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. 2018 Mar:516:165-175.
doi: 10.1016/j.virol.2018.01.012.

Characterization of interaction between Trim28 and YY1 in silencing proviral DNA of Moloney murine leukemia virus

Andreia Lee  1 , Oya CingÖz  2 , Yosef Sabo  2 , Stephen P Goff  3
Affiliations

Characterization of interaction between Trim28 and YY1 in silencing proviral DNA of Moloney murine leukemia virus

Andreia Lee et al. Virology. 2018 Mar.

Abstract

Moloney Murine Leukemia Virus (M-MLV) proviral DNA is transcriptionally silenced in embryonic cells by a large repressor complex tethered to the provirus by two sequence-specific DNA binding proteins, ZFP809 and YY1. A central component of the complex is Trim28, a scaffold protein that regulates many target genes involved in cell cycle progression, DNA damage responses, and viral gene expression. The silencing activity of Trim28, and its interactions with corepressors are often regulated by post-translational modifications such as sumoylation and phosphorylation. We defined the interaction domains of Trim28 and YY1, and investigated the role of sumoylation and phosphorylation of Trim28 in mediating M-MLV silencing. The RBCC domain of Trim28 was sufficient for interaction with YY1, and acidic region 1 and zinc fingers of YY1 were necessary and sufficient for its interaction with Trim28. Additionally, we found that residue K779 was critical for Trim28-mediated silencing of M-MLV in embryonic cells.

Keywords: Moloney murine leukemia virus; Phosphorylation; Protein-protein interaction; SUMO; Transcriptional silencing; Trim28; YY1.

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Figures

Fig. 1.
Fig. 1.. Interaction between rYY1 and rTrim28.
Lanes 1–4: rYY1 was incubated with anti-YY1 or control rabbit antiserum, and antibodies were then loaded onto protein A/G beads. The beads were incubated with rTrim28, then washed and analyzed by Western blot, probed with anti-Trim28, or anti-YY1 antibodies as indicated at left. Lanes 1 and 2: 10% of total input recombinant proteins. Lane 3: IP with anti-YY1 antibody. Lane 4: IP with control rabbit antibody. No rTrim28 was detected with anti-YY1 IP. Lanes 5–8: rFlag-HP1 was loaded onto anti-Flag beads. Beads were incubated with rTrim28, then washed and analyzed by Western blot, probed with anti-Trim28, or anti-Flag antibodies as indicated at left. Lanes 5 and 6: 10% of total input recombinant proteins. Lane 7: IP with anti-Flag antibody. Lane 8: IP with control rabbit antibody. rTrim28 was readily detected bound to rFlag-HP1.
Fig. 2.
Fig. 2.. Co-IP of YY1 with Trim28 mutants containing deletions of major domains.
(A) Schematic of the Trim28 wild-type and mutant proteins. All mutants were HA-tagged at the N-terminus. Domains known to interact with other repressor partners are noted above the domain. The extent of interaction of Trim28 mutants with endogenous YY1 is indicated in the right column. A strong interaction is denoted by “++”, no interaction is indicated by “–”, and mutants that displayed poor expression are noted by n/a. (B) HA-Trim28 constructs were overexpressed in F9 cells, endogenous YY1 was immunoprecipitated with anti-YY1 antibody, and bound proteins were examined by Western blot probed with anti-HA antibody. For each mutant, groups of three lanes are shown: 2% of the total protein input to the IPs, YY1 IP, and control IP. Examples of positive and a poorly expressed mutant protein are shown. The bands corresponding to the Trim28 mutants positive for binding are marked in the input lanes by an asterisk.
Fig. 3.
Fig. 3.. Co-IP of Trim28 and YY1 mutants.
(A) Schematic of YY1 wild-type and mutant proteins. All mutants were myc-tagged at the N-terminus. Domains known to interact with other repressor partners are noted above the domain. Extent of interaction of YY1 mutants with endogenous Trim28 is indicated in the right column. A strong interaction is noted by “++”, a weak interaction is noted by “+”, and no interaction is indicated by “–”. (B) Upper panel: Myc-YY1 constructs were overexpressed in F9 cells, and cell lysates were prepared for co-IP experiments. Myc-YY1 proteins were immunoprecipitated with anti-myc beads, and bound proteins were examined by Western blot probed with an anti-Trim28 antibody. The band running below the Trim28 bands in the Myc IP lanes represent residual anti-myc antibody. The bands corresponding to the YY1 mutants are marked at the left of the input lanes by an asterisk. Mutants analyzed on this blot include the four mutants in which the AR2, GA, spacer, and ZNFs regions were independently deleted. Lower panel: Mutants analyzed on this blot include the mutants in which two or more regions were deleted.
Fig. 4.
Fig. 4.. Mutations of major sumoylation sites on Trim28.
(A) Schematic of Trim28 protein with potentially sumoylated lysine residues indicated. (B) Mutant Trim28 was expressed and endogenous Trim28 was knocked down in F9 cells, and cell lysates were prepared and analyzed by Western blot. The “No KD” lane shows basal Trim28 levels in F9 cells. The empty vector (EV) lane shows the level of endogenous Trim28 expression in all KD lines, and the remaining lanes show expression levels of exogenous Trim28. (C) Western blots monitoring interaction of mutant Trim28 with YY1 by co-IP. YY1 was pulled-down with anti-YY1 antibody and the western blot was probed for Trim28. Co-IP results for four mutants are shown in the top panel and for the three remaining mutants on the bottom panel. (D) NIH3T3, untreated F9 cells, and Trim28 mutant F9 lines were infected with the M-MLV-GFP virus, and GFP expression was measured by flow cytometry. Uninfected cells were used to set our gates for counting “GFP-positive” expressing cells. NIH3T3 represent maximal M-MLV expression levels, and F9 cells represent maximal repression of M-MLV expression. Bars report the percentage of GFP-positive cells detected. Values represent the mean percentage of three biological replicates, and error bars represent the standard error of the mean. **denotes p < 0.01 significance of difference from EV control by student’s t-test. p values for samples with no significant difference from EV are indicated.
Fig. 5.
Fig. 5.. SUMO conjugation of Trim28.
(A) Immunoprecipitation of HA-tagged Trim28 and probing for Flag-tagged SUMO1. Wild-type Flag-SUMO1 (WT) or Flag-SUMO1GG (GG) was stably co-expressed with wild-type HA-Trim28 (WT) or HA-Trim286KR (6KR) in 293T cells. The input lanes demonstrate the expression levels of Flag-SUMO1, Flag-SUMO1GG, HA-Trim28, or HA-Trim286KR. Trim28 was immunoprecipitated using anti-HA magnetic beads and bound proteins were analyzed by Western blot probed with anti-Flag antibodies to detect sumoylation levels onTrim28. The asterisk marks the position of the unconjugated monomeric SUMO1. (B) Immunoprecipitation of HA-tagged Trim28 and probing for Flag-tagged SUMO2. SUMO2 is indicated by “2” and SUMO2GG is indicated by “2gg, ” and immunoprecipitation of HA-Trim28 with SUMO1 was included as a control in the first two lanes.
Fig. 6.
Fig. 6.. Mutation of two major phosphorylation sites of Trim28.
(A) Schematic of Trim28 protein with the major phosphorylation sites marked. Serine sites were mutated to alanine or to an acidic residue that mimics a constitutively phosphorylated residue. (B) Mutant Trim28 was expressed in F9 cells, endogenous Trim28 was knocked down, and cell lysates were prepared and analyzed by Western blot. The “No KD” lane shows basal Trim28 levels in F9 cells. The empty vector (EV) lane shows the level of endogenous Trim28 expression in all KD lines, and the remaining lanes show expression levels of exogenous Trim28. (C) Western blot monitoring interaction of mutant Trim28 with YY1 by co-IP. YY1 was immunoprecipitated with anti-YY1 antibody and the bound proteins were analyzed by western blot probed for Trim28. (D) Untreated F9 cells, and lines expressing empty vector (EV), wild-type Trim28 (WT), or Trim28 mutants, all with KD of endogenous Trim28, were infected with M-MLV-Luc virus, and luciferase activities were measured after 48 h. Relative light units (RLU) are values expressed relative to F9 control. Values indicate the mean of four biological replicates, and error bars represent the standard error of the mean. Lower panels: Lysates were analyzed by western blot with anti-Trim28 antibodies to monitor KD and expressed levels of Trim28. Anti-tubulin antibodies were used to report tubulin levels as a loading control.
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