1 Chromatin Boundries Observe DNA loops attached to nuclear scaffold DNA loops are 30-90 kb in length DNA is attached to Nuclear Matrix Attachment region.

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1 Chromatin Boundries Observe DNA loops attached to nuclear scaffold DNA loops are kb in length DNA is attached to Nuclear Matrix Attachment region is called Matrix Attachment Region (or MAR) MAR Scaffold Loop (30-60 kb) Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

2 Chromatin Boundries MARs bp; 70% A/T rich; topoisomerase II recognition site Also called Scaffold Attachment Region (SAR) No effect in transient transfection Confer position independence on integrated genes MAR Gene Strong Expression EpisomalIntegrated Gene StrongVariable Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

3 Assays to identify MARS Isolate nuclei extract histones 1. In vivo- restriction digest extract matrix-bound DNA 2. In vitro- degrade all DNA Add DNA fragments to MAR Determine which DNA fragments bind Sequence DNA In vitro In vivo Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

4 Are MARS insulators? MARs Some mammalian MARs will function as insulators in Drosophila Not all MARs have insulator activity Insulators Mammalian MARs can function as insulators in Drosophila Classic insulator is scs and scs’ marking boundries of the heat shock locus in Drosophila –Eliminates “position effects” gypsy is also an insulator Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

5 LCRs- Review by Higgs Cell 95:299 (1998) A. Overview 1.Chromosomal environment- differential expression depending on where located- 2. Position effects observed in  translocations  transgenic mice  cell culture 3. Great quote “We hide our ignornace by using terms “open vs closed”, “permissive vs non-permissive, “euchromatin vs heterochromatin”” Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

6 4. Open chromatin  uncondensed, replicate early, near center of nucleus, histones hyperacetylated, DNaseI sensitive, undermethylated 5. DNaseI sensitivity –a. general DNaseI sensitivity- DNA 10-fold more accessible to DNaseI, covering long regions –b. DNaseI hypersensitivity- DNA 100-fold more sensitive to DNaseI Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

7 Questions- What defines the origins of replication? What directs nuclear sublocalization? What sequences define boundries of open and closed chromatin? What organizes higher order chromatin structures? Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

8 Human  -globin gene cluster Five erythroid-specific genes Arranged in order of expression LCR is upstream cluster of 5 (actually 7) HS sites Each HS site binds numerous factors Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

9 Human  -globin gene cluster- Transgenics Each HS site binds numerous factors Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

10 Human  -globin gene cluster- Transgenics Remove LCR, low level expression of all genes in cluster LCR confers copy number dependence > 10 additional LCRs identified Upstream cluster of 5 HS sites Each HS site binds numerous factors Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

11 Hispanic deletion Transcriptionally inactive 100kb up and downstream DNAseI resistant late replicating Conclude- LCR opens chromatin (is this true??) Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

12 Models and observations Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

13 Proposed LCR functions Open chromatin prevent variegated heterochromatization Affects timing of replication Keeps promoters histone-free Change subcellular localization of  locus LCR transcription affects rest of locus expression Recruit HATs Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

14 Role of intragenic transcription? Increase in intragenic transcripts correlates with globin gene activity –e.g.  and  intragenic transcripts higher when  and  transcripts higher Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

15 Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

16 Human  -globin gene cluster- Questions What regulatory properties does the LCR confer? (gene regulation?) Is replication timing directed by gene activity? Is chromatin structure a cause or consequence of globin gene activation? Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.

17 Model: Stage-specific activation within subdomains by an LCR holocomplex “Early” “Late” Putative chromatin boundries (small spheres) Advanced Molecular and Cellular Genetics BIO 4751 Spring 2003 Gary A. Bulla, Ph.D.