Transcriptional repression by the insulator protein CTCF involves histone deacetylases

Abstract

The highly conserved zinc-finger protein, CTCF, is a candidate tumor suppressor protein that binds to highly divergent DNA sequences. CTCF has been connected to multiple functions in chromatin organization and gene regulation including chromatin insulator activity and transcriptional enhancement and silencing. Here we show that CTCF harbors several autonomous repression domains. One of these domains, the zinc-finger cluster, silences transcription in all cell types tested and binds directly to the co-repressor SIN3A. Two distinct regions of SIN3A, the PAH3 domain and the extreme C-terminal region, bind independently to this zinc-finger cluster. Analysis of nuclear extract from HeLa cells revealed that CTCF is also capable of retaining functional histone deacetylase activity. Furthermore, the ability of regions of CTCF to retain deacetylase activity correlates with the ability to bind to SIN3A and to repress gene activity. We suggest that CTCF driven repression is mediated in part by the recruitment of histone deacetylase activity by SIN3A.

Figure 1

Recombinant CTCF constructs. Either full-length CTCF [CTCF-(FL)] or the indicated deletions were fused C-terminal to the Gal4 DNA binding domain or to GST.

Figure 2

CTCF harbors several autonomous silencing domains. Transient co-transfections were carried out with the reporter construct p17mer6xs-tk-CAT and with expression plasmids coding for the indicated Gal–CTCF fusions. Cell lines used for transfection were the chicken erythroblasts (HD-3) and African Green Monkey kidney cells (COS-1, CV-1). The CAT activity achieved is expressed as fold repression relative to the CAT activity seen after co-transfection with the expression plasmid coding for the DNA binding domain of Gal4 (Gal). Absolute CAT activities after co-transfection of reporter and the Gal expression plasmid were 19% conversion of chloramphenicol to the acetylated form (HD-3), 23% (COS-1) and 68% (CV-1). The standard error is indicated.

Figure 3

Binding of CTCF to SIN3A. (A) In vitro translated SIN3A binds to GST–CTCF fusions. The indicated GST fusions were expressed in E.coli, affinity purified and incubated with a mixture of in vitro translated and [³⁵S]methionine-labeled SIN3A. Lane 1 contains 10% of the input used for the precipitations. The positive control, GST–N-CoR, bound SIN3A (lane 8), whereas GST alone (lane 2) resulted in no binding. (B) Endogenous SIN3A protein from HeLa nuclear extract binds to GST–CTCF. The indicated GST–CTCF fusions were used to precipitate proteins from HeLa nuclear extracts. The precipitates were analyzed by a western-blot using an antibody directed against SIN3A.

Figure 4

The zinc-finger domain of CTCF interacts with two regions of SIN3A. (A) Schematic representation of mSIN3A indicating the characteristic PAH domains (53). The deletion constructs used are indicated below. (B) Binding of mSIN3A deletions to CTCF in vitro. The indicated ³⁵S-labeled full-length SIN3A (SIN3A-FL) and SIN3A deletions were incubated with the GST domain (G) or with the fusion of GST with the CTCF zinc-finger domain (B). The input (I) is 10% of the in vitro translated product used in the assay. The in vitro translated C-terminal region of SIN3A (C-TERM) was incubated with GST or the GST fusion with full-length CTCF (GST-FL) or with GST fused to the CTCF zinc-finger domain (GST–ZF).

Figure 5

GST–CTCF binds HDAC activity from HeLa cells. HeLa nuclear extract was incubated with GST fusions and bound HDAC activity was determined. (A) As a positive control for HDAC binding, a fusion of GST with the retinoblastoma protein (GST–RB) was used (20). For negative controls, GST bound to beads or empty beads have been used. The fusion protein of GST and full-length CTCF (GST–CTCF) binds HDAC activity. (B) Individual domains of CTCF (Fig. 1) were fused to GST and tested for HDAC binding. In this experiment approximately one-tenth of the GST proteins tested in (A) were used.

Figure 6

CTCF mediated repression can be relieved by TSA. Pools of NIH 3T3 cells stably transfected with the reporter construct p17mer6xs-tk-CAT were used for transient expression of Gal or Gal fusion proteins. Transfected cells were treated for 12 h with 10 ng/ml TSA as indicated. Fold repression is calculated relative to the CAT activity after expression of the Gal DNA binding domain (Gal).