Transgenic expression of CBBP, a CXC domain protein, establishes paramutation in maize

Abstract

Paramutation is the ability of specific DNA sequences to communicate in trans to establish meiotically heritable expression states. Paramutation at the maize b1 locus is mediated by seven unique noncoding transcribed tandem repeats of 853 bp that are required to establish and maintain the meiotically heritable expression and distinct chromatin states associated with b1 paramutation. In this study, we report the identification of a CXC-domain protein CBBP (CXC domain b1-repeat binding protein) that binds to a defined region within the b1 tandem repeat sequence in vivo and in vitro. When CBBP is expressed from a transgene in maize, it can induce a silent state at the b1 locus that is heritable in progeny no longer containing the transgene, and the silent epiallele is capable of silencing an active epiallele, characteristic of paramutation. Accumulation of the CBBP protein correlates with b1 silencing in transgenic and nontransgenic plants. The ability of CBBP to form multimers and to bind to the b1 tandem repeats suggests a model for counting the number of b1 repeats. In contrast to previously identified proteins involved in paramutation, CBBP does not share similarity to the known components of the Arabidopsis RNAi heterochromatin silencing pathway. Thus, this study defines another class of protein that is involved in heritable gene silencing.chromatin | gene silencing P aramutation was initially described at the maize r1 locus (1) as an interaction between specific alleles that leads to heritable changes in expression. Since that time, several other examples of paramutation were identified in maize and in other species, including other plants, fungi, and animals (see refs. 2-5 for review). Recent findings demonstrated an essential role for RNAi in paramutation, as four cloned genes involved in paramutation encode proteins closely related to RNAi pathway components (6-9).Herein we focus on paramutation at b1, a locus that encodes a transcription factor required to activate the purple anthocyanin pigment biosynthetic pathway. Two epialleles that participate in paramutation, B-I and B′, have the same DNA sequences (10) but distinct epigenetic states (11). B-I has a high transcription rate, resulting in homozygous dark purple plants, whereas B′ plants are lightly pigmented because of a reduced transcription rate. In heterozygous plants, B-I is always changed (paramutated) to B′. The new B′ epiallele is fully capable of paramutating naïve B-I epialleles in subsequent generations (11). The B′ epiallele is extremely stable; changes from B′ to B-I have never been observed in wild-type genetic backgrounds. In contrast, B-I is unstable and can spontaneously change to B′ at variable frequencies of 0.1-10% (11, 12).The key sequences required for b1 paramutation are seven unique tandem repeats of an 853-bp noncoding DNA located ∼100 kb upstream of the b1 transcription start site (10). Although this sequence and the number of repeats are identical in B-I and B′ epialleles, the DNA methylation pattern and chromatin structure d...

RNA-mediated trans -communication can establish paramutation at the b1 locus in maize

“…Although the B′* state established by the transgene is not as paramutagenic as "natural" paramutation, it is similar to that observed for B′ alleles with fewer repeats (20), the state established by the binding of the CBBP protein (11), and most other paramutation systems, which are often not 100% efficient (46). Thus, the generation of a hairpin dsRNA or siRNAs from the b1TR sequence can establish a heritable chromatin state in trans at the endogenous locus.…”

required to maintain repression2 Is a Novel Protein That Facilitates Locus-Specific Paramutation in Maize

“…Perhaps the CTR of RMR2 directs nucleic acid binding, while the poorly conserved N-terminal region coordinates contacts with other peptides. These speculative assignments remain to be tested, but interestingly, a DNA binding protein associating with the B1-I upstream repeats appears to play a role in facilitating paramutation at b1 (Brzeska et al, 2010). In A. thaliana, a DNA binding protein (SSH1/DTF1) required for both DNA methylation-dependent and methylation-independent gene silencing (Liu et al, 2011) was recently found in coimmunoprecipitations with NRPD1 (Law et al, 2011).…”

Noncoding RNA and Polycomb recruitment

“…This interaction was mapped to a specific domain in CLF, the CXC domain, found in a number of other plant proteins. Although the function of this domain has not been extensively characterized, a CXC protein within the maize protein CBBP, that has a role in paramutation, was found to mediate DNA binding (Brzeska et al 2010).…”