Newly Characterized Region of CP190 Associates with Microtubules and Mediates Proper Spindle Morphology in Drosophila Stem Cells

Abstract

CP190 is a large, multi-domain protein, first identified as a centrosome protein with oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a striking redistribution of CP190 to centrosomes and the mitotic spindle, in addition to the population at chromosomes. Here, we investigate CP190 in detail by performing domain analysis in cultured Drosophila S2 cells combined with protein structure determination by X-ray crystallography, in vitro biochemical characterization, and in vivo fixed and live imaging of cp190 mutant flies. Our analysis of CP190 identifies a novel N-terminal centrosome and microtubule (MT) targeting region, sufficient for spindle localization. This region consists of a highly conserved BTB domain and a linker region that serves as the MT binding domain. We present the 2.5 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The ability of the linker region to robustly localize to MTs requires BTB domain-mediated dimerization. Deletion of the linker region using CRISPR significantly alters spindle morphology and leads to DNA segregation errors in the developing Drosophila brain neuroblasts. Collectively, we highlight a multivalent MT-binding architecture in CP190, which confers distinct subcellular cytoskeletal localization and function during mitosis.

Mechanisms of Enhancer-Promoter Interactions in Higher Eukaryotes

“…The C2H2 proteins, including Pita, dCTCF, and Su(Hw), interact with BTB-containing proteins (bric-a-brac, tramtrack, and broad complex), such as Mod(mdg4) and CP190 [ 150 , 166 , 167 , 168 ]. The CP190 protein contains a classical N-terminal BTB domain that forms homodimers and is conserved among higher eukaryotes [ 169 , 170 , 171 ]. In contrast, the BTB domain of Mod(mdg4) belongs to an insect-specific group [ 172 , 173 ].…”

Opbp is a new architectural/insulator protein required for ribosomal gene expression

“…CP190 (a 1096 amino acid protein) contains an N-terminal BTB/POZ domain, an aspartic-acid-rich D-region, four C2H2 zinc finger motifs and a C-terminal E-rich domain ( 29 , 34 ). The BTB domain of CP190 forms stable homodimers that may be involved in protein–protein interactions ( 29 , 35 , 36 ). In addition to these motifs, CP190 also contains a centrosomal targeting domain (M) responsible for its localization to centrosomes during mitosis ( 37 ).…”

The Centrioles,Centrosomes, Basal Bodies, and Cilia ofDrosophila melanogaster

“…Curiously, one of the first centrosomal antigens to be followed in this way, CP190, first identified through a monoclonal antibody, Bx63 ( Frasch et al 1986 ), may not have its major functions at the centrosome. The Bx63 antigen was later purified as a MT-associated protein (MAP) ( Kellogg and Alberts 1992 ) and indeed does have a MT-binding domain and some function in spindle assembly ( Plevock et al 2015 ). However, its major role is as a chromatin insulator that is critical to direct the formation of chromosomal loops to organize transcription units in the interphase nucleus [reviewed by Ahanger et al (2013) ].…”