Maintenance of neuronal laterality in Caenorhabditis elegans through MYST histone acetyltransferase complex components LSY-12, LSY-13 and LIN-49

Abstract

Left/right asymmetrically expressed genes permit an animal to perform distinct tasks with the right vs. left side of its brain. Once established during development, lateralized gene expression patterns need to be maintained during the life of the animal. We show here that a histone modifying complex, composed of the LSY-12 MYST-type histone acetyltransferase, the ING-family PHD domain protein LSY-13, and PHD/bromodomain protein LIN-49, is required to first initiate and then actively maintain lateralized gene expression in the gustatory system of the nematode Caenorhabditis elegans. Similar defects are observed upon postembryonic removal of two C2H2 zinc finger transcription factors, die-1 and che-1, demonstrating that a combination of transcription factors, which recognize DNA in a sequence-specific manner, and a histone modifying enzyme complex are responsible for inducing and maintaining neuronal laterality.

Figure 1.—

lsy-12, a MYST-type histone acetyltransferase, affects ASE laterality. (A) Genes known to be involved in controlling ASEL/R laterality (Hobert 2006; Didiano et al. 2010). (B) Schematic representation of phenotype of representative “2 ASEL” and “2 ASER” mutants. (C) Effects of lsy-12 on ASEL/R laterality markers. A subset of the defects were already reported, upon the initial identification of the lsy-12 locus (Sarin et al. 2007). Numbers below the panels indicate the penetrance of the phenotype, i.e., the fraction of animals that display the phenotype shown in the fluorescent image above. Data on other lsy-12 alleles were reported in Sarin et al. (2007, 2008, 2010). Animals that express the die-1 reporter fosmid also contain a ASEL/R-expressed red fluorescent reporter (che-1∷mCherry) for cell identification. A list of transgenes used in the study is provided in the File S1. Animals were scored as adults. (D) lsy-12 encodes a MYST-type histone acetyltransferase. Analysis of ESTs, expression tiling array clones, PCR specific rescue, and RT–PCR (see File S1 for more details) revealed that R07B5.8 and R07B5.9 are one genetic locus encoding at least two major splice isoforms. The 3′, polyadenylated end of yk82d06 and EX1785569 provide evidence for the existence of the lsy-12b, while other clones provide evidence of the lsy-12a isoform. RT–PCR data suggest that additional splice variants may be produced by the lsy-12 locus (some possibly even including the more upstream predicted gene T11A5.1), but we have not been able to conclusively identify the start and end of such alternative transcripts (data not shown; see also www.wormbase.org). The arrow indicates the predicted translational start site. lsy-12 expression constructs are shown in the lower part of the panel, with the bottom one being a negative control. Staggered red lines indicate that these constructs were generated by in vivo recombineering of co-injected, overlapping PCR fragments (Boulin et al. 2006), some of which were generated by an in vitro PCR fusion approach (Hobert 2002). The generation of constructs is described in File S1. Rescuing data is quantified in Table S1.

Figure 2.—

lsy-12 is continually required in ASE but may also act early. (A) lsy-12 is required for the initial manifestation of asymmetry, as assessed by gcy-5∷gfp expression (otIs220 transgene). In wild-type animals, gcy-5 expression is first observed exclusively in ASER in threefold-stage embryos; in lsy-12(ot563) animals, expression of gcy-5∷gfp is bilateral from the onset. (B) Temperature-shift experiments indicate a sustained requirement for lsy-12 activity. lsy-12(ot563) animals that had been grown for several generations at either 15° or 25° were plated and temperature shifted up to 25° or down to 15° at the following timepoints: Embryo, 2-cell; pre-comma; two- to threefold; threefold (those embryos were collected from dissected adult) and postembryonic, L1; L2; L3; L4; and 2-day-old adult. All animals were then scored as 3-day-old adults.

Figure 3.—

The MYST-complex component lsy-13 affects ASE laterality. (A) lsy-13 controls ASE laterality. The upper panel shows the structure of the lsy-13 locus and the lsy-13 null allele. The gene structure is confirmed by EST clones (www.wormbase.org). All other panels show the head regions of adult animals. Numbers below the panels indicate the penetrance of the phenotype, i.e. the fraction of animals that display the phenotype shown in the fluorescent image above. The die-1 and lsy-6 wild-type control images are the same as in other figures and shown for comparison only. The red fluorescent marker in the lsy-6 panel (ceh-36^prom∷dsRed2) allows identification of the ASE neurons. Animals that express the die-1 reporter fosmid also contain a ASEL/R-expressed red fluorescent reporter (che-1∷mCherry). A list of transgenes used in the study is provided in File S1. We note that lsy-13 function can be maternally supplied (homozygous offspring of a heterozygous lsy-13 parent does not display a mutant phenotype). In contrast to the lin-49 null mutant animals, lsy-13(ok1475) null mutant animals are viable and display no obvious morphological abnormalities. (B) A reporter gene which contains 2.8 kb of 5′ sequences to the first exon of lsy-13, generated by PCR fusion (Hobert 2002), is broadly expressed, including in the two ASE neurons, marked with a red fluorescent reporter gene. Primer sequences for the construct are provided in File S1. (C) Model for lsy-12/lsy-13/lin-49 function, based on the phenotypic similarities between the genes shown here. che-1 directly regulates expression of terminal differentiation genes—both symmetrically and asymmetrically expressed ones—as well as regulators of the bistable feedback loop (Etchberger et al. 2007, 2009). Left/right asymmetrically expressed genes, contain cis-regulatory elements (indicated by “?”) in addition to the CHE-1 binding site (the ASE motif) that restrict CHE-1 activity to ASEL or ASER (Etchberger et al. 2009). Genetically, die-1 controls the activity of the factors that restrict che-1 activity, but it is not known whether die-1 fulfills this function directly (through binding to these additional motifs) or indirectly through the regulation of other factors. Since die-1 autoregulates its own transcription (L. Cochella and O. Hobert, unpublished data), the HAT complex also impinges on die-1 expression itself.