free questions before

What are the four main questions this lecture aims to answer?

The four main questions are: 1. What is a nucleosome? 2. How do chromatin remodelers shape chromatin? 3. Why is remodeling important for transcription? 4. How are remodeling activities affected in disease?

What is the fundamental repeating unit of chromatin, and what does it consist of?

The fundamental repeating unit of chromatin is the nucleosome, which consists of an octamer of core histone proteins (two each of H2A, H2B, H3, and H4) wrapped by approximately 147 base pairs of DNA.

What are the core histone proteins and what is the linker histone?

The core histones are Histone 2A (H2A), Histone 2B (H2B), Histone 3 (H3), and Histone 4 (H4). The linker histone is Histone 1 (H1).

What is the structure of the histone tails, and why is this important?

The N-terminal tails protrude from the nucleosome for H3, H4, and H2B, while both N- and C-terminal tails protrude for H2A. These tails are intrinsically disordered, meaning they are highly flexible and can move dynamically to perform different functions, and they are heavily post-translationally modified.

What class of proteins controls the spatial positioning of nucleosomes, and how do they function?

The spatial positioning of nucleosomes is controlled by a class of proteins called chromatin 'remodelers'. These enzymes shuffle, evict, or remodel nucleosomes through ATPase domains.

What are the four major subfamilies of Snf2 ATPase chromatin remodelers?

The four major subfamilies are SWI/SNF, CHD, ISWI, and INO80.

What are the general functions of the CHD, ISWI, and SWI/SNF remodeler families?

CHD3-5-containing NuRD complexes are involved in gene repression. ISWI-containing complexes are responsible for sliding nucleosomes to arrange their regular spacing along chromatin. SWI/SNF complexes are involved in gene activation.

What were the first described subunits of the SWI/SNF (BAF) complex, and how were they identified?

SNF2 and SNF5 were the first described subunits. They were identified in yeast as regulators of SUC2 gene activation in 1981. Mutants of these genes had disrupted nucleosome organisation patterns at the SUC2 promoter, and the phenotype could be rescued by mutating H2A or H2B coding genes.

What was demonstrated by the 1999 'sliding assay' using recombinant SWI/SNF?

The assay demonstrated that in the presence of ATP, the SWI/SNF (BAF) complex is capable of shuffling nucleosomes along a DNA template, which was measured by changes in restriction digestion of the DNA template.

What are the three sub-complexes of the mammalian BAF family and what are their ATPase subunits?

The three sub-complexes are cBAF (canonical BAF), PBAF (PBRM1 BAF), and ncBAF (non-canonical BAF). SMARCA2 or SMARCA4 are the ATPase dependent enzymatic subunits of these complexes.

Where are the different BAF sub-complexes specifically enriched in the genome?

All BAF complexes overlap with accessible active regions of the genome. cBAF is specifically enriched at enhancers (primed and active), while PBAF and ncBAF have a stronger preference for promoters than cBAF.

What was revealed by the cryo-EM structural model of the endogenous human BAF complex?

The cryo-EM structure revealed that the huge (>2MDA) BAF complex wraps entirely around the nucleosome to facilitate its sliding/remodeling activity. It also revealed the position of disease-relevant variants in relation to the nucleosome interface (SMARCB1), protein-protein interactions (ARID1A/B), and enzymatic activity (SMARCA4).

How does the BAF complex facilitate transcription factor (TF) binding?

BAF directs accessibility to facilitate TF binding. Knockout or inhibition of the catalytic SMARCA4 subunit leads to rapid loss of accessibility, which is tightly coupled to loss of lineage-specific TF binding (OCT4, SOX2, and ESRB).

What happens when the BAF complex is artificially recruited to a reporter gene locus?

Artificially recruiting the BAF complex to a reporter gene locus rapidly increased accessibility at this site (within 8-20 minutes), which led to reporter gene activation. Crucially, transcriptional repressors of the Polycomb family were evicted from this reporter promoter along with their associated repressive histone modifications.

What are the three key mechanisms of the BAF complex in transcription?

1. BAF is responsible for chromatin remodeling and accessibility at active gene promoters and enhancers. 2. This facilitates transcription factor binding. 3. It also facilitates the eviction of repressors (like Polycomb complexes).

What is the overall role of the BAF complex in cell biology?

The BAF complex is a critical player in cell fate decisions and differentiation.

What is the result of homozygous loss of SMARCB1, and in which cancer is this a primary driver?

Homozygous loss of SMARCB1 is the primary driver event in Malignant Rhabdoid Tumours (MRT), which are extremely aggressive cancers of early childhood.

How genetically simple are Malignant Rhabdoid Tumours (MRT)?

MRTs have an extremely low rate of mutation, with loss of SMARCB1 being essentially the sole recurrent event. In some cases, there were no other identified mutations, demonstrating that high mutation rates are dispensable for the genesis of these cancers.

What did mouse models reveal about the tumour suppressive role of SMARCB1?

Homozygous SMARCB1 knockout mice are embryonically lethal. However, SMARCB1 heterozygous mice are viable and develop aggressive sarcomas that closely resemble human MRTs, with the second allele of SMARCB1 spontaneously lost, highlighting its key tumour suppressive role.

What happens when SMARCB1 expression is restored in MRT cell lines?

Restoring SMARCB1 expression restores the BAF complex to the correct sites of the genome, restores active enhancer features (H3K4me1 and H3K27ac), restores accessibility, and halts proliferation of these cells, highlighting the tumour suppressive role of SMARCB1.

What is the relationship between Polycomb (PRC2) and BAF in the context of cancer?

There is an antagonistic relationship between Polycomb (PRC2) and BAF. In cancers with BAF loss (like MRT), PRC2 can be an excellent drug target to restore balance to the epigenome and the equilibrium between gene activation and repression. Inhibiting the methyltransferase EZH2 (a PRC2 component) can lead to durable tumor regression.

What recurrent genetic alteration is found in Small Cell Carcinoma of the Ovary (SCCOHT)?

The catalytic subunits of BAF (SMARCA2 and SMARCA4) are recurrently deleted or silenced in SCCOHT.

What is the role of the SS18-SSX fusion oncoprotein in synovial sarcoma?

The SS18-SSX fusion oncoprotein is the driving event in synovial sarcoma. The SSX protein part of the fusion has an affinity for Polycomb repressed loci, which redirects the BAF complex from active genes to these repressed sites. This drives aberrant transcription and activates oncogenic gene expression programs.

What is the consequence of removing the SS18-SSX fusion oncoprotein from synovial sarcoma cells?

Removing the fusion oncoprotein (with shRNA) causes the BAF complex to be retargeted to its normal target genes. This induces accessibility (ATAC-seq), coincides with activation of enhancers (H3K27ac), and ultimately causes the synovial sarcoma cells to cease proliferation.

Which BAF subunit is a dependency in synovial sarcoma and is being targeted therapeutically?

CRISPR screens revealed that synovial sarcoma cells are dependent on the BAF subunit BRD9. A compound called dBRD9 was developed to degrade BRD9 and showed promising activity in vitro and in vivo, leading to clinical trials.

In what contexts might inhibiting the BAF complex be a beneficial therapeutic strategy?

Inhibiting the BAF complex could improve cancer in specific cases including: 1. Synovial sarcoma where BAF activity is targeted to the wrong regions. 2. Cancers where SMARCA4 is deleted, as there is a synthetic lethal relationship between SMARCA2 and SMARCA4. 3. Cancers that depend on lineage-specific TF activity (AR, PAX3-FOXO1).

What is the significance of ARID1B haploinsufficiency?

Haploinsufficiency of ARID1B (a member of the SWI/SNF complex) is a frequent cause of intellectual disability and is one of the main causes of Coffin-Siris syndrome, accounting for 76% of identified mutations in that disorder.

What type of mutations in SMARCA2 cause Nicolaides-Baraitser syndrome, and what is their effect?

Heterozygous de novo missense mutations in SMARCA2 cause Nicolaides-Baraitser syndrome. These mutations cluster within sequences encoding ultra-conserved motifs in the catalytic ATPase region and likely are associated with disrupted ATPase activity, rather than impairing complex assembly.

Besides ARID1B and SMARCA2, what other BAF complex subunits are mutated in chromatinopathies?

Many others have been reported, including ARID1A, SMARCB1, SMARCE1, SMARCA4, and subunits associated with autism like SMARCC1, SMARCC2, and PBR