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A CRISPR/Cas9-mediated screen identifies regulators of early plasma cell differentiation

ABSTRACT: The differentiation of B cells into antibody-secreting cells depends on cell division-coupled, epigenetic and other cellular processes that are incompletely understood. We have developed a CRISPR/Cas9-based screen that models an early stage of T cell-dependent plasma cell differentiation and measures B cell survival or proliferation versus the formation of CD138+ plasmablasts. Here, we refined and extended this screen to more than 500 candidate genes that are highly expressed in plasma cells. Among known genes whose deletion preferentially affected plasmablast formation were the transcriptional regulators Prdm1, Irf4 and Pou2af1, and the Ern1 gene encoding the ER stress sensor IRE1a. Moreover, we newly identified several genes involved in NF-kB or p38 signaling (Pim2, Nfkbia, Mapk14), vesicle trafficking (Arf4, Preb) and epigenetic regulators that form part of the NuRD complexes (Hdac1, Mta2, Mbd2). Defective plasmablast formation caused by Ern1 deletion could not be rescued by spliced XBP1 (XBP1s), a transcription factor dependent on and downstream of IRE1a, suggesting that in early plasma cell differentiation IRE1a acts independently of XBP1s. The deletion of ARF4, a small GTPase required for ER-to-Golgi transport, impaired plasmablast formation by blocking antibody secretion. Plasmablast formation after Hdac1 deletion was consistently reduced by about 50%, while deletion of the closely related Hdac2 gene had no effect. Hdac1 knock-out led to strongly perturbed protein expression of antagonistic transcription factors that specify plasma cell versus B cell identity (by decreasing IRF4 and BLIMP1 and increasing BACH2 and PAX5). Taken together, our results highlight specific and non-redundant roles for Ern1, Arf4 and Hdac1 in the early steps of plasma cell differentiation.

INSTRUMENT(S): Q Exactive HF-X, Q Exactive Plus

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): B Cell, Blood Plasma

DISEASE(S): B-cell Lymphoma

SUBMITTER: Oliver Popp

LAB HEAD: Van Trung Chu

PROVIDER: PXD038201 | Pride | 2022-12-22

REPOSITORIES: Pride

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			Action	DRS
	Animal_20180407_OP_MM_ChuVanThrung_Plex_fr01.raw	Raw
	Animal_20180407_OP_MM_ChuVanThrung_Plex_fr02.raw	Raw
	Animal_20180407_OP_MM_ChuVanThrung_Plex_fr03.raw	Raw
	Animal_20180407_OP_MM_ChuVanThrung_Plex_fr04.raw	Raw
	Animal_20180407_OP_MM_ChuVanThrung_Plex_fr05.raw	Raw

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Project description:This experiment was conducted to identify mRNA transcripts alteration in liver of Ern1 liver specific knockout mice. The following abstract from the submitted manuscript describes the major findings of this work. The IRE1a-XBP1s axis regulates the COPII-mediated secretory program in response to nutrient availability. Lin Liu, Jie Cai, Xijun Liang, Qian Zhou, Huimin Wang, Chenyun Ding, Yuangang Zhu, Liwei Xiao, Tingting Fu, Zhisheng Xu, Jing Liu, Yujing Yin, Lei Fang, Bin Xue, Yan Wang, Aibin He, Yong Liu, Xiao-Wei Chen, and Zhenji Gan The cytoplasmic coat protein complex-II (COPII) is an evolutionarily conserved machinery that is essential for efficient trafficking of protein and lipid cargos. How the COPII machinery is regulated to meet the metabolic demand in response to alterations of nutritional state remains largely unexplored, however. Here, we show that dynamic changes of COPII-mediated secretion parallel the activation of XBP1s, the critical transcription factor in handling cellular ER stress, in both live cells and mouse liver upon physiological fluctuations of nutrient availability. Using live-cell imaging approaches, we demonstrate that XBP1s is sufficient to promote COPII-dependent secretion, mediating the nutrient stimulatory effects. ChIP-seq and RNA-seq analyses reveal that nutritional signals induce dynamic XBP1s occupancy of promoters of COPII secretion-related genes, thereby driving COPII-directed secretory process. Liver-specific disruption of the IRE1a-XBP1s signaling branch results in diminished COPII-mediated secretion. Reactivation of XBP1s in mice lacking hepatic IRE1a restores COPII-mediated lipoprotein secretion, and reverses the fatty liver and hypolipidemia phenotypes. Thus, our results demonstrate a previously unappreciated mechanism in the metabolic control of liver protein and lipid secretion: the IRE1a-XBP1s axis functions as a nutrient-sensing regulatory nexus that integrates nutritional states and the COPII secretory program.

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A CRISPR/Cas9-mediated screen identifies regulators of early plasma cell differentiation

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