lncRNA LINC00941 modulates MTA2/NuRD occupancy to suppress premature human epidermal differentiation.

Numerous long non-coding RNAs (lncRNAs) were shown to have a functional impact on cellular processes such as human epidermal homeostasis. However, the mechanism of action for many lncRNAs remains unclear to date. Here, we report that lncRNA LINC00941 regulates keratinocyte differentiation on an epigenetic level through association with the NuRD complex, one of the major chromatin remodelers in cells. We find that LINC00941 interacts with NuRD-associated MTA2 and CHD4 in human primary keratinocytes. LINC00941 perturbation changes MTA2/NuRD occupancy at bivalent chromatin domains in close proximity to transcriptional regulator genes, including the EGR3 gene coding for a transcription factor regulating epidermal differentiation. Notably, LINC00941 depletion resulted in reduced NuRD occupancy at the EGR3 gene locus, increased EGR3 expression in human primary keratinocytes, and increased abundance of EGR3-regulated epidermal differentiation genes in cells and human organotypic epidermal tissues. Our results therefore indicate a role of LINC00941/NuRD in repressing EGR3 expression in non-differentiated keratinocytes, consequentially preventing premature differentiation of human epidermal tissues.

Heat stress at the bicellular stage inhibits sperm cell development and transport into pollen tubes.

For successful double fertilization in flowering plants (angiosperms), pollen tubes deliver two non-motile sperm cells towards female gametes (egg and central cell, respectively). Heatwaves, especially during the reproduction period, threaten male gametophyte (pollen) development, resulting in severe yield losses. Using maize (Zea mays) as a crop and grass model system, we found strong seed set reduction when moderate heat stress was applied for two days during the uni- and bicellular stages of pollen development. We show that heat stress accelerates pollen development and impairs pollen germination capabilities when applied at the unicellular stage. Heat stress at the bicellular stage impairs sperm cell development and transport into pollen tubes. To understand the course of the latter defects, we used marker lines and analyzed the transcriptomes of isolated sperm cells. Heat stress affected the expression of genes associated with transcription, RNA processing and translation, DNA replication, and the cell cycle. This included the genes encoding centromeric histone 3 (CENH3) and α-tubulin. Most genes that were mis-regulated encode proteins involved in the transition from metaphase to anaphase during pollen mitosis II (PM II). Heat stress also activated spindle assembly check point and meta- to anaphase transition genes in sperm cells. In summary, mis-regulation of the identified genes during heat stress at the bicellular stage results in sperm cell development and transport defects ultimately leading to sterility.

Cytosolic RGG RNA-binding proteins are temperature sensitive flowering time regulators in Arabidopsis.

mRNA translation is tightly regulated by various classes of RNA-binding proteins (RBPs) during development and in response to changing environmental conditions. In this study, we characterize the arginine-glycine-glycine (RGG) motif containing RBP family of Arabidopsis thaliana representing homologues of the multifunctional translation regulators and ribosomal preservation factors Stm1 from yeast (ScStm1) and human SERBP1 (HsSERBP1). The Arabidopsis genome encodes three RGG proteins named AtRGGA, AtRGGB and AtRGGC. While AtRGGA is ubiquitously expressed, AtRGGB and AtRGGC are enriched in dividing cells. All AtRGGs localize almost exclusively to the cytoplasm and bind with high affinity to ssRNA, while being capable to interact with most nucleic acids, except dsRNA. A protein-interactome study shows that AtRGGs interact with ribosomal proteins and proteins involved in RNA processing and transport. In contrast to ScStm1, AtRGGs are enriched in ribosome-free fractions in polysome profiles, suggesting additional plant-specific functions. Mutant studies show that AtRGG proteins differentially regulate flowering time, with a distinct and complex temperature dependency for each AtRGG protein. In conclusion, we suggest that AtRGGs function in fine-tuning translation efficiency to control flowering time and potentially other developmental processes in response to environmental changes.

Casein kinase 1 and 2 phosphorylate Argonaute proteins to regulate miRNA-mediated gene silencing.

MicroRNAs (miRNAs) together with Argonaute (AGO) proteins form the core of the RNA-induced silencing complex (RISC) to regulate gene expression of their target RNAs post-transcriptionally. Argonaute proteins are subjected to intensive regulation via various post-translational modifications that can affect their stability, silencing efficacy and specificity for targeted gene regulation. We report here that in Caenorhabditis elegans, two conserved serine/threonine kinases - casein kinase 1 alpha 1 (CK1A1) and casein kinase 2 (CK2) - regulate a highly conserved phosphorylation cluster of 4 Serine residues (S988:S998) on the miRNA-specific AGO protein ALG-1. We show that CK1A1 phosphorylates ALG-1 at sites S992 and S995, while CK2 phosphorylates ALG-1 at sites S988 and S998. Furthermore, we demonstrate that phospho-mimicking mutants of the entire S988:S998 cluster rescue the various developmental defects observed upon depleting CK1A1 and CK2. In humans, we show that CK1A1 also acts as a priming kinase of this cluster on AGO2. Altogether, our data suggest that phosphorylation of AGO within the cluster by CK1A1 and CK2 is required for efficient miRISC-target RNA binding and silencing.

The archaeal Lsm protein from Pyrococcus furiosus binds co-transcriptionally to poly(U)-rich target RNAs.

Posttranscriptional processes in Bacteria include the association of small regulatory RNAs (sRNA) with a target mRNA. The sRNA/mRNA annealing process is often mediated by an RNA chaperone called Hfq. The functional role of bacterial and eukaryotic Lsm proteins is partially understood, whereas knowledge about archaeal Lsm proteins is scarce. Here, we used the genetically tractable archaeal hyperthermophile Pyrococcus furiosus to identify the protein interaction partners of the archaeal Sm-like proteins (PfuSmAP1) using mass spectrometry and performed a transcriptome-wide binding site analysis of PfuSmAP1. Most of the protein interaction partners we found are part of the RNA homoeostasis network in Archaea including ribosomal proteins, the exosome, RNA-modifying enzymes, but also RNA polymerase subunits, and transcription factors. We show that PfuSmAP1 preferentially binds messenger RNAs and antisense RNAs recognizing a gapped poly(U) sequence with high affinity. Furthermore, we found that SmAP1 co-transcriptionally associates with target RNAs. Our study reveals that in contrast to bacterial Hfq, PfuSmAP1 does not affect the transcriptional activity or the pausing behaviour of archaeal RNA polymerases. We propose that PfuSmAP1 recruits antisense RNAs to target mRNAs and thereby executes its putative regulatory function on the posttranscriptional level.

RBPome identification in egg-cell like callus of Arabidopsis.

RNA binding proteins (RBPs) have multiple and essential roles in transcriptional and posttranscriptional regulation of gene expression in all living organisms. Their biochemical identification in the proteome of a given cell or tissue requires significant protein amounts, which limits studies in rare and highly specialized cells. As a consequence, we know almost nothing about the role(s) of RBPs in reproductive processes such as egg cell development, fertilization and early embryogenesis in flowering plants. To systematically identify the RBPome of egg cells in the model plant Arabidopsis, we performed RNA interactome capture (RIC) experiments using the egg cell-like RKD2-callus and were able to identify 728 proteins associated with poly(A+)-RNA. Transcripts for 97 % of identified proteins could be verified in the egg cell transcriptome. 46 % of identified proteins can be associated with the RNA life cycle. Proteins involved in mRNA binding, RNA processing and metabolism are highly enriched. Compared with the few available RBPome datasets of vegetative plant tissues, we identified 475 egg cell-enriched RBPs, which will now serve as a resource to study RBP function(s) during egg cell development, fertilization and early embryogenesis. First candidates were already identified showing an egg cell-specific expression pattern in ovules.

Characterization of the nuclear import of the human CHD4-NuRD complex.

Chromatin remodeling enzymes form large multiprotein complexes that play central roles in regulating access to the genome. Here, we characterize the nuclear import of the human CHD4 protein. We show that CHD4 enters the nucleus by means of several importin-α proteins (1, 5, 6 and 7), but independently of importin ß1. Importin α1 directly interacts with a monopartite 'KRKR'-motif in the N-terminus of CHD4 (amino acids 304-307). However, alanine mutagenesis of this motif only leads to an ∼50% reduction in nuclear localization of CHD4, implying that there are additional import mechanisms. Interestingly, we could show that CHD4 was already associated with the nucleosome remodeling deacetylase (NuRD) core subunits, such as MTA2, HDAC1 and RbAp46 (also known as RBBP7), in the cytoplasm, suggesting an assembly of the NuRD core complex before nuclear import. We propose that, in addition to the importin-α-dependent nuclear localization signal, CHD4 is dragged into the nucleus by a 'piggyback' mechanism using the import signals of the associated NuRD subunits.

Elongation factor 1 is a component of the Arabidopsis RNA polymerase II elongation complex and associates with a subset of transcribed genes.

Elongation factors modulate the efficiency of mRNA synthesis by RNA polymerase II (RNAPII) in the context of chromatin, thus contributing to implement proper gene expression programmes. The zinc-finger protein elongation factor 1 (ELF1) is a conserved transcript elongation factor (TEF), whose molecular function so far has not been studied in plants. Using biochemical approaches, we examined the interaction of Arabidopsis ELF1 with DNA and histones in vitro and with the RNAPII elongation complex in vivo. In addition, cytological assays demonstrated the nuclear localisation of the protein, and by means of double-mutant analyses, interplay with genes encoding other elongation factors was explored. The genome-wide distribution of ELF1 was addressed by chromatin immunoprecipitation. ELF1 isolated from Arabidopsis cells robustly copurified with RNAPII and various other elongation factors including SPT4-SPT5, SPT6, IWS1, FACT and PAF1C. Analysis of a CRISPR-Cas9-mediated gene editing mutant of ELF1 revealed distinct genetic interactions with mutants deficient in other elongation factors. Moreover, ELF1 associated with genomic regions actively transcribed by RNAPII. However, ELF1 occupied only c. 33% of the RNAPII transcribed loci with preference for inducible rather than constitutively expressed genes. Collectively, these results establish that Arabidopsis ELF1 shares several characteristic attributes with RNAPII TEFs.

A specific type of Argonaute phosphorylation regulates binding to microRNAs during C. elegans development.

Argonaute proteins are at the core of the microRNA-mediated gene silencing pathway essential for animals. In C. elegans, the microRNA-specific Argonautes ALG-1 and ALG-2 regulate multiple processes required for proper animal developmental timing and viability. Here we identified a phosphorylation site on ALG-1 that modulates microRNA association. Mutating ALG-1 serine 642 into a phospho-mimicking residue impairs microRNA binding and causes embryonic lethality and post-embryonic phenotypes that are consistent with alteration of microRNA functions. Monitoring microRNA levels in alg-1 phosphorylation mutant animals shows that microRNA passenger strands increase in abundance but are not preferentially loaded into ALG-1, indicating that the miRNA binding defects could lead to microRNA duplex accumulation. Our genetic and biochemical experiments support protein kinase A (PKA) KIN-1 as the putative kinase that phosphorylates ALG-1 serine 642. Our data indicate that PKA triggers ALG-1 phosphorylation to regulate its microRNA association during C. elegans development.

The human RNA polymerase I structure reveals an HMG-like docking domain specific to metazoans.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth, and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk, and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This "dock II" domain resembles a truncated HMG box incapable of DNA binding which may serve as a downstream transcription factor-binding platform in metazoans. Biochemical analysis, in situ modelling, and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG box domain-containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

Retinoschisin and novel Na/K-ATPase interaction partners Kv2.1 and Kv8.2 define a growing protein complex at the inner segments of mammalian photoreceptors.

The RS1 gene on Xp 22.13 encodes retinoschisin which is known to directly interact with the retinal Na/K-ATPase at the photoreceptor inner segments. Pathologic mutations in RS1 cause X-linked juvenile retinoschisis (XLRS), a hereditary retinal dystrophy in young males. To further delineate the retinoschisin-Na/K-ATPase complex, co-immunoprecipitation was performed with porcine and murine retinal lysates targeting the ATP1A3 subunit. This identified the voltage-gated potassium (Kv) channel subunits Kv2.1 and Kv8.2 as direct interaction partners of the retinal Na/K-ATPase. Colocalization of the individual components of the complex was demonstrated at the membrane of photoreceptor inner segments. We further show that retinoschisin-deficiency, a frequent consequence of molecular pathology in XLRS, causes mislocalization of the macromolecular complex during postnatal retinal development with a simultaneous reduction of Kv2.1 and Kv8.2 protein expression, while the level of retinal Na/K-ATPase expression remains unaffected. Patch-clamp analysis revealed no effect of retinoschisin-deficiency on Kv channel mediated potassium ion currents in vitro. Together, our data suggest that Kv2.1 and Kv8.2 together with retinoschisin and the retinal Na/K-ATPase are integral parts of a macromolecular complex at the photoreceptor inner segments. Defective compartmentalization of this complex due to retinoschisin-deficiency may be a crucial step in initial XLRS pathogenesis.

Photoswitching of Feedback Inhibition by Tryptophan in Anthranilate Synthase.

The artificial regulation of enzymatic activity by light is an important goal of synthetic biology that can be achieved by the incorporation of light-responsive noncanonical amino acids via genetic code expansion. Here, we apply this concept to anthranilate synthase from Salmonella typhimurium (stTrpE). This enzyme catalyzes the first step of tryptophan biosynthesis, and its activity is feedback-inhibited by the binding of the end-product of the pathway to an allosteric site. To put this feedback inhibition of stTrpE by tryptophan under the control of light, we individually replaced 15 different amino acid residues with the photosensitive noncanonical amino acid o-nitrobenzyl-O-tyrosine (ONBY). ONBY contains a sterically demanding caging group that was meant to cover the allosteric site. Steady-state enzyme kinetics showed that the negative effect of tryptophan on the catalytic activity of the two variants stTrpE-K50ONBY and stTrpE-Y455ONBY was diminished compared to the wild-type enzyme by 1 to 2 orders of magnitude. Upon light-induced decaging of ONBY to the less space-consuming tyrosine residue, tryptophan binding to the allosteric site was restored and catalytic activity was inhibited almost as efficiently as observed for wild-type stTrpE. Based on these results, direct photocontrol of feedback inhibition of stTrpE-K50ONBY and stTrpE-Y455ONBY could be achieved by irradiation during the reaction. Molecular modeling studies allowed us to rationalize the observed functional conversion from the noninhibited caged to the tryptophan-inhibited decaged states. Our study shows that feedback inhibition, which is an important mechanism to regulate key metabolic enzymes, can be efficiently controlled by the purposeful use of light-responsive noncanonical amino acids.

Molecular insights into intra-complex signal transmission during stressosome activation.

The stressosome is a pseudo-icosahedral megadalton bacterial stress-sensing protein complex consisting of several copies of two STAS-domain proteins, RsbR and RsbS, and the kinase RsbT. Upon perception of environmental stress multiple copies of RsbT are released from the surface of the stressosome. Free RsbT activates downstream proteins to elicit a global cellular response, such as the activation of the general stress response in Gram-positive bacteria. The molecular events triggering RsbT release from the stressosome surface remain poorly understood. Here we present the map of Listeria innocua RsbR1/RsbS complex at resolutions of 3.45 Å for the STAS domain core in icosahedral symmetry and of 3.87 Å for the STAS domain and N-terminal sensors in D2 symmetry, respectively. The structure reveals a conformational change in the STAS domain linked to phosphorylation in RsbR. Docking studies indicate that allosteric RsbT binding to the conformationally flexible N-terminal sensor domain of RsbR affects the affinity of RsbS towards RsbT. Our results bring to focus the molecular events within the stressosome complex and further our understanding of this ubiquitous signaling hub.

H3K9 demethylases IBM1 and JMJ27 are required for male meiosis in Arabidopsis thaliana.

Dimethylation of histone H3 lysine 9 (H3K9me2), a crucial modification for heterochromatin formation and transcriptional silencing, is essential for proper meiotic prophase progression in mammals. We analyzed meiotic defects and generated genome-wide profiles of H3K9me2 and transcriptomes for the mutants of H3K9 demethylases. Moreover, we also identified proteins interacting with H3K9 demethylases. H3K9me2 is usually found at transposable elements and repetitive sequences but is absent from the bodies of protein-coding genes. In this study, we show that the Arabidopsis thaliana H3K9 demethylases IBM1 and JMJ27 cooperatively regulate crossover formation and chromosome segregation. They protect thousands of protein-coding genes from ectopic H3K9me2, including genes essential for meiotic prophase progression. In addition to removing H3K9me2, IBM1 and JMJ27 interact with the Precocious Dissociation of Sisters 5 (PDS5) cohesin complex cofactors. The pds5 mutant shared similar transcriptional alterations with ibm1 jmj27, including meiosis-essential genes, yet without affecting H3K9me2 levels. Hence, PDS5s, together with IBM1 and JMJ27, regulate male meiosis and gene expression independently of H3K9 demethylation. These findings uncover a novel role of H3K9me2 removal in meiosis and a new function of H3K9 demethylases and cohesin cofactors in meiotic transcriptional regulation.

Phosphorylation of the FACT histone chaperone subunit SPT16 affects chromatin at RNA polymerase II transcriptional start sites in Arabidopsis.

The heterodimeric histone chaperone FACT, consisting of SSRP1 and SPT16, contributes to dynamic nucleosome rearrangements during various DNA-dependent processes including transcription. In search of post-translational modifications that may regulate the activity of FACT, SSRP1 and SPT16 were isolated from Arabidopsis cells and analysed by mass spectrometry. Four acetylated lysine residues could be mapped within the basic C-terminal region of SSRP1, while three phosphorylated serine/threonine residues were identified in the acidic C-terminal region of SPT16. Mutational analysis of the SSRP1 acetylation sites revealed only mild effects. However, phosphorylation of SPT16 that is catalysed by protein kinase CK2, modulates histone interactions. A non-phosphorylatable version of SPT16 displayed reduced histone binding and proved inactive in complementing the growth and developmental phenotypes of spt16 mutant plants. In plants expressing the non-phosphorylatable SPT16 version we detected at a subset of genes enrichment of histone H3 directly upstream of RNA polymerase II transcriptional start sites (TSSs) in a region that usually is nucleosome-depleted. This suggests that some genes require phosphorylation of the SPT16 acidic region for establishing the correct nucleosome occupancy at the TSS of active genes.

Hepatocyte expressed chemerin-156 does not protect from experimental non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a rapidly growing liver disease. The chemoattractant chemerin is abundant in hepatocytes, and hepatocyte expressed prochemerin protected from NASH. Prochemerin is inactive and different active isoforms have been described. Here, the effect of hepatocyte expressed muChem-156, a highly active murine chemerin isoform, was studied in the methionine-choline deficient dietary model of NASH. Mice overexpressing muChem-156 had higher hepatic chemerin protein. Serum chemerin levels and the capability of serum to activate the chemerin receptors was unchanged showing that the liver did not release active chemerin. Notably, activation of the chemerin receptors by hepatic vein blood did not increase in parallel to total chemerin protein in patients with liver cirrhosis. In experimental NASH, muChem-156 had no effect on liver lipids. Accordingly, overexpression of active chemerin in hepatocytes or treatment of hepatocytes with recombinant chemerin did not affect cellular triglyceride and cholesterol levels. Importantly, overexpression of muChem-156 in the murine liver did not change the hepatic expression of inflammatory and profibrotic genes. The downstream targets of chemerin such as p38 kinase were neither activated in the liver of muChem-156 producing mice nor in HepG2, Huh7 and Hepa1-6 cells overexpressing this isoform. Recombinant chemerin had no effect on global gene expression of primary human hepatocytes and hepatic stellate cells within 24 h of incubation. Phosphorylation of p38 kinase was, however, increased upon short-time incubation of HepG2 cells with chemerin. These findings show that muChem-156 overexpression in hepatocytes does not protect from liver steatosis and inflammation.

Chemerin Overexpression in the Liver Protects against Inflammation in Experimental Non-Alcoholic Steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is marked by macrophage infiltration and inflammation. Chemerin is a chemoattractant protein and is abundant in hepatocytes. The aim of this study was to gain insight into the role of hepatocyte-produced prochemerin in NASH. Therefore, mice were infected with adeno-associated virus 8 to direct hepatic overexpression of prochemerin in a methionine-choline deficient dietary model of NASH. At the end of the study, hepatic and serum chemerin were higher in the chemerin-expressing mice. These animals had less hepatic oxidative stress, F4/80 and CC-chemokine ligand 2 (CCL2) protein, and mRNA levels of inflammatory genes than the respective control animals. In order to identify the underlying mechanisms, prochemerin was expressed in hepatocytes and the hepatic stellate cells, LX-2. Here, chemerin had no effect on cell viability, production of inflammatory, or pro-fibrotic factors. Notably, cultivation of human peripheral blood mononuclear cells (PBMCs) in the supernatant of Huh7 cells overexpressing chemerin reduced CCL2, interleukin-6, and osteopontin levels in cell media. CCL2 was also low in RAW264.7 cells exposed to Hepa1-6 cell produced chemerin. In summary, the current study showed that prochemerin overexpression had little effect on hepatocytes and hepatic stellate cells. Of note, hepatocyte-produced chemerin deactivated PBMCs and protected against inflammation in experimental NASH.

Controlling Enzymatic Activity by Modulating the Oligomerization State via Chemical Rescue and Optical Control.

Selective switching of enzymatic activity has been a longstanding goal in synthetic biology. Drastic changes in activity upon mutational manipulation of the oligomerization state of enzymes have frequently been reported in the literature, but scarcely exploited for switching. Using geranylgeranylglyceryl phosphate synthase as a model, we demonstrate that catalytic activity can be efficiently controlled by exogenous modulation of the association state. We introduced a lysine-to-cysteine mutation, leading to the breakdown of the active hexamer into dimers with impaired catalytic efficiency. Addition of bromoethylamine chemically rescued the enzyme by restoring hexamerization and activity. As an alternative method, we incorporated the photosensitive unnatural amino acid o-nitrobenzyl-O-tyrosine (ONBY) into the hexamerization interface. This again led to inactive dimers, but the hexameric state and activity could be recovered by UV-light induced cleavage of ONBY. For both approaches, we obtained switching factors greater than 350-fold, which compares favorably with previously reported activity changes that were caused by site-directed mutagenesis.

Balancing of mitochondrial translation through METTL8-mediated m3C modification of mitochondrial tRNAs.

Mitochondria contain a specific translation machinery for the synthesis of mitochondria-encoded respiratory chain components. Mitochondrial tRNAs (mt-tRNAs) are also generated from the mitochondrial DNA and, similar to their cytoplasmic counterparts, are post-transcriptionally modified. Here, we find that the RNA methyltransferase METTL8 is a mitochondrial protein that facilitates 3-methyl-cytidine (m3C) methylation at position C32 of the mt-tRNASer(UCN) and mt-tRNAThr. METTL8 knockout cells show a reduction in respiratory chain activity, whereas overexpression increases activity. In pancreatic cancer, METTL8 levels are high, which correlates with lower patient survival and an enhanced respiratory chain activity. Mitochondrial ribosome profiling uncovered mitoribosome stalling on mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons. Further analysis of the respiratory chain complexes using mass spectrometry revealed reduced incorporation of the mitochondrially encoded proteins ND6 and ND1 into complex I. The well-balanced translation of mt-tRNASer(UCN)- and mt-tRNAThr-dependent codons through METTL8-mediated m3C32 methylation might, therefore, facilitate the optimal composition and function of the mitochondrial respiratory chain.

The Arabidopsis condensin CAP-D subunits arrange interphase chromatin.

Condensins are best known for their role in shaping chromosomes. Other functions such as organizing interphase chromatin and transcriptional control have been reported in yeasts and animals, but little is known about their function in plants. To elucidate the specific composition of condensin complexes and the expression of CAP-D2 (condensin I) and CAP-D3 (condensin II), we performed biochemical analyses in Arabidopsis. The role of CAP-D3 in interphase chromatin organization and function was evaluated using cytogenetic and transcriptome analysis in cap-d3 T-DNA insertion mutants. CAP-D2 and CAP-D3 are highly expressed in mitotically active tissues. In silico and pull-down experiments indicate that both CAP-D proteins interact with the other condensin I and II subunits. In cap-d3 mutants, an association of heterochromatic sequences occurs, but the nuclear size and the general histone and DNA methylation patterns remain unchanged. Also, CAP-D3 influences the expression of genes affecting the response to water, chemicals, and stress. The expression and composition of the condensin complexes in Arabidopsis are similar to those in other higher eukaryotes. We propose a model for the CAP-D3 function during interphase in which CAP-D3 localizes in euchromatin loops to stiffen them and consequently separates centromeric regions and 45S rDNA repeats.