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Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.

ABSTRACT: A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear. Here, we uncover DNA mimicry by the ciliary kinesin KIF7 as a mechanism for the recruitment of GLI to microtubules, wherein the coiled-coil dimerization domain of KIF7, characterized by its striking shape, size and charge similarity to DNA, forms a complex with the DNA-binding zinc fingers in GLI, thus revealing a mode of tethering a DNA-binding protein to the cytoskeleton. GLI increases KIF7 microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive GLI tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We retooled this coiled-coil-based GLI-KIF7 interaction to inhibit the nuclear and cilium localization of GLI. This strategy can potentially be exploited to downregulate erroneously activated GLI in human cancers.

SUBMITTER: Haque F

PROVIDER: S-EPMC10016618 | biostudies-literature | 2022 Jul

REPOSITORIES: biostudies-literature

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Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.

Haque Farah F Freniere Christian C Ye Qiong Q Mani Nandini N Wilson-Kubalek Elizabeth M EM Ku Pei-I PI Milligan Ronald A RA Subramanian Radhika R

Nature cell biology 20220620 7

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. By contrast, the principal effectors of Hedgehog signalling, the GLI transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How GLI is tethered to microtubules remains unclear. Here, we uncover DNA mimicry by the ciliary kinesin KIF7 as a mechanism for the recruitment of GLI to microtubules, wherein the coiled-coil dimerization domain of ...[more]

PMID: 35725768

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Project description:Deficits in DNA damage-repair pathways are the root cause of several human cancers. In mammalian cells, DNA double-strand break repair is carried out by multiple mechanisms, including homologous recombination (HR). The partner and localizer of BRCA2 (PALB2), which is an essential factor for HR, binds to the breast cancer susceptibility 1 (BRCA1) protein at DNA double-strand breaks. At the break site, PALB2 also associates with the breast cancer susceptibility 2 (BRCA2) protein to form a multiprotein complex that facilitates HR. The BRCA1-PALB2 interaction is mediated by association of predicted helical coiled-coil regions in both proteins. PALB2 can also homodimerize through the formation of a coiled coil by the self-association of helical elements at the N-terminus of the PALB2 protein, and this homodimerization has been proposed to regulate the efficiency of HR. We have produced a segment of PALB2, designated PALB2cc (PALB2 coiled coil segment) that forms ?-helical structures, which assemble into stable homodimers. PALB2cc also forms heterodimers with a helical segment of BRCA1, called BRCA1cc (BRCA1 coiled coil segment). The three-dimensional structure of the homodimer formed by PALB2cc was determined by solution NMR spectroscopy. This PALB2cc homodimer is a classical antiparallel coiled-coil leucine zipper. NMR chemical-shift perturbation studies were used to study dimer formation for both the PALB2cc homodimer and the PALB2cc/BRCA1cc heterodimer. The mutation of residue Leu24 of PALB2cc significantly reduces its homodimer stability, but has a more modest effect on the stability of the heterodimer formed between PALB2cc and BRCA1cc. We show that mutation of Leu24 leads to genomic instability and reduced cell viability after treatment with agents that induce DNA double-strand breaks. These studies may allow the identification of distinct mutations of PALB2cc that selectively disrupt homodimeric versus heterodimeric interactions, and reveal the specific role of PALB2cc homodimerization in HR.

Project description:The proteins SFPQ (splicing Factor Proline/Glutamine rich) and NONO (non-POU domain-containing octamer-binding protein) are mammalian members of the Drosophila Behaviour/Human Splicing (DBHS) protein family, which share 76% sequence identity in their conserved 320 amino acid DBHS domain. SFPQ and NONO are involved in all steps of post-transcriptional regulation and are primarily located in mammalian paraspeckles: liquid phase-separated, ribonucleoprotein sub-nuclear bodies templated by NEAT1 long non-coding RNA. A combination of structured and low-complexity regions provide polyvalent interaction interfaces that facilitate homo- and heterodimerisation, polymerisation, interactions with oligonucleotides, mRNA, long non-coding RNA, and liquid phase-separation, all of which have been implicated in cellular homeostasis and neurological diseases including neuroblastoma. The strength and competition of these interaction modes define the ability of DBHS proteins to dissociate from paraspeckles to fulfil functional roles throughout the nucleus or the cytoplasm. In this study, we define and dissect the coiled-coil interactions which promote the polymerisation of DBHS proteins, using a crystal structure of an SFPQ/NONO heterodimer which reveals a flexible coiled-coil interaction interface which differs from previous studies. We support this through extensive solution small-angle X-ray scattering experiments using a panel of SFPQ/NONO heterodimer variants which are capable of tetramerisation to varying extents. The QM mutant displayed a negligible amount of tetramerisation (quadruple loss of function coiled-coil mutant L535A/L539A/L546A/M549A), the Charged Single Alpha Helix (ΔCSAH) variant displayed a dimer-tetramer equilibrium interaction, and the disulfide-forming variant displayed constitutive tetramerisation (R542C which mimics the pathological Drosophila nonAdiss allele). We demonstrate that newly characterised coiled-coil interfaces play a role in the polymerisation of DBHS proteins in addition to the previously described canonical coiled-coil interface. The detail of these interactions provides insight into a process critical for the assembly of paraspeckles as well as the behaviour of SFPQ as a transcription factor, and general multipurpose auxiliary protein with functions essential to mammalian life. Our understanding of the coiled coil behaviour of SFPQ also enhances the explanatory power of mutations (often disease-associated) observed in the DBHS family, potentially allowing for the development of future medical options such as targeted gene therapy.

Dataset Information

Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.

Publications

Cytoskeletal regulation of a transcription factor by DNA mimicry via coiled-coil interactions.

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