Partial cytological diploidization of neoautotetraploid meiosis by induced cross-over rate reduction.

Polyploids, which arise from whole-genome duplication events, have contributed to genome evolution throughout eukaryotes. Among plants, novel features of neopolyploids include traits that can be evolutionarily or agriculturally beneficial, such as increased abiotic stress tolerance. Thus, in addition to being interesting from an evolutionary perspective, genome duplication is also increasingly recognized as a promising crop improvement tool. However, newly formed (neo)polyploids commonly suffer from fertility problems, which have been attributed to abnormal associations among the multiple homologous chromosome copies during meiosis (multivalents). Here, we test the long-standing hypothesis that reducing meiotic cross-over number may be sufficient to limit multivalent formation, favoring diploid-like bivalent associations (cytological diploidization). To do so, we developed Arabidopsis thaliana lines with low cross-over rates by combining mutations for HEI10 and TAF4b. Double mutants showed a reduction of ~33% in cross-over numbers in diploids without compromising meiotic stability. Neopolyploids derived from the double mutant show a cross-over rate reduction of about 40% relative to wild-type neotetraploids, and groups of four homologs indeed formed fewer multivalents and more bivalents. However, we also show that the reduction in multivalents comes with the cost of a slightly increased frequency of univalents and that it does not rescue neopolyploid fertility. Thus, while our results do show that reducing cross-over rates can reduce multivalent frequency in neopolyploids, they also emphasize that there are additional factors affecting both meiotic stability and neopolyploid fertility that will need to be considered in solving the neopolyploid fertility challenge.

Development and Clinical Validation of the LymphMonitor Technology to Quantitatively Assess Lymphatic Function.

Current diagnostic methods for evaluating the functionality of the lymphatic vascular system usually do not provide quantitative data and suffer from many limitations including high costs, complexity, and the need to perform them in hospital settings. In this work, we present a quantitative, simple outpatient technology named LymphMonitor to quantitatively assess lymphatic function. This method is based on the painless injection of the lymphatic-specific near-infrared fluorescent tracer indocyanine green complexed with human serum albumin, using MicronJet600TM microneedles, and monitoring the disappearance of the fluorescence signal at the injection site over time using a portable detection device named LymphMeter. This technology was investigated in 10 patients with unilateral leg or arm lymphedema. After injection of a tracer solution into each limb, the signal was measured over 3 h and the area under the normalized clearance curve was calculated to quantify the lymphatic function. A statistically significant difference in lymphatic clearance in the healthy versus the lymphedema extremities was found, based on the obtained area under curves of the normalized clearance curves. This study provides the first evidence that the LymphMonitor technology has the potential to diagnose and monitor the lymphatic function in patients.

Interaction of the NRF2 and p63 transcription factors promotes keratinocyte proliferation in the epidermis.

Epigenetic regulation of cell and tissue function requires the coordinated action of transcription factors. However, their combinatorial activities during regeneration remain largely unexplored. Here, we discover an unexpected interaction between the cytoprotective transcription factor NRF2 and p63- a key player in epithelial morphogenesis. Chromatin immunoprecipitation combined with sequencing and reporter assays identifies enhancers and promoters that are simultaneously activated by NRF2 and p63 in human keratinocytes. Modeling of p63 and NRF2 binding to nucleosomal DNA suggests their chromatin-assisted interaction. Pharmacological and genetic activation of NRF2 increases NRF2-p63 binding to enhancers and promotes keratinocyte proliferation, which involves the common NRF2-p63 target cyclin-dependent kinase 12. These results unravel a collaborative function of NRF2 and p63 in the control of epidermal renewal and suggest their combined activation as a strategy to promote repair of human skin and other stratified epithelia.

A Modular Ligation Strategy for Asymmetric Bivalent Nucleosomes Trimethylated at K36 and K27.

In nature, individual histones in the same nucleosome can carry identical (symmetric) or different (asymmetric) post-translational modification (PTM) patterns, increasing the combinatorial complexity. Embryonic stem cells exhibit "bivalent" nucleosomes, some of which are marked by an asymmetric arrangement of H3K36me3 (an activating PTM) and H3K27me3 (a repressive PTM). Here we describe a modular synthetic method to access such asymmetrically modified nucleosomes and show that H3K36me3 inhibits the activity of the methyltransferase PRC2 locally while still prolonging its chromatin binding time.

Engineered Multivalent Sensors to Detect Coexisting Histone Modifications in Living Stem Cells.

The regulation of fundamental processes such as gene expression or cell differentiation involves chromatin states, demarcated by combinatorial histone post-translational modification (PTM) patterns. The subnuclear organization and dynamics of chromatin states is not well understood, as tools for their detection and modulation in live cells are lacking. Here, we report the development of genetically encoded chromatin-sensing multivalent probes, cMAPs, selective for bivalent chromatin, a PTM pattern associated with pluripotency in embryonic stem cells (ESCs). cMAPs were engineered from a set of PTM-binding (reader) proteins and optimized using synthetic nucleosomes carrying defined PTMs. Applied in live ESCs, cMAPs formed discrete subnuclear foci, revealing the organization of bivalent chromatin into local clusters. Moreover, cMAPs enabled direct monitoring of the loss of bivalency upon treatment with small-molecule epigenetic modulators. cMAPs thus provide a versatile platform to monitor chromatin state dynamics in live cells.

DNA binding by PHF1 prolongs PRC2 residence time on chromatin and thereby promotes H3K27 methylation.

Polycomb repressive complex 2 (PRC2) trimethylates histone H3 at lysine 27 to mark genes for repression. We measured the dynamics of PRC2 binding on recombinant chromatin and free DNA at the single-molecule level using total internal reflection fluorescence (TIRF) microscopy. PRC2 preferentially binds free DNA with multisecond residence time and midnanomolar affinity. PHF1, a PRC2 accessory protein of the Polycomblike family, extends PRC2 residence time on DNA and chromatin. Crystallographic and functional studies reveal that Polycomblike proteins contain a winged-helix domain that binds DNA in a sequence-nonspecific fashion. DNA binding by this winged-helix domain accounts for the prolonged residence time of PHF1-PRC2 on chromatin and makes it a more efficient H3K27 methyltranferase than PRC2 alone. Together, these studies establish that interactions with DNA provide the predominant binding affinity of PRC2 for chromatin. Moreover, they reveal the molecular basis for how Polycomblike proteins stabilize PRC2 on chromatin and stimulate its activity.

Single-molecule kinetic analysis of HP1-chromatin binding reveals a dynamic network of histone modification and DNA interactions.

Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required.

ß-Carboline Amides as Intrinsic Directing Groups for C(sp2)-H Functionalization.

Many site-selective palladium-catalyzed C-H functionalization methods require directing groups. We report here ß-carboline amides as intrinsic directing groups for C(sp2)-H functionalization. Various substrates including the natural product alangiobussinine and the marinacarboline core structure were functionalized using carboline-directed δ-C(sp2)-H alkynylations. This transformation proceeds under mild conditions and is compatible with a wide variety of ß-arylethamines. δ-Alkynylation of ß-arylethamines via a six-membered palladacycle is favored over γ-C(sp2)-H bond functionalization when both positions are accessible. The versatility of ß-carboline amides as directing groups is evidenced by other δ-C(sp2)-H functionalizations such as alkenylation, arylation, and C-N bond formation.

Multivalency governs HP1α association dynamics with the silent chromatin state.

Multivalent interactions between effector proteins and histone post-translational modifications are an elementary mechanism of dynamic chromatin signalling. Here we elucidate the mechanism how heterochromatin protein 1α (HP1α), a multivalent effector, is efficiently recruited to the silent chromatin state (marked by trimethylated H3 at Lys9, H3K9me3) while remaining highly dynamic. Employing chemically defined nucleosome arrays together with single-molecule total internal reflection fluorescence microscopy (smTIRFM), we demonstrate that the HP1α residence time on chromatin depends on the density of H3K9me3, as dissociated factors can rapidly rebind at neighbouring sites. Moreover, by chemically controlling HP1α dimerization we find that effector multivalency prolongs chromatin retention and, importantly, accelerates the association rate. This effect results from increased avidity together with strengthened nonspecific chromatin interactions of dimeric HP1α. We propose that accelerated chromatin binding is a key feature of effector multivalency, allowing for fast and efficient competition for binding sites in the crowded nuclear compartment.

A novel Nrf2-miR-29-desmocollin-2 axis regulates desmosome function in keratinocytes.

The Nrf2 transcription factor controls the expression of genes involved in the antioxidant defense system. Here, we identified Nrf2 as a novel regulator of desmosomes in the epidermis through the regulation of microRNAs. On Nrf2 activation, expression of miR-29a and miR-29b increases in cultured human keratinocytes and in mouse epidermis. Chromatin immunoprecipitation identified the Mir29ab1 and Mir29b2c genes as direct Nrf2 targets in keratinocytes. While binding of Nrf2 to the Mir29ab1 gene activates expression of miR-29a and -b, the Mir29b2c gene is silenced by DNA methylation. We identified desmocollin-2 (Dsc2) as a major target of Nrf2-induced miR-29s. This is functionally important, since Nrf2 activation in keratinocytes of transgenic mice causes structural alterations of epidermal desmosomes. Furthermore, the overexpression of miR-29a/b or knockdown of Dsc2 impairs the formation of hyper-adhesive desmosomes in keratinocytes, whereas Dsc2 overexpression has the opposite effect. These results demonstrate that a novel Nrf2-miR-29-Dsc2 axis controls desmosome function and cutaneous homeostasis.