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Biol/Chem 473

Biol/Chem 473. Schulze lecture 8: Chromatin remodelling. Spreading of heterochromatin in flies (and humans!). K9 of H3 (H3K9me). Spreading of silenced chromatin on a homeotic gene. E(z) (SET). K27 of H3 H3K27me. Polycomb. Polycomb. Polycomb. Polycomb-complexed chromatin.

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Biol/Chem 473

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  1. Biol/Chem 473 Schulze lecture 8: Chromatin remodelling

  2. Spreading of heterochromatin in flies (and humans!) K9 of H3 (H3K9me)

  3. Spreading of silenced chromatin on a homeotic gene E(z) (SET) K27 of H3 H3K27me Polycomb Polycomb Polycomb Polycomb-complexed chromatin

  4. Heterochromatin vs. euchromatin adapted from Jenuwein & Allis (2001) Science 293, 1074

  5. Heterochromatin vs. euchromatin H3K4me adapted from Jenuwein & Allis (2001) Science 293, 1074

  6. Heterochromatin vs. euchromatin H3K9me adapted from Jenuwein & Allis (2001) Science 293, 1074

  7. Regulation of homeotic genes H3K27me Repressed homeotic gene active homeotic gene adapted from Jenuwein & Allis (2001) Science 293, 1074

  8. Three big questions… • How are the PcG proteins targeted to their cognate sequences? • What is the maintenance “mark” that functions to keep specific genes active/inactive? • How is the maintenance mark maintained through cell division?

  9. Deposition of histones during replication During replication, parental histones are distributed randomly onto the replicated DNA strands, and newly synthesized histones fill the gaps. http://www.umassmed.edu/faculty/show.cfm?start=0&faculty=912

  10. Enzymes that regulate chromatin • Two classes of enzymes that regulate chromatin structure: histone modifiers (last two lectures) and chromatin remodelers (today). • Histone modifiers don’t alter nucleosome position; they make passive marks that recruit more active functions (histone code). • Chromatin remodelers hydrolize ATP to actively remodel chromatin: shift nucleosome position with respect to DNA, exposing or occluding regulatory sequences. • These enzymes function within larger complexes of subunits that collectively act to enhance and/or target the remodeling activity.

  11. How to plough through chromatin?

  12. Ground state is restrictiveSilent state is MORE restrictive GROUND STATE Struhl, 1999

  13. Chromatin remodeling is an active process • Chromatin remodeling describes the energy-dependent (ATP) displacement or reorganization of nucleosomes that occurs in conjunction with activation (or repression) of genes for transcription • Chromatin remodelers also play a role in recombination and repair

  14. Chromatin remodelers move nucleosomes around • Remodeling improves access to DNA or histone binding sites recognized by transcriptional regulators or histone modifiers • This remodeling process can lead to activation or repression

  15. Why do you need ATP to move nucleosomes around? • Bending DNA around a bunch of histones depends on DNA sequence to some extent • Some sequences bend more easily than others • Nucleosomes do have strong affinity for DNA and even have sequence preferences • This preference may be exploited in nature to organize a chromatin landscape energetically conducive (or not) to gene expression • (Widom et al. (2006) Nature 442(17):772 – computational paper) • But that landscape must change as gene expression requirements change… • …and this requires energy

  16. Stamp collecting: classifying chromatin remodelers • Chromatin remodeling complexes are classified based on protein motifs found in addition to the ATPase domain, or on how the ATPase domain itself is structured • This classification is purely structural, designed to make it easier for us humans to sort them all out – it may not accord with functional criteria • There is a great deal of mixing and matching of subunits between various groups of remodelers (combinatorials again….)

  17. SWI2/SNF2 subfamily ATPase BROMO ATPase SANT ISWI subfamily ATPase CHD/Mi2 subfamily CHROMO DNA binding ATPase ATPase Ino80 subfamily SWI2/SNF2 ATPase SUPERFAMILY

  18. SWI2/SNF2 subfamily ATPase BROMO Originally isolated genetically in budding yeast as mutants in mating type switching and sucrose non-fermenting functions BROMO from Drosophila: Brahma ATPase SANT ISWI subfamily SANT SWI3 ADA2 N-CoR TFIIIB ATPase CHD/Mi2 subfamily CHROMO DNA binding ATPase ATPase Ino80 subfamily SWI2/SNF2 ATPase SUPERFAMILY

  19. Chromatin remodeling complexes SWI2/SNF2 subfamily ISWI subfamily hBrm

  20. Conservation of SWI/SNF subfamily complexes

  21. Pc-G PRC1 SWI/SNF PRC2 Cloned Members of the Pc-G and trx-G trx-G Genetically SWI/SNF subfamily members isolated as activators of gene expression (PRE/TRE) Homeotic gene expression trx Mll, Mll2 trl brmBrm, Brg1 mo Baf155, Baf170 osa Baf250 snr1 Baf47/Snf5 z ash-1 Ash1l ash-2 Ash2l lid kis lawc Pc M33/MPc1, MPc2, MPc3 ph Rae28/Mph1 Psc Mel18, Bmi-1 Scm Scmh1, Scmh2 PclM96 Pho YY1 Su(z)2 Mel18, Bmi-1 dRING Ring1a, Ring1b E(z)Enx-1, -2/Ezh-1, -2 esceed Asx Asxl1, Asxl2 crm

  22. Method: nucleosomal mobility assay using EMSA(electrophoretic mobility shift assay)

  23. Experiment: how do SWI/SNF subfamily chromatin remodelers shift nucleosomes? • Amplify template (~215bp –bit longer than normal so you can assay movement) • Purify histones • Reconstitute end-labeled mononucleosomes in a test tube (bunch of chemistry) • Nucleosomes at the end of a DNA fragment will migrate on a gel more rapidly than those in the middle of a DNA fragment • Extract those stable nucleosomal fragments • Run again in the presence of SWI/SNF, with (+) or without (-) ATP • What happens?

  24. SWI/SNF sub family remodelers can slide nucleosomes along DNA Ramachandran et al. (2003) J. Biol. Chem. 278(49):48590

  25. Binds DNA (sequence dependant OR independent) Recognizes acetylated lysines in histone tails Binds DNA AND protein Binds DNA (sequence dependant OR independent) Binds DNA and maybe also histone tails what’s this doing in the nucleus??? dunno Protein domains in a SWI/SNF complex

  26. Drosophila polytene chromosomes allow visualization of chromosomal activity during interphase Chromosomes replicate without nuclear division: endoreplication

  27. Drosophila polytene chromosomes allow visualization of chromosomal activity during interphase Chromosomes replicate without nuclear division: endoreplication

  28. Where does Brahma localize in the interphase nucleus? • The Drosophila SWI/SNF ATPase Brahma localizes to active regions of the genome • There is little or no colocalization with the homeotic repressor Polycomb Armstrong et al. (2002) Embo J. 21(19):5245

  29. SWI/SNF and cancer • The tumor suppressor BRCA1 co-purifies with the human SWI/SNF Brg1 complex • Is the SWI/SNF remodeling complex functioning as a transcriptional co-activator? Bochar et al. (2000) Cell 102:257

  30. “Imitation switch” – isolated first in Drosophila by homology with SWI/SNF from yeast Chromatin remodeling complexes SWI2/SNF2 subfamily ISWI subfamily hBrm

  31. Associates with acetylated histones Once upon a time, a DNA binding domain. Now? A comparison of SWI/SNF and ISWI subfamily ATPases BRAHMA ATPase somewhere in here ISWI

  32. ISWI complexes are VERY diverse Nucleosome remodeling factor Chromatin accessibility complex ATP-dependent chromatin assembly and remodeling factor Nucleolar remodeling complex

  33. Transcriptional activation of some homeotic genes involved in replication of heterchromatin? Bozhenok et al. (2002)Embo J 21(9) 2231 keeping sister chromatids together during mitosis rDNA repression ISWI complexes are VERY diverse

  34. Where does ISWI localize in the interphase nucleus? • Distribution of ISWI and RNA PolII do NOT overlap (much) • Do ISWI remodelers play a role in chromatin repression? Deuring et al. (2000) Mol. Cell 5:355

  35. Common method for analyzing chromatin structure Method: microccocal digestion measures nucleosome repeat lengths

  36. Silent vs. active chromatin show different micrococcal nuclease digestion patterns • Transgene in heterochromatin or euchromatin • Cut genomic DNA with micrococcal nuclease (cuts between nucleosomes) • Blot DNA and incubate with transgene sequence probe (Southern!) MNase Cryderman et al. (1999) NAR 27(16): 3364

  37. ISWI ATPases can induce regular OR irregular nucleosomal spacing • The implication is that ISWI chromatin remodelers can organize chromatin into a more repressive configuration, OR a more active one • Since yeast has two ISWI ATPases with different effects on chromatin in vitro, they may have evolved contrasting activities Gelbart et al. (2001) Mol. Cell Biol. 21(6): 2098

  38. Summary: SWI/SNF and ISWI • Two types of ATPases that form large multi-subunit chromatin remodeling complexes. • These complexes use the energy of ATP to remodel nucleosomal DNA. • SWI/SNF subfamily members are more often associated with activation of chromatin but they can silence as well (I didn’t show you evidence but it’s out there…) • ISWI subfamily members correlate with repressed chromatin but they can activate as well. • Chromatin remodelers contrast with chromatin modifiers: make sure you know how!

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