We fused the epitope-recognizing fragment of heavy-chain antibodies from Camelidae sp. with fluorescent proteins to generate fluorescent, antigen-binding nanobodies (chromobodies) that can be expressed in living cells. We demonstrate that chromobodies can recognize and trace antigens in different subcellular compartments throughout S phase and mitosis. Chromobodies should enable new functional studies, as potentially any antigenic structure can be targeted and traced in living cells in this fashion.

It is well established that all camelids have unique antibodies circulating in their blood. Unlike antibodies from other species, these special antibodies are devoid of light chains and are composed of a heavy-chain homodimer. These so-called heavy-chain antibodies (HCAbs) are expressed after a V-D-J rearrangement and require dedicated constant gamma-genes. An immune response is raised in these so-called heavy-chain antibodies following classical immunization protocols. These HCAbs are easily purified from serum, and the antigen-binding fragment interacts with parts of the target that are less antigenic to conventional antibodies. Since the antigen-binding site of the dromedary HCAb is comprised in one single domain, referred to as variable domain of heavy chain of HCAb (VHH) or nanobody (Nb), we designed a strategy to clone the Nb repertoire of an immunized dromedary and to select the Nbs with specificity for our target antigens. The monoclonal Nbs are well produced in bacteria, are very stable and highly soluble, and bind their cognate antigen with high affinity and specificity. We have successfully developed recombinant Nbs for research purposes, as probe in biosensors, to diagnose infections, and to treat diseases like cancer or trypanosomosis.

Trithorax (Trx) is a member of the trithorax group (trxG) of epigenetic regulators, which is required to maintain active states of Hox gene expression during development. We have purified from Drosophila embryos a trithorax acetylation complex (TAC1) that contains Trx, dCBP, and Sbf1. Like CBP, TAC1 acetylates core histones in nucleosomes, suggesting that this activity may be important for epigenetic maintenance of gene activity. dCBP and Sbf1 associate with specific sites on salivary gland polytene chromosomes, colocalizing with many Trx binding sites. One of these is the site of the Hox gene Ultrabithorax (Ubx). Mutations in either trx or the gene encoding dCBP reduce expression of the endogenous Ubx gene as well as of transgenes driven by the bxd regulatory region of Ubx. Thus Trx, dCBP, and Sbf1 are closely linked, physically and functionally, in the maintenance of Hox gene expression.

Rapid induction of the Drosophila melanogaster heat shock gene hsp70 is achieved through the binding of heat shock factor (HSF) to heat shock elements (HSEs) located upstream of the transcription start site (reviewed in ref. 3). The subsequent recruitment of several other factors, including Spt5, Spt6 and FACT, is believed to facilitate Pol II elongation through nucleosomes downstream of the start site. Here, we report a novel mechanism of heat shock gene regulation that involves modifications of nucleosomes by the TAC1 histone modification complex. After heat stress, TAC1 is recruited to several heat shock gene loci, where its components are required for high levels of gene expression. Recruitment of TAC1 to the 5'-coding region of hsp70 seems to involve the elongating Pol II complex. TAC1 has both histone H3 Lys 4-specific (H3-K4) methyltransferase (HMTase) activity and histone acetyltransferase activity through Trithorax (Trx) and CREB-binding protein (CBP), respectively. Consistently, TAC1 is required for methylation and acetylation of nucleosomal histones in the 5'-coding region of hsp70 after induction, suggesting an unexpected role for TAC1 during transcriptional elongation.