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En'Cas'ing The Stress: Engineering a Human Cell Line Knockout of Heat Shock Response Coordinator Genes Using CRISPR Cas9 System
Author(s) -
Canales Gabriela IslaInez,
Pani Bibhusita,
Nudler Evgeny
Publication year - 2016
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.30.1_supplement.811.5
Subject(s) - hsf1 , microbiology and biotechnology , heat shock factor , biology , heat shock , proteostasis , heat shock protein , genetics , gene , hsp70
The Heat Shock Response (HSR) is an evolutionarily conserved response to high temperatures and other stresses that controls adaptive proteostasis, and is primarily regulated by the factor, Heat shock transcription factor 1 (HSF1). In mammalian cells, HSF1 is converted from an inactive monomeric form to an active trimer in response to heat stress. A ribonucleoprotein complex comprising of eukaryotic translation elongation factor eEF1A1, and a long noncoding RNA HSR1 are the key components of HSF1 activation. Once activated, HSF1 is recruited to Heat Shock Protein (HSP) promoter regions, upregulating chaperone activity in the cell. Along with mediating initiation of HSR, eEF1A1 is also a vital component of protein synthesis machinery. Interestingly, another isoform of eEF1A, called eEF1A2, is expressed in some specialized terminally differentiated cells of skeletal muscle, heart, pancreatic islets and motor neurons, all of which are prone to protein aggregation. The two isoforms are 92% identical and are reciprocally regulated. To better understand the role of eEF1A1 and HSF1 proteins in humans, we use a CRISPR‐Cas9 nickase system to knockout HSF1 and eEF1A1 in a human cell line. We showed that the hTERT‐immortalized, normal diploid foreskin fibroblast cell line, BJ‐5ta, produces both eEF1A isoforms. This will allow us to perform eEF1A1 knockout in these cells. We hypothesize that HSF1 knockout cell line will survive under normal conditions but express very low thermotolerance. Conjointly, we hypothesize that the elimination of eEF1A1 may be compensated by the upregulation of eEF1A2. If viable, the eEF1A1 knockout cell line will be used for screening mutants of eEF1A2 restoring activation of HSR. Both HSF1 and eEF1A1 knockout lines will also be used for future studies to improve upon the current model of the HSR pathway and potentially reveal therapeutic targets for diseases like ALS, Alzheimer's disease, Parkinson's disease, type 2 diabetes, and amyloidosis. Support or Funding Information This investigation was supported in part by a MARC Undergraduate Student Training in Academic Research (U‐STAR) National Research Service Award (NRSA) Institutional Research Training Grant (2 T34 GM008663) from the National Institutes of Health, National Institute for General Medical Sciences. This research was supported in part by a grant to UMBC from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program.