Biology subjects

Bartholomew, B.

Publications and source records attributed to Bartholomew, B..

4 recordsLinked to original sources

bioRxiv2026

Sequence-dependent binding modes of INO80 control +1 nucleosome positioning

Cellular self-organisation counteracts entropy at the expenditure of energy. In case of the first level of nuclear DNA organisation, this relates to regular nucleosome arrays and interspaced nucleosome-depleted regions (NDRs), for example at promoters or replication origins1-5. The organisation of nucleosomes as building blocks of chromatin is orchestrated by the collective activities of ATP-dependent chromatin remodellers6-9. Yet, how remodellers achieve positional specificity, in particular regarding the promoter-proximal +1 nucleosomes, remains unclear. Here, we show that the S. cerevisiae chromatin remodeller INO80 unexpectedly distinguishes DNA sequence asymmetry within the +1 nucleosome of the SWH1 gene through distinct inhibited and active nucleosome-binding modes. In structural and biochemical analyses of INO80 on nucleosomes with the endogenous sequence, we identified an inhibited binding mode where the entire INO80 remodelling unit flipped on the +1 nucleosome. INO80 adopted this remodelling-incompetent binding mode when facing the promoter, but a remodelling-competent mode when facing the gene body. This directional read-out of intra-nucleosomal DNA sequence asymmetry, together with extranucleosomal NDR sequence features, prevented nucleosome sliding into the NDR while permitting array formation over the gene. Our work shows how DNA features contribute to ATP-dependent self-organisation of promoter chromatin by INO80.

biochemistry↗

bioRxiv2024

Conformational switching of Arp5 subunit differentially regulates INO80 chromatin remodeling

The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80s ATPase activity to nucleosome movement. Our investigation reveals that the grappler domain of Arp5 interacts with the acidic pocket of nucleosomes through two distinct mechanisms: an arginine anchor or a hydrophobic/acidic patch. These two modes of binding serve distinct functions within INO80 as shown in vivo by mutations in these regions resulting in varying phenotypes and in vitro by diverse effects on nucleosome mobilization. Our findings suggest that the hydrophobic/acidic patch of Arp5 is likely important for dimer exchange by INO80, while the arginine anchor is crucial for mobilizing nucleosomes.

biochemistry↗

bioRxiv2022

An RNA binding module of SWI/SNF is required for activation of cell-type specific enhancers and super-enhancers in early development

The mammalian SWI/SNF complex is an ATP-dependent chromatin remodeler and master regulator in development that when mutated is the cause for several human diseases including cancer. Although SWI/SNF is highly enriched at enhancers and its basic chromatin remodeling activities have been studied for over 30 years, there is little known about how it regulates enhancer activity or enhancer-promoter interactions. We find a putative RNA binding module located near the C-terminus of the catalytic subunit of SWI/SNF required for SWI/SNF recruitment to cell-type specific enhancers and super-enhancers in naive and cell lineage primed pluripotent cells. The AT-hook is required for acquisition of the active histone marks H3K27ac and H3K4me1 and recruitment of the MLL3/4 co-activator to these enhancers and super-enhancers. Consistent with changes in enhancer architecture, loss of the AT-hook interferes with activation of genes involved in cell lineage priming as well as genes normally activated in naive pluripotent cells.

genomics↗

bioRxiv2019

De novo Identification of Essential Protein Domains from CRISPR/Cas9 Tiling-sgRNA Knockout Screens

High-throughput CRISPR/Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. To facilitate de novo identification of essential protein domains from such screens, we developed ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refers to the protein regions that are associated with strong sgRNA dropout effect in the screens. We used ProTiler to analyze a published CRISPR tiling screen dataset, and identified 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlapped with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also revealed unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which we validated experimentally. Surprisingly, the CKHS regions were negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR/Cas9 mediated amino acids loss.

bioinformatics↗