HiCAR: Analysis of Open Chromatin Associated Long‐range Chromatin Interaction Using Low‐Input Materials

Wei, Xiaolin; Tran, Duc; Diao, Yarui

doi:10.1002/cpz1.899

Cited by 1 publication

(2 citation statements)

References 53 publications

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“…The full protocol for the latest developed HiCAR workflow was published by Wei et al 57 . Collection of 400,000 cells were digested and fixed with 1% formaldehyde for 10 min, followed by quenching with 125 mM glycine and washed with cold TBS (20 mM Tris-HCl, 150 mM NaCl, pH 7.4) once.…”

Section: Methodsmentioning

confidence: 99%

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MED1 IDR acetylation reorganizes the transcription preinitiation complex, rewires 3D chromatin interactions and reprograms gene expression

Lin,

Roeder,

Barrows

et al. 2024

Preprint

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With our current appreciation of the complexity of eukaryotic transcription, whose dysregulation drives diseases including cancer, it is becoming apparent that identification of key events coordinating multiple aspects of transcriptional regulation is of special importance. To elucidate how assembly of RNA polymerase II (Pol II) with Mediator complex preinitiation complexes (PICs) and formation of transcription-permissive 3D chromatin organization are coordinated, we studied MED1, a representative subunit of the Mediator complex that acts to establish functional preinitiation complexes (PICs) that forms biomolecular condensates through an intrinsically disordered region (IDR) to facilitate transcription, and is implicated in the function of estrogen receptor α (hereafter ER) in ER-positive breast cancer (ER+ BC) cells. We found that MED1 is acetylated at 6 lysines in its IDR and, further, that MCF7 ER+ BC cells in which endogenous MED1 is replaced by an ectopic 6KR (non-acetylatable) mutant (6KR cells) exhibit enhanced cell growth and elevated expression of MED1-dependent genes. These results indicate an enhanced function of 6KR MED1 that may be attributed to two mechanisms: (1) reorganized PIC assembly, as indicated by increased MED1 and Pol II, decreased MED17, and equivalent ERα occupancies on chromatin, particularly at active enhancers and promoters; (2) sub-TAD chromatin unfolding, as revealed by HiCAR (Hi-C on accessible regulatory DNA) analyses. Furthermore, in vitro assays demonstrate distinct physio-chemical properties of liquid-liquid phase separation (LLPS) for 6KR versus 6KQ MED1 IDRs, and for non-acetylated versus CBP-acetylated WT MED1 IDR fragments. Related, Pol II CTD heptads are sequestered in 6KR and control WT MED1 IDR condensates, but not 6KQ and CBP-acetylated WT MED1 IDR condensates. These findings, in conjunction with recent reports of PIC structures, indicate that MED1 coordinates reorganization of the PIC machinery and the rewiring of regional chromatin organization through acetylation of its IDR. This study leads to an understanding of how the transition in phase behavior of a transcription cofactor acts as a mechanistic hub integrating linear and spatial chromatin functions to support gene expression, and have potential therapeutic implications for diseases involving MED1/Mediator-mediated transcription control.

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Section: Methodsmentioning

confidence: 99%

“…The sequencing was performed with Illumina NovaSeq SP (100 bp two ends) by Genomics Resource Center in Rockefeller University. All PCR primer/oligo sequences were reported by Wei, et al 57 .…”

Section: Methodsmentioning

confidence: 99%