Molecular Characterization of Nepali Potato Cultivars Using Randomly Amplified Polymorphic DNA (Rapd) Markers

Randomly amplified polymorphic DNA (RAPD) was used to study the genetic diversity of four local cultivars of potato. Amplification with ten arbitrary decamer primers produced 29 different marker bands of which 69.0% were polymorphic. The size range of the amplified DNAs ranged between 370 bp and 2500 bp. On average, 17.5 alleles per genotype were amplified using the RAPD primers. With the selected primers sufficient polymorphism could be detected to allow identification of individual genotypes. A dendrogram displaying the relative genetic similarities between the genotypes showed a range of 55.2-69.0% similarity

Molecular Characterization of Nepali Potato Cultivars using Randomly Amplified Polymorphic DNA (RAPD) Markers

Randomly amplified polymorphic DNA (RAPD) was used to study the genetic diversity of four local cultivars of potato. Amplification with ten arbitrary decamer primers produced 29 different marker bands of which 69.0% were polymorphic. The size range of the amplified DNAs ranged between 370 bp and 2500 bp. On average, 17.5 alleles per genotype were amplified using the RAPD primers. With the selected primers sufficient polymorphism could be detected to allow identification of individual genotypes. A dendrogram displaying the relative genetic similarities between the genotypes showed a range of 55.2-69.0% similarity

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

DYNAMICS OF NUCLEOSOME REMODELING BY ATP-DEPENDENT CHROMATIN REMODELERS

Chromatin is highly regulated nucleoprotein complex facilitating the dynamic balance between genome packaging and accessibility. The central workhorses regulating the dynamic nature of chromatin are ATP-dependent chromatin remodelers- ISWI, SWI/SNF, INO80, and CHD/Mi2. All chromatin remodelers transduce the energy from ATP hydrolysis to translocate on DNA, break histone-DNA contacts, and mobilize nucleosomes. However, the final outcomes of nucleosome remodeling are diverse - nucleosome sliding, dimer exchange, nucleosome disassembly, and nucleosome conformation alteration. This study sheds light on how different chromatin remodelers catalyze various structural transformations. We provide novel insights into the nucleosome dynamics, the role of histone octamer dynamics on nucleosome remodeling by ISW2, mechanism of dimer exchange by INO80 and mechanism of nucleosome disassembly by the coordinated action of RSC and histone chaperone Nap1. We also provide insights on how aberrant SWI/SNF complexes affect fundamental enzymatic properties such as ATPase and processive nucleosome remodeling. ISW2 remodelers sense and respond to the length of linker DNA separating the nucleosome and centers nucleosome. Histone octamers are perceived as a mostly static structure whereas DNA deforms itself to fit nucleosome. We have found change in histone octamer conformation as a novel step in ISW2 mobilizing DNA through the nucleosome. We provide evidence for an induced fit mechanism where histone-histone and histone-DNA interactions change in respond to remodeler, and these changes promote DNA entry into the nucleosome. Our data supports a model in which DNA translocation causes the change in histone octamer conformation, followed by DNA entry into nucleosome and resetting of the histone octamer core. We also move a step ahead and show that SANT domain promotes the entry of DNA into nucleosome and resets the histone octamer core allowing processive nucleosome mobilization. INO80 nucleosome remodeling provides two outcomes- nucleosome centering and dimer exchange. INO80 exchanges H2A.Z-H2B dimer for H2A-H2B. We show that INO80 is incredibly slow at centering nucleosome compared to ISW2. We also provide evidence for a mechanism where INO80 persistently displaces DNA from the dimer interface, unlike ISW2, facilitating dimer exchange. In another instance, we show that kinetic step sizes are modulated by a combination of enzyme and DNA sequence properties, and are not hardwired into the enzyme. ISW2 has been previously shown to translocate DNA with a kinetic step sizes of ~7bp and ~3bp. We show that kinetic step sizes may vary depending on nucleosomal location where we monitor DNA movement. Next, we studied the mechanism of nucleosome disassembly by RSC in the presence of Nap1. We found that Nap1 promotes the disassembly of the distal nucleosome that RSC collides with rather than the proximal nucleosomes it mobilizes. SWI/SNF tops the list of the frequently mutated epigenetic factor in cancer with its subunits mutated in more than 20% of all cancers. Loss of hSnf5 is a driver mutation in pediatric rhabdoid tumors. Our lab has previously identified that the deletion of Snf5 causes yeast SWI/SNF to lose an entire module comprised of Snf5, Swp82, and Taf14. In this study, we establish the properties of aberrant SWI/SNF complex formed in the absence of Snf5 module. The deletion resulted in lower ATP hydrolysis and nucleosome mobilization activities of the mutant SWI/SNF. We found that Snf5 module is necessary to couple ATP hydrolysis with DNA translocation. We studied the role of accessory domain AT-hooks in the ATPase subunit of SWI/SNF and found similar results. Interestingly, AT hook and SnAC domains, and Snf5 subunit were found to communicate with the same region in ATPase domain physically. These studies provide valuable mechanistic insights into chromatin structure and function and highlight how different chromatin remodelers catalyze different chromatin remodeling outcomes. We also provide new insights on how the activity of the core ATPase motor is regulated either by accessory domains on the same subunit or by accessory subunits as a part of the larger complex

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

Summary: 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. : Mutation of SWI/SNF chromatin remodeling complex subunits contributes to cancer and neurological disorders. Sen et al. report that loss of the Snf5 subunit alters the architecture and function of SWI/SNF in a yeast model system. These findings may reflect changes that occur in pediatric rhabdoid tumors with mutated Snf5. Keywords: Chromatin remodeling, SWI/SNF, Snf5, SMARCB1, BAF47, INI

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling.

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking

Dynamic regulation of transcription factors by nucleosome remodeling

Abstract The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes