Boundaries of eliminated heterochromatin of Tetrahymena are positioned by the DNA-binding protein Ltl1

Abstract

During differentiation of the Tetrahymena thermophila somatic nucleus, its germline-derived DNA undergoes extensive reorganization including the removal of ∼50 Mb from thousands of loci called internal eliminated sequences (IESs). IES-associated chromatin is methylated on lysines 9 and 27 of histone H3, marking newly formed heterochromatin for elimination. To ensure that this reorganized genome maintains essential coding and regulatory sequences, the boundaries of IESs must be accurately defined. In this study, we show that the developmentally expressed protein encoded by Lia3-Like 1 (LTL1) (Ttherm_00499370) is necessary to direct the excision boundaries of particular IESs. In ΔLTL1 cells, boundaries of eliminated loci are aberrant and heterogeneous. The IESs regulated by Ltl1 are distinct from those regulated by the guanine-quadruplex binding Lia3 protein. Ltl1 has a general affinity for double stranded DNA (Kd ∼ 350 nM) and binds specifically to a 50 bp A+T rich sequence flanking each side of the D IES (Kd ∼ 43 nM). Together these data reveal that Ltl1 and Lia3 control different subsets of IESs and that their mechanisms for flanking sequence recognition are distinct.

Programmed genome rearrangements in ciliates

“…In Tetrahymena, flanking sequences are known to have a significant role in the elimination of a number of IESs [70,71]. Together, Lia3p and Lia3-Like 1 (LTL1) regulatory proteins interact with flanking regulatory sequences to determine MDS-IES boundaries for several IESs for excision [72][73][74]. These data highlight the importance of both IES pointer sequences and their flanking regions in identifying MDS-IES boundaries.…”

Roles of Noncoding RNAs in Ciliate Genome Architecture

“…Meanwhile, a second boundary-defining protein, Ltl1, was found to be required for the excision of another subset of IESs. These Ltl1-dependent IESs also share an IR sequence at their boundaries that is distinct from the LIA3-dependent IESs' IR sequence [ 82 ]. Together, these studies suggest a model for IES boundary recognition that involves a number of boundary-defining proteins, each identifying a unique IR sequence flanking a subset of IESs.…”