Fluorescent proteins with excitation and emission maxima in the near-infrared would be especially useful because of the low light absorbance of mammalian tissues in this optical window. Verkhusha and colleagues present a bright, photostable fluorescent protein with optimized near-infrared spectra that does not require the addition of exogenous co-factors. Imaging biological processes in mammalian tissues will be facilitated by fluorescent probes with excitation and emission bands within the near-infrared optical window of high transparency1. Here we report a phytochrome-based near-infrared fluorescent protein (iRFP) with excitation and emission maxima at 690 nm and 713 nm, respectively. iRFP does not require an exogenous supply of the chromophore biliverdin and has higher effective brightness, intracellular stability and photostability than earlier phytochrome-derived fluorescent probes. Compared with far-red GFP-like proteins, iRFP has a substantially higher signal-to-background ratio in a mouse model due to its infrared-shifted spectra.

Four papers in this issue tackle the hot topic of chromatin remodelling, specifically, how methyl marks on chromatin are 'read' by the proteins that interact with them. Two report on BPTF (bromodomain and PHD domain transcription factor), a subunit of NURF, the nucleosome remodelling factor. It contains a domain known as a PHD finger, which is shown to bind to histone H3 trimethylated at lysine 4 (H3K4) and to maintain proper activity at developmentally critical HOX genes. The accompanying structural study of the complex explains how the site specificity for H3K4 is achieved. The two other papers reveal that the PHD domain of tumour suppressor ING2 also recognizes trimethylated H3K4, and link the histone mark to repression of transcription. The four papers together establish certain PHD finger domains as previously unrecognized chromatin-binding modules. In a News and Views piece, Peter B. Becker discusses what these papers tell us about the function of the chemical modifications of histone tails. One of four papers establishing certain PHD domains as effectors of trimethylated histone H3K4, a chromatin mark generally associated with active transcription. This paper describes the structure of the PHD finger from the tumour suppressor ING2 complexed with H3K4me3. Covalent modifications of histone tails have a key role in regulating chromatin structure and controlling transcriptional activity. In eukaryotes, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with active chromatin and gene expression1,2,3,4. We recently found that plant homeodomain (PHD) finger of tumour suppressor ING2 (inhibitor of growth 2) binds H3K4me3 and represents a new family of modules that target this epigenetic mark5. The molecular mechanism of H3K4me3 recognition, however, remains unknown. Here we report a 2.0 Å resolution structure of the mouse ING2 PHD finger in complex with a histone H3 peptide trimethylated at lysine 4. The H3K4me3 tail is bound in an extended conformation in a deep and extensive binding site consisting of elements that are conserved among the ING family of proteins. The trimethylammonium group of Lys 4 is recognized by the aromatic side chains of Y215 and W238 residues, whereas the intermolecular hydrogen-bonding and complementary surface interactions, involving Ala 1, Arg 2, Thr 3 and Thr 6 of the peptide, account for the PHD finger's high specificity and affinity. Substitution of the binding site residues disrupts H3K4me3 interaction in vitro and impairs the ability of ING2 to induce apoptosis in vivo. Strong binding of other ING and YNG PHD fingers suggests that the recognition of H3K4me3 histone code is a general feature of the ING/YNG proteins. Elucidation of the mechanisms underlying this novel function of PHD fingers provides a basis for deciphering the role of the ING family of tumour suppressors in chromatin regulation and signalling.

A vast colour palette of monomeric fluorescent proteins has been developed to investigate protein localization, motility and interactions. However, low brightness has remained a problem in far-red variants, which hampers multicolour labelling and whole-body imaging techniques. In the present paper, we report mKate2, a monomeric far-red fluorescent protein that is almost 3-fold brighter than the previously reported mKate and is 10-fold brighter than mPlum. The high-brightness, far-red emission spectrum, excellent pH resistance and photostability, coupled with low toxicity demonstrated in transgenic Xenopus laevis embryos, make mKate2 a superior fluorescent tag for imaging in living tissues. We also report tdKatushka2, a tandem far-red tag that performs well in fusions, provides 4-fold brighter near-IR fluorescence compared with mRaspberry or mCherry, and is 20-fold brighter than mPlum. Together, monomeric mKate2 and pseudo-monomeric tdKatushka2 represent the next generation of extra-bright far-red fluorescent probes offering novel possibilities for fluorescent imaging of proteins in living cells and animals.

Four spectrally distinct near-infrared fluorescent proteins based on bacterial phytochromes are described, expanding the possibilities for multicolor in vivo imaging experiments in nontransparent organisms. Near-infrared fluorescent proteins (FPs) are in high demand for in vivo imaging. We developed four spectrally distinct near-infrared FPs—iRFP670, iRFP682, iRFP702 and iRFP720—from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging with standard approaches in living cells and mice. The four new iRFPs and the previously engineered iRFP713 allow multicolor imaging with spectral unmixing in living mice.