During development of the ciliated protozoan Tetrahymena thermophila, germline-derived DNA undergoes extensive reorganization including the removal of ~50Mbp in segments called internal eliminated sequences (IES’s) from the differentiating somatic genome. The eliminated sequences are targeted for excision by small-RNA-directed heterochromatin formation. To ensure that the reorganized, gene-dense genome maintains all coding and regulatory sequences, the boundaries of the eliminated DNAs must be accurately defined. We have identified developmentally expressed proteins that define the boundaries of specific subsets of IESs. First, we discovered that the guanine-quadruplex binding protein, Lia3, specifically regulates the rearrangement of loci that are flanked by G-rich (5’-AAAAAGGGGG-3’) boundary controlling sequences. Second, we characterized the Lia3-like protein 1 (Ltl1) and found that the IES’s regulated by Ltl1, which include the D IES, are distinct from those regulated by the Lia3. Initial binding assays show that Ltl1 has a high affinity for double-stranded DNA, but does not bind single-stranded DNA. The IESs regulated by these two proteins comprise only a fraction of the thousands of eliminated loci. These results suggest that multiple proteins are required to accurately control the specification of heterochromatin domains that are targeted for removal from the somatic genome.