The H2BE76K mutation drives breast cancer development by enhancing the transcription of ADAM19. The H2BE76K mutation drives breast cancer development by enhancing the transcription of ADAM19

Study was conducted to understand the effect of the cancer associated H2BE76K mutation. Using CRISPR/Cas9 Knock-in cell lines expressing FLAG-tagged WT and E76K mutant H2B, we conducted gene expression profiling experiments and studied the effect of the H2BE76K mutation on H2B occupancy using CUT&RUN sequencing. Overall design: CRISPR/Cas9 cell lines expressing FLAG tagged Wildtype (WT) H2B and H2BE76K were generated to study the H2BE76K mutation in breast invasive carcinoma. Two WT and two mutant E76K cell lines as well as the Parental MDA-MB-231 cell lines were used for experiments. Gene expression of WT and E76K mutant lines was profiled by RNA-seq. Genomic localization of FLAG-tagged WT and E76K H2B proteins were profiled by CUT&RUN sequencing. ATAC-seq was performed to detect any difference of chromosomal accessibility between H2BE76K and WT.

本研究旨在解析癌症相关H2BE76K突变的生物学效应。本研究采用表达FLAG标签野生型（Wildtype, WT）及E76K突变型H2B的CRISPR/Cas9敲入细胞系，开展基因表达谱分析实验，并借助CUT&RUN测序（CUT&RUN sequencing）探究H2BE76K突变对H2B染色质占据情况的影响。实验整体设计：为研究浸润性乳腺癌（breast invasive carcinoma）中的H2BE76K突变，我们构建了表达FLAG标签野生型H2B与H2BE76K突变型H2B的CRISPR/Cas9细胞系。本次实验共使用2株野生型细胞系、2株E76K突变型细胞系，以及亲本MDA-MB-231细胞系。通过RNA测序（RNA-seq）对野生型与E76K突变型细胞系的基因表达水平进行了谱分析；通过CUT&RUN测序对FLAG标记的野生型及E76K突变型H2B蛋白的基因组定位特征进行了谱分析；此外还通过转座酶可及性测序（ATAC-seq）检测H2BE76K与野生型细胞系之间的染色体可及性差异。