BRG1 Brahma-Related Gene 1 Non-Small Cell Lung Cancer Taylor Eatmon BIOL 445: Cancer Biology

Research on S. cerevisiae identified genes that coordinately function to regulate transcription. In 1984, two independent genetic screens on S. cerevisiae aimed to identify genes that, when mutated, affect the mating-type switch and sucrose fermentation pathways. So what this means is that scientists screened for mutants that had mating-type switch and sucrose-non-fermentable phenotypes. In yeast, mating occurs between haploids, which can either be a or Alpha (opposites normally), so mutants switched type. And then second, they looked for mutants that could not ferment sucrose for energy as normal. Ultimately, this research showed the genes identified function coordinately to regulate transcription. The genes they found were grouped into the SWI/SNF family. SWI/SNF Family: SWItch/Sucrose Non-Fermentable. BAF Complex: human analog of SWI/SNF, BRG-Associated Factor. BRG1 is a subunit of the BAF complex Each BAF complex contains either BRG1 or BRM (brahma gene in drosophila). They play complementary roles and are thought to have a synthetic-lethal relationship, which is going to be important when discussing treatment for the cancer.

The BAF Complex uses energy from ATP hydrolysis to remodel chromatin structure by an unknown mechanism. The BAF complex uses energy from ATP hydrolysis to remodel chromatin structure and regulate transcription. Now just as a review from one of our recent lectures: We know that chromatin = DNA + histones. Due to the scale and compaction problem that is present because of how much DNA we carry around, the nucleosomes (the subunit of chromatin, 147 bp of DNA wrapping around the 8 histones) mediates the problem. We also know that remodeling our chromatin structure is a way of epigenetically modifying transcription of our genes. So we can condense that chromatin to decrease transcription and therefore expression, or we can open it for the opposite effects. And this is exactly what the BAF complex does. It is a nucleosome remodeling complex in eukaryotes. It is a group of proteins that associate to remodel the way that DNA is packaged. Function: Histone-modifying enzymes recognize and covalently mark (by acetylation, methylation, phosphorylation, ribosylation and ubiquitination) specific residues of the histone tails ATP-dependent chromatin remodeling complexes specifically recognize these histones marks, and through ATP hydrolysis unwrap, mobilize, exchange or eject the nucleosome, and subsequently recruiting a transcriptional apparatus to nucleosomal DNA Different effects of the ATP dependent chromatin remodeling activity of remodelers on nucleosomal DNA: upon hydrolysis of ATP, a protected region of chromatin can become available to DNA binding protein complexes, such as transcription factors (in green). Nucleosomes can be unwrapped, mobilized or ejected to allow these processes. In some cases ATP dependent remodeling complexes can use ATP to introduce histone variants within the nucleosome by a process called dimer exchange. (DBP stands for DNA-binding protein) *sliding or dissociation* A proposed mechanism: The other is known as the "bulge" or "loop-recapture" mechanism and it involves the dissociation of DNA at the edge of the nucleosome with reassociation of DNA inside the nucleosome, forming a DNA bulge on the octamer surface. The DNA loop would then propagate across the surface of the histone octamer in a wave-like manner, resulting in the repositioning of DNA without changes in the total number of histone-DNA contacts.

BRG1 is an ATPase and the central catalytic subunit of the BAF Complex. ~ 140 kb in length BRG1 stands for brahma related gene. The gene is found on P arm of Chromosome 19 (red box) and is approx.140 kb in length. The BRG1 protein is the central catalytic subunit of the BAF complex. It is an ATPase with helicase activity. BRG1 has an ATP binding pocket to bind ATP and hydrolyze it to gain energy for the BAF complex to function. This disrupts DNA-histone interaction which promotes sliding of the nucleosome.

Depending on the subunits present, the BAF complex has various targets in multiple pathways and processes. Explain picture Pathways: Hedgehog, Wnt, Rb, Cell motility, etc. DNA ds break repair

The mouse model shows that BRG1 is required for proper early development. +/- with exencephaly +/- Animal model is the mouse. BRG1 is essential for proper development early on in life. Null homozygotes die as blastocysts prior to implantation because they cannot hatch out of the zona pellucida (the thick transparent membrane surrounding a mammalian ovum before implantation). Null hets may develop exencephaly and have a predisposition to tumors. Explain left pic: high res photo at E17.5. Explain right picture: In Vitro Blastocyst Outgrowths Bright field photographs of heterozygous (A and C) or homozygous (B and D) Brg1 blastocysts after being cultured for 7 days in serum-supplemented media. Blastocysts were either placed directly into culture (A and B) or first treated with Pronase (mixture of proteases) to remove zona pellucidae (C and D). (A) In the absence of Pronase, heterozygous explants hatched from the zona pellucidae (ZP), trophectoderm (TE) attached to and spread out across the bottom of tissue culture wells, and the inner cell masses (ICM) underwent extensive proliferation. (B) In contrast, homozygotes failed to hatch and died. Pyknotic blastomeres are evident (arrow). (C) In the presence of Pronase, heterozygous explants exhibited robust outgrowth of the TE and ICM. (D) In contrast, homozygotes attached to the bottom of the dish, but the TE and ICM died with little or no outgrowth. Pyknotic cells are evident (arrow). BRG1 is also required for zygotic genome activation, and proliferation and/or differentiation of neuronal, lymphocyte, heart, and blood cells. Hets – 30 months, WT – 60 months WT +/+

BRG1 can be mutated in multiple locations and in various ways. BRG1 can be mutated in different ways and in different places along the gene. Nonsense mutations result in truncated proteins, cant associate with the complex. Missense mutations; protein can associate sometimes but cannot Common BRG1-inactivating mutations lead to truncated proteins that cannot power the BAF complex. Locations of point mutations in human cancer cell lines – all along the gene in various domains. Bromodomain recognizes acetylated lysine resides of histone tails

BRG1 inactivation results in the inability to control transcription and promotes Non-Small Cell Lung Cancer aggressiveness. 1 out of every 4 cases is lung cancer, NSSLC is the most common type of lung cancer. BRG1 mutations are found in 30% of NSSLC cases. Adenocarcinoma – start in early versions of cells that would normally secrete substances such as mucus Squamous cell carcinoma – start in early versions of squamous cells which are flat cells that line the inside of the airways in the lungs. Large cell carcinoma – can appear in any part of the lungs, fast-growing cancer Details of link between BRG1 mutation and tumor growth still remain unclear. BUT, Brg1 mutations have been found to coexist with mutations in genes such as KRAS, p53. Suggest tumor suppressor function and suppress transcription of mutated genes? Still unknown, research being done. X ray pic: This elderly man with a history of emphysema – note the hyper inflated lungs – presented with a cough. His chest x-ray shows a subtle rounded lesion above the right hilum (arrows). CT-guided biopsy showed that this was a carcinoma. Also involved in other cancers – pancreatic, breast, prostate.

A synthetic lethality-based strategy for treatment of BRG1-deficient tumor cells is still being developed. Best treatment option is surgery, if it is caught early on.

References https://en.wikipedia.org/wiki/SMARCA4#/media/File:Protein_SMARCA4_PDB_2grc.png http://www.genecards.org/cgi-bin/carddisp.pl?gene=SMARCA4#aliases_descriptions http://mcb.asm.org/content/20/6/1899/F1.expansion.html http://www.nature.com/ng/journal/v46/n5/full/ng.2922.html http://cancerres.aacrjournals.org/content/60/21/6171 https://www.ncbi.nlm.nih.gov/pmc/?term=25115300[PMID]&report=imagesdocsum http://www.cell.com/trends/genetics/fulltext/S0168-9525%2814%2900079-1 http://www.webmd.com/lung-cancer/ss/slideshow-lung-cancer-overview https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924208/figure/F1/ http://www.humpath.com/spip.php?article1927 http://clincancerres.aacrjournals.org/content/20/1/21.figures-only http://www.cell.com/molecular-cell/fulltext/S1097-2765%2800%2900127-1#TBL1 https://www.cancer.org/cancer/non-small-cell-lung-cancer/about/key-statistics.html http://www.svuhradiology.ie/case-study/lung-cancer-cxr/ http://cancerres.aacrjournals.org/content/73/17/5508