleus Lamins are targeted to the INM Lamin A Distribution Confocal Microscopy How chromatin is structured and organized within the nucleus Lamin B2 protein NH2 CLVM COOH CaaX motif Farnesyl transferase S NH2 CLVM COOH Cleavage and methylation S NH2 C COOCH3 How chromatin is structured and organized within the nucleus Pre Lamin A protein NH2 SYLLGNSSPRTQSPQNCSIM COOH Farnesylation cleavage and methylation S NH2 SYLLGNSSPRTQSPQNC COOCH3 Specific cleavage S LLGNSSPRTQSPQNC COOCH3 NH2 SY COOH Mature Lamin A protein How chromatin is structured and organized within the nucleus RER ONM Cytoplasm ONM LBR LAP2 LAP1 Emerin C C E BAF E N C HP1 N Heterochrom atin BAF N C A C B1 B2 ONM INM N Nucleoplasm How chromatin is structured and organized within the nucleus Mutations in LMNA produce an intriguingly diverse spectrum of diseases including Muscular dystrophies Neuropathies Dilated cardiomyopathy Familial partial lipodystrophy Skeletal malformations Atypical Werner s syndrome premature aging Hutchinson Gilford Progeria Syndrome HGPS or precocious aging syndrome Laminopathies How chromatin is structured and organized within the nucleus Distribution of known mutations in LMNA that result in Progerias Nucleus of normal cells Nucleus of HGPS cells How chromatin is structured and organized within the nucleus 5 1 2 3 4 5 6 7 8 9 10 11 Cryptic splice site 11 12 Normal splicing HGPS splicing Deletion of 150 bp 12 3 How chromatin is structured and organized within the nucleus Pre Lamin A protein NH2 SYLLGNSSPRTQSPQNCSIM COOH Farnesylation cleavage and methylation S NH2 SYLLGNSSPRTQSPQNC COOCH3 Specific cleavage S LLGNSSPRTQSPQNC COOCH3 NH2 SY COOH Mature Lamin A protein How chromatin is structured and organized within the nucleus Protease Wild type Lamin A Protease Lamin A 50 Progerin Lamin A is not tethered to the INM by a farnesyl group Lamin 50 progerin remains tethered to the INM by a farnesyl group How chromatin is structured and organized within the nucleus Nat Med 2005 April 11 4 440 445 Progerin is less mobile than Lamin A How chromatin is structured and organized within the nucleus Cytoplasm Nucleus from normal cells ONM C C E BAF E N C HP1 N BAF N C ONM INM C E BAF E N N Nucleoplasm How chromatin is structured and organized within the nucleus Cytoplasm Nucleus from HGPS cells ONM C C E BAF E N C HP1 N BAF N C ONM INM C E BAF E N N Nucleoplasm How chromatin is structured and organized within the nucleus Cytoplasm Nucleus from HGPS cells ONM ONM INM Nucleoplasm How chromatin is structured and organized within the nucleus Cytoplasm Nucleus from HGPS cells ONM ONM INM Misshapen nuclear envelope Disorganized heterochromatin Nucleoplasm Outline Localization of Heterochromatin in Nucleus Nuclear Lamina Mis localization and Progeria Basic Structure of Chromatin Histone Code How chromatin is structured and organized within the nucleus Heterochromatin Euchromatin Nucleolus II Chromosome Organization and Gene Regulation II In eukaryotes DNA is assembled into chromatin Thus to understand how transcription is modulated we need to study How chromatin is structured and organized within the nucleus How chromatin s structural integrity is regulated by remodeling enzymes during gene activation and repression II Chromosome Organization and Gene Regulation II II Chromosome Organization and Gene Regulation II II Chromosome Organization and Gene Regulation II II Chromosome Organization and Gene Regulation II A typical nucleosome contains 147bp of DNA wrapped around the histone octamer Each octamer consists of two central H3 H4 heterodimers which form a H3 H4 tetramer flanked on either sides by an H2A H2B heterodimer Each core histone has a compact domain and a flexible N terminal tail of 30 residues II Chromosome Organization and Gene Regulation II Histone Modifications II Chromosome Organization and Gene Regulation II The tails are also believed to stabilize intra and internucleosomal DNA interaction For example the H4 tail interact with the H2A H2B interface of an adjacent nucleosome Such interactions may facilitate nucleosome compaction and transcriptional repression The tails of H4 in particular are known to be acetylated during gene activation Acetylation neutralize the K basic charge which in turn is speculated to loosen contacts between the tails and other histones or DNA II Chromosome Organization and Gene Regulation II Histone Code Code Reader Writer Complex Hypothesis