Functional Dissection of C. elegans bZip-Protein CEBP-1 Reveals Novel Structural Motifs Required for Axon Regeneration and Nuclear Import

Abstract

The basic leucine-zipper (bZIP) domain transcription factors CCAAT/enhancer-binding proteins (C/EBP) have a variety of roles in cell proliferation, differentiation, and stress response. In the nervous system, several isoforms of C/EBP function in learning and memory, neuronal plasticity, neuroinflammation, and axon regeneration. We previously reported that the Caenorhabditis elegans C/EBP homolog, CEBP-1, is essential for axon regeneration. CEBP-1 consists of 319 amino acids, with its bZIP domain at the C-terminus and a long N-terminal fragment with no known protein motifs. Here, using forward genetic screening with targeted genome editing, we have identified a unique domain in the N-terminus that is critical for its in vivo function. Additionally, we characterized three nuclear localization signals (NLS) in CEBP-1 that act together to mediate CEBP-1’s nuclear import. Moreover, the Importin-α, IMA-3, can bind to CEBP-1 via one of the NLS. ima-3 is ubiquitously expressed in all somatic cells, and ima-3 null mutants are larval lethal. Using Cre-lox dependent neuron-specific deletion strategy, we show that ima-3 is not critical for axon development, but is required for axon regeneration in adults. Together, these data advance our understanding of CEBP-1’s function, and suggest new regulators that remain to be identified to expand the CEBP-1 protein interactome.

Forward genetic screening identifies novel roles for N-terminal acetyltransferase C and histone deacetylase in C. elegans development

“…S1C ). This allowed for the identification of a novel functional domain within CEBP-1 14 . Interestingly, despite screening more animals and expanding the mutagenized genome, our screen with PEEL-induced selection found no new genes (Supplementary Figs.…”

“…In the second suppressor screen, we designed a PEEL-induced selection scheme (Supplementary Fig. S1B ) 14 . Briefly, we mutagenized L4 animals of CZ24853: nipi-3(ju1293); juEx7152[nipi-3( + ); Phsp::peel-1; Pmyo-2::GFP] using ethyl methane sulphonate (EMS, three rounds of mutagenesis with concentration between 25 and 40 mM) following a standard protocol.…”

Femtosecond laser microdissection for isolation of regenerating C. elegans neurons for single-cell RNA sequencing

“…Upon axon injury, CEBP-1 and ETS-4 function downstream to DLK-1/p38 and cAMP pathway, respectively, to form a complex which induces expression of svh-2 gene 29 . Stimulation of SVH-2 receptor tyrosine kinase promotes axon regeneration through activation of the JNK pathway 31,57 . Our data indicated an up-regulation of svh-2 gene in both wild-type and dlk-1 (0) animals following axotomy, although the change was not statistically significant (data not shown).…”

“…The transparent body of C. elegans further allows analysis of such phenotypes, including axon regrowth rate, guidance, and fusion at a single neuron resolution in vivo with fluorescence microscopy. Past nerve regeneration studies in C. elegans have led to the discovery of many conserved nerve regeneration pathways [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] , notably the dual leucine zipper kinase (DLK-1/p38) signaling cascade, whose role in neural development, regeneration, and degeneration has been confirmed in numerous vertebrate and invertebrate species 7,[27][28][29][30][31] .Nerve regeneration research in C. elegans has relied on costly and time-consuming mutant screens that test individual genes for function in axon regeneration (Supplementary Note 1). The rapidly evolving high-throughput sequencing methods offer an opportunity to accelerate progress.…”

Femtosecond laser microdissection isolating regeneratingC. elegansneurons for single cell RNA sequencing

“…The transparent body of C. elegans further allows analysis of such phenotypes, including axon regrowth rate, guidance, and fusion at a single neuron resolution in vivo with fluorescence microscopy. Past nerve regeneration studies in C. elegans have led to the discovery of many conserved nerve regeneration pathways [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26] , notably the dual leucine zipper kinase (DLK-1/p38) signaling cascade, whose role in neural development, regeneration, and degeneration has been confirmed in numerous vertebrate and invertebrate species 7,[27][28][29][30][31] .…”