LIA4 Encodes a Chromoshadow Domain Protein Required for Genomewide DNA Rearrangements in Tetrahymena thermophila

Abstract

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 (⌬LIA4) 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.

Phosphorylation of an HP1-like protein is a prerequisite for heterochromatin body formation in Tetrahymena DNA elimination

“…These defects are most probably a consequence of the block of DNA elimination because various DNA elimination defective mutants show similar phenotypes (30)(31)(32). Consistent with the previous observations that DNA elimination is required for viable progeny production (26,27,31), none of the pairs from JUB4 KO strains produced viable progeny, whereas ∼80% of the pairs isolated from WT cells produced viable progeny (Fig. S2E).…”

“…Alternatively, because phosphorylation and/or RNA binding of HP1 modulate its affinity to chromatin in some eukaryotes (13)(14)(15)37), Pdd1p phosphorylation may also modulate the physical dynamics of Pdd1p, which may be required for other heterochromatin proteins to be properly arranged on chromatin for heterochromatin body formation. Several proteins are known to be required for the formation of heterochromatin bodies in Tetrahymena (26,27,32), and the phosphorylation of Pdd1p may be essential for the chromatin localization of such proteins.…”

DRH1, a p68-related RNA helicase gene, is required for chromosome breakage in Tetrahymena

“…The two micro- and two macronuclei developmental arrest point, which we observed upon mating Δ DRH1 mic strains, is a common phenotype described for several strains lacking genes required for developmentally programmed DNA rearrangements (Shieh and Chalker, 2013; Rexer and Chalker, 2007; Motl and Chalker, 2011; Malone et al, 2005; Horrell and Chalker, 2014; Noto et al, 2010; Mochizuki et al, 2002; Bednenko et al, 2009). To determine whether the developmental arrest observed upon loss of DRH1 expression was associated with defects in genome rearrangements, we assessed IES excision and chromosome breakage efficiency in Δ DRH1 mic strains post-conjugation.…”

Heterochromatin aggregation during DNA elimination in Tetrahymena is facilitated by a prion-like protein

“…For this purpose, we chose six heterochromatin body components: Jub1p, Lia1p, Lia4p, Lia5p, Pdd1p and Pdd2p, all of which are exclusively expressed during conjugation and are required for the formation of heterochromatin bodies (Coyne et al, 1999;Horrell and Chalker, 2014;Kataoka and Mochizuki, 2015;Nikiforov et al, 1999;Rexer and Chalker, 2007;Shieh and Chalker, 2013). We expressed these proteins using the MTT1 promoter in vegetative cells as HA-tagged proteins and analyzed their localization (Fig.…”

“…The first group consists of proteins required for the formation of heterochromatin at IESs, including the RNAi machinery components Dcl1p, Twi1p, Giw1p and Ema1p, the histone methyltransferase Ezl1p and the HP1-like protein Pdd1p (Aronica et al, 2008;Coyne et al, 1999;Liu et al, 2004Liu et al, , 2007Malone et al, 2005;Mochizuki and Gorovsky, 2005;Noto et al, 2010). The second group includes proteins that are dispensable for heterochromatin formation but are required for its assembly into heterochromatin bodies: Jub1p, Lia1p, Lia4p, Lia5p, Pdd2p, Tku80p and Tpb2p Rexer and Chalker, 2007;Horrell and Chalker, 2014;Shieh and Chalker, 2013;Liu et al, 2007;Cheng et al, 2010;Lin et al, 2012;Vogt and Mochizuki, 2013;Kataoka and Mochizuki, 2015). Although some of the mechanisms responsible for the former RNAi-directed heterochromatin formation process have been elucidated at the biochemical level, the latter heterochromatin body formation process remains elusive.…”