Gro/TLE/Grg transcriptional corepressors regulate many developmental and signaling pathways, but their repressive mechanisms are not fully understood. We investigated Gro/TLE/Grg-mediated repression by using various chromatin substrates, both in vitro and in vivo, and present evidence for a newly discovered activity for this class of transcriptional corepressor.
In the absence of recruitment by a transcription factor, we found that Grg3 readily binds mononucleosome particles, at least in part via the interaction with the tails of histones H3 and H4. However, we discovered another level of Grg3 interaction with chromatin: with a dinucleosome subunit, helping to generate a condensed structure. In vivo studies suggest that the yeast Tup1 homolog bridges across adjacent nucleosomes (Ducker and Simpson, 2000
). Since we found that N-terminally deleted Grg3, deficient in tetramerization (Chen et al., 1998
), also elicits chromatin condensation, it is unlikely that condensation is mediated by bridging across nucleosomes by different Grg3 subunits in a native tetramer. We suggest that chromatin condensation may be generated by an individual Grg3 subunit interacting simultaneously with H3 tails of adjacent nucleosomes or with a new structure generated by the interface of adjacent nucleosomes. The ability of different domains of Tup1 to interact independently with core histones (Edmondson et al., 1996
) is consistent with the former possibility.
By contrast, aggregation of separate nucleosome arrays requires an intact tetramerization domain of Grg3, as does the generation of recruitment-based, nuclease-resistant chromatin in vitro and transcriptional repression in vivo. Deletion of the tetramerization domain diminished, but did not eliminate, FoxA1 interactions, making it difficult to assess the extent to which the ability to aggregate arrays is necessary for the repressed chromatin structure upon FoxA1-based recruitment. Regardless, the middle region of Grg3 becomes exposed to V8 protease when full-length Grg3 binds to nucleosome arrays. Thus, chromatin binding induces a conformational change in the tetramer so that the subunits become exposed. This could facilitate the bridging of Grg3 between arrays to generate the aggregates. Secondarily, the conformational change in Grg3 induced by chromatin binding may indirectly facilitate stable interactions with FoxA, since we found that the middle region of free Grg3 impairs the protein from binding to FoxA factors.
Significantly, chromatin binding by Grg3 is indispensable for FoxA1- and Hes1-based genomic site recruitment; neither transcription factor could recruit Grg3 to free DNA. The existence of positioned nucleosomes at the alb1
enhancer in liver cells (McPherson et al., 1993
; Chaya et al., 2001
) may facilitate the ability of FoxA factors to recruit Grg3. In summary, we suggest that Gro/TLE/Grg proteins scan chromatin domains for being able to transiently bind nucleosomes, which stabilizes an open tetramer and consequently allows the tetramer to bind a recruiting transcription factor. Since Groucho/TLE/Grg proteins are thought to be refractory to binding highly acetylated chromatin (Chen and Courey, 2000
), associated HDAC activity (Chen et al., 1999
) could enable stable transcription factor-based recruitment.
Furthermore, we found that Grg3 generates a compact chromatin structure that is resistant to nucleases only when binding both chromatin and a recruiting transcription factor. We found that FoxA1-Grg3-array complexes are resistant to three different nucleases over the distance of approximately 3−4 nucleosomes in vitro. Strikingly, this corresponds to the same region over which Grg3 was recruited to the alb1 enhancer by FoxA1 in vivo, causing repression. Three to four nucleosomes spans the size of many enhancer and promoter elements. We therefore suggest that the recruitment-based blanketing of Grg3 over such a chromatin domain is part of the mechanism for transcriptional repression by the Gro/TLE/Grg corepressors.
Functionally, we found that recruitment-based “blanketing” of local chromatin by Grg3 results in impaired recruitment of a transcriptional activator, TBP, and RNA polymerase II. This distinguishes the mechanism of repression by Grg with that of Sir complexes (see Introduction). Strikingly, Grg3 impaired the enhanced recruitment of NF-1, TBP, and pol II that normally occurs when the H2.35 liver cell line is shifted to differentiating culture conditions. Interestingly, local levels of linker histone were not significantly affected by Grg3, indicating that the Grg3-bound chromatin neither excludes linker histone nor is excluded by it.
When Grg recruitment sites are separated in chromatin, as for the afp
enhancers, Grgs can apparently span a larger chromatin domain; though this terminates distal to recruitment sites (, −2000, −1000 regions). Although Gro/TLE/Grg proteins can repress genes distal to their site of recruitment (Chen and Courey, 2000
), we suggest that by blanketing an enhancer, or an extended enhancer domain (as for afp
), Gro/TLE/Grg proteins indirectly suppress distal enhancer-promoter interactions. This is opposed to long-range spreading of a repressive and resistant chromatin structure from a recruitment site, as seen with Sir proteins (Pirrotta and Gross, 2005
). The yeast homolog Tup1 was found to spread along an entire STE6 gene on an episome (Ducker and Simpson, 2000
). Interestingly, the linker regions of the episomal Tup1-STE6 chromatin remained sensitive to MNase, similar to the linker region sensitivity to nucleases of the condensed chromatin generated by Grg3 alone on our nucleosome arrays (, ).
In conclusion, our studies emphasize the importance of reconstituting regulatory complexes on chromatin templates of different complexity and assessing the concordance with endogenous genes, to unveil mechanisms of genetic control. The mechanisms and consequences of chromatin binding that we found for Grg are different than those of other corepressor classes. Understanding how the condensed chromatin structure generated by Grg alone, in vitro, is converted to a resistant chromatin domain by transcription factor recruitment could unveil new molecular targets for antagonizing corepressor activity.