Regulation of episomal gene expression by KRAB/KAP1-mediated histone modifications

Abstract

KAP1 is an essential cofactor of KRAB zinc finger proteins, a family of vertebrate-specific epigenetic repressors of largely unknown functions encoded in the hundreds by the mouse and human genomes. So far, KRAB/KAP1-mediated gene regulation has been studied within the environment of chromosomal DNA. Here we demonstrate that KRAB/KAP1 regulation is fully functional within the context of episomal DNA, such as adeno-associated viral and nonintegrated lentiviral vectors, and is correlated with histone modifications typically associated with this epigenetic regulator.

FIG. 1.

Schematic diagram of the vectors used in the study. The rAAV vector encodes EGFP under the control of the mPGK promoter, with teto sequences in one copy downstream of the EGFP gene. ITR, AAV inverted terminal repeats for AAV2; β-globin intron, beta-globin mRNA slice donor/splice acceptor; hGH polyA, the polyadenylation site from the bovine growth hormone gene. The LV are self-inactivated; the 5′ U3 region is wild type except for that in LV-pFUT, which comes from cytomegalovirus (CMV). The teto sequences in pFUT are located in the deleted part of the 3′ U3 region. All vectors bear the encapsidation signal, the Rev-responsive element (RRE), the central polypurine tract with its termination sequence (cPPT/cTS), and the woodchuck hepatitis virus responsive element (WPRE). The internal promoter is the one from Ub C (Ubiquitin) for LV-pFUT, the one from human elongation factor 1 alpha (EF1α) for LV-tTRKrabRed, and the one from cytomegalovirus for LV-Cre. IRES, internal ribosome entry site derived from the encephalomyocarditis virus; tTRKRAB, cDNA encoding the tetracycline transrepressor (tTR) from Escherichia coli Tn10 fused to the KRAB domain of human Kox1; Cre, Cre recombinase; dsRed2, variant red fluorescent protein modified with six point mutations that improve solubility by reducing the tendency to form aggregates.

FIG. 2.

In vitro regulation of EGFP expression from AAV and IDLV by tTRKRAB. 293T cells carrying 20 copies of LV-tTR-KRAB-Red were transduced in the presence or absence of Dox with either rAAV encoding mPGK-eGFP-tetO at a multiplicity of infection (MOI) of 1.5 or LV-pFUT or IDLV at an MOI of 30 or 40, respectively. 293T cells containing no LV-tTR-KRAB-Red vector were transduced under the same conditions in the presence of Dox. The cells were FACS analyzed at 48 h posttransduction. For the LV-tTR-KRAB-Red-transduced cells, the percentage of EGFP-positive cells is gated on the dsRed-positive population.

FIG. 3.

In vivo regulation of EGFP expression from AAV vector by tTRKRAB. Transgenic mice expressing the tTRKRAB protein under the control of the hPGK promoter were injected intravenously with rAAV. (A) At 3 weeks postinjection, EGFP fluorescence was measured with a Xenogen IVIS imaging system on live animals treated (4) or not treated (3) with Dox. A noninjected animal was used as a negative control (1), and a tTRKRAB transgenic mouse injected with a nonregulatable AAV encoding EGFP under the control of the hPGK promoter without the teto sequence was used as a positive control (2). The color scale next to the images indicates the signal intensity. (B) At 4 months postinjection, animals were sacrificed and GFP expression analyzed by immunofluorescence with an antibody directed against EGFP on different tissues (liver, heart, and muscle). The animal numbers are the same as those in the whole-animal images: panels 1 and 2 are the negative and positive controls, respectively, and panels 3 and 4 are hPGK::tTRKRAB mice injected with the regulatable AAV, treated (4) or not (3) with Dox.

FIG. 4.

tTRKRAB-mediated regulation of AAV and IDLV is KAP1 dependent. (A) GFP expression in primary embryonic fibroblasts derived from mice homozygous for a floxed KAP1 allele first transduced with LV-tTRKRAB-Red and LV-Cre, followed by transduction with AAV, LV-pFUT, or IDLV, as indicated under the graphics. Cells were cultured with or without Dox and FACS analyzed 48 h after the last transduction. (B) PCR analysis of primary embryonic fibroblasts after transduction with LV-tTRKrabRed and/or LV-Cre to check for KAP1 excision. The flox/flox band represents a null allele.

FIG. 5.

tTRKRAB/KAP1 silencing on episomal DNA is mediated by histone modifications. 293T cells carrying 20 copies of the LV-tTRKrabRed vector were transduced with AAV at an MOI of 0.1 or with LV-pFUT or IDLV at an MOI of 1 and cultured in the presence or absence of Dox. The histone trimethylation profile (H3 tri-methyl K9) and KAP1 binding on AAV and LV promoters were investigated by ChIP and quantitative SYBR green PCR. The cellular promoter of the muscle-specific master regulator MyoD gene served as a control for methylation, and that for ZNF77 served as a control for KAP1 binding.