Mutation of TRIM28-Lys304 to Gln Attenuates its Interaction with KRAB-ZNFs and Promotes Differentiation of Erythroleukemic K562 Cells

TRIM28/KAP1/TIF1β is a key epigenetic modier. Genetic ablation of trim28 is embryonic lethal although RNAi-mediated knockdown in somatic cells yields viable cells. Reduction in TRIM28 abundance at the cellular or organismal level results in polyphenism. Posttranslational modications such as phosphorylation and sumoylation have been shown to regulate TRIM28 activity. Moreover, the methylation of DNA, RNA and histones and acetylation of histones are key epigenetic modications that regulate gene expression. A number of lysine residues of TRIM28 are subject to acetylation, but how acetylation of TRIM28 affects its functions remains poorly understood. an on comparable wild-type counterpart and and interactions with heterochromatin-binding The and was activated of tail H2B H4 self-association hence nucleosome an acetylation-mimic H3 tail stability of wrapped DNA within individual nucleosomes These studies validity of the acetyl-lysine mimic, TRIM28-K304Q, for studying TRIM28 activities in vivo. How is the interaction between TRIM28 and KRAB-ZNFs regulated in vivo? One via the acetylation of TRIM28-K304 as we have demonstrated in our present study. Our observation of reduced binding of the KRAB domain by TRIM28-K304Q, was for either mutant K266Q or K340Q, suggests that the specic acetylation of K304 may impact the interaction between the KRAB domain and the TRIM28 homodimer (dimerization is mediated by the RBCCs). Molecular modeling failed to pinpoint the interaction at atomic resolution based on published structural studies and our unpublished observations. As such, further structural studies are needed. It is possible that the unstructured C-terminal residues (813–835) or sumoylation of TRIM28 may inuence the interaction. Our present results suggest that acetylation/deacetylation of TRIM28-K304 may regulate its interaction with KRAB-ZNFs in a manner.


Background
The evolutionarily conserved, vertebrate-speci c tripartite motif protein 28 (TRIM28) also known as KRABassociated protein 1 (KAP1) and transcription intermediary factor-β (TIF1β), is an essential developmental regulator-speci cally, a universal transcriptional co-repressor for the KRAB-ZNFs, a large family of transcriptional repressors in vertebrates. The mammalian genome encodes >350 KRAB-ZNFs (1-3) involved in growth and differentiation (4,5), the majority of which consists of sequence-speci c DNAbinding C2H2-type zinc-nger modules and a KRAB domain that recruits TRIM28.
TRIM28 is a maternal-effect protein required for maintenance of genomic imprinting (20,21). The early embryonic lethality resulting from the dysregulation of genomic imprinting in mice lacking maternal TRIM28 attests to its key roles in embryonic development (20,22). Moreover, TRIM28 not only maintains DNA methylation at imprinted regions during early genome-wide reprogramming but also regulates DNA methylation at imprinted gene promoters after reprogramming (23).
Posttranslational phosphorylation and sumoylation of TRIM28 contribute to the regulation of its functions (19,(24)(25)(26)(27)(28)(29). Recruitment of TRIM28 by KRAB-ZNFs enhances its sumoylation and gene repression activity (30). Phosphorylation and sumoylation of TRIM28 are critical for modulating the increase in production of type I interferons and proin ammatory cytokines during infection with in uenza virus (31,32). The acetylation of lysine residues in histones, certain transcription factors, chromatin remodelers, metabolic enzymes, and many regulatory proteins is another widely recognized example of posttranslational modi cation that signi cantly alters protein function and cellular regulation. TRIM28 can undergo acetylation in multiprotein complexes that consist TRIM28 and its associated proteins such as NuRD, HP1, SETDB1, KRAB-ZNFs, and DNMTs (33). Although acetylation of histones is widely known to play key roles in regulating gene expression (34)(35)(36) , the impact of TRIM28 acetylation on its activity remains poorly understood. To address this dearth of knowledge, we targeted three acetylation sites of human TRIM28 (K266, 304 and 340) or mouse Trim28 (K267, 305 and 341) in the coiled-coil domain.
Upon mutation of the individual Trim28 lysines to Gln or Arg, only K305Q was compromised with respect to the interaction with KRAB-ZNFs. The gene expression ensemble of the erythroleukemic cell line K562 harboring an endogenous mutation of K304Q differed considerably from that of the parent K562 line. We propose that TRIM28-K304Q could be used as an acetylation mimic for systematic investigation of the potential effects of acetylation/deacetylation on phenotypic changes at both the cellular and organismal levels in a spatiotemporal manner.

Results
The acetylation mimic TRIM28-K304Q has relatively weak interaction with the KRAB domain Acetylation of human TRIM28-K266, -K304 and -K340 (equivalent to mouse Trim28-K267, -305 and -K341) in the RBCC region has been identi ed by MS (33). We produced the acetylation-mimicking mutants of mouse Trim28, namely K267Q, K305Q and K341Q, as well as acetylation-defective mutants K267R and K305R. These constructs were expressed in human embryonic kidney 293T cells to test their interaction with Gal4 DNA-binding domain (Gal4 DBD )-KRAB and KRAB-ZNF. In a pull-down analysis, only K305Q, but not K267Q or K341Q, had decreased interaction with recombinant Gal4 DBD -KRAB (Fig. 1A). Co-IP of recombinant FLAG-tagged Trim28-K267Q, -K267R, -K305Q or -K305R and hemagglutinin (HA)tagged ZFP57 also demonstrated that K305Q had impaired interaction with recombinant ZFP57 (Fig.  1B). These results may re ect the binding of the KRAB domain by steady-state dimers formed between recombinant and endogenous TRIM28s: K305Q-K305Q, K304-K305Q, and K304-K304 (TRIM28-K304 is endogenous TRIM28). It is likely that both the K305Q homodimer and K304-K305Q heterodimer contributed to the decreased binding to the KRAB domain. In 293T cells, wild-type HA-Trim28 was coexpressed with wild-type or FLAG-K267R, -K267Q, -K305R, or -K305Q. The protein complexes brought down by HA beads were detected by anti-FLAG (Fig. S1A). These results indicated that these mutants can associate with wild-type Trim28. 293T cells were transfected with mouse FLAG-Trim28-K305Q, followed by IP and LC-MS/MS analysis, which identi ed a speci c peptide (residues 32-69) from human TRIM28 (Fig. S1B). The functional luciferase assay demonstrated that K304Q mutant had decreased transcription-suppressor activity (Fig. 1C, upper panel). Western blotting revealed similar protein abundance in each reaction (Fig.1C, lower panel). These results suggested that the acetylation mimic Trim28-K305Q had decreased interaction with the KRAB domain, both physically and functionally.
Generation of TRIM28-K304Q and TRIM28 knockout K562 cells by CRISPR/Cas9-mediated genomic editing To explore the physiological consequences of TRIM28 acetylation, we analyzed TRIM28 acetylation in the human leukemia cell line K562 by IP with an antibody against N-terminal TRIM28 (Fig. S2A) followed by LC-MS/MS analysis. Several lysines in the coiled-coil domain located at the same heptad position were acetylated, including K304 (Fig. S2B). To investigate the function of TRIM28 acetylation, human TRIM28-K304Q was knocked-in to K562 cells ( Fig. 2A). A SacI restriction site was inserted for genome examination. Several possible clones were further checked by droplet digital PCR (ddPCR) (Fig. S3A). Clones Q1 and Q2 each had three K304Q alleles. After con rmation via Sanger sequencing and western blotting, only Q1 was the correct K304Q knock-in clone, and Q2 lacked TRIM28 expression owing to deletion of certain sequences (Fig. 2B, Fig. S3B). The TRIM28 protein from Q1 was immunoprecipitated with anti-TRIM28-N followed by LC-MS/MS. The TRIM28 sequence coverage was 84%, and the K304Q mutation was con rmed (Fig. 2C). To further study TRIM28 function, we created TRIM28 knockout (KO) K562 cells using a pair of single guide RNAs (sgRNAs) and Cas9 (Fig. S4A). A clone with homozygous knockout was con rmed by quantitative ddPCR (Fig.S4B). A K562 cell proliferation assay with wild-type TRIM28, K304Q, and TRIM28-KO cells revealed that K304Q cells grew slower than the wild-type cells, and TRIM28-KO cells grew very slowly (Fig. S4C). The sensitivity of these cells to each of the two histone deacetylase (HDAC) inhibitors SAHA (suberoylanilide hydroxamic acid) and imatinib was analyzed by trypan blue staining, which revealed that wild-type cells were more sensitive to imatinib than the other two mutant cells. These results were consistent with the relatively higher proliferation rate of wild-type cells compared with the mutants. TRIM28-KO cells were more sensitive to SAHA than either the wild-type or K304Q cells (Fig. S4D). TRIM28-K304Q cells became attached cells, whereas the wild-type cells remained unattached (Fig. S4E). Trim28-KO cells were transfected with wild-type Trim28, Trim28-K305Q, -K305R, or -R310Q, and the interaction between each Trim28 protein and GAL4 DBD -KRAB domain was assessed via IP. The interaction between the GAL4 DBD -KRAB domain and Trim28-K305Q was substantially lower than that of wild-type Trim28 (Fig. S4F).
Hemoglobin gene expression in TRIM28-K304Q mutant K562 cells TRIM28 contributes to the regulation of genetic imprinting through its binding to KRAB-ZNFs (14).
TRIM28 is also involved in regulating erythroid cell maturation (37)(38)(39)(40)(41). The expression of each of the HBE (ε-globin) and HBG (γ-globin) genes was analyzed by quantitative PCR (qPCR; Fig. 3A). Interestingly, compared with wild-type cells, their expression was moderately increased in K304Q cells and highly increased in TRIM28-KO cells. To further demonstrate that TRIM28 represses HBE and HBG expression, we performed ChIP of the LCR (Locus Control Region) of the globin gene (i.e., HS (hypersensitive site)), HBE and HBG promoters. The results clearly showed reduced binding of TRIM28-K304Q to each of the HS3, HBE, and HBG promoters (Fig. 3B).

RNA-sequencing of wild-type, TRIM28-K304Q and TRIM28-KO K562 cells
To assess global gene expression in wild-type, TRIM28-K304Q, and TRIM28 KO cells, we performed RNA sequencing (RNA-seq). The K304Q and KO cells had similar gene expression pro les, but each pro le differed from that of wild-type cells (Fig. 4A). In K304Q cells, 442 genes were upregulated and 491 downregulated compared with wild-type cells (Fig. 4B). A Gene Ontology (GO) analysis of K304Q and KO cells revealed the upregulation of DNA-binding proteins such as ZNFs ( Fig. 4C and Table S1). Interestingly, the megakaryocytic repressor IKAROS family transcription factors IKZF2 and IKZR3 and the paternally imprinted genes PEG3 and DLK1 were upregulated, and the embryonic and fetal globin repressor SOX6, the maternally imprinted gene MEG3, and melanoma antigen family members MAGEC1, MAGEC2, MAGEB1 and MAGEA1 were downregulated (Table S1). To further verify the up-or downregulation of these genes, we performed RT-qPCR (Fig. 4D), which con rmed the changes in expression of certain genes identi ed by RNA-seq of TRIM28-K304Q cells. The imprinting gene that encodes the long noncoding RNA H19 was signi cantly upregulated in TRIM28-K304Q cells, although H19 was not listed in RNA-seq data. Furthermore, the TRIM28-K304Q-induced perturbation of ZNF expression highlights the important roles of dynamic regulation of acetylation of TRIM28 (as exempli ed by the acetylation mimic TRIM28-K304Q) on gene expression. KEGG pathway analysis revealed signi cant changes in gene expression pertaining to pathways such as platelet activation, thyroid hormone signaling, and miRNAs in cancer and PI3K/AKT signaling (Fig. 5A). Integrin β3 is involved in these pathways, and we veri ed the upregulation of its mRNA and protein in TRIM28-K304Q cells (Fig. 5B). Fibronectin, which interacts physically with integrin β3, was also highly upregulated in K304Q cells (Fig. 5B), which might explain the attached phenotype of K304Q cells (Fig.  S4D). Five upregulated and ve downregulated genes in K304Q cells were chosen to highlight their roles in the differentiation potential of TRIM28-K304Q K562 cells (Table 1) (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52). The expression of each of leukemia inhibitory factor receptor (LIFR), activin A receptor (ACVR1C), and inhibin beta E (INHBE) was veri ed by RT-qPCR (Fig. 5C). Their expression levels in wild-type and TRIM28-K304Q cells were consistent with the RNA-seq results, and LIFR and ACVR1C levels increased whereas INHBE level decreased in TRIM28-K304Q cells. Interestingly, drugs such as the DNMT inhibitor decitabine and HDAC inhibitor SAHA, were used to induce K562 cell differentiation into erythrocytes, resulting in upregulation of LIFR and ACVR1C mRNAs and repression of INHBE mRNA (Fig. 5D). Several ZNFs, e.g., ZNF382, ZNF527, ZNF568, ZNF667, and ZNF829, that were upregulated in TRIM28-K304Q cells were also induced by decitabine in wild-type K562 cells (Fig. S5). Induction of HBE, HBG, and H19 expression by decitabine in wild-type K562 cells is shown in Fig. 5E. Taken together, these results suggested that TRIM28-K304Qinduced or decitabine-induced fetal globin expression in K562 cells may converge on the expression or repression of downstream soluble mediators or transcription factors. The induction of certain ZNFs by TRIM28-K304Q may in fact alter the methylation of promoters/enhancers for ZNF genes and thereby perturb the output of ZNF networks.
Identi cation of proteins that interact with TRIM28 and TRIM28-304Q To identify proteins that interact with each of TRIM28 and TRIM28-304Q, we performed IP experiments using anti-TRIM28-N-conjugated protein A beads. The bead-bound TRIM28 or TRIM28-K304Q was washed and eluted with the antibody-generating peptide, and each eluent was analyzed by MS. The majority of TRIM28-interacting proteins were ZNFs; among them, 88 interacted with TRIM28, 54 interacted with both wild-type and TRIM28-K304Q, and 7 interacted with TRIM28-k304Q (Table 2). A number of non-ZNFs that also were speci c wild-type TRIM28-interacting proteins were also identi ed (Table S2) (Table S2). These results suggested that wild-type-TRIM28 participates in methylation of each of H3K9 and m6A RNA as well as the nuclear exosome decay pathway, whereas the K304Q mutant retains its ability to interact with HP1 and some RNA-binding proteins. In addition, we also analyzed the phosphorylation sites of wild-type and TRIM28-K304Q (Table  S3). The phosphorylated S473 and S824 were detected in K304Q mutant, indicating the phosphorylation was not affected by K304Q modi cation.

TRIM28-K304Q mutant interacts relatively weakly with ZNF445 to increase H19 expression
To explore the mechanism by which TRIM28-K304Q regulates H19 expression, we performed ChIP using anti-TRIM28-N and anti-ZNF445. TRIM28 and ZNF445 were enriched at the H19 promoter in wild-type K562 cells but not in K304Q mutant cells (Fig. 6A). TRIM28-associated proteins were analyzed by LC-MS/MS; one such protein was the ICR (imprinting control region)-binding protein, ZNF445 (Fig. 6B, Table  2S, panel A). Co-IP demonstrated that TRIM28-K304Q interacted weakly with ZNF445 (Fig. 6C). Knockdown of ZNF445 in wild-type K562 cells activated H19 expression (Fig. 6D) and decreased the enrichment of TRIM28 at the H19 promoter and the LCR-HS2 (Fig. 6E). These results suggested that the association of TRIM28 with ZNF445 is at least partially responsible for repressing H19 expression and that the weakened interaction between TRIM28-K304Q and ZNF445 can derepress H19 expression.

Discussion
Lysine acetylation is a reversible posttranslational modi cation of proteins that may regulate their interactions with other macromolecules. Lysine acetylation targets protein complexes such as those containing TRIM28 and others involved in chromatin remodeling, DNA replication, transcription, posttranscriptional mRNA processing, intermediary metabolism and protein turnover (33,36). For example, lysine acetylation within the core histone tail domains has been shown to inhibit nucleosome formation, an effect likely related to its role in facilitating transcription. Experimentally, the effects of acetylation have been studied using recombinant mutants in which lysine residues are substituted with glutamine as a mimic of acetylated lysine, or with arginine as a mimic of unacetylated lysine. The presence of an acetylation-mimic residue within the tail domains of H2B and H4 substantially inhibits self-association and hence nucleosome formation, whereas an acetylation-mimic residue in the H3 tail affects the stability of wrapped DNA within individual nucleosomes (34,35). These studies underscore the validity of the acetyl-lysine mimic, TRIM28-K304Q, for studying TRIM28 activities in vivo. How is the interaction between TRIM28 and KRAB-ZNFs regulated in vivo? One possibility is via the acetylation of TRIM28-K304 as we have demonstrated in our present study. Our observation of reduced binding of the KRAB domain by TRIM28-K304Q, which was not the case for either mutant K266Q or K340Q, suggests that the speci c acetylation of K304 may impact the interaction between the KRAB domain and the TRIM28 homodimer (dimerization is mediated by the RBCCs). Molecular modeling failed to pinpoint the interaction at atomic resolution based on published structural studies (6,7,53,54) and our unpublished observations. As such, further structural studies are needed. It is possible that the unstructured C-terminal 23 residues (813-835) or sumoylation of TRIM28 may in uence the interaction. Our present results suggest that acetylation/deacetylation of TRIM28-K304 may regulate its interaction with KRAB-ZNFs in a spatiotemporal manner.
TRIM28 regulates the reprogramming of induced pluripotent stem cells and differentiation of pluripotent stem cells. TRIM28 interactions with KRAB-ZNFs alter DNA methylation patterns in addition to H3K9me3 to facilitate gene repression during reprogramming. Interaction between TRIM28 and certain pluripotencyassociated ZNFs such as ZNF114, ZNF483 and ZNF589/SZF1 maintains pluripotency of induced pluripotent stem cells. Disruption of any of these interactions causes the pluripotent cells to undergo differentiation (55). Consistent with these results, our present data suggest that TRIM28 interacts more strongly with ZNF589/SZF1 than does TRIM28-K304Q (Table 2). These results are consistent with our nding that TRIM28-K304Q-K562 cells undergo differentiation whereas the parent cells do not.
In addition to the widely recognized co-repressor function of TRIM28 for KRAB-ZNFs, TRIM28 can also interact with SMARCAD1 (SWI/SNF-related, matrix-associated actin-dependent regulator of chromatin, subfamily A, containing DEAD/H box 1) through the CUE (coupling of ubiquitin conjugation to ER degradation) domain of SMARCAD1 (15,56). SMARCAD1 not only binds to and regulates TRIM28 target genes in embryonic stem cells but also appears to compete with KRAB-ZNFs for interaction with TRIM28 (15). Further investigation of both the distinct and overlapping roles of SMARCAD1-TRIM28 and KRAB-ZNFs-TRIM28 or KRAB-ZNFs-TRIM28-K304Q could illuminate their roles in gene speci city and modes of regulation. Interaction between SMARCAD1 and TRIM28-K304Q with the consequent effect(s) on gene expression remains an interesting issue.
During the development of human erythrocytes, there is a switch from expression of the embryonic εglobin gene to the fetal -globin gene in utero, and the -globin gene is silenced postpartum as β-globin gene expression becomes predominant. BCL11A and SOX6 cooperate to silence -globin expression in adult human erythroid progenitors (50). Consistent with these results and the increased expression ofglobin in TRIM28-K304Q K562 cells, SOX6 expression was found to be reduced in these cells. Using a recombinant construct of BCL11A for tandem a nity chromatography in murine erythroleukemia cells and K562 cells, TRIM28 was found to be among the many BCL11A-associated proteins identi ed (57). The role of TRIM28 in the context of associating with BCL11A has not been addressed. Interestingly, inducible deletion of trim28 in adult mouse hematopoietic cells revealed that Trim28 is essential for the cell-autonomous development of immature erythroblasts in bone marrow (40). Trim28 is recruited by the orphan nuclear receptor TR2/TR4 (NR2C1/NR2C2) heterodimer to repress embryonic/fetal globin gene expression. Exactly how the activity of TRIM28 is regulated in vivo to facilitate repression or activation of embryonic and fetal globin genes remains unclear. The observed decreased interaction between TRIM28-K304Q and KRAB-ZNFs suggests that K304 is vital for the TRIM28-mediated modulation of embryonic and fetal globin genes. Alternatively, TRIM28-K304Q may have failed to be recruited by TR2/TR4 in our experimental system.
It has been reported that maternal TRIM28 not only maintains DNA methylation at imprinted regions in the germline during early genome-wide reprogramming but also regulates DNA methylation at imprinted gene promoters after reprogramming (23). The long noncoding RNA H19 has a role in the negative regulation of body weight and cell proliferation in a mouse model (58). H19 also plays a role in the regulation of the imprinted gene network (58). In our present study, H19 RNA was upregulated in TRIM28-K304Q or TRIM28-KO K562 cells, and the upregulation correlated with the propensity of mutant K562 cells to differentiate. Consistent with a previous report (59), our results showed that, compared with wildtype TRIM28, TRIM28-K304Q had a weaker interaction with ZNF445, leading to upregulation of H19 expression (Fig. 6). In addition, TRIM28-mediated hypomethylation of the differentially methylated region (DMR) of H19/IGF2:IG-DMR (i.e., ICR1) might result in the decreased paternal expression of IGF2 and increased maternal expression of H19 (60). We have con rmed that H19 RNA is dramatically induced by a DNMT inhibitor (Fig. 5E). In contrast to H19, the imprinted gene MEG3 was downregulated in both TRIM28 KO and TRIM28-K304Q K562 cells. It is known that knockdown of TRIM28 results in differential expression of H19 and Gtl2 (Meg3) in sheep broblasts (61). However, MEG3 expression is low or undetectable in multiple types of primary tumors and cancer cell lines (62)(63)(64)(65)

Conclusions
K562 cells in culture undergo spontaneous differentiation when the cells reach relatively high density. Their differentiation can also be induced by drugs such as HDAC inhibitors (e.g., SAHA and sodium butyrate), DMSO, hemin, rapamycin, imatinib, 5-aza-cytidine/decitabine, and ribavirin (73, and unpublished observations). HDAC inhibitors may promote the opening of chromatin in the region of the fetal globin gene, thereby inducing its expression. The connections between our present results for TRIM28-K304Q-induced and decitabine-triggered differentiation are likely a consequence of the hypomethylation of fetal and embryonic globin enhancers or promoters despite the apparent mechanistic differences, i.e., TRIM28-K304Q has reduced binding a nity for KRAB-ZNFs and DNMTs whereas decitabine inhibits DNMTs. Additionally, the acetylation-mimic TRIM28-K304Q-mediated regulation of ZNFs and differentiation-related genes (Table1) correlates with decitabine-induced genes in K562 cells (Fig. 5). This suggests that TRIM28 acetylation is involved in the differentiation of K562 cells. Further insights into the convergence or divergence of mechanisms of drugs capable of inducing fetal and embryonic globin expression may have therapeutic implications.

Cell lines and plasmids
Human erythroleukemic K562 cells were purchased from American Type Culture Collection (ATCC, CCL-243) and cultured in RPMI 1640 medium supplemented with 15% fetal calf serum (Gibco), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Gibco) at 37°C with 5% CO 2 in a humidi ed incubator. Human embryonic kidney 293T cells were purchased from ATCC (CRL-3216) and cultured in Dulbecco's modi cation of Eagle medium with 10% fetal calf serum, 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Gibco) at 37°C with 5% CO 2 in a humidi ed incubator. The mouse FLAG-Trim28 expression plasmid was constructed as described (27). The EcoRI-SalI fragment from FLAG-Trim28 was ligated into pCMV-HA-N (Clontech) for HA-Trim28 expression. The TRIM28 mutants were generated with the Q5 Site-Directed Mutagenesis kit (NEB) and primers shown in Table S4A. HA-ZFP57 was cloned via PCR and the sequence con rmed. The ZNF445-DDK-Myc expression plasmid was purchased from OriGene. The GAL4 DBD -KRAB expression vector and 5×GAL4 DBS -E1bTATA-luciferase reporter were kindly obtained from Dr. Frank J. Rauscher, III.
CRISPR-mediated KO of TRIM28 and generation of mutant TRIM28-K304Q in K562 cells TRIM28 KO was performed by nucleofection of a Cas9 ribonucleoprotein (RNP). Cas9 sgRNAs, targeting TRIM28, were designed using the CRISPR Design Tool at www.benchling.com. Only sgRNAs with high predicted off-target scores (more precise) were selected. The sgRNAs were synthesized by in vitro transcription and puri ed by denaturing PAGE as described (69). Each puri ed sgRNA was refolded into a functional structure in buffer containing 20 mM HEPES pH 7.5, 150 mM KCl, 10% glycerol, 1 mM 2mercaptoethanol, and 1 mM MgCl 2 .
The TRIM28-K304Q mutant was produced by co-nucleofection of Cas9 RNP and a synthetic DNA repair template for homology-directed repair. A 1490-nt wild-type sequence of TRIM28, including the K304 site, were ampli ed by PCR using the following primer sets: TRIM28 genomic DNA (gDNA) forward primer 5'-CTCTACATCTTCCCAATAAATGGCCCAGTG-3' and reverse primer 5'-TGTGAACAAAGCAGAACCCTCTGCCTCAGT-3'. The PCR reaction contained 200 ng genomic DNA and Taq polymerase. The PCR DNA fragment was ligated into pGEM-T Easy (Promega) to construct a pGEM-T Easy/hTRIM28 wild-type plasmid.
Site-directed mutagenesis was then performed to introduce the K304Q mutation in the plasmid using the primer set of the Q5 Site-Directed Mutagenesis kit (New England Biolabs) and primer sets (hTRIM28-K304Q site-directed forward 5'-GCTCAATAAGCGGGGCCGTGTG and reverse 5'-TCCTGCATGATCTGCAGGATGGCC). The resulting plasmid, pGEM-T Easy/hTRIM28-K304Q, carried the K304Q (AAG>CAG) mutation as well as a nearby silent mutation encoding a SacI restriction sequence for screening. The DNA repair template was ampli ed from the plasmid by PCR using the primer sets K304Q homology-directed repair (HDR) template forward 5'-CTACCTAGCCTGACCTGCTGTG and reverse 5'-CTCACCCCAGACAGCATCATCA. Recombinant Cas9 was puri ed as described (DOI: 10.1002/cpmb.43).
Cas9 RNP was prepared by incubating puri ed recombinant Cas9 and sgRNA at 1:1.2 molar ratio at 37°C for 10 min. The DNA repair template was then added to the RNP mixture. Nucleofection of human CML K562 cells was performed in a Lonza 4D Nucleofector system using SE Cell Line 4D-Nucleofector TM kit and FF-120 pulse (Lonza). After nucleofection, the cells were incubated at 37°C for 48 h, and single cells were sorted by a BD FACSJazz automated cell sorter at the Flow Cytometry Core Facility of the Institute of Biomedical Sciences, Academia Sinica. The same primer set (TRIM28 gDNA primers) that is complementary to the anking regions of the HDR region was used to amplify the target region of individual single clones. The co-integrated SacI restriction site was used to screen for the insertion of the K304Q mutation.
Identi cation of associated proteins from immunoprecipitated TRIM28 and TRIM28-K304Q Peptide identi cation by MS was performed by the Mass Spectrometry Common Facility at the Institute of Biological Chemistry, Academia Sinica, using a LTQ-Orbitrap Velos system (Thermo Fisher). Data interpretation and correlations between the spectra and amino acid sequences within a human EST database and customized FLAG-Trim28 sequence were analyzed using Mascot (Matrix Science software package).

RNA-sequencing
Total cellular RNA was extracted using TRIzol Reagent (Invitrogen  (Table S4B). The ampli cation conditions were 60 cycles of 95°C for 10 s and 60°C for 15 s. The results were analyzed by the 2 -∆∆Ct relative quantitation method. All experiments were independently repeated three times.

Chromatin-immunoprecipitation (ChIP)
K562 cells were cultured to a density of 2 × 10 6 /ml and harvested for crosslinking with 1% formaldehyde in medium at room temperature for 10 min. Glycine was added ( nal concentration, 125 mM) to quench unreacted formaldehyde for 5 min. Cells were pelleted by centrifugation, and the pellet was washed with Chromatin complexes were eluted with 100 μl elution buffer (1% SDS, 0.1 M NaHCO 3 , containing 1 μl proteinase K) for 2 h at 62°C with mixing, with subsequent incubation at 95°C for 10 min to de-crosslink the chromatin complexes. Samples were cooled to room temperature, and the supernatant was transferred to a new tube. After extraction by phenol/chloroform, the DNA was precipitated by ethanol and analyzed by qPCR with speci c primers (Table S4C).

Statistical analysis
All data are presented as the mean ± SD of at least three independent experiments. Statistical signi cance (*P < 0.05, **P < 0.01 or ***P < 0.001) was determined by the one-tailed Student's t-test.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information les.

Competing interests
The authors declare that they have no competing interests. CJC conceived and coordinated the study and wrote the paper. All authors read and approved the nal manuscript.