The fibronectin type-III (FNIII) domain of ATF7IP contributes to efficient transcriptional silencing mediated by the SETDB1 complex

Abstract

Background The histone methyltransferase SETDB1 (also known as ESET) represses genes and various types of transposable elements, such as endogenous retroviruses (ERVs) and integrated exogenous retroviruses, through a deposition of trimethylation on lysine 9 of histone H3 (H3K9me3) in mouse embryonic stem cells (mESCs). ATF7IP (also known as MCAF1 or AM), a binding partner of SETDB1, regulates the nuclear localization and enzymatic activities of SETDB1 and plays a crucial role in SETDB1-mediated transcriptional silencing. In this study, we further dissected the ATF7IP function with its truncated mutants in Atf7ip knockout (KO) mESCs. Results We demonstrated that the SETDB1-interaction region within ATF7IP is essential for ATF7IP-dependent SETDB1 nuclear localization and silencing of both ERVs and integrated retroviral transgenes, whereas its C-terminal fibronectin type-III (FNIII) domain is dispensable for both these functions; rather, it has a role in efficient silencing mediated by the SETDB1 complex. Proteomic analysis identified a number of FNIII domain-interacting proteins, some of which have a consensus binding motif. We showed that one of the FNIII domain-binding proteins, ZMYM2, was involved in the efficient silencing of a transgene by ATF7IP. RNA-seq analysis of Atf7ip KO and WT or the FNIII domain mutant of ATF7IP-rescued Atf7ip KO mESCs showed that the FNIII domain mutant re-silenced most de-repressed SETDB1/ATF7IP-targeted ERVs compared to the WT. However, the silencing activity of the FNIII domain mutant was weaker than that of the ATF7IP WT, and some of the de-repressed germ cell-related genes in Atf7ip KO mESCs were not silenced by the FNIII domain mutant. Such germ cell-related genes are targeted and silenced by the MAX/MGA complex, and MGA was also identified as another potential binding molecule of the ATF7IP FNIII domain in the proteomic analysis. This suggests that the FNIII domain of ATF7IP acts as a binding hub of ATF7IP-interacting molecules possessing a specific interacting motif we named FAM and contributes to one layer of the SETDB1/ATF7IP complex-mediated silencing mechanisms. Conclusions Our findings contributed to further understanding the function of ATF7IP in the SETDB1 complex, revealed the role of the FNIII domain of ATF7IP in transcriptional silencing, and suggested a potential underlying molecular mechanism for it.

Repression of germline genes by PRC1.6 and SETDB1 in the early embryo precedes DNA methylation-mediated silencing

“…Consistent with a direct role for MGA/MAX-mediated SETDB1 recruitment, MAX was recently shown to interact with SETDB1 via co-immunoprecipitation, and Ddx4 and Dazl promoters showed reduced H3K9me3 in Max KD pESCs 29 . However, a recent study suggests that SETDB1 may indirectly interact with PRC1.6, via its known binding partner ATF7IP 93 . ATF7IP contains a C-terminal fibronectin type-III (FNIII) domain, which was shown to interact with MGA in pESCs via mass spectrometry.…”

The Updating of Biological Functions of Methyltransferase SETDB1 and Its Relevance in Lung Cancer and Mesothelioma

“…Recently, the location of SETDB1 inside the nucleus was discovered to be mediated by ATF7IP, which increases the ubiquitination of SETDB1 to promote its enzymatic activity [ 84 , 85 , 86 ]. The fibronectin type-III (FNIII) domain of ATF7IP plays a certain role in the transcriptional repression function mediated by the SETDB1-ATF7IP complex.…”

“…The fibronectin type-III (FNIII) domain of ATF7IP plays a certain role in the transcriptional repression function mediated by the SETDB1-ATF7IP complex. However, the FNIII domain seems to be unrelated to the nuclear localization of SETDB1 and to the ATF7IP-dependent integrated retroviral transgenes silencing [ 86 ]. Above all, by alternating the conformation of the X chromosome through SETDB1-ATF7IP-MBD1 complex, SETDB1 also plays an important role in XCI.…”

Somatic Genetic Mosaicism in the Apolipoprotein E-null Mouse Aorta

“…One of the partners of ATF7IP is the histone methyltransferase SETDB1, a repressor of proinflammatory cytokine expression in macrophages. 43,44 Phospholipase B domain containing 1 (PLBD1) was first identified as 11…”

“…One of the partners of ATF7IP is the histone methyltransferase SETDB1, a repressor of proinflammatory cytokine expression in macrophages. 43,44 Phospholipase B domain containing 1 (PLBD1) was first identified as a neutrophil phospholipase B precursor potentially involved in proinflammatory lipid release. 45 Accordingly, PLBD1 expression is increased in a variety of inflammatory conditions.…”