DNA-binding specificity and in vivo targets of Caenorhabditis elegans nuclear factor I.

The conserved nuclear factor I (NFI) family of transcription factors is unique to animals and essential for mammalian development. The Caenorhabditis elegans genome encodes a single NFI family member, whereas vertebrate genomes encode 4 distinct NFI protein subtypes (A, B, C, and X). NFI-1-deficient worms exhibit abnormalities, including reduced lifespan, defects in movement and pharyngeal pumping, and delayed egg-laying. To explore the functional basis of these phenotypes, we sought to comprehensively identify NFI-1-bound loci in C. elegans. We first established NFI-1 DNA-binding specificity using an in vitro DNA-selection strategy. Analysis yielded a consensus motif of TTGGCA(N)(3)TGCCAA, which occurs 586 times in the genome, a 100-fold higher frequency than expected. We next asked which sites were occupied by NFI-1 in vivo by performing chromatin immunoprecipitation of NFI-1 followed by microarray hybridization. Only 55 genomic locations were identified, an unexpectedly small target set. In vivo NFI-1 binding sites tend to be upstream of genes involved in core cellular processes, such as chromatin remodeling, mRNA splicing, and translation. Remarkably, 59 out of 70 (84%) of the C. briggsae orthologs of the identified targets contain conserved NFI binding sites in their promoters. These experiments provide a foundation for understanding how NFI-1 is recruited to unexpectedly few in vivo sites to perform its developmental functions, despite a vast over-representation of its binding motif.

The genomic distribution and function of histone variant HTZ-1 during C. elegans embryogenesis.

In all eukaryotes, histone variants are incorporated into a subset of nucleosomes to create functionally specialized regions of chromatin. One such variant, H2A.Z, replaces histone H2A and is required for development and viability in all animals tested to date. However, the function of H2A.Z in development remains unclear. Here, we use ChIP-chip, genetic mutation, RNAi, and immunofluorescence microscopy to interrogate the function of H2A.Z (HTZ-1) during embryogenesis in Caenorhabditis elegans, a key model of metazoan development. We find that HTZ-1 is expressed in every cell of the developing embryo and is essential for normal development. The sites of HTZ-1 incorporation during embryogenesis reveal a genome wrought by developmental processes. HTZ-1 is incorporated upstream of 23% of C. elegans genes. While these genes tend to be required for development and occupied by RNA polymerase II, HTZ-1 incorporation does not specify a stereotypic transcription program. The data also provide evidence for unexpectedly widespread independent regulation of genes within operons during development; in 37% of operons, HTZ-1 is incorporated upstream of internally encoded genes. Fewer sites of HTZ-1 incorporation occur on the X chromosome relative to autosomes, which our data suggest is due to a paucity of developmentally important genes on X, rather than a direct function for HTZ-1 in dosage compensation. Our experiments indicate that HTZ-1 functions in establishing or maintaining an essential chromatin state at promoters regulated dynamically during C. elegans embryogenesis.

X chromosome repression by localization of the C. elegans dosage compensation machinery to sites of transcription initiation.

Among organisms with chromosome-based mechanisms of sex determination, failure to equalize expression of X-linked genes between the sexes is typically lethal. In C. elegans, XX hermaphrodites halve transcription from each X chromosome to match the output of XO males. Here, we mapped the binding location of the condensin homolog DPY-27 and the zinc finger protein SDC-3, two components of the C. elegans dosage compensation complex (DCC). We observed strong foci of DCC binding on X, surrounded by broader regions of localization. Binding foci, but not adjacent regions of localization, were distinguished by clusters of a 10-bp DNA motif, suggesting a recruitment-and-spreading mechanism for X recognition. The DCC was preferentially bound upstream of genes, suggesting modulation of transcriptional initiation and polymerase-coupled spreading. Stronger DCC binding upstream of genes with high transcriptional activity indicated a mechanism for tuning DCC activity at specific loci. These data aid in understanding how proteins involved in higher-order chromosome dynamics can regulate transcription at individual loci.