ISWI Flashcards
What is the role of NURF
NURF(Nucleosome remodelling factor) plays a critical role in regulating transcriptional regulation downstream of multiple prominent signalling pathways and is required for proper development.
BPTF has been shown to bind stably to SMARCA1.13, which is devoid of enzymatic activity. The role of this active complex remains unclear(Oppkoifer et al)
What is the role of CECR2, what complex is it found in.
Protein required for proper neural action in mice
It is found in the heterodimeric SNF2L complex named CERF
What is the function of ACF and WICH? What do the acronyms stand for? What proteins compose it?
Both have been found to facilitate DNA replication and promote DNA repair. ACF complex also is involved in general nucleosome assembly
ACF- ATP-utilizing chromatin assembly and remodelling factor
WICH- WSTF-ISWI chromatin remodelling complex (Williams Syndrome Transcription Factor)
ACF consists of BAZ1A and SMARCA5, WICH consists of BAZ1B and SMARCA5
What makes up the RSF complex? What does it stand for? What is it implicated in?
It is made up with RSF1 and SMARCA5. Remodelling and spacing factor. It is generally implicated in transcriptional regulation and more recently in responding DNA damage. It assembles centromeric chromatin
What makes up the NoRC complex? WHat does it stand for?What is it associated it?
It is made up of BAZ2A(Tip5) and SMARCA5. It stands for Nuclear remodelling complex. NoRC promotes genome stability and plays a critical role in repressing ribosomal gene transcription
What is the CHRAC complex consists of?
It consists the auxiliary subunits CHRAC-15/17 and ACF and SMARCA5. It plays a role in RNAP II transcription. CHRAC stands for Chromatin assembly complex.
Can uncomplexed ISWI catalytic subunits remodel?
Uncomplexed ISWI catalytic subunits from multiple species including human SMARCA5 can reposition nucleosomes without the need of a regulatory subunit. This is an activity dependent on an intact histone H4 N-terminal tail and coupled to a transient deformation of the histone octamer.
However, addition of regulatory subunits significantly increases the rate and directs the product distribution of the remodelling reaction.
How did the ACF complex show us that regulatory subunits are important for the ISWI complexes
-When bound to BAZ1A/ACF, the ATPase and remodelling activities of SMARCA5 become tightly regulated by the length of extranucleosomal DNA present in the nucleosome substrate(referred to as linker DNA)
- As a mononucleosome is engaged on opposite sides by two ACF complexes, the histone octamer slides preferentially to the longer linker DNA and the mononucleosomes assume a “centre” configuration equilibrium with lengths of extanucleosomal DNA protruding from each side.
In Oppkoifer et al, what was shown about the previously described regulatory subunits with the ISWI remodellers
Oppkoifer et al showed that SMARCA1 and SMARCA5 can interact with each others previoulst described subunits. Although one expects there to be a preference for previously described interactions, it was seen that relative recovery of regulatory subunits from IPs largely matched the mRNA level for that subunit. This means that abundance is an important factor in determining incorporation of regulatory subunits into ISWi complexes. this makes sense given the high identity between SNF2L and SNF2H.
Previous interactions were discovered by purifying factors responsible for specific chromatin assembly and remodelling activities, so alternative active complexes may have been lost during purification procedures.
The approach used in Oppkoifer et al was an unbiased approach of immunoprecipitating endogenous SMARCA1 and SMARCA5 and identifying Co-purifying proteins by mass spec. A negative control was CRISPR-CAS9 mediated genome editing to abrogate expression of SMARCA1 and SMARCA5
When was ISWI discovered
Originally purified from Drosophila as the ATPase motor of nucleosome remodelling factor (NURF) complex(Tsukiyama et al)
What does the N-terminal catalytic ATPase domain and C-terminal HAND-SANT-SLIDe domain do
Mediates DNA interactions and responsible for extranucleosome DNA binding and H4 tail interactions (ALkhatib and Landry, Clapier 2001, Grune, Toto)
Furthermore, ISWI remodelling is mediated by two additionally auto regulatory motifs AutoN and NegC that lie adjacent to the ATPase domain (Clapier and Cairns)
Describe the ISWI complexes composition
Typically heterodimers of an ISWi protein(SNF2H or SNF2L) and a protein containing a BAZ(Bromodomain adjacent to zinc finger) domain that are either a member of the BAZ transcription family or closely related (RSF1 and BPTF)
In addition to BAZ domains, most of these partner proteins also include AT hooks and/or DDT domains that are thought to facilitate DNA binding (doers 2001)
The PHD Zinc finger domain in BPTF interacts with the histone H3K4me3 modification.
What have mutations in Drosophila ISWI or Nurf301(BPTF) result in
Decreased histone H1 levels and a general decondensation of the male X chromosome
What can you say about ISWI mediated nucleosome spacing and histone H1 deposition
Taken together, they promote gene repression and higher order chromatin structures, likely presenting a key role for these proteins in the transition from a progenitor to a differentiated cell fate
How do ISWI proteins regulate transcription through maintenance of NFRs(nucleosome free regions
In Drosophila, ISWI genome wide binding data demonstrated that the protein is located in genie and intervening regions. Binding occurs at ~300bp downstream of the TSS where it helps position the distal nucleosomes.(Sala et al 2011)
In yeast, Isw2 binds near promoter regions to move nucleosomes close to NFRs and repress the generation of both coding and non-coding transcripts( Whitehouse, Zentner)
- Isw1b complex is recruited to H36me3 marks in gene bodies where it reassembles chromatin over the coding regions after transcription(Maltby et al)
- In humans genome wide binding studies of SNF2H and SNF2L have indicated the binding profile near the TSS and at a large number of genes: they generate positioned nucleosomes at these binding areas.
-SNF2H is particularly adept at positioning nucleosomes adjacent to CTCF binding sites and its depletion reduces organization distal of the +3 nucleosome
-Depletion of ACF1(BAZ1A),RSF-1,WSTF(BAZ1B) and Tip5(BAZ2A) had minimal effects compared to the depletion of SNF2H suggesting functional redundancy amongst the different complexes
-In contrast, SNF2L has a minor effect on nucleosome positioning at CTCF binding sites but shows small reductions in nucleosome occupancy that are proximal to other transcription factor binding sites(Wiechens )
- NURF complex interacts with TFs to regulate target gene expression
