https://openworks.mdanderson.org/sumexp23/1029/thumbnail.jp

Green, Michael R

Luke, Sarah S

Yang, Haopeng

English

OpenWorks @ MD Anderson

Introduction• CREBBP is frequently mutated in germinal center-derived B-cell lymphomas such as follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL).• CREB-binding protein (CREBBP) and its paralog E1A-binding protein P300 (EP300) are acetyltransferases that regulate gene expression by acetylating histone and non-histone proteins.• The CREBBP lysine acetyltransferase (KAT) domain point mutant showed a dramatic reduction on the level of H3K27Ac, leading to loss of function in transcription regulation.• The Green Lab previously performed molecular characterization using isogenic CRISPR/Cas9-modified lymphoma cells and identified H3K27Ac loss at enhancers associated with MHC II.• A dCas9-p300 fused protein construct was developed to activate genes from the promoter and the enhancer by guiding acetyltransferase to specific loci.Title of the Poster Presentation Goes HereAuthors of the Poster Presentation Goes HereThe University of Texas MD Anderson Cancer Center Enhancers activation by CRISPR/Cas9-based acetyltransferase rescues loss of function in CREBBP point mutantSarah S. Luke, Haopeng Yang Ph.D., Michael R. Green Ph.D.Department of Lymphoma - Myeloma, University of Texas MD Anderson Cancer CenterFig. 1: Structure of the dCas9-p300 complex and the process of target localization to promote enhancers and transcription.Created with BioRender.comFig 2:  The comparison of different promoter A) EF1α  and B) SFFV in driving eGFP expression in multiple lymphoma cell line.B)Objective• Optimize dCas-p300 construct in lymphoma cell lines with a high transduction efficiency.• Activate genes that were reduced in CREBBP point mutant by dCas-p300 system.Methods• A dCas9-P300 complex was optimized to serve as a source for gene activation.• Cloning was used to change the structure of the dCas9-P300 complex from its original and universal promoter, Elongation Factor 1 Alpha (EF1α), to a B-cell specific promoter, Spleen Focus-Forming Virus (SFFV).• Since the enhancer region is larger than the promoter, 2 guide RNAs (gRNAs) with GFP-labelled and mCherry-labelled were used to investigate the efficiency.AcknowledgementsI would like to express my gratitude to the members of the Green Lab for guiding and mentoring me throughout my Summer Research Internship. Additionally, I would like to acknowledge the University Outreach Program and Bryn Mawr College for giving me the opportunity to explore my interests in the field of science research and Oncology at MD Anderson Cancer Center.A)Results• Flow cytometry showed the highly transduced efficiency in B cell lymphoma with over 85% double positive population in both CD74 and MHC II guides transduction.• The expression of CD74 and HLA-DR (direct target of CIITA) increased dramatically in a dose dependent manner.Conclusion• Switching the promoter from EF1α to SFFV significantly increased the transduction efficiency in B cells.• The data obtained indicated that the use of two gRNA constructs resulted in the activation of a gene’s enhancer region via increasing the acetylation of their locus and promoting the transcription, which partially rescues the loss of function in the CREBBP point mutant.References• Mondello, Patrizia, et al. "Selective inhibition of HDAC3 targets synthetic vulnerabilities and activates immune surveillance in lymphoma." Cancer discovery 10.3 (2020): 440-459.• Hilton, Isaac B., et al. "Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers." Nature biotechnology 33.5 (2015): 510-517.• Shrimp, Jonathan H., et al. "Chemical control of a CRISPR-Cas9 acetyltransferase." ACS chemical biology 13.2 (2018): 455-460.mCherryGFPmCherryGFPAPC (CD74)Pacific Blue (HLA-DR)GFP +GFP +NegDouble +mCherry +mCherry +Double +NegA)A) B)B) Fig 3:The flowcytometry histogram of CD74 (B) indicated an increased expression in the double positive population (A) compared with any single positive population or double negative population.Fig 4:Similar to CD74, the flowcytometry chart of HLA-DR indicated the activation of CIITA by showing an increased expression of  its direct target (HLA-DR) in double positive population compared with double negative population.

Enhancers activation by CRISPR/Cas9-based acetyltransferase rescues loss of function in CREBBP point mutant

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1029&context=sumexp23

Enhancers activation by CRISPR/Cas9-based acetyltransferase rescues loss of function in CREBBP point mutant

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