Common Effects of Acidic Activators on Large-Scale Chromatin Structure and Transcription

Abstract

Large-scale chromatin decondensation has been observed after the targeting of certain acidic activators to heterochromatic chromatin domains. Acidic activators are often modular, with two or more separable transcriptional activation domains. Whether these smaller regions are sufficient for all functions of the activators has not been demonstrated. We adapted an inducible heterodimerization system to allow systematic dissection of the function of acidic activators, individual subdomains within these activators, and short acidic-hydrophobic peptide motifs within these subdomains. Here, we demonstrate that large-scale chromatin decondensation activity is a general property of acidic activators. Moreover, this activity maps to the same acidic activator subdomains and acidic-hydrophobic peptide motifs that are responsible for transcriptional activation. Two copies of a mutant peptide motif of VP16 (viral protein 16) possess large-scale chromatin decondensation activity but minimal transcriptional activity, and a synthetic acidic-hydrophobic peptide motif had large-scale chromatin decondensation activity comparable to the strongest full-length acidic activator but no transcriptional activity. Therefore, the general property of large-scale chromatin decondensation shared by most acidic activators is not simply a direct result of transcription per se but is most likely the result of the concerted action of coactivator proteins recruited by the activators' short acidic-hydrophobic peptide motifs.

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“…It was previously reported that targeting of either VP16 or p65 caused dramatic decondensation of non-centromeric reporter arrays (Carpenter et al, 2005). We also observed some decondensation of the HAC when targeted by p65 or VP16 (Fig.…”

“…This domain promotes the opening of the local chromatin structure and results in a substantial increase in local RNA polymerase activity (Carpenter et al, 2005). TetR-EYFPp65 is an analogous construct that contains a minimal C-terminal trans-activation domain derived from the NF-kB subunit p65.…”

“…TetR-EYFPp65 is an analogous construct that contains a minimal C-terminal trans-activation domain derived from the NF-kB subunit p65. This domain induces an open chromatin environment but is less efficient at promoting transcriptional activity (Carpenter et al, 2005).…”

“…The coding sequence of the p65 C-terminal acidic activation domain was amplified from NYE108 (Carpenter et al, 2005) by using p65-RE_F (59-TCC-GGAGCGTCGACCCCGGGG-39) and p65-RE_R (59-TCCGGATTAGGAGCT-GATCTGACTCAGC-39). The PCR products were subcloned into pGEM-T Easy vectors (Promega).…”

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“…Excellent work has been done in this field in recent years [46][47][48] . Some researchers write custom software or macros for non-high-throughput image analysis programs, but their general applicability is limited 8,13,24,40,42,[49][50][51][52][53][54][55][56][57] . A more user-friendly solution is proprietary commercial software, typically bundled with an expensive image acquisition instrument and targeted to the pharmaceutical drug screening market 58 (for screening multiwell plates) or to clinical pathology laboratories 59 (for histological slides).…”