Centromeric transcription maintains centromeric cohesion in human cells

Centromeric transcription has been shown to play an important role in centromere functions. However, lack of approaches to specifically manipulate centromeric transcription calls into question that the proposed functions are a direct consequence of centromeric transcription. By monitoring nascent RNAs, we found that several transcriptional inhibitors exhibited distinct, even opposing, efficacies on the suppression of ongoing gene and centromeric transcription in human cells, whereas under the same conditions, total centromeric RNAs were changed to a lesser extent. The inhibitor suppressing ongoing centromeric transcription weakened centromeric cohesion, whereas the inhibitor increasing ongoing centromeric transcription strengthened centromeric cohesion. Furthermore, expression of CENP-B DNA-binding domain or CENP-B knockdown moderately increased centromeric transcription without altering gene transcription; as a result, centromeric cohesion was accordingly strengthened. Targeting of the Kox1-KRAB domain with CENP-B DB to centromeres specifically decreased centromeric transcription and weakened centromeric cohesion. Thus, based on these findings, we propose that a major function of centromeric transcription is to maintain centromeric cohesion in human cells.

ZNF354C is a transcriptional repressor that inhibits endothelial angiogenic sprouting

Zinc finger proteins (ZNF) are a large group of transcription factors with diverse functions. We recently discovered that endothelial cells harbour a specific mechanism to limit the action of ZNF354C, whose function in endothelial cells is unknown. Given that ZNF354C has so far only been studied in bone and tumour, its function was determined in endothelial cells. ZNF354C is expressed in vascular cells and localises to the nucleus and cytoplasm. Overexpression of ZNF354C in human endothelial cells results in a marked inhibition of endothelial sprouting. RNA-sequencing of human microvascular endothelial cells with and without overexpression of ZNF354C revealed that the protein is a potent transcriptional repressor. ZNF354C contains an active KRAB domain which mediates this suppression as shown by mutagenesis analysis. ZNF354C interacts with dsDNA, TRIM28 and histones, as observed by proximity ligation and immunoprecipitation. Moreover, chromatin immunoprecipitation revealed that the ZNF binds to specific endothelial-relevant target-gene promoters. ZNF354C suppresses these genes as shown by CRISPR/Cas knockout and RNAi. Inhibition of endothelial sprouting by ZNF354C is dependent on the amino acids DV and MLE of the KRAB domain. These results demonstrate that ZNF354C is a repressive transcription factor which acts through a KRAB domain to inhibit endothelial angiogenic sprouting.

Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay, and hyperphagia/obesity. This disorder is caused by the absence of paternally-expressed gene products from chromosome 15q11-q13. We previously demonstrated that knocking out ZNF274, a KRAB-domain zinc finger protein capable of recruiting epigenetic machinery to deposit the H3K9me3 repressive histone modification, can activate expression from the normally silent maternal allele of SNORD116 in neurons derived from PWS iPSCs. However, ZNF274 has many other targets in the genome in addition to SNORD116. Depleting ZNF274 will surely affect the expression of other important genes and disrupt other pathways. Here we used CRISPR/Cas9 to delete ZNF274 binding sites at the SNORD116 locus to determine whether activation of the maternal copy of SNORD116 could be achieved without altering ZNF274 protein levels. We obtained similar activation of gene expression from the normally silenced maternal allele in neurons derived from PWS iPSCs, compared to ZNF274 knockout, demonstrating that ZNF274 is directly involved in the repression of SNORD116. These results suggest that interfering with ZNF274 binding at the maternal SNORD116 locus is a potential therapeutic strategy for PWS.

KRAB domain of ZFP568 disrupts TRIM28-mediated abnormal interactions in cancer cells

Interactions of KRAB (Krüppel-associated box)-associated protein KAP1 [also known as TRIM28 (tripartite motif containing protein 28)] with DNA-binding KRAB zinc finger (KRAB-ZF) proteins silence many transposable elements during embryogenesis. However, in some cancers, TRIM28 is upregulated and interacts with different partners, many of which are transcription regulators such as EZH2 in MCF7 cells, to form abnormal repressive or activating complexes that lead to misregulation of genes. We ask whether a KRAB domain—the TRIM28 interaction domain present in native binding partners of TRIM28 that mediate repression of transposable elements—could be used as a tool molecule to disrupt aberrant TRIM28 complexes. Expression of KRAB domain containing fragments from a KRAB-ZF protein (ZFP568) in MCF7 cells, without the DNA-binding zinc fingers, inhibited TRIM28–EZH2 interactions and caused degradation of both TRIM28 and EZH2 proteins as well as other components of the EZH2-associated polycomb repressor 2 complex. In consequence, the product of EZH2 enzymatic activity, trimethylation of histone H3 lysine 27 level, was significantly reduced. The expression of a synthetic KRAB domain significantly inhibits the growth of breast cancer cells (MCF7) but has no effect on normal (immortalized) human mammary epithelial cells (MCF10a). Further, we found that TRIM28 is a positive regulator of TRIM24 protein levels, as observed previously in prostate cancer cells, and expression of the KRAB domain also lowered TRIM24 protein. Importantly, reduction of TRIM24 levels, by treatment with either the KRAB domain or a small-molecule degrader targeted to TRIM24, is accompanied by an elevated level of tumor suppressor p53. Taken together, this study reveals a novel mechanism for a TRIM28-associated protein stability network and establishes TRIM28 as a potential therapeutic target in cancers where TRIM28 is elevated. Finally, we discuss a potential mechanism of KRAB-ZF gene expression controlled by a regulatory feedback loop of TRIM28–KRAB.

DUF3669, a “domain of unknown function” within ZNF746 and ZNF777, oligomerizes and contributes to transcriptional repression

Background ZNF746 and ZNF777 belong to a subset of the large Krüppel-associated box (KRAB) zinc finger (ZNF) transcription factor family. They contain, like four other members in human, an additional conserved domain, the “domain of unknown function 3669” (DUF3669). Previous work on members of this subfamily suggested involvement in transcriptional regulation and aberrant ZNF746 overexpression leads to neuronal cell death in Parkinson’s disease. Results Here we demonstrate that N-terminal protein segments of the ZNF746a major isoform and ZNF777 act in concert to exert moderate transcriptional repression activities. Full potency depended on the intact configuration consisting of DUF3669, a variant KRAB domain and adjacent sequences. While DUF3669 contributes an intrinsic weak inhibitory activity, the isolated KRAB-AB domains did not repress. Importantly, DUF3669 provides a novel protein-protein interaction interface and mediates direct physical interaction between the members of the subfamily in oligomers. The ZNF746 protein segment encoded by exons 5 and 6 boosted repressor potency, potentially due to the presence of an acceptor lysine for sumoylation at K189. Repressor activity of the potent canonical ZNF10 KRAB domain was not augmented by heterologous transfer of DUF3669, pointing to the importance of context for DUF3669’s impact on transcription. Neither ZNF746a nor ZNF777 protein segments stably associated with TRIM28 within cells. Isoform ZNF746b that contains, unlike the major isoform, a full-length KRAB-A subdomain, displayed substantially increased repressor potency. This increase is due to canonical mechanisms known for KRAB domains since it did not take place in HAP1 knockout models of TRIM28 and SETDB1. A glycine to glutamic acid replacement that complies with a bona fide conserved “MLE” sequence within KRAB-A led to a further strong gain in repressor potency to levels comparable to those of the canonical ZNF10 KRAB domain. Each gain of repressive activity was accompanied by an enhanced interaction with TRIM28 protein. Conclusion DUF3669 adds a protein-protein interaction surface to a subgroup of KRAB-ZNF proteins within an N-terminal configuration with variant KRAB and adjacent sequences likely regulated by sumoylation. DUF3669 contributes to transcriptional repression strength and its homo- and hetero-oligomerization characteristics probably extended the regulatory repertoire of KRAB-ZNF transcription factors during amniote evolution.

KAP1 is an antiparallel dimer with a functional asymmetry

KAP1 (KRAB domain–associated protein 1) plays a fundamental role in regulating gene expression in mammalian cells by recruiting different transcription factors and altering the chromatin state. In doing so, KAP1 acts both as a platform for macromolecular interactions and as an E3 small ubiquitin modifier ligase. This work sheds light on the overall organization of the full-length protein combining solution scattering data, integrative modeling, and single-molecule experiments. We show that KAP1 is an elongated antiparallel dimer with an asymmetry at the C-terminal domains. This conformation is consistent with the finding that the Really Interesting New Gene (RING) domain contributes to KAP1 auto-SUMOylation. Importantly, this intrinsic asymmetry has key functional implications for the KAP1 network of interactions, as the heterochromatin protein 1 (HP1) occupies only one of the two putative HP1 binding sites on the KAP1 dimer, resulting in an unexpected stoichiometry, even in the context of chromatin fibers.

Structure of KAP1 tripartite motif identifies molecular interfaces required for retroelement silencing

Transcription of transposable elements is tightly regulated to prevent genome damage. KRAB domain-containing zinc finger proteins (KRAB-ZFPs) and KRAB-associated protein 1 (KAP1/TRIM28) play a key role in regulating retrotransposons. KRAB-ZFPs recognize specific retrotransposon sequences and recruit KAP1, inducing the assembly of an epigenetic silencing complex, with chromatin remodeling activities that repress transcription of the targeted retrotransposon and adjacent genes. Our biophysical and structural data show that the tripartite motif (TRIM) of KAP1 forms antiparallel dimers, which further assemble into tetramers and higher-order oligomers in a concentration-dependent manner. Structure-based mutations in the B-box 1 domain prevent higher-order oligomerization without significant loss of retrotransposon silencing activity, indicating that, in contrast to other TRIM-family proteins, self-assembly is not essential for KAP1 function. The crystal structure of the KAP1 TRIM dimer identifies the KRAB domain binding site in the coiled-coil domain near the dyad. Mutations at this site abolished KRAB binding and transcriptional silencing activity of KAP1. This work identifies the interaction interfaces in the KAP1 TRIM responsible for self-association and KRAB binding and establishes their role in retrotransposon silencing.