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Proteins that possess a chromo domain are well‐known for their roles in heterochromatin assembly and maintenance. The Heterochromatin Protein 1 (HP1) family, with a chromo domain and carboxy‐terminal chromo shadow domain, targets heterochromatin through interaction with histone H3 methylated on lysine 9 (H3K9me2/3). The structural and functional diversity of these proteins observed in both fission yeast and metazoans correlate with chromatin specialization. To expand these studies, we examined chromo domain proteins in the ciliate Tetrahymena thermophila, which has functionally diverse and developmentally regulated heterochromatin domains. We identified thirteen proteins similar to HP1. Together they possess only a fraction of the possible chromo domain subtypes and most lack a recognizable chromo shadow domain. Using fluorescence microscopy to track chromatin localization of tagged proteins through the life cycle, we show evidence that in T. thermophila this family has diversified with biological roles in RNAi‐directed DNA elimination, germline genome structure, and somatic heterochromatin. Those proteins with H3K27me3 binding sequence characteristics localize to chromatin in mature nuclei, whereas those with H3K9me2/3 binding characteristics localize to developing nuclei undergoing DNA elimination. Findings point to an expanded and diversified family of chromo domain proteins that parallels heterochromatin diversity in ciliates.

Extensive DNA elimination occurs as part of macronuclear differentiation during Tetrahymena sexual reproduction. The identification of sequences to excise is guided by a specialized RNA interference (RNAi) machinery that targets the methylation of histone H3 lysine 9 (K9) and K27 on chromatin associated with these internal eliminated sequences (IESs). This modified chromatin is reorganized into heterochromatic subnuclear foci, which is a hallmark of their subsequent elimination. Here, we demonstrate that Lia4, a chromoshadow domain-containing protein, is an essential component in this DNA elimination pathway. LIA4 knockout ( ΔLIA4 ) lines fail to excise IESs from their developing somatic genome and arrest at a late stage of conjugation. Lia4 acts after RNAi-guided heterochromatin formation, as both H3K9 and H3K27 methylation are established. Nevertheless, without LIA4 , these cells fail to form the heterochromatic foci associated with DNA rearrangement, and Lia4 accumulates in the foci, indicating that Lia4 plays a key role in their structure. These data indicate a critical role for Lia4 in organizing the nucleus during Tetrahymena macronuclear differentiation.

There is a need for improved methods to analyze transcriptional control of mammalian stem cell genes. We propose that embryonic stem cells (ESCs) will have broad utility as a model system, because they can be manipulated genetically and then differentiated into many cell types in vitro, avoiding the need to make mice. Results are presented demonstrating the utility of ESCs for analyzing cis-acting sequences using Olig2 as a model gene. Olig2 is a transcription factor that plays a key role in the development of a ventral compartment of the nervous system and the oligodendrocyte lineage. The functional role of an upstream region (USR) of the Olig2 gene was investigated in ESCs engineered at the undifferentiated stage and then differentiated into ventral neural cells with sonic hedgehog and retinoic acid. Deletion of the USR from the native gene via gene targeting eliminates expression in ventral neural cells differentiated in cell culture. The USR is also essential for regulated expression of an Olig2 transgene inserted at a defined foreign chromosomal site. A subregion of the USR has nonspecific promoter activity in transient transfection assays in cells that do not express Olig2. Taken together, the data demonstrate that the USR contains a promoter for the Olig2 gene and suggest that repression contributes to specific expression. The technology used in this study can be applied to a wide range of genes and cell types and will facilitate research on cis-acting DNA elements of mammalian genes. Disclosure of potential conflicts of interest is found at the end of this article.

Summary The somatic nucleus of Oxytricha trifallax contains over 15,000 different chromosomes, most containing a single gene. Analysis of this 50 Mb genome uncovers novel regulatory strategies and adaptive potential when gene copy number and allelic frequency are no longer constrained by genetic linkage.