A proline-rich TGF-beta-responsive transcriptional activator interacts with histone H3.

Abstract

The molecular mechanisms involved in the regulation of gene expression by transforming growth factor-13 (TGF-13) have been analyzed. We show that TGF-~ specifically induces the activity of the proline-rich trans-activation domain of CTF-1, a member of the CTF/NF-I family of transcription factors. A TGF-13-responsive domain (TRD) in the proline-rich transcriptional activation sequence of CTF-I was shown to mediate TGF-13 induction in NIH-3T3 cells. Mutagenesis studies indicated that this domain is not the primary target of regulatory phosphorylations, suggesting that the growth factor may regulate a CTF-l-interacting protein. A two-hybrid screening assay identified a nucleosome component, histone H3, as a specific CTF-l-interacting protein in yeast. Furthermore, the CTF-1 trans-activation domain was shown to interact with histone H3 in both transiently and stably transfected mammalian cells. This interaction requires the TRD, and it appears to be upregulated by TGF-f~ in vivo. Moreover, point mutations in the TRD that inhibit TGF-f~ induction also reduce interaction with histone H3. In vitro, the trans-activation domain of CTF-1 specifically contacts histone H3 and oligomers of histones H3 and H4, and full-length CTF-1 was shown to alter the interaction of reconstituted nucleosomal cores with DNA. Thus, the growth factor-regulated trans-activation domain of CTF-1 can interact with chromatin components through histone H3. These findings suggest that such interactions may regulate chromatin dynamics in response to growth factor signaling.

The repression of nuclear factor I/CCAAT transcription factor (NFI/CTF) transactivating domain by oxidative stress is mediated by a critical cysteine (Cys-427)

“…The C-terminal part of the TAD (amino acids 486-499) displays transcriptional activity but it is less active than the whole TAD. This region has been described previously as a TRD [9]. It is noticeable that it is not sensitive to H # O # (compared with the intact TAD).…”

“…This cysteine, contrary to Cys-405, seems to be easily modified by either oxidizing or alkylating reagents, suggesting a different exposure of these residues within the TAD protein sequence. Moreover, this TAD has been shown to interact directly with several proteins involved in the control of transcription, such as the TATA-box-binding protein TBP [22], the co-activator CBP\ p300 [23] and the histones H1 and H3 [9,24]. This TAD was also shown to enhance synergistically the activation of transcription mediated by the oestrogen receptor [25] and the aryl hydrocarbon receptor (AhR) [10] pathways.…”

“…pRSV.Gal.CTF, pRSV.Gal.Sp1 and pRSV.Gal.Oct expressed fusion proteins containing the Gal4 DBD fused to the TAD of the human NFI\CTF, Sp1 and Oct2 transcription factors respectively. They were derived from the pGal(399-499), pGalOct2 and pGalSp1 plasmids (described in [9]) in which the simian virus 40 (SV40) promoters have been replaced by Rous sarcoma virus (RSV) promoters because the SV40 promoter is very sensitive to oxidative stress whereas that of RSV is not [4]. Amino acid point mutations in the TAD of NFI\CTF were produced using a double PCR technique with mutated oligonucleotides [13].…”

“…The last 13 residues of NFI\CTF were identified as the TGFβ-responsive domain (TRD) [9]. In fibroblasts, the TGFβ effect is associated with an increase in cytosolic calcium [7].…”

Expression of the Smooth Muscle Myosin Heavy Chain Gene Is Regulated by a Negative-acting GC-rich Element Located between Two Positive-acting Serum Response Factor-binding Elements

“…However, several studies indicated that, at least in some circumstances, CTF/NF1 functions to regulate transcription via a mechanism that is likely to involve higher order chromatin structures (45,46). Such an activity would likely be missed in a transient transfection reporter gene analysis where "naked" plasmid DNA is utilized (35).…”