The human factors YY1 and LSF repress the human immunodeficiency virus type 1 long terminal repeat via recruitment of histone deacetylase 1

Abstract

Enigmatic mechanisms restore the resting state in activated lymphocytes following human immunodeficiency virus type 1 (HIV-1) infection, rarely allowing persistent nonproductive infection. We detail a mechanism whereby cellular factors could establish virological latency. The transcription factors YY1 and LSF cooperate in repression of transcription from the HIV-1 long terminal repeat (LTR). LSF recruits YY1 to the LTR via the zinc fingers of YY1. The first two zinc fingers were observed to be sufficient for this interaction in vitro. A mutant of LSF incapable of binding DNA blocked repression. Like other transcriptional repressors, YY1 can function via recruitment of histone deacetylase (HDAC). We find that HDAC1 copurifies with the LTR-binding YY1-LSF repressor complex, the domain of YY1 that interacts with HDAC1 is required to repress the HIV-1 promoter, expression of HDAC1 augments repression of the LTR by YY1, and the deacetylase inhibitor trichostatin A blocks repression mediated by YY1. This novel link between HDAC recruitment and inhibition of HIV-1 expression by YY1 and LSF, in the natural context of a viral promoter integrated into chromosomal DNA, is the first demonstration of a molecular mechanism of repression of HIV-1. YY1 and LSF may establish transcriptional and virological latency of HIV, a state that has recently been recognized in vivo and has significant implications for the long-term treatment of AIDS.

Figures

FIG. 1

YY1 and LSF associate in vivo and in vitro, in the absence of a DNA-binding site or other factors. (A) Immunoprecipitations of Jurkat nuclear extracts using either α-YY1, α-LSF, or a nonspecific rabbit polyclonal antiserum. Mock immunoprecipitations (IP) were performed in the absence of antibody. Precipitates were assayed by Western blot using α-LSF. Approximately 75% of the LSF protein recovered by α-LSF is also immunoprecipitated by α-YY1. To demonstrate the recognition of YY1, a Western blot of input nuclear extract is displayed at the right. (B) EMSA was performed using the RCS-binding site and the indicated amounts of LSF and YY1; total amount of protein was normalized by the addition of bovine serum albumin. The mobility of the native RCS complex formed by nuclear extract is displayed at the right. Nonspecific interactions with the RCS are indicated. (C) Complexes supershifted by the addition of either α-YY1 or α-LSF. Addition of α-YY1 had no effect on the LSF complexes in the absence of YY1 protein (not shown).

FIG. 2

Mapping of the YY1-LSF interaction domains. (A) Representation of wild-type (wt) LSF and LSF deletion mutants used to identify the region of interaction between LSF with YY1. ΔX, deletion up to codon X; XΔ, deletion after codon X; XX, mutated single codons. The amount of LSF bound to GST-YY1 varied from 2.5 to 7% of the input, depending on the experiment. All values were normalized to the amount of wild-type LSF bound to GST-YY1 in the experiment. (B) Representative autoradiographs showing input LSF constructs and LSF constructs retained by GST-YY1 and by GST. (C) Graphical representation of the YY1 chimeras, all of which contained the GST tag. All constructs also contained the N-terminal region of YY1 (amino acids 1 to 294) except YY1 Zn Fingers, which lacked this region. YY1 is the full-length wild-type YY1 molecule. Nonshaded regions represent GFI-1 zinc fingers (related to Krüppel zinc finger proteins). GFI contains only GFI-1 zinc fingers, Chi 1 contained the first YY1 zinc finger, Chi 2 contained the first two YY1 zinc fingers, Chi 5 contained the last two YY1 zinc fingers, Chi 7 contained the second YY1 zinc finger only, and YY1 Zn Fingers contained all four YY1 zinc fingers without the YY1 amino-terminal region. The first two zinc fingers of YY1 are required for optimal binding of LSF. Chi 1, Chi 2, Chi 7, and YY1 bound LSF, whereas Chi 5, GFI-1, and GST exhibited background levels of LSF binding. A lane containing only a diluted aliquot of labeled LSF serves as a marker. When normalized for protein concentration, a construct expressing only the YY1 zinc fingers fused to GST binds LSF as avidly as intact GST-YY1. Background levels of binding varied between experiments, as shown.

FIG. 3

Repression by YY1 and LSF requires functional LSF and HDAC interaction-competent YY1. Expression of an integrated LTR-CAT reporter in HeLa-CD4-LTR cells, when activated by 200 ng of pAR-Tat, was inhibited by 2.5 μg of CMV-YY1 or 2.5 μg of both CMV-YY1 and CMV-LSF; 2.5 μg of CMV-LSF had no effect on expression of CAT; 2.5 μg of dnLSF (pCMV-LSF 234QL/236KE), incapable of binding DNA but capable of forming inactive multimers, blocked inhibition of Tat-activated LTR expression by 2.5 μg of YY1; 2.5 μg of CMV-YY1Δ154-199, incapable of interacting with HDAC, was unable to inhibit Tat-activated expression. All transfections received a total of 5 μg of CMV promoter-driven plasmid. Data are from at least four independent transfections, normalized for expression of cotransfected β-actin–luciferase.

FIG. 4

YY1, LSF, and HDAC1 copurify with RCS-binding activity. (A) Activities of crude nuclear extract and elution fractions from an RCS DNA affinity chromatography column. Shown are results of EMSA using the RCS probe (top panel), and of Western blotting using rabbit polyclonal α-YY1 (second panel), rabbit polyclonal α-CP2 (LSF) (third panel), and rabbit polyclonal α-HDAC1/2 (bottom panel). EMSA was performed with 4 μg of nuclear extract (NE) and 20 ng of DNA affinity column eluate. Western blotting was performed with 20 μg of nuclear extract and 200 ng of DNA affinity column eluate. An arrow indicates the YY1-specific complex, as validated by α-YY1 interference in EMSA. Positions of molecular weight markers are indicated in kilodaltons. (B) HDAC activity of DNA affinity chromatography fractions correlates with the presence of the YY1-LSF complex.

FIG. 5

YY1 directly affects production of HIV in vitro. Production of HIV-1 is inhibited by YY1 but not by YY1Δ154-199 lacking the HDAC interaction domain following transfection of HeLa cells with 0.5 μg of the CXCR4 prototypic clone pNL4-3 (left) or 1 μg of the CCR5 prototypic clone pYU-2 (right). Data are representative of three transfections.

FIG. 6

Model of recruitment by LSF of YY1 and then HDAC to the HIV promoter.