Bilateral asymmetry in C. elegans can arise from left-right asymmetric cell lineages. The single left-right unpaired MI neuron descends from the right side of an otherwise left-right symmetric cell lineage that on the left generates the
e3D epithelial cell.
We sought mutations that transform MI into an
e3D-like cell or
e3D into an MI-like cell, thereby generating symmetry in these cell lineages. We recovered 16 mutations in 7 genes that transform MI into an
e3D-like cell. We previously showed that the establishment of the MI-
e3D asymmetry requires asymmetric expression of a transcriptional cascade in which the Otx homeodomain protein CEH-36 is expressed in the MI grandmother cell but not in the
e3D grandmother cell and that CEH-36 promotes asymmetric expression of two bHLH proteins, NGN-1 and HLH-2, in the MI mother cell but not in the
e3D mother cell (Nakano et al., Development 137, 4017, 2010). Here we show that another isolate,
n5357, is a gain-of-function allele of the gene
his-9, which encodes a replication-dependent histone H3 protein. This
his-9(gf) mutation acts cell-autonomously in the MI mother cell and affects a process downstream of or in parallel to the expression of CEH-36, NGN-1 and HLH-2.
Replication-dependent histone H3-H4 proteins are deposited onto nucleosomes by the CAF-1 complex during DNA replication. The human CAF-1 complex is composed of three subunits,
p150,
p60 and
p48. Inactivation of T06D10.2, Y71G12B.1 and
rba-1, which encode proteins homologous to
p150,
p60 and
p48, respectively, transformed MI into an
e3D-like cell. That a gain-of-function mutation in a histone H3 gene phenocopied the loss of CAF-1 function suggests that the mutant H3 protein inhibits CAF-1-mediated nucleosome formation. We performed a nucleosome assembly reaction to monitor CAF-1 activity in vitro and observed that corresponding mutant histone H3 proteins indeed inhibited CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is required to generate the MI-
e3D asymmetry. We suggest that during S phase of the MI mother cell CAF-1 assembles nucleosomes on which the NGN-1/HLH-2 complex recruits histone-modifying enzymes to generate a chromatin state necessary to specify MI and that CAF-1-mediated nucleosome formation and the asymmetric localization of the NGN-1/HLH-2 complex drive left-right asymmetric epigenetic regulation, leading to the establishment of the MI-
e3D asymmetry.