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PBRM1 directs PBAF to pericentromeres and centromeres to constrain transcription and protect genome integrity

ABSTRACT: The specialized structure of the centromere is critical for effective chromosome segregation, but its repetitive nature and configuration makes it vulnerable to rearrangements. Centromere fragility can drive tumorigenesis, but protective mechanisms preventing fragility are still not fully understood. To identify core functions of the tumor suppressor PBRM1, a subunit of the PBAF remodelling complex, we created a panel of isogenic cell lines. We found that compromised centromere integrity is a general feature of cells lacking PBRM1. This defect creates a dependence on the spindle assembly checkpoint, which can be therapeutically exploited. We map the SMARCA4 subunit of PBAF across centromeric and pericentromeric chromatin and find PBRM1-dependent association with specific sequences. We further find that loss of PBRM1 leads to altered transcription of pericentromere-associated genes and transposable elements. These data identify PBAF as a protector of centromere structure with profound consequences for genome integrity.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human) Mus Musculus (mouse)

SUBMITTER: Graeme Benstead-Hume

LAB HEAD: Jyoti Choudhary

PROVIDER: PXD043209 | Pride | 2025-01-06

REPOSITORIES: pride

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			Action	DRS
	20180808_KL_Downs_lab_LMS_set01_01.raw	Raw
	20180808_KL_Downs_lab_LMS_set01_02.raw	Raw
	20180808_KL_Downs_lab_LMS_set01_03.raw	Raw
	20180808_KL_Downs_lab_LMS_set01_04_rep.raw	Raw
	20180808_KL_Downs_lab_LMS_set01_05_rep.raw	Raw

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Project description:PBRM1 is the 2nd-most frequently inactivated gene in clear cell renal cell cancer (RCC) but the oncogenic mechanisms, and hence methods for correction, are unclear. PBRM1 is a subunit of the PBAF coactivator complex that transcription factors use to reposition nucleosomes (‘open chromatin’) for gene activation. We therefore looked for transcription factors that recruit endogenous PBRM1 in kidney lineage/RCC cells, as a waypoint to identifying pathways impacted by PBRM1 loss. Unbiased immunoprecipitation/mass-spectrometry analyses of the endogenous PBRM1 interactome in kidney lineage cells revealed PAX8, a master transcription factor essential for proximal tubule epithelial fates, as the major transcription factor recruiting PBRM1/PBAF. The reverse analyses of the PAX8 interactome confirmed recruitment specifically of PBRM1/PBAF, and not the functionally similar BAF coactivator complex. More conspicuous in the PAX8 hub in RCC cells, however, were several corepressors, e.g. DNMT1, which oppose coactivators to repress instead of activate genes. Accordingly, key PAX8 target genes, e.g., GATA3, LHX1, WT1, and ~1000 other downstream kidney epithelial genes, but not PAX8 or PAX2, demonstrated loss of the histone lysine 27 acetylation (H3K27ac) activation mark, increase in CpG methylation repression marks, and lower expression in RCC vs normal kidney cortex, with the greatest repression in cases with bi-allelic PBRM1 inactivation. PBRM1 re-introduction into RCC cells, or depletion of the corepressor DNMT1 using siRNA or a clinical drug decitabine, rebalanced composition and function of the PAX8 transcription factor hub to coactivators, to thereby activate terminal epithelial-fates in vitro and in vivo. In sum, PBRM1 loss in RCC cells skews coregulator composition of the PAX8 master transcription factor hub toward corepressors and repression instead of activation of the downstream terminal epithelial-program; this oncogenic action could be reversed by pharmacologic corepressor depletion/inhibition.

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PBRM1 directs PBAF to pericentromeres and centromeres to constrain transcription and protect genome integrity

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