Mechanistic Analysis of Chromatin Remodeling Enzymes: a Dissertation

The inherently repressive nature of chromatin presents a sizeable barrier for all nuclear processes in which access to DNA is required. Therefore, eukaryotic organisms ranging from yeast to humans rely on a battery of enzymes that disrupt the chromatin structure as a means of regulating DNA transactions. These enzymes can be divided into two broad classes: those that covalently modify histone proteins, and those that actively disrupt nucleosomal structure using the free energy derived from ATP hydrolysis. The latter group, huge, multisubunit ATP-dependent chromatin remodeling factors, are emerging as a common theme in all nuclear processes in which access to DNA is essential. Although transcription is the process for which a requirement for chromatin remodeling is best documented, it is now becoming clear that other processes like replication, recombination and DNA repair rely on it as well. A growing number of ATP-dependent remodeling machines has been uncovered in the last 10 years. Although they differ in their subunit composition, organism or tissue restriction, substrate specificity, and regulating/recruiting partners, it has become increasingly evident that all ATP-dependent chromatin remodeling factors share a similar underlying mechanism. This mechanism is the subject of the studies presented in this thesis. Chromatin-remodeling factors seem to bind both the histone and DNA components of nucleosomes. From a fixed position on nucleosomes, the remodeling factors appear to translocate on the DNA, generating torsional stress on the double helix. This activity has several consequences, including the distortion of the DNA structure on the surface of the histone octamer, the disruption of histone-DNA interactions, and the mobilization of the nucleosome core with respect to the DNA. The work presented in this thesis, along with data reported by other groups, supports the hypothesis that yeast SWI/SNF chromatin remodeling complex and the recombinational repair factor, Rad54p, both employ similar mechanisms to regulate gene transcription, and facilitate homologous DNA pairing and recombination, respectively

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SWI-SNF-Mediated Nucleosome Remodeling: Role of Histone Octamer Mobility in the Persistence of the Remodeled State

SWI-SNF is an ATP-dependent chromatin remodeling complex that disrupts DNA-histone interactions. Several studies of SWI-SNF activity on mononucleosome substrates have suggested that remodeling leads to novel, accessible nucleosomes which persist in the absence of continuous ATP hydrolysis. In contrast, we have reported that SWI-SNF-dependent remodeling of nucleosomal arrays is rapidly reversed after removal of ATP. One possibility is that these contrasting results are due to the different assays used; alternatively, the lability of the SWI-SNF-remodeled state might be different on mononucleosomes versus nucleosomal arrays. To investigate these possibilities, we use a coupled SWI-SNF remodeling–restriction enzyme assay to directly compare the remodeling of mononucleosome and nucleosomal array substrates. We find that SWI-SNF action causes a mobilization of histone octamers for both the mononucleosome and nucleosomal array substrates, and these changes in nucleosome positioning persist in the absence of continued ATP hydrolysis or SWI-SNF binding. In the case of mononucleosomes, the histone octamers accumulate at the DNA ends even in the presence of continued ATP hydrolysis. On nucleosomal arrays, SWI-SNF and ATP lead to a more dynamic state where nucleosomes appear to be constantly redistributed and restriction enzyme sites throughout the array have increased accessibility. This random positioning of nucleosomes within the array persists after removal of ATP, but inactivation of SWI-SNF is accompanied by an increased occlusion of many restriction enzyme sites. Our results also indicate that remodeling of mononucleosomes or nucleosomal arrays does not lead to an accumulation of novel nucleosomes that maintain an accessible state in the absence of continuous ATP hydrolysis

Rad54p is a chromatin remodeling enzyme required for heteroduplex DNA joint formation with chromatin

In eukaryotic cells, the repair of DNA double-strand breaks by homologous recombination requires a RecA-like recombinase, Rad51p, and a Swi2p/Snf2p-like ATPase, Rad54p. Here we find that yeast Rad51p and Rad54p support robust homologous pairing between single-stranded DNA and a chromatin donor. In contrast, bacterial RecA is incapable of catalyzing homologous pairing with a chromatin donor. We also show that Rad54p possesses many of the biochemical properties of bona fide ATP-dependent chromatin-remodeling enzymes, such as ySWI/SNF. Rad54p can enhance the accessibility of DNA within nucleosomal arrays, but it does not seem to disrupt nucleosome positioning. Taken together, our results indicate that Rad54p is a chromatin-remodeling enzyme that promotes homologous DNA pairing events within the context of chromatin

Utilizing murine inducible telomerase alleles in the studies of tissue degeneration/regeneration and cancer.

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-Lox TERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 PtenL/L p53L/L LSL-mTERTL/L and thymic T-cell lymphoma model G4 Atm-/- mTERTER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures of performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells

LXH254, a Potent and Selective ARAF-Sparing Inhibitor of BRAF and CRAF for the Treatment of MAPK-Driven Tumors

Purpose: Targeting RAF for anti-tumor therapy in RAS-mutant tumors holds promise. Herein we describe in detail novel properties of the type II RAF inhibitor LXH254. Experimental Design: LXH254 was profiled in biochemical, in vitro, and in vivo assays including examining the activities of the drug in a large panel of cancer-derived cell lines, and a comprehensive set of in vivo models. In addition, activity of LXH254 was assessed in cells where different sets of RAF paralogs were ablated, or that expressed kinase-impaired and dimer-deficient variants of ARAF. Results: We describe an unexpected paralog selectivity of LXH254, which is able to potently inhibit BRAF and CRAF, but has less activity against ARAF. LXH254 was active in models harboring BRAF alterations, including atypical BRAF alterations co-expressed with mutant K/NRAS, and NRAS mutants, but had only modest activity in KRAS mutants. In RAS mutant lines loss of ARAF, but not BRAF or CRAF, sensitized cells to LXH254. ARAF-mediated resistance to LXH254 required both kinase function and dimerization. Higher concentrations of LXH254 were required to inhibit signaling in RAS-mutant cells expressing only ARAF relative to BRAF or CRAF. Moreover, specifically in cells expressing only ARAF, LXH254 caused paradoxical activation of MAPK signaling in a manner similar to dabrafenib. Lastly, in vivo, LXH254 drove complete regressions of isogenic variants of RAS mutant cells lacking ARAF expression, while parental lines were only modestly sensitive. Conclusions: LXH254 is a novel RAF-inhibitor able to inhibit dimerized BRAF and CRAF as well as monomeric BRAF while largely sparing ARAF