The transcriptional cofactor MIER1-beta negatively regulates histone acetyltransferase activity of the CREB-binding protein
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Mier1 encodes a novel transcriptional regulator and was originally isolated as a fibroblast growth factor early response gene. Two major protein isoforms have been identified, MIER1α and β, which differ in their C-terminal sequence. Previously, we demonstrated that both isoforms recruit histone deacetylase 1 (HDAC1) to repress transcription. To further explore the role of MIER1 in chromatin remodeling, we investigated the functional interaction of MIER1 with the histone acetyltransferase (HAT), Creb-binding protein (CBP).
Using GST pull-down assays, we demonstrate that MIER1 interacts with CBP and that this interaction involves the N-terminal half (amino acids 1–283) of MIER1, which includes the acidic activation and ELM2 domains and the C-terminal half (amino acids 1094–2441) of CBP, which includes the bromo-, HAT, C/H3 and glutamine-rich domains. Functional analysis, using HEK293 cells, shows that the CBP bound to MIER1 in vivo has no detectable HAT activity. Histone 4 peptide binding assays demonstrate that this inhibition of HAT activity is not the result of interference with histone binding.
Our data indicate that an additional mechanism by which MIER1 could repress transcription involves the inhibition of histone acetyltransferase activity.
KeywordsChromatin Remodel Histone Acetyltransferase Activity Histone Binding Growth Factor Early Response Sant Domain
MIER1 is a newly described transcriptional regulator that functions in anterioposterior patterning in the Xenopus embryo  and as an inhibitor of anchorage-independent growth of breast carcinoma cells . Two major protein isoforms, MIER1α and β, have been identified  and structurally, these two isoforms share a number of domains with other transcriptional regulators, including ELM2 , SANT  and acid activation domains. At the molecular level, MIER1 can both activate and repress transcription. The former involves the N-terminal acidic activation domain  while repression occurs by at least two distinct mechanisms: displacement of transcription factors, like Sp1, from their cognate binding sites  and recruitment of the chromatin remodeling enzyme, HDAC1 through its ELM2 domain . Recently, studies have shown that the SANT domain also plays a crucial role in chromatin remodeling; in particular, this domain is required for efficient histone acetylation . In this report, we extended our investigation of MIER1 in chromatin remodeling by examining its ability to interact with CBP and regulate its HAT activity.
The GST fusion and myc-tagged hmi-er1β (GenBank: NM_001077701) sequences were constructed using pGEX-4T-1 and pCS3+MT plasmids, respectively and their production has been described elsewhere [7, 8]. The full-length mouse CBP (GenBank: NM_001025432) in pRc/RSV was a kind gift from Dr. Roland Kwok (University of Michigan). CBP 1–1096 and CBP1094–2441 were constructed by PCR amplification of the full-length sequence using 5'-ggggatccatggccgagaacttgctggacg-3' (forward) with 5'-cgggatccctacataagtgcctggcgtagctcctcg-3' (reverse) and 5'-ggggatccgcacttatgccaactctagaag-3' (forward) with 5'-ccggatccctacaaaccctccacaaactttt-3' (reverse), respectively. The PCR products were digested with BamH1 and inserted into the BamH1 site of the pCMV-Tag2B vector (Stratagene). Anti-myc hybridoma supernatant was prepared from 9E10 cells (ATCC)  grown in hybridoma serum-free media (Invitrogen, Inc.) supplemented with 1% OptiMab monoclonal antibody production enhancer (Invitrogen, Inc.). GST pull-down assays were performed as in , using 0.35 μg of GST or equimolar amounts of GST fusion proteins and 100,000 cpm of 35S-labeled in vitro translation products. Transient transfections were performed as in . HAT assays were performed as in ; briefly, cell lysates were subjected to immunoprecipitation with the indicated antibody and the washed beads incubated with 100 nCi [14C]acetyl-CoA (51 mCi/mmol, Amersham), 30 μM H4 biotinylated peptide (Upstate Biotechnology Inc.) and 300 nM trichostatin A (Sigma) in HAT buffer  for 45 min at 30°C. The supernatants were collected and incubated with streptavidin-agarose (Pierce) at 4°C for 20 min; the 14C incorporated into the bound H4 peptide was determined by liquid scintillation counting.
Results and discussion
The N-terminal half of MIER1 interacts with the C-terminal half of CBP
Binding of MIER1 results in inhibition of CBP HAT activity
As expected, no HAT activity was detectable in immunoprecipitates from cells transfected with empty vector or mier1β (Figure 2B, lanes 2–3), however high levels of HAT activity were measured in those from cells expressing CBP alone (Fig. 2B, lane 4). When CBP was co-immunoprecipitated with MIER1β on the other hand, no detectable HAT activity was recovered in the pellet (Fig. 2B, lane 5). The presence of CBP in the co-IP was verified in a parallel sample subjected to Western blot analysis with anti-flag (Fig. 2A, panel iii, lane 3). These data show that when associated with MIER1β, CBP has no detectable HAT activity.
MIER1 does not interfere with histone binding to CBP
Together, our data show that MIER1 physically interacts with CBP and inhibits its HAT activity; this inhibition is not the result of interference with histone binding but is possibly due to a direct effect on the HAT catalytic domain.
This work was supported by a grant from the Canadian Institutes of Health Research to LLG and GDP. The authors thank Krista Butt for her expert technical assistance.
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