Sumoylation of the Transcriptional Intermediary Factor 1β (TIF1β), the Co-repressor of the KRAB Multifinger Proteins, Is Required for Its Transcriptional Activity and Is Modulated by the KRAB Domain

Abstract

Small ubiquitin-related modifier (SUMO) has emerged as a key post-translational modulator of protein functions. Here we show that TIF1␤, a developmental regulator proposed to act as a universal co-repressor for the large family of KRAB domaincontaining zinc finger proteins, is a heavily SUMO-modified substrate. A combined analysis of deletion and punctual mutants identified TIF1␤ as a multilysine acceptor for SUMO which specifically targets six lysine residues (Lys 554 , Lys 575 , Lys 676 , Lys 750 , Lys 779 , and Lys 804 ) within the TIF1␤ C-terminal repressive region. Reporter gene assays indicate that TIF1␤ requires SUMO-modification for its repressive activity. Indeed, sumoylation-less mutants failed to recapitulate TIF1␤-dependent repression. TIF1␤ homodimerization properties and interaction with the KRAB domain are preserved in the mutants with lysine to arginine substitutions as confirmed by in vivo bioluminescence resonance energy transfer (BRET). Using histone deacetylase (HDAC) inhibitors, we also demonstrate that TIF1␤ sumoylation is a prerequisite for the recruitment of HDAC and that TIF1␤ SUMO-dependent repressive activity involves both HDAC-dependent and HDAC-independent components. Finally, we report that, in addition to relying on the integrity of its PHD finger and on its self-oligomerization, TIF1␤ sumoylation is positively regulated by its interaction with KRAB domain-containing proteins. Altogether, our results provide new mechanistic insights into TIF1␤ transcriptional repression and suggest that KRAB multifinger proteins not only recruit TIF1␤ co-repressor to target genes but also increase its repressive activity through enhancement of its sumoylation.The transcriptional intermediary factor 1␤ (TIF1␤, 2 KRIP-1, KAP-1, TRIM 28) is an essential developmental regulator belonging to the TIF1 family and is believed to act as the universal transcriptional co-repressor for the large family of vertebrate-specific Krüppel-associated box (KRAB) domain-containing zinc finger transcription factors (1-7). All TIF1 family members (TIF1␣, ␤, ␥, ␦, and bonus) are characterized by an N-terminal tri-partite motif (named TRIM or RBCC) composed of a RING (really interesting new genes) finger followed by two B-boxes and a coiled-coil domain as well as a C-terminal bi-partite motif encompassing a PHD (Plant HomeoDomain) finger and a bromo domain (2, 8 -11). The RBCC motif of TIF1␤ is known to homodimerize and to specifically interact with the KRAB domain of KRAB multifinger proteins (12-14). The TIF1␤ PHD finger and bromo domain were reported to cooperate in transcriptional repression via recruitment of repressive enzymatic activities such as histone deacetylase complexes (NurD/Mi2-␣) and a methyltransferase protein (SETDB1) (15, 16). Moreover, TIF1␤ (like all TIF1 family members) displays the canonical amino acid motif, PXVXL, interacting with HP1 heterochromatin proteins (HP1␣, ␤, and ␥) involved in gene silencing (17)(18)(19). Recently, the TIF1␤/HP1 interaction was shown to be essential for histone modificat...

Structural insights into human KAP1 PHD finger–bromodomain and its role in gene silencing

“…Unlike other SUMO E3 ligases-such as the RING/U-box and HECT domain E3s 39 , which commonly function to bring together an activated E2 SUMO and a substrate to promote SUMO conjugation-the KAP1 PHD finger-bromodomain is an intramolecular SUMO E3 ligase, thus ensuring ordered and stoichiometric site-specific SUMOylation at several lysine residues within the bromodomain and in the region N-terminal to the PHD finger, which together are required for KAP1's co-repression activity 33,41,42 .…”

Tripartite Motif Containing 28 (Trim28) Can Regulate Cell Proliferation by Bridging HDAC1/E2F Interactions

“…pcDNA3-FLAG-HA-TIF1␤ and pcDNA3.1-Myc-TIF1␤ (22) were kind gifts from Dr. Muriel Aubry (Université de Montréal). pcDNA3-E2F3 (23), pcDNA3-E2F4 (23), pGL3-p27 (24) and pGL2-E2F1 (25) have been previously described.…”

TRIM28 is required by the mouse KRAB domain protein ZFP568 to control convergent extension and morphogenesis of extra-embryonic tissues

“…GAL4DBD-ZFP568 transcriptional repression in the presence of ectopic FLAG-TRIM28 chatwo was slightly reduced compared with when similar amounts of wild-type FLAG-TRIM28 were transfected (Fig. 6A, compare blue and red bars), but was not as severely affected compared with similar experiments using a sumoylation-deficient FLAG-TRIM28 6KR previously described to impair TRIM28 repressive activity (Mascle et al, 2007) (Fig. 6A, compare red and green bars).…”

“…pCDNA3.1-Gal4DBD-TRIM28, pCDNA3.1-Gal4DBD-TRIM28 6KR , pGL35XUAS firefly luciferase and pRL Renilla luciferase are described in Mascle et al (Mascle et al, 2007). Other constructs were generated using primers as indicated in supplementary material Table S1.…”