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Dbf4-Dependent Kinase Finetunes INO80 Function at Chromosome Replication Origins

ABSTRACT: The highly conserved Dbf4-Dependent Kinase (DDK) plays a pivotal role in the nucleus during S phase, where it directly phosphorylates the replicative helicase, the minichromosome maintenance (MCM) complex. This leads to the initiation of chromosome replication. However, aside from the MCM complex, few other targets have been identified to date, leaving DDK an understudied kinase. Here, we describe a two-pronged mass spectrometry-based approach and define the nuclear DDK-dependent phosphoproteome, which consists of approximately 400 phosphorylation events. Within this network, we found that DDK directly phosphorylates the Arp8 subunit of the multi-subunit chromatin remodeler complex INO80. Arp8 phosphorylation stabilises INO80’s intramolecular complex integrity, which finetunes its nucleosome spacing activity at replication origins. This adjustment of origin chromatin architecture stimulates replication and is important for the response to replication stress. Our results represent a significant advance in our understanding of the molecular mechanisms underlying the regulation of replication origins.

INSTRUMENT(S):

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Shibojyoti Lahiri

LAB HEAD: Axel Imhof

PROVIDER: PXD053426 | Pride | 2026-04-06

REPOSITORIES: Pride

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			Action	DRS
	20230521_MSCK11_DSBU_Frac_09_01_BA1_1_1605.d.zip	Other
	20230521_MSCK11_DSBU_Frac_09_02_BA1_1_1610.d.zip	Other
	20230521_MSCK11_DSBU_Frac_09_03_BA1_1_1615.d.zip	Other
	20230521_MSCK11_DSBU_Frac_10_01_BA2_1_1606.d.zip	Other
	20230521_MSCK11_DSBU_Frac_10_02_BA2_1_1611.d.zip	Other

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Project description:Embryonic stem cell (ESC) self-renewal and pluripotency is controlled by the coordinated action of transcription factors and chromatin regulators. Compared to the pluripotency transcription factors, the function of the chromatin regulators, especially the ATP-dependent chromatin remodelers, remains poorly understood in ESCs. Here, we show that INO80, a SWI/SNF family chromatin remodeling complex, is essential for ESC self-renewal, pluripotency, somatic cell reprogramming, and embryonic development. Ino80, the ATPase of the complex, forms an auto-regulatory loop with the ESC master transcription factors Oct4, Nanog, and Sox2. More importantly, it co-occupies the enhancer regions of most key pluripotency genes with the master transcription factors, and positively regulates their expression by maintaining an open chromatin structure. Our data suggests that INO80 is an integral component of the pluripotency transcription network, and plays a critical role in both the maintenance and establishment of pluripotency Mouse J1 cells were infected with lentiviral-shNT or lentiviral-Ino80, RNA was extracted at 2, 4 or 6 days after infection. agent: non-targeting shRNA: NS_Day_2_1, NS_Day_2_2, NS_Day_4_1, NS_Day_4_2, NS_Day_6_1, NS_Day_6_2 agent: Ino80 shRNA: shIno80_Day_2_1, shIno80_Day_2_2, shIno80_Day_4_1, shIno80_Day_4_2, shIno80_Day_6_1, shIno80_Day_6_2 time: 48 hours: NS_Day_2_1, NS_Day_2_2, shIno80_Day_2_1, shIno80_Day_2_2 time: 96 hours: NS_Day_4_1, NS_Day_4_2, shIno80_Day_4_1, shIno80_Day_4_2 time: 144 hours: NS_Day_6_1, NS_Day_6_2, shIno80_Day_6_1, shIno80_Day_6_2 cell line: mouse J1 ESC: NS_Day_2_1, NS_Day_2_2, shIno80_Day_2_1, shIno80_Day_2_2, NS_Day_4_1, NS_Day_4_2, shIno80_Day_4_1, shIno80_Day_4_2, NS_Day_6_1, NS_Day_6_2, shIno80_Day_6_1, shIno80_Day_6_2 biological replicate: shIno80_Day_2_1, shIno80_Day_2_2 biological replicate: shIno80_Day_4_1, shIno80_Day_4_2 biological replicate: shIno80_Day_6_1, shIno80_Day_6_2 biological replicate: NS_Day_2_1, NS_Day_2_2 biological replicate: NS_Day_4_1, NS_Day_4_2 biological replicate: NS_Day_6_1, NS_Day_6_2

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Dbf4-Dependent Kinase Finetunes INO80 Function at Chromosome Replication Origins

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