SUMMARY Centromeres are specialized chromatin domains specified by the centromere-specific CENP-A nucleosome. The stable inheritance of vertebrate centromeres is an epigenetic process requiring deposition of new CENP-A nucleosomes by HJURP. We show HJURP is recruited to centromeres through a direct interaction between the HJURP centromere targeting domain and the Mis18α-β C-terminal coiled-coil domains. We demonstrate Mis18α and Mis18β form a heterotetramer through their C-terminal coiled-coil domains. Mis18α-β heterotetramer formation is required for Mis18BP1 binding and centromere recognition. S. pombe contains a single Mis18 isoform that forms a homotetramer, showing tetrameric Mis18 is conserved from fission yeast to humans. HJURP binding disrupts the Mis18α-β heterotetramer and removes Mis18α from centromeres. We propose stable binding of Mis18 to centromeres in telophase licenses them for CENP-A deposition. Binding of HJURP deposits CENP-A at centromeres and facilitates the removal of Mis18, restricting CENP-A deposition to a single event per cell cycle.

The Mis18 complex specifies the site of new CENP-A nucleosome assembly by recruiting the CENP-A specific assembly factor HJURP (Holliday junction recognition protein). The human Mis18 complex consists of Mis18α, Mis18β and Mis18 binding protein 1 (Mis18BP1/hsKNL2). Although Mis18α and Mis18β are highly homologous proteins, we find that their conserved YIPPEE domains mediate distinct interactions that are essential to link new CENP-A deposition to existing centromeres. We find that Mis18α directly interacts with the N-terminus of Mis18BP1; whereas, Mis18β directly interacts with CENP-C during G1 phase, revealing that these proteins have evolved to serve distinct functions in centromeres of higher eukaryotes. The N-terminus of Mis18BP1, containing both the Mis18α and CENP-C binding domains, is necessary and sufficient for centromeric localization. Therefore, the Mis18 complex contains dual CENP-C recognition motifs that are combinatorially required to generate robust centromeric localization that leads to CENP-A deposition.

Tumor heterogeneity drives disease progression, treatment resistance, and patient relapse, yet remains largely underexplored in invasion and metastasis. Here, we investigated heterogeneity within collective cancer invasion by integrating DNA methylation and gene expression analysis in rare purified lung cancer leader and follower cells. Our results showed global DNA methylation rewiring in leader cells and revealed the filopodial motor MYO10 as a critical gene at the intersection of epigenetic heterogeneity and three-dimensional (3D) collective invasion. We further identified JAG1 signaling as a previously unknown upstream activator of MYO10 expression in leader cells. Using live-cell imaging, we found that MYO10 drives filopodial persistence necessary for micropatterning extracellular fibronectin into linear tracks at the edge of 3D collective invasion exclusively in leaders. Our data fit a model where epigenetic heterogeneity and JAG1 signaling jointly drive collective cancer invasion through MYO10 up-regulation in epigenetically permissive leader cells, which induces filopodia dynamics necessary for linearized fibronectin micropatterning.

Metastatic medullary thyroid cancer (MTC) is incurable and FDA-approved kinase inhibitors that include oncogenic RET as a target do not result in complete responses. Association studies of human MTCs and murine models suggest that the CDK/RB pathway may be an alternative target. The objective of this study was to determine if CDKs represent therapeutic targets for MTC and to define mechanisms of activity. Using human MTC cells that are either sensitive or resistant to vandetanib, we demonstrate that palbociclib (CDK4/6 inhibitor) is not cytotoxic to MTC cells but that they are highly sensitive to dinaciclib (CDK1/2/5/9 inhibitor) accompanied by reduced CDK9 and RET protein and mRNA levels. CDK9 protein was highly expressed in 83 of 83 human MTCs and array-comparative genomic hybridization had copy number gain in 11 of 30 tumors. RNA sequencing demonstrated that RNA polymerase II-dependent transcription was markedly reduced by dinaciclib. The CDK7 inhibitor THZ1 also demonstrated high potency and reduced RET and CDK9 levels. ChIP-sequencing using H3K27Ac antibody identified a superenhancer in intron 1 of RET. Finally, combined inhibition of dinaciclib with a RET kinase inhibitor was synergistic. In summary, we have identified what we believe is a novel mechanism of RET transcription regulation that potentially can be exploited to improve RET therapeutic targeting.

Increased p21 activated kinase (PAK) signaling and expression has been identified in the invasive fronts of aggressive papillary thyroid cancers (PTCs), including those with RET/PTC, BRAF V600E, and mutant RAS expression. Functionally, thyroid cancer cell motility in vitro is dependent on Group 1 PAKs particularly PAK1. In the present study, we hypothesize that BRAF, a central kinase in PTC tumorigenesis and invasion, regulates thyroid cancer cell motility in part through PAK activation. Using three well-characterized human thyroid cancer cell lines, we demonstrated in all cell lines that BRAF knockdown reduced PAK phosphorylation of direct downstream targets. In contrast, inhibition of MEK activity either pharmacologically or with siRNA did not reduce PAK activity indicating MEK is dispensable for PAK activity. Inhibition of cell migration through BRAF loss is rescued by overexpression of either constitutive active (CA) MEK1 or PAK1, demonstrating that both signaling pathways are involved in BRAF-regulated cell motility. To further characterize BRAF-PAK signaling, immunofluorescence and immunoprecipitation demonstrated that both exogenously overexpressed and endogenous PAK1 and BRAF co-localize and physically interact, and that this interaction was enhanced in mitosis. Finally, we demonstrated that acute induction of BRAFV600E expression in vivo in murine thyroid glands results in increased PAK expression and activity confirming a positive signaling relationship in vivo. In conclusion, we have identified a signaling pathway in thyroid cancer cells which BRAF activates and physically interacts with PAK and regulates cell motility.