Chromatin Remodeling Activities

• ATP-Dependent:• Swi/snf• RSC• Ino80.com• Brahma• ISWI• CHD

• ATP-Independent:• HAT• HDAT

SWI/SNF

• Swi= switch genesactivating HOendonuclease

• Involved in matingtype selection by DNArecombination

• Snf=sucrose non-fermenting

• Required for sucroseuptake and utilization

SWI/SNF

• Isolated as an 11 subunit complex in yeast• Contains an DNA-dependent ATPase subunit that

is stimulated by both DNA and nucleosomes• The swi2/snf2 ATPase subunit is found in all three

known ATP-dependent chromatin remodelingcomplexes

• None of the eleven subunits are required forviability in yeast!

SWI/SNF

• 5 subunits were identified genetically:swi3/snf2, swi1, swi2, snf5 and snf6

• The remaining six subunits were found inthe biochemically isolated complex: Swp82,Swp73, Swp 61, Swp59, TAFII30 andsnfl1

SWI/SNF

• The swi/snf complex can bind both DNA andnucleosomes in the nM range

• DNA binding is through the minor groove• Nucleosome binding is not through the minor

groove; rather the complex is thought to bind tothe nucleosome directly

• There is no known DNA binding specificity• The complex can allow increased restriction

enzyme accessibility and DNAaseI sensitivity tonucleosomes in vitro

SWI/SNF

• Has been shown in vitro to facilitate themovement of histones in cis and trans

• Generates nucleosome-free regions in vitro• Can bring about local changes in DNA twist

leading to the formation of “accessible”nucleosomes

• This property is shared by all three ATP-dependent remodeling activities: ISWI and CHD

SWI/SNF

• Genome-wide array analysis shows thatonly 5% of genes are dependent on theATPase activity of the complex (snf2disruption)

• Interestingly 5% of the genes in yeast arefurther activated by the knockout; hence arepressor function might apply to theseproteins

The RSC Complex

• RSC was identified molecularly bysearching for proteins that are in a complexwith sth1 (an ATPase with high homologyto swi2/snf2 found by low stringencyhybridization screens)

• The complex has 15 subunits, some sharehomology to the swi/snf components

Comparison of ATP-dependent Remodelingcomplexes

RSC Complex

• 4 subunits that share homology with swi/snf genes arerequired for viability Sth1, Rsc8, Rsc6 and Sfh1

• Disruption of any of these four genes results in G2/Marrest

• RSC is 10 times more abundant than swi/snf• RSC can be found in four different complexes of varying

subunit composition• Rsc1 and Rsc2 do not seem to be present in the same

complexes and may have partially redundant functions

RSC Complex

• Catalyzes the transfer of histone octamers fromone DNA to another

• Forms a RSC-nucleosome complex that is moresusceptible to nuclease digestion and restrictionenzymes

• This susceptibility persists after removal of ATPand RSC

• This ‘remodeled’ state is reversible by the furtheraddition of RSC and ATP

RSC Complex Summary

• It is likely that RSC is the primarychromatin remodeling activity in vivo

• Required for mitotic growth and is veryabundant

Ino80.com

• A novel complex purified from yeast based onhomology of its primary subunit to swi2/snf2

• There are both Drosophila and human homologuesof the primary subunit; Ino80

• The homologues share three regions of homologyan ATPase domain: a TELY motif at the N-terminus and a GTIE motif at the C-terminus

• Ino80 does not contain a SANT or bromo domain

Ino80.com

• Purified as a 12 subunit complex• Has DNA helicase-homologues Rvb1 and 2• Helicase activity is 3’ to 5’• Several actin-related subunits• Null mutants of ino80 show DNA repair

phenotypes, sensitive to UV damage etc.

Comparison of ATP-dependent Remodelingcomplexes

The Brahma Complex

• Found in Drosophila via genetic screens forsuppressors of polycomb (a repressor of homeoticgenes)

• Found biochemically as a large multi-subunitcomplex in Drosophila

• The BRM subunit has ATPase activity and withseven other subunits form the complex

• Initially these subunits were named BAPs untiltheir genetic identity was determined

The Brahma Complex

• Notable subunits include trithorax proteins whichare also activators of homeotic genes

• Moria/Bap155 (MOR), OSA/Eyelid, snr1 (snf5related1)

• MOR can dimerize through its C-terminal leucinezipper

• MOR binds to BRM through its SANT domain• Conserved region II of BRM is the site that BRM

binds through the SANT domain

BRM-MOR Interactions

• Swi3 interacts with conserved region II onswi2/snf2

• RSC components sth1 and Rsc8 interactthrough the same shared domains

• Thus the ATPase-containing primarysubunit interacts with a conservedcomponent in the complexes through thesame internal protein domains

BAP111

• The Bap111 subunit is not found in theyeast swi/snf or RSC yeast complexes

• Humans do posses a homologue of thissubunit BAF57 and thus is unique tometazoans

• Bap111 contains a high mobility groupdomain found on many chromatin proteinsand can bind DNA through this motif

Human swi/snf-RSC Complexes

• First identified by isolating a high molecularweight complex that contained a protein withhomology to snf2/swi2

• This human gene homologue can replace the yeastswi2 gene in vivo (in yeast)

• At least two separate complexes exist eachcontaining an ATPase subunit either BRG1 orhBRM and a set of related subunits

• Purification of these complexes from different celllines show further subunit heterogeneity

Human swi/snf-RSC

• BAF vs PBAF complexes• BAF complexes contain either hBRM or BRG1 ATPases• PBAF contain only BRG1 as the ATPase-containing

primary subunit and BAF180• BAF180 contains 6 bromodomains, 2 BAH domains and

one HMG domain• BAF180 bears striking homology to three separate RSC

proteins; Rsc1, 2 and 4• Thus PBAF is thought to represent the human counterpart

of RSC

Human swi/snf and mitosis

• Chromatin must undergo extensive condensation duringmitosis what happens to the remodeling activities?

• The BRG1 complex is present during mitosis and seems tobe regulated by phosphorylation of the BRG1 and hswi3subunits

• Phosphorylation of these proteins is thought to inactivatethe complex and thus be important for mitosis to continue

• The hBRM complexes are also phosphorylated but thismodification may result in their degradation during mitosis

The ISWI subfamily

• Members of this subfamily Imitation of switch havehomology to swi2/snf2 only in the ATPase domain

• The ATPase activity in these complexes is stimulated bynucleosomes and not DNA

• ISWI contains a SANT domain and may bind DNAthrough this domain

• SANT (Swi3, Ada2, N-CoR and TFIIIB) a domain foundin many transcriptional corepressors and activators

• ISWI is essential for viability in Drosophila

ISWI Complexes

Drosophila ISWI

• Three ATP dependent nucleosome remodeling activitiesisolated from Drosophila embryos

• NURF (nucleosome remodeling factor) can cause themovement of nucleosomes along DNA

• CHRAC (chromatin assembly complex) in addition toNURF activity it can generate regularly spacednucleosomes and allow restriction enzyme cleavage

• ACF (ATP-utilizing chromatin assembly and remodelingfactor) can generate regularly spaced nucleosomes in anhistone-chaperone dependent fashion

Yeast ISWI

• Yeast homologues to the Drosophila ISWIare Iswi1p and Iswi2p

• Both complexes have nucleosome-stimulated ATPase activity and canregularly space nucleosomes

• Iswi1 plays a role in certain stress-responses• Iswi2 important for meiosis

Human ISWI complexes

• RSF (remodeling and spacing factor)• WCRF 600-700kD complex• hACF 300kD complex• hCHRAC shares homology with Drosophila

subunits

Comparison of ATP-dependent Remodelingcomplexes

The CHD subfamily• In addition to swi2/snf2 ATPase activity these proteins

contain a chromo (chromatin organization modifier)domain and a DNA binding domain

• Proteins containing chromo domains bind toheterochromatin possibly through methylated histones

• Complexes that contain CHD proteins show both ATPdependent nucleosome rearrangement and histonedeacetylase activities

• Some CHD proteins individually possess histonedeacetylase activity and ATP-dependent chromatinremodeling activity

The CHD subfamily

• Some complexes have additional activitiesincluding Me-CpG DNA binding activity

• CHD complexes are likely to be involved inchromatin inactivation and repression oftranscription

CDH Complexes

Histone Acetylation by HATs

• Histone acetylation of the N-terminal tails is oftenassociated with gene activation

• Histone acetyltransferases have been identifiedfrom yeast to man

• The first in yeast was Gcn5 a transcritpionalcoactivator

• Others now identified include; CBP (CREBbinding protein) PCAF (CBP-associated factor)hTAFII250 TFIIIC SRC1 (steroid co-activator)

• They are recruited to promoter regions by DNA-binding activators

HAT Activities

HAT’s are found in large complexes

Activators Recruit HAT Complex

Figure 13.31