Biology subjects

Medina-Pritchard, B.

Publications and source records attributed to Medina-Pritchard, B..

5 recordsLinked to original sources

bioRxiv2024

Rules of engagement for condensins and cohesins guide mitotic chromosome formation

During mitosis, interphase chromatin is rapidly converted into rod-shaped mitotic chromosomes. Using Hi-C, imaging, proteomics and polymer modeling, we determine how the activity and interplay between loop-extruding SMC motors accomplishes this dramatic transition. Our work reveals rules of engagement for SMC complexes that are critical for allowing cells to refold interphase chromatin into mitotic chromosomes. We find that condensin disassembles interphase chromatin loop organization by evicting or displacing extrusive cohesin. In contrast, condensin bypasses cohesive cohesins, thereby maintaining sister chromatid cohesion while separating the sisters. Studies of mitotic chromosomes formed by cohesin, condensin II and condensin I alone or in combination allow us to develop new models of mitotic chromosome conformation. In these models, loops are consecutive and not overlapping, implying that condensins do not freely pass one another but stall upon encountering each other. The dynamics of Hi-C interactions and chromosome morphology reveal that during prophase loops are extruded in vivo at [~]1-3 kb/sec by condensins as they form a disordered discontinuous helical scaffold within individual chromatids.

molecular biology↗

bioRxiv2024

PLK1-Mediated Phosphorylation Cascade Activates the Mis18 Complex to Ensure Centromere Inheritance

Accurate chromosome segregation requires the attachment of spindle microtubules to centromeres, which are epigenetically defined by the enrichment of CENP-A nucleosomes. During DNA replication, existing CENP-A nucleosomes undergo dilution as they get redistributed among the two DNA strands. To preserve centromere identity, CENP-A levels must be restored in a cell-cycle controlled manner orchestrated by the Mis18 complex. Here we provide a comprehensive mechanistic basis for PLK1-mediated licensing of CENP-A loading. We demonstrate that PLK1 interacts with Mis18 and Mis18BP1 subunits of the Mis18 complex by recognising self-primed phosphorylations of Mis18 (S54) and Mis18BP1 (T78 and S93) through its Polo-box binding domain. Disrupting these PLK1 phosphorylations perturbed the centromere recruitment of HJURP and new CENP-A loading. Biochemical and functional analyses show that phosphorylation of Mis18 and subsequent PLK1 binding is required to activate the Mis18/{beta} complex for robust Mis18/{beta}-HJURP interaction. Thus, our study reveals key molecular events underpinning the licensing role of PLK1 in ensuring accurate centromere inheritance. One-Sentence SummaryPLK1 phosphorylation cascade licenses CENP-A loading by facilitating HJURP centromere recruitment via Mis18/{beta} activation.

cell biology↗

bioRxiv2021

Structural Basis for Mis18 Complex Assembly: Implications for Centromere Maintenance

The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18, Mis18y and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18, 2 Mis18y and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18 can associate with centromeres and deposit CENP-A independently of Mis18y, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

cell biology↗

bioRxiv2019

Structural Basis for CAL1-Mediated Centromere Maintenance

Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting is achieved by HJURP and the Mis18 complex, respectively. Here, using crystal structures, we show how Drosophila CAL1, an evolutionarily distinct CENP-A chaperone, targets CENP-A to the centromere receptor CENP-C without the requirement of the Mis18 complex: while the N-terminal CAL1 fragment (CAL11-160) wraps around CENP-A/H4 through multiple physical contacts, the C-terminal CAL1 fragment (CAL1893-914) directly binds CENP-C cupin dimer. Our work shows CAL1, though divergent at the primary structure, employs evolutionarily conserved and adaptive structural principles to recognise CENP-A/H4 and CENP-C providing insights into the minimalistic principles underlying centromere maintenance.

biochemistry↗

bioRxiv2019

Epigenetic inheritance of centromere identity in a heterologous system

The centromere is an essential chromosomal region required for accurate chromosome segregation. Most eukaryotic centromeres are defined epigenetically by the histone H3 variant, CENP-A, yet how its self-propagation is achieved remains poorly understood. Here we developed a heterologous system to reconstitute epigenetic inheritance of centromeric chromatin by ectopically targeting the Drosophila centromere proteins dCENP-A, dCENP-C and CAL1 to LacO arrays in human cells. Dissecting the function of these three components uncovers the key role of self-association of dCENP-C and CAL1 for their mutual interaction and dCENP-A deposition. Importantly, we identify the components required for dCENP-C loading onto chromatin, involving a cooperation between CAL1 and dCENP-A nucleosomes, thus closing the epigenetic loop to ensure dCENP-C and dCENP-A replenishment during the cell division cycle. Finally, we show that all three Drosophila factors are sufficient for dCENP-A propagation and propose a model for the epigenetic inheritance of centromere identity.

cell biology↗