Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.

The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF

Disparity in the DNA translocase domains of SWI/SNF and ISW2

An ATP-dependent DNA translocase domain consisting of seven conserved motifs is a general feature of all ATP-dependent chromatin remodelers. While motifs on the ATPase domains of the yeast SWI/SNF and ISWI families of remodelers are highly conserved, the ATPase domains of these complexes appear not to be functionally interchangeable. We found one reason that may account for this is the ATPase domains interact differently with nucleosomes even though both associate with nucleosomal DNA 17–18 bp from the dyad axis. The cleft formed between the two lobes of the ISW2 ATPase domain is bound to nucleosomal DNA and Isw2 associates with the side of nucleosomal DNA away from the histone octamer. The ATPase domain of SWI/SNF binds to the same region of nucleosomal DNA, but is bound outside of the cleft region. The catalytic subunit of SWI/SNF also appears to intercalate between the DNA gyre and histone octamer. The altered interactions of SWI/SNF with DNA are specific to nucleosomes and do not occur with free DNA. These differences are likely mediated through interactions with the histone surface. The placement of SWI/SNF between the octamer and DNA could make it easier to disrupt histone–DNA interactions

CAL1 is the Drosophila CENP-A assembly factor

Centromeres are specified epigenetically by the incorporation of the histone H3 variant CENP-A. In humans, amphibians, and fungi, CENP-A is deposited at centromeres by the HJURP/Scm3 family of assembly factors, but homologues of these chaperones are absent from a number of major eukaryotic lineages such as insects, fish, nematodes, and plants. In Drosophila, centromeric deposition of CENP-A requires the fly-specific protein CAL1. Here, we show that targeting CAL1 to noncentromeric DNA in Drosophila cells is sufficient to heritably recruit CENP-A, kinetochore proteins, and microtubule attachments. CAL1 selectively interacts with CENP-A and is sufficient to assemble CENP-A nucleosomes that display properties consistent with left-handed octamers. The CENP-A assembly activity of CAL1 resides within an N-terminal domain, whereas the C terminus mediates centromere recognition through an interaction with CENP-C. Collectively, this work identifies the “missing” CENP-A chaperone in flies, revealing fundamental conservation between insect and vertebrate centromere-specification mechanisms

SWI/SNF-NUCLEOSOME INTERACTIONS AND DISASSEMBLY OF NUCLEOSOMES: NOVEL METHODOLOGIES FOR MAPPING PROTEIN-PROTEIN AND PROTEIN-DNA INTERACTION

AN ABSTRACT OF THE DISSERTATION OF MEKONNEN LEMMA DECHASSA, for the Doctor of Philosophy degree in MOLECULAR BIOLOGY, MICROBIOLOGY, AND BIOCHEMISTRY, presented on December 18, 2008, at Southern Illinois University Carbondale. TITLE: SWI/SNF-NUCLEOSOME INTERACTIONS AND DISASSEMBLY OF NUCLEOSOMES: NOVEL METHODOLOGIES FOR MAPPING PROTEIN-PROTEIN AND PROTEIN-DNA INTERACTION MAJOR PROFESSOR: Dr. Blaine Bartholomew The SWI/SNF complex disrupts and mobilizes chromatin in an ATP-dependent manner. A site-directed photoaffinity crosslinking approach in which photoreactive moieties attached at specific sites within histone octamer was developed and used to map the interactions of SWI/SNF with the histone octamer face of the nucleosome. We identified the subunits that contact the nucleosomal histone proteins. The catalytic Swi2/Snf2 and Snf5 subunit were found to interact with a large surface of nucleosomal histone proteins. Affinity proteolysis, using FeEDTA that is attached to DNA or the histone octamer as a probe, was applied to identify the domains and motifs of SWI/SNF subunits that interact with DNA and nucleosome. It was found that motifs in the N-terminal lobe of ATPase/helicase domain of Sw2/Snf2 are in close contact with both DNA and histone octamer. Crosslinking and peptide mapping by chemical proteolysis revealed that the region in the C-terminal lobe of ATPase/helicase domain of Swi2/Snf2 is bound to the internal nucleosome region that is two helical turns from the dyad axis. The impact on nucleosome remodeling by adjacent nucleosomes and the recruitment of SWI/SNF by transcriptional activator was examined using a high resolution histone-DNA contact mapping and a single molecule MAP-IT technique. The data shows that the presence of adjacent nucleosomes promotes nucleosome eviction and the recruitment of SWI/SNF by Gal4-VP16 to dinucleosomes restricted the nucleosome mobilization in one direction. Finally, based on the data from this study and previous reports, the mechanism how recruitment and neighboring nucleosomes alter the outcome of SWI/SNF remodeling is discussed