Independent and Complementary Functions of Caf1b and Hir1 for Chromatin Assembly in <i>Tetrahymena thermophila</i>

Histones and DNA associate to form the nucleosomes of eukaryotic chromatin. Chromatin assembly factor 1 (CAF-1) complex and histone regulatory protein A (HIRA) complex mediate replication-couple (RC) and replication-independent (RI) nucleosome assembly, respectively. CHAF1B and HIRA share a similar domain but play different roles in nucleosome assembly by binding to the different interactors. At present, there is limited understanding for the similarities and differences in their respective functions. <i>Tetrahymena thermophila</i> contains transcriptionally active polyploid macronuclei (MAC) and transcriptionally silent diploid micronuclei (MIC). Here, the distribution patterns of Caf1b and Hir1 exhibited both similarities and distinctions. Both proteins localized to the MAC and MIC during growth, and to the MIC during conjugation. However, Hir1 exhibited additional signaling on parental MAC and new MAC during sexual reproduction and displayed a punctate signal on developing anlagen. Caf1b and Hir1 only co-localized in the MIC with Pcna1 during conjugation. Knockdown of <i>CAF1B</i> impeded cellular growth and arrested sexual reproductive development. Loss of <i>HIR1</i> led to MIC chromosome defects and aborted sexual development. Co-interference of <i>CAF1B</i> and <i>HIR1</i> led to a more severe phenotype. Moreover, <i>CAF1B</i> knockdown led to the up-regulation of <i>HIR1</i> expression, while knockdown of <i>HIR1</i> also led to an increase in <i>CAF1B</i> expression. Furthermore, Caf1b and Hir1 interacted with different interactors. These results showed that CAF-1 and Hir1 have independent and complementary functions for chromatin assembly in <i>T. thermophila</i>.