Cyclommatus metallifer, focusing on doublesex gene function and its interaction with juvenile hormone (JH) signaling. doublesex genes encode transcription factors that orchestrate male and female specific trait development, and JH acts as a mediator between nutrition and mandible growth. We found that the Cmdsx gene regulates sex differentiation in the stag beetle. Knockdown of Cmdsx by RNA-interference in both males and females produced intersex phenotypes, indicating a role for Cmdsx in sex-specific trait growth. By combining knockdown of Cmdsx with JH treatment, we showed that female-specific splice variants of Cmdsx contribute to the insensitivity of female mandibles to JH: knockdown of Cmdsx reversed this pattern, so that mandibles in knockdown females were stimulated to grow by JH treatment. In contrast, mandibles in knockdown males retained some sensitivity to JH, though mandibles in these individuals did not attain the full sizes of wild type males. We suggest that moderate JH sensitivity of mandibular cells may be the default developmental state for both sexes, with sex-specific Dsx protein decreasing sensitivity in females, and increasing it in males. This study is the first to demonstrate a causal link between the sex determination and JH signaling pathways, which clearly interact to determine the developmental fates and final sizes of nutrition-dependent secondary-sexual characters.Sexual dimorphisms in trait expression are widespread among animals and are especially pronounced in ornaments and weapons of sexual selection, which can attain exaggerated sizes. Expression of exaggerated traits is usually male-specific and nutrition sensitive. Consequently, the developmental mechanisms generating sexually dimorphic growth and nutrition-dependent phenotypic plasticity are each likely to regulate the expression of extreme structures. Yet we know little about how either of these mechanisms work, much less how they might interact with each other. We investigated the developmental mechanisms of sex-specific mandible growth in the stag beetle  Cyclommatus metallifer. We examined the regulation and function of a key sex determination gene in animals, doublesex (dsx), and its interaction with juvenile hormone (JH), an important insect hormone known to regulate insect polyphenisms including the regulation of the disproportionate growth of male stag beetle mandibles. We found that Cmdsx changes mandibular responsiveness to JH in a sex-specific pattern. Based on these results, we hypothesize that sex-specific regulation of JH responsiveness is a developmental link between nutrition and sexual differentiation in stag beetles.Sexual dimorphisms such as the exaggerated antlers of deer, the enormous clawed chelae of crabs, and the horns and mandibles of beetles, are widespread across animal taxa and have fascinated biologists for centuries. Much recent work has uncovered the importance of the role of the sex-determination pathway in the expression of sexually dimorphic traits. However, critical interactions between this pathway and other growth regulatory mechanisms – for example, the physiological mechanisms involved in nutrition-dependent expression of these traits – are less well understood. In this study, we provide evidence of a developmental link between nutrition-sensitivity and sexual differentiation in the giant mandibles of the sexually dimorphic stag beetle,  The evolution of sex-specific traits in animals has long fascinated biologists. How is growth regulated so that it differs dramatically between males and females? Sexual dimorphisms are widespread across diverse animal taxa and include exaggerated sexually selected traits like the antlers of deer, the enormous clawed chelae of crabs, and the elaborate trains of peacocks Sex-specific exaggerated traits often develop in a condition-dependent manner, so that not all individuals produce the trait even in the same sex Doublesex (dsx) is conserved in structure and function in all insect species where it has been examined dsx gene is transcribed in both sexes, but then differentially spliced to produce a male-specific or a female-specific mRNA  and rhinoceros beetles demonstrate a functional role for dsx in sex-specific growth of horns Recent studies in model organisms such as the fruit fly, nematode, medaka fish, and mouse, implicate a group of highly conserved proteins known as DM, or DNA binding motif proteins, as major effectors of sexual differentiation  transcript was obtained by degenerate PCR and subsequent Rapid Amplification of cDNA Ends PCR (RACE-PCR). Expression analyses of Cmdsx were carried out by Reverse Transcription PCR (RT-PCR) and real-time quantitative PCR (qPCR) to reveal the spatio-temporal expression pattern and sex-specificity of the Cmdsx transcripts during the prepupal period, which is known to be the critical period when mandibular tissues proliferate to their final adult size (dsx during sex-specific morphogenesis was investigated by gene knockdown using RNA interference (RNAi) against the Cmdsx transcripts. In addition to this, to investigate the putative interaction between dsx and endocrine (JH) signaling during mandibular growth, we ectopically applied JH analog to dsxRNAi individuals.Here, we investigated the developmental mechanisms of sex-specific mandible growth in the stag beetle ult size  [5]. TheCmdsx transcript was obtained by degenerate PCR and subsequent RACE-PCR  containing primarily noncoding sequence and showing no similarity to dsx exons in any other insects, only occurs in splice variant A . Cmdsx variants C and D are similar to each other overall, with the exception of exon 6 which is only found in CmdsxC (doublesex gene of Onthophagus taurusO. taurus (OtDsxM) also share a 25 amino acid sequence at their 3′ end (O. taurus (OtDsxF1 and OtDsxF2), respectively . In the range of the observed body size, dsxRNAi males possessed smaller mandibles, and the mandible-size difference became larger as body size increases , and in dsxRNAi males the number of spines increased from zero to four   . FinallyAi males .We have previously shown that JH titer during the prepupal period is correlated with adult male body size and mandible size in the stag beetle, and that ectopic application of JH to the prepupal male induces male mandible proliferation Cmdsx. To test for a functional role of Cmdsx in causing sex differences in the sensitivity of mandibular cells to JH, we applied a JH analog (JHA) to RNAi (GFP or dsx) treated males and females (GFPRNAi) females, JHA application did not induce mandible growth  males, JHA application induced significant mandible growth  when mandibular cells proliferate maximally in males Cmdsx expression coincides precisely with inhibition of mandibular cellular proliferation  when cells in male mandibles are especially sensitive to the growth-promoting effects of JH Cmdsx during mandible growth. Considering the results of Cmdsx knockdown, we suggest that male-specific Cmdsx transcripts may promote mandible growth, and female-specific transcripts inhibit mandible growth, in part by enhancing or repressing the sensitivity of mandibular cells to JH.Examination of  pattern . Recent feration . ExpressCmdsx caused mandibles to be sensitive to JHA, where they otherwise would not have been, suggesting that normal expression of female-specific isoforms of CmDsx contributes to insensitivity of female mandibles to JH. Because JH acts to stimulate cell proliferation during this developmental stage, such a mechanism would repress excessive growth of this structure in females. In males, knockdown of Cmdsx combined with topical application of JHA stimulated some mandible growth, but not as much as in control animals with application of JHA. This indicates that male mandibles retained some sensitivity to JH even in their lowered expression of Cmdsx. We suggest that some sensitivity of mandibular cells to JH is the default developmental state for these animals. In normal males, male-specific CmDsx isoforms may increase the sensitivity of mandibular cells to JH, contributing to rapid and disproportionate growth of these exaggerated structures. In this case, knockdown of Cmdsx would remove this extra-sensitivity, restoring mandibular cells to their default state and producing males with large, but not extreme, mandible sizes. Another possibility is that dsx and JH act in parallel to regulate mandible growth in males. If this were the case, then Cmdsx and JH would act independently to stimulate exaggerated growth of male mandibles, and their effects would simply be additive. Thus, crosstalk between dsx and JH in males will need to be investigated in future studies.Our results demonstrate, for the first time in any insect, a functional link between Dsx expression and JH signaling. Knockdown animals had significantly altered responses to topical JHA application, compared with control animals . In femaAlthough there have been many previous reports of sex-specific JH actions on secondary-sexual characters in various insect lineages dsx regulation of sexual dimorphism has also been reported for horned beetles (Onthophagus taurus) dsx in sex differentiation, including development of sex-specific exaggerated traits, were shown. Horns in O. taurus are dimorphic in two ways: females do not produce horns , and males smaller than a threshold body size produce only rudimentary horns . Kijimoto et al showed that only large males expressed the male specific isoform of Otdsx (OtdsxM); small males did not OtdsxM may be sensitive to nutrition, as well as sex C. metallifer, where horns do not exhibit male dimorphism  and where we find at best minimal evidence of nutrition-dependent expression of dsx . Expression of Cmdsx in mandibles of large males was at most 1.3 times that of mandibles in small males . Based on these results we suggest that endocrine pathways sensitive to nutrition may interact with the sex determination pathway both upstream dsx.A recent study on dsxRNAi males to induce full growth of mandibles may indicate the action of other regulatory pathways for mandible growth. One likely candidate is the insulin-signaling pathway, because this pathway is known to regulate body and organ size in insects in accordance with nutritional conditions Trypoxylus dichotomus) are known to be more sensitive to insulin signals than other metric traits  Onthophagus nigriventris) Cmdsx may contribute to sex differences in sensitivity to these signals as well  were purchased from Hercules-Hercules, Sapporo, Japan. Detail rearing and breeding methods are described in the Supporting Information , elongation factor 1 alpha (EF-1a) and ribosomal protein L32 (rpl32). Primer sequences for degenerate PCR are listed in http://blast.ncbi.nlm.nih.gov/Blast.cgi). To further confirm the orthologues, we made multiple alignments of dsx genes including the orthologues from the other insect species, and constructed neighbor-joining trees of protein sequences (for Cmdsx) or mRNA sequences  using ClustalX program http://www.clustal.org/)  , S4, S5.al.org/) .C. metallifer dsx transcript sequence using the following RACE primers  and the SMART RACE cDNA Amplification Kit . The amplified cDNA fragments were subcloned and sequenced as described before. The protein domains were predicted by using CDD (conserved domain database) on NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).Rapid amplification of cDNA ends (RACE) -PCR was performed to obtain the full length Cmdsx transcripts in small and large male and female prepupae were examined during the period of maximal mouthpart growth with RT-PCR and real-time qPCR. Briefly, individuals were reared under high versus low nutrition conditions that result in small versus large prepupae; details for this can be found in our previous study The expression patterns of dsx was accomplished by knockdown of the dsx transcript by RNA interference (RNAi) during prepupal development. To silence all Cmdsx transcripts, double-stranded RNA (dsRNA) against a 352 bp region of Cmdsx common to all four splice variants  was performed with body size as a covariate using R 3.0.1 software GFPRNAi and dsxRNAi samples .For statistical test of Cmdsx expression levels using real-time qPCR in the prepupal mandibles of males and females injected with dsRNA against GFP (control) or Cmdsx. Primers for real-time qPCR were designed to the common region shared by all isoforms . To prevent overestimation of transcripts by detecting injected dsRNA, we designed the primers to amplify a region that had no overlap with the region used in dsRNA synthesis.RNAi efficiency was also examined by measurement of Cmdsx on JH action, we combined a Cmdsx knockdown experiment with ectopic application of the JH analog (JHA). According to previous study GFP or Cmdsx into the dorsal thorax of late 3rd instar C. metallifer larvae as described above. Then, when the knockdown experimental prepupae reached stage 2, five µg of the JH analog fenoxycarb diluted in 10 µl of acetone (Wako) was applied to the dorsal thorax according to previous study To investigate the effects of GFPRNAi males, dsxRNAi males, GFPRNAi females, dsxRNAi females), analysis of covariance (ANCOVA) was performed with body size as a covariate using R 3.0.1 software GFPRNAi males; F = 2.9354, P = 0.1029 in dsxRNAi males; F = 2.4669, P = 0.1507 in GFPRNAi females, F = 0.6751, P = 0.4185 in dsxRNAi females). Statistical significance of JHA application effects was adjusted for multiple comparisons by using Benjamini & Hochberg method To estimate the effect of JHA application on relative mandible size for each of the four RNAi categories  A dsx gene tree based on the conserved DM domain amino acid sequences region using the neighbor-joining method with bootstrap support above the branches.Alignment of Dsx sequences. (A) Alignment of the conserved amino acid sequences of the predicted CmDsx protein and those of other insects. Only the two most conserved regions are presented and include the DNA binding (DM) domain and the dsx dimer domain (dashed black box). Identical amino acids are highlighted in black. Putative conserved residues that distinguish the Dsx DM domain from the DM domain of other proteins are shown by arrowheads. The dashed blue box indicates weak similarity of sequence among CmDsxA, CmDsxB and OtDsxM. The solid blue box indicates conserved 25 amino acid sequence in CmDsxB and OtDsxM. Cm: (TIF)Click here for additional data file.Figure S2Cmdsx in prepupal GFPRNAi and dsxRNAi females (above) and males (below) in mandibles. The Y-axes show relative expression levels, which are specific to each panel. Averages and 95% confidence intervals of three technical replicates are indicated. Asterisks indicate significant differences between dsxRNAi and GFPRNAi samples .Effect of RNAi knockdown. Relative expression change of (TIF)Click here for additional data file.Figure S3Phylogenetic tree of GAPDH.(TIF)Click here for additional data file.Figure S4Phylogenetic tree of EF-1a.(TIF)Click here for additional data file.Figure S5Phylogenetic tree of RPL32.(TIF)Click here for additional data file.Table S1List of degenerate primer sequences for gene cloning.(XLS)Click here for additional data file.Table S2List of primer sequences for real-time qPCR.(XLS)Click here for additional data file.Table S3Sample sizes of surviving animals in combination experiment of dsx RNAi and JHA treatment.(XLS)Click here for additional data file.Text S1Supporting materials and methods.(DOC)Click here for additional data file.


The use of biting to obtain food items attached to the substratum is an ecologically widespread and important mode of feeding among aquatic vertebrates, which rarely has been studied. We did the first evolutionary analyses of morphology and motion kinematics of the feeding apparatus in Indo-Pacific members of an iconic family of biters, the marine angelfishes (f. Pomacanthidae). We found clear interspecific differences in gut morphology that clearly reflected a wide range of trophic niches. In contrast, feeding apparatus morphology appeared to be conserved. A few unusual structural innovations enabled angelfishes to protrude their jaws, close them in the protruded state, and tear food items from the substratum at a high velocity. Only one clade, the speciose pygmy angelfishes, showed functional departure from the generalized and clade-defining grab-and-tearing feeding pattern. By comparing the feeding kinematics of angelfishes with wrasses and parrotfishes (f. Labridae) we showed that grab-and-tearing is based on low kinematics disparity. Regardless of its restricted disparity, the grab-and-tearing feeding apparatus has enabled angelfishes to negotiate ecological thresholds: Given their widely different body sizes, angelfishes can access many structurally complex benthic surfaces that other biters likely are unable to exploit. From these surfaces, angelfishes can dislodge sturdy food items from their tough attachments. Angelfishes thus provide an intriguing example of a successful group that appears to have evolved considerable trophic diversity based on an unusual yet conserved feeding apparatus configuration that is characterized by limited functional disparity. Structural and functional attributes of the feeding apparatus are considered important features in promoting the impressive evolutionary diversification and ecological success of teleosts, or modern bony fishes Data on how the feeding apparatus is configured, but not on how it actually moves, has led to the concept of ‘many-to-one mapping’, which explains the relationship between structural redundancy and functional convergence The importance of ecomorphological quantification of biting has often been noted Marine angelfishes (f. Pomacanthidae) are especially worthy of detailed analyses to redress this research imbalance. Historically, it is only their morphology that has been examined Field observations suggest that angelfishes occupy a diverse range of trophic niches, ranging from spongivory and herbivory to planktivory Here, we study feeding apparatus structure and function among Indo-Pacific marine angelfishes. A phylogeny for the family permits us to attain a balanced analysis of interspecific variation in feeding apparatus form and function across a predominantly biting marine fish lineage. First, we evaluate the level of structural variation in the angelfish feeding apparatus, compared with earlier studies Given that angelfishes appear to occupy a wide range of ecological niches, we hypothesise that their feeding apparatus is characterized by a high level of structural diversity. Moreover, we hypothesize that this structural diversity is reflected by clear differences in biting kinematics across the family. Finally, we hypothesise that feeding kinematics are correlated with, and mechanically linked with, variation in trophic niches, with the alternative being many-to-one mapping between feeding morphology and kinematics.All results reported in this paper were generated via research endorsed by a Great Barrier Reef Marine Park collection and research permit (G01/257_1) and by a James Cook University Ethics Approval (A657/01).Taxon selection followed a phylogeny derived from 12S and 16S mDNA Centropyge [Centropyge] bicolor and Apolemichthys trimaculatus are both gracile omnivores that feed on zoobenthos; Genicanthus melanospilos is a zooplanktivore that occasionally bites attached invertebrate food items; Centropyge [Xiphypops] bispinosa is a herbivore; Pygoplites diacanthus is an omnivore that feeds on attached invertebrates; Chaetodontoplus duboulayi is an omnivore that feeds on sponges and tunicates; Pomacanthus [Euxiphipops] sexstriatus is a herbivore that feeds on calcareous and turf algae; Pomacanthus [Arusetta] semicirculatus is a carnivore that feeds on sturdy invertebrate food items (e.g. poriferan sponges and tunicates).The following species were sampled  covered with a mixed epifauna of sponges, turf algae, ascidians, tubeworms, and tunicates were collected from local coastal marine pylons. A major advantage of this food-treatment was that all taxa would feed on it. This minimizes the risk of introducing critical prey-type or prey-size effects into the resulting dataset. For husbandry purposes, both rock oyster shell epifauna and live ghost shrimp (Acetes sp.) collected in adjacent waters were provided. Prior to experiments, reflective markers were glued to the fish skin as reference markers for motion analysis. These markers were carefully attached over joints of interest in the jaws, suspensorium, pectoral girdle and the craniovertebral joint, and as reference markers at the bases of the dorsal, pectoral and pelvic fins , and total length (TL), standard length (SL) and head length (HL) measurements were taken . SpecimeThe correspondence between previously inferred trophic niches tTOT) were cropped from protrusion-onset (tS), via time of bite (tB) to completed jaw retraction (tC) using Virtual Dub (v.1.7.4). The nine reflective markers  and then log-transformed. Variables were omitted from analysis only if there was significant auto-correlation between variables. The choice of which of two auto-correlated variables to remove was guided by biomechanical evaluations. This resulted in a final dataset of 23 informative variables that was subjected to further analyses. We verified that the resulting dataset did not violate assumptions for parametric analyses using the data normality examination in Systat v. 12. A MANOVA was used to evaluate the extent of variation in the dataset, and we then ran a principal component analysis (PCA) on the correlation matrix of the kinematic dataset . Therefore, a discriminate function analysis (DFA) was used to examine the extent to which kinematic variables could segregate taxa across available 2D-kinematic functional space (Statistica v.6.0). The canonical correspondence component of DFA is highly sensitive to subtle variation, and thus a powerful method when aiming to identify and maximize the display of variation among predefined groups Functional disparity is defined as the range of diversity in a clade All angelfishes have heads that are laterally compressed, with cheek regions (suspensoria) that are reduced anteriorly, and oral jaws that can protrude far in front of the face because of their flexible and loose suspension . Major sCentropyge [Xiphypops], where a more obliquely horizontal resting angle leads to a more downwards directed protrusion of the jaws  with highly variable duration (0.054–0.3 s) leading to a maximum protrusion averaging 23% head length (HL), but only reaching 14% HL in the planktivore Genicanthus. Following maximum protrusion, a distinct and rapid jaw closure around the food item (0.012–0.059 s) is facilitated by rotation at the intramandibular joint. The jaw closure designates the time of bite (tB). Almost immediately following the time of bite, angelfishes tear off the food that is captured between their bristle-like teeth using a rapid jaw retraction (0.2–0.9 m s−1). The retraction of the jaws is augmented by a sideways head-jerk and pectoral-fin propelled rearward movement of the fish.All bites from all individuals are characterised by a slow phase of jaw protrusion . A principal component analysis (PCA) on the correlation-matrix of the kinematics dataset returned two statistically informative PC axes . However, hypothesis testing using pair-wise comparisons only recovered statistically significant differences between Genicanthus and all other taxa.A MANOVA on the transformed kinematics variables indicated significant variance in the dataset  on the same dataset, to determine if interspecific differences were too subtle to be recovered by the PCA. Seven canonical correspondence axes were returned of which the first three alone explained 78% of the dataset variance ; Table 2In the analysis comparing prey-capture functional disparity of angelfishes and labrids, four out of 16 PC axes explained more than 10% each of the total variance in the dataset, and these axes are shown in Among Indo-Pacific marine angelfishes, an unusual combination of structural and functional traits result in a novel and evolutionary conserved ‘grab-and-tearing’ feeding method. The morphology and kinematics underpinning this novel feeding method seems fundamentally different from other biters and from modern bony fishes in general. Comparisons with labrid reef fishes showed that the kinematic disparity of the grab-and-tearing feeding apparatus in angelfishes is restricted. The diverse trophic ecology of angelfishes appear better explained by gut morphological than by skull morphological disparity. However, given the descriptive nature of our skull morphological analysis, the notion of low structural disparity in the angelfish skull remains somewhat conjectural until a formal morphospace analysis is in place. Regardless, our kinematics and trophic ecological results reflect a contrasting trend in how morphology and kinematics underpin ecological patterns and the evolution of trophic diversity in aquatic feeding vertebrates.There was little variation seen in angelfish feeding apparatus morphology, and the function of the feeding apparatus varied little from a previous study of a generalised angelfish species The few structural novelties are either synapomorphies relative to other bony fishes The few interspecific differences in angelfish feeding kinematics were far outweighed by much more subtle variation in the duration, onset-timing and velocity of kinematics that are ancestral to bony fishes. Interestingly, magnitude variables influenced species-segregation less than timing variables. The latter variables are under intrinsic behavioural control, and therefore likely to be less susceptible to morphological and phylogenetic constraints Genicanthus compared with its obligate biting sister taxa A common idea is that structural novelty leads to functional decoupling of associated structures The ‘grab-and-tearing’ feeding system arose once and underwent a limited amount of change during the 38 million years of evolution in this ecologically successful family. How is the high level of ecological versatility, clearly reflected by gut morphology and field observations, explained by such stereotyped feeding morphology and biting kinematics? The unusual and highly versatile feeding apparatus, combined with the evolution of an order of magnitude variation in body size, appears to have permitted angelfishes to negotiate several ecological thresholds Pomacanthus [Euxiphipops] clade as well as in the pygmy angelfish subgenus Centropyge [Xiphypops]Our analyses of angelfish gut morphology substantiated earlier notions of herbivory having arisen independently in the large-bodied P. [Euxiphipops] to feed on sturdy calcareous or foliaceous algae The high diversity in angelfish gut morphology combines with an overriding role of body size on the trophic evolution of the family. Benefits of being large are most obvious among spongivores and robust omnivores, where an associated increase in the forcefulness of grab-and-tearing facilitates the rupturing or dislodging of structurally resilient sponges and tunicates Centropyge [Xiphypops], the physical force-production constraints inherent to a small body size have led to gracile combing or shearing strategies and therefore feeding on delicate foliaceous algae. Their oblique jaw protrusion means that the body can remain parallel with the substratum during feeding, which likely improves the predator-avoidance response Centropyge [Centropyge] feed on attached colonial invertebrates close to shelter or tucked away within the reef-matrix Pygmy angelfishes show the only major morphological divergence within the family. In Centropyge clades, support the elevation of C. [Xiphypops] to full generic status. The clade of herbivorous, small-bodied and heavily hybridizing Centropyge [Xiphypops] and their planktivorous sister-taxon Genicanthus recently underwent pronounced and rapid speciation. Together, they comprise 25% of all angelfishes. Interestingly, this could be a product of increased disparity as a result of hybridization The clear differences in jaw structure and function, and in relative gut length, between the two Genicanthus differed from all other angelfishes by having a reduced degree of motion in most of its ancestral feeding mechanisms. Restricted movements of its diminutive mouth, coupled with the shortest and least differentiated gin the family corresponds with a secondary functional reversal to planktivory vs. Labridae, 60 myo; Our study is one of the first to quantify the kinematics underpinning the biting feeding mode, and the first to sample kinematics in a systematic manner across a monophyletic clade mainly comprised of biters. The kinematics disparity characterizing angelfish biting was only half of that seen in labrid wrasses and parrotfishes, which have repeatedly evolved biting as well as ram-suction feeding strategies. Our disparity measurements must be considered preliminary because of the small number of species sampled. Moreover, ancestry-calibrated comparative analyses were impossible, as no angelfish fossils are available. Regardless, two lines of inference support our disparity estimates: The uneven phylogenetic sampling of labrid feeding kinematics, and the substitution of missing data-points with median values  likely rA general lack of morphological change in angelfishes since the Eocene reflects the status of most reef fish families Figure S1Summary of feeding kinematics in f. Pomacanthidae and f. Labridae. Labrids have more variable kinematics, as indicated by standard deviation whiskers, and typically higher mean values than pomacanthids. Abbreviations: Amplitudes; MDDep, mandible depression; OPRot, opercular rotation; NCEle, neurocranial elevation; GPExp, gape expansion; JAWprot, jaw protrusion; Maximum-timing; MDDepTim, mandible depression; OPTim, opercular rotation; NCTim, neurocranial elevation; GPTim, gape expansion; PROTtim, jaw protrusion; Durations; MDdepDur, lower jaw depression; OPDur, opercular rotation; NCDur, neurocranial elevation; GPDur, gape expansion; GPCLDur, gape occlusion; PROTdur, jaw protrusion; RETdur, jaw retraction; BITEdur, total prey capture event. Data as listed in (TIF)Click here for additional data file.Table S1Uncorrected variables for comparative kinematics analyses. Data used came from: aPresent study, bcAverage of values reported in defg(DOC)Click here for additional data file.Video S1Centropyge [Centropyge] bicolor feeding on Ghost shrimp (Acetes). The video was recorded at 200 fps to view 8 times slower than real-time.(MPG)Click here for additional data file.Video S2Apolemichthys trimaculatus feeding on a sponge. The video was recorded at 200 fps to view 8 times slower than real-time.(MPG)Click here for additional data file.Video S3Genicanthus melanospilos feeding, first on attached- then a free-floating piece of squid (Loligo). The video was recorded at 500 fps to view 20 times slower than real-time.(MPG)Click here for additional data file.Video S4Centropyge [Xiphypops] bispinosa feeding on turf algae. The video was recorded at 200 fps to view 8 times slower than real-time.(MPG)Click here for additional data file.Video S5Pygoplites diacanthus feeding on a sponge. The video was recorded at 200 fps to view 8 times slower than real-time.(MPG)Click here for additional data file.Video S6Chaetodontoplus duboulayi feeding on a sponge. The video was recorded at 200 fps to view 8 times slower than real-time.(MPG)Click here for additional data file.Video S7Pomacanthus [Euxiphipops] sexstriatus feeding on a sponge. The video was recorded at 200 fps to view 8 times slower than real-time.(MPG)Click here for additional data file.Video S8Pomacanthus [Arusetta] semicirculatus feeding on a sponge. The video was recorded at 200 fps to view 8 times slower than real-time.(MPG)Click here for additional data file.


Copy Number Variations (CNVs) and Single Nucleotide Polymorphisms (SNPs) have been the major focus of most large-scale comparative genomics studies to date. Here, we discuss a third, largely ignored, type of genetic variation, namely changes in tandem repeat number. Historically, tandem repeats have been designated as non functional “junk” DNA, mostly as a result of their highly unstable nature. With the exception of tandem repeats involved in human neurodegenerative diseases, repeat variation was often believed to be neutral with no phenotypic consequences. Recent studies, however, have shown that as many as 10% to 20% of coding and regulatory sequences in eukaryotes contain an unstable repeat tract. Contrary to initial suggestions, tandem repeat variation can have useful phenotypic consequences. Examples include rapid variation in microbial cell surface, tuning of internal molecular clocks in flies and the dynamic morphological plasticity in mammals. As such, tandem repeats can be useful functional elements that facilitate evolvability and rapid adaptation. The scientific community has previously dismissed repetitive regions as nonfunctional, “junk” DNA. However, recent evidence with the assistance of whole genome sequencing illuminates the significant role repeats might play in genomes. In the 1960s, scientists identified these repetitive elements as the explanation for the negative correlation between an organism’s phenotypic complexity and its genome size . Two categories of repetitive regions exist—interspersed repeats and tandem repeats (TRs). Interspersed repeats, the more predominant type of repeat, are remnants of transposons dispersed throughout the genome. Such elements are responsible for the diverse array of genome sizes amongst various species . On the On the basis of unit length  23] wherei.e., coding) repeats.In many instances, repeat mutations, especially in gene deserts, have neutral effects and remain undetected. Cases like these may have contributed to the previous notion that repeats were nonfunctional junk or selfish DNA ,26,27. Hcis-regulatory elements [cis-regulatory sites than in coding sequences in part, due to the high prevalence of TRs in regulatory regions, particularly the AC dinucleotide repeat [Saccharomyces cerevisiae indicates that about 25% of all promoters contain TRs. Variation in these repeats is associated with variable gene expression within and between yeast species [Phenotypic evolution has been mostly attributed to changes in gene expression and regulation due to variation in (non-coding) elements ,29. Upone repeat . The aut species  .Additional studies uncover a link between variable TRs in promoters and changes in gene expression and/or function. This suggests that instead of being neutral sequences without a functional value, TRs may actually be significant contributors to functional variation in gene expression. In the following section, we report the studies that reveal a link between variable TRs and changes in gene expression. We focus particularly on studies which reveal the mechanism through which repeats in non-coding regions affect downstream gene expression. A summary of numerous correlation studies, in which gene expression variation has only been associated with variable promoter TRs, can be found in the following reference .The high prevalence of TRs in regulatory regions, in many organisms, suggests that repeat variation might be a common, evolutionarily conserved, mechanism regulating gene expression. A large number of studies report correlations between TRs and changes in gene expression . Other sNeisseria meningitidis, changes in the number of TAAA repeats located in the nadA promoter induces variable expression of the NadA adhesin protein. This highly frequent switching of phenotypic outcome, also referred to as phase variation, is mediated by the loss or gain of binding sites for the regulatory protein IHF [nadA promoter is discussed in the following paragraph. Rapid phenotypic switching mediated by intragenic  repeats will be discussed in a later section of this review. Variable TRs in promoters can affect gene expression by altering the number of transcription factor binding sites. Frequent TR variability of repeat-containing transcription factor binding sites results in changes in the number of binding sites. An example of such a mechanism was first uncovered in pathogenic bacterial species. In tein IHF . A seconin vitro [cis-regulatory elements [Changes in transcription factor binding sites as a result of unstable TRs have also been described in eukaryotes. A variable TCC repeat in the promoter region of the human epidermal growth factor receptor (a proto-oncogene) induces changes in gene expression by altering the number of binding sites for the transcriptional regulator Sp1 . A changin vitro . Though elements .nadA promoter of N. meningitidis (see above), changes in the number of TAAA repeats also induce changes in spacing between distal and proximal binding sites (flanking the TAAA repeat) for the NadR repressor protein [N. meningitidis membrane protein PorA correlates with the number of guanidine residues within the -10 and -35 spacer region in the upstream promoter. A mere loss or gain of one nucleotide in this polyGuanidine TR leads to significant changes in PorA transcription [Haemophilus influenzae, promoter TRs modulate the expression of genes involved in the formation of adhesive appendages or fimbriae. Variation in a TA dinucleotide repeat in the bidirectional promoter of two genes hifA and hifB alters the critical spacing between the -10 and -35 consensus sequence, thus affecting the bidirectional transcription initiation of both genes [Bordetella pertussis [Mycoplasma hyorhinis [Variable TRs in promoters not only affect the number of transcription factor binding sites, but can also induce changes in spacing between critical promoter elements . In the  protein . Additiocription . Similarth genes . Severalertussis , and Mycyorhinis .N. meningitidis genes are expected to be phase variable as a result of unstable TRs [Genome-wide sequencing suggests there are many more pathogenic bacteria, than the ones mentioned above, in which tandem repeat-mediated phase variation is operable. In fact, more than 30 able TRs . It seems that this form of gene expression regulation  is common among several pathogenic bacterial species. This rapid phenotypic switching, results in a heterogeneous bacterial population in which some individuals are better suited to environmental changes  and consequently have better chances of survival.DNA is intricately organized in the nucleus and packaged into structures called nucleosomes, in which DNA is wrapped around an octamer of four core histones, to form chromatin. Chromatin structure is highly dynamic and has large implications in the regulation of gene expression, especially since histone complexes affect accessibility of transcription factors to DNA. et al. illustrates TR enrichment in nucleosome-free regions of yeast promoter regions and supports this finding with preliminary observations in human promoters , in rare cases, it may exist as Z-DNA . This conformation is drastically different from the normal B-DNA, that it might block access of transcription factors to DNA. Several studies suggest a role for TRs as elements that assist in Z-DNA formation. Alternating purines and pyrimidines, which favor the Z conformation, are found as TR sequences in the genome. Authors Naylor and Clark find evidence for a CA repeat lying upstream of the rat prolactin gene that enables Z-DNA formation. Additionally, this repeat inhibits gene transcription by potentially deterring transcriptional efficacy of RNA polymerase II . SpecifiFunctional roles for TRs present in introns and untranslated regions have been mostly uncovered by studies investigating the pathogenic mechanisms involved in neurodegenerative diseases. These studies provide insight into how variable intergenic TRs can act as modulators of gene expression. In this section, we focus on such studies and highlight the mechanism through which variable TRs in introns and untranslated regions can lead to changes in gene expression. DMPK gene causes myotonic dystrophy type 1 (DM1) and the expansion of a CCTG repeat in intron 1 of the ZNF9 gene causes myotonic dystrophy type 2 (DM2). The expanded RNAs adopting a stable hairpin structure, bind to and alter the activity of RNA processing proteins CUG-BP1 and MNBL [i.e., downstream targets) involved in different cellular processes. Tandem repeats in introns and untranslated regions can influence gene expression by modulating the activity of RNA-binding proteins. The mechanistic role of TRs in recruiting RNA-binding proteins has been proposed to explain the overlapping clinical features of two genetically unrelated neurological diseases. Expansion of a CTG repeat in the 3' UTR of the and MNBL . This leBRD2) and epilepsy in humans uncovered the role of variable intronic repeats in regulating gene expression through alternative mRNA splicing pattern. In intron 2 of the BRD2 gene, a polymorphic GT repeat is strongly associated with epilepsy. Gradual shortening from 13 GT repeats to 1 GT repeat results in a higher proportion of (non-productive) alternatively spliced BRD2 transcripts over normal transcripts. These alternative mRNAs produce non-functional BRD2 proteins [A study investigating the link between expression of the Bromodomain-Containing Gene  might be a common and conserved regulatory mechanism. The central question that remains is how can TR variability promote evolvability? And what are the effects on an organismal level? The studies described in this section indicate that variable TRs in regulatory regions can strongly impact gene expression and/or function through various mechanisms. The high prevalence of these repeats in diverse genomes suggest that this type of gene expression regulation  enable phenotypic evolution, ultimately impacting the population; rapid changes in TRs create phenotypic variants within a population, leading to fast adaptation  and increased survival. et al. reports that yeast promoters containing TRs have evolved faster since their expression patterns are more divergent between and within yeast strains compared to promoters without repeats [In eukaryotes, a recent study provides experimental evidence on the role of TRs as drivers of rapid evolution. Vinces  repeats . Promote repeats . This stMMP3 gene contains a variable polyT tract that is rapidly evolving in the primate lineage. In humans, variation in this repeat results in variable expression of the MMP3 gene, associated with risk of heart disease: the 5T allele results in higher MMP3 expression and is associated with myocardial infarction and aneurysm, whereas the 6T allele drives a lower MMP3 expression and is associated with coronary artery disease [MMP3 gene expression and based on its associated phenotype in humans, it clearly exhibits a functional value.In complex organisms, such studies are difficult to undertake, though some reports indicate that this might be the case for particular genes. The promoter of the  disease . In EuroTandem repeats in coding regions are mostly known for their association with a number of human neurodegenerative diseases. Yet their abundance in functional regions of the genome and their enrichment in certain gene categories, suggest that repeats in coding sequences might play a beneficial role. In this section, we review studies revealing functionally beneficial roles of long (minisatellite) and short (microsatellite) coding repeats.Minisatellites are enriched in cell surface genes and conserved through evolution from yeast to humans . VariousS. cerevisiae FLO1 gene encodes a cell-surface adhesin that mediates cell-cell and cell-surface adhesion. The gene contains a repeat tract consisting of tandemly repeated units of approximately 100 nt, which are variable across different yeast strains [FLO1 repeat length illustrate that the intensity of the adhesion directly correlates with the length of the repeat  [The  strains . Phenotyh other)  , prone to O-glycosylation. The addition of covalently linked sugar groups renders the adhesins, and subsequently the cell wall, hydrophobic. Thus, an increased number of repeats enhances buoyancy of yeast strains. Additionally, transformation of S. cerevisiae strains, that generally sediment, with flor FLO11 repeat tract and promoter enable yeast cells to form buoyant biofilms [biofilms .Candida albicans. The ALS gene family encodes adhesins similar in structure to the baker’s yeast flocculins that mediate biofilm formation. The length of the central repeat domain correlates with adhesion propensity  [ALS genes can generate cell-surface variability required to evade the host’s immune response [Similar observations have also been made in the human pathogen e cells) . Recombiresponse . Trinucleotide repeats, in particular polyglutamine (polyQ) repeats, are involved in a number of human neurodegenerative diseases , with numerous studies illustrating the conferral of such diseases upon repeat expansions exceeding a particular threshold. Though TRs involved in human diseases will not be discussed extensively here, it is important to note that severity and time of onset of these neurodegenerative diseases depend on the repeat expansion: longer repeats correlate with earlier disease onset and more severe symptoms .Considering the little we know, such devastating effects of TR mutations have often overshadowed our knowledge of TRs. However, the predominance of repetitive DNA sequences in functional parts of the genomes, despite such a high propensity for mutations and apparent low information content, hint at a greater role for TRs. In fact, though the genetic code is degenerate, amino acid repeats tend to be coded by pure DNA repeats, illustrating the conservation of repetitive regions on the DNA level as well as the protein level. These unstable repeats, might be tolerated in some coding sequences because they hold the potential for the acquisition of a beneficial novel/improved function . TRs may not merely be “junk DNA” as was previously assumed. We will highlight key studies that provide experimental evidence for this hypothesis in the following section.Neisseria gonorrhoeae, repeat variations in the P. II family of cell surface genes allow for evasion of the host immune system [Intragenic TRs can also mediate phase variation . In the bacterium, e system . Changese system . This swlic1 gene of Haemophilus influenzae (enables the addition of phosphorylcholine moieties to cell membrane lipopolysaccharides (LPS)), which contains a variable intragenic CAAT repeat. Formation of a complete, functioning Lic1 protein or a truncated mutant depends on variation of an intragenic CAAT repeat. Differences in LPA structures promote different bacterial variants enabling survival of the bacterium in the host [Similar occurrences are seen in the the host . mod genes), are also phase variable. The modA gene, in Neisseria and Haemophilus species, contains either an AGTC or AGCC repeat whose mutations directly alter the rate of phase variation [modA gene contains two potential start codons depending on repeat number but only the one that yields the longest transcript gives rise to a functional protein. Variation in ModA repeat number thus has consequences on the expression of its downstream targets that include outer membrane proteins, iron transporters and heat-shock proteins [modA gene leads to biofilm thickness and better survival rates of N. gonorrhoeae cells in human cervical epithelial cells [Phase variable genes are not merely limited to cell-surface genes; recent evidence suggests that regulatory genes, such as those encoding DNA-methyltransferases  repeats have strong implications for neurodegenerative diseases . In Drost genome . Overallt genome ,62 and rHere we showcase several studies regarding the possible functional role of polyQ repeats, ranging from its involvement in morphological variation to protein regulation. Organisms possess circadian clocks, or innate biological timing mechanisms that synchronize various cellular processes with the external environment. The typical circadian cycle, also referred to as a period, occurs over a span of 24 h. The circadian rhythm, cued by environmental factors , is crucial to an organism’s survival and well-being; maximizing ideal circadian patterns may confer a fitness advantage.Neurospora crassa, intragenic CAG repeats control expression of the transcription factor white collar-1 (WC-1), responsible for expression of an important regulator of the circadian rhythm [N. crassa leads to differences in circadian periods; TR frequencies exhibit a latitudinal cline: longer repeats (corresponding to shorter circadian rhythms) are commonly seen amongst strains collected from lower latitudes [In the species n rhythm . The varatitudes .CLOCK gene, a regulator of circadian patterns, contains a variable polyQ repeat in its C-terminus. Statistical correlation studies of the CLOCK gene unveil that TR variation differs between two bird species, the migratory bluethroat and the non-migratory blue tit; TR is more unstable in the blue tits population [Tandem repeat variation has also been linked to differences in bird migratory patterns . The avipulation . These oSuch studies additionally support the beneficial role of unstable repeats, especially in diversifying gene pools, which may allow for better adaptation of organisms.Higher-order eukaryotes unveil another possible role for repeats. In these organisms, TRs are enriched in genes involved in determining body morphology, suggesting that unstable repeats underlie phenotypic diversity .Alx-4 and Runx-2. Though the Alx-4 gene does not contain a pure polyQ repeat but rather a PQ tract, a 51-nt deletion of this region is found in dogs with an additional rear claw . The Runx-2 gene, a transcription factor controlling osteoblast development, contains two polymorphic repeats—a polyQ repeat and a polyalanine repeat. Here, the ratio of glutamine to alanine repeats correlates with the degree of dorsoventral nose bend and midface length in dog breeds [Runx-2 repeat region by Sears et al. suggests that the ratio of the two repeats correlates to onset of transcriptional activation [A fascinating study attributed morphological variation, occurring over short evolutionary timescales, to variations in TRs. The great diversity of morphology among dog breeds cannot merely be adequately explained by point mutations; the paper by Fondon and Garner provides compelling evidence associating variable TRs with changes in dog skull morphology occurring over a span of approximately fifty years . In compg breeds . A furthtivation . The rolThe morphological diversification of dog breeds over approximately 50 years, an extremely short time span from an evolutionary standpoint, is the poignant aspect of this study by Fondon and Garner. Even despite strong selection against genetic diversity as a result of domestication and inbreeding, TR involvement in controlling development and body morphology suggests that variable TR may play a critical role in the rapid evolution of dog skeletal morphology.Runx-2 repeats measured expression of its target and showed that mRNA of the Runx-2 target increased with longer Runx-2 repeats [et al. provide evidence that increasing repeat number in vitro and in vivo leads to increased rates of mRNA transcription [A further exploration of the  repeats . Another repeats . Gerber cription . Though human TATA-binding protein (TBP) gene has been shown to induce the neurodegenerative disease spinocerebellar ataxia (SCA17) [et al. demonstrate that polyQ expansion inversely relates to TBP dimerization—the longer the repeat, the lower the frequency of TBP dimers (The expansion of polyQ tracts in the transcription factor  (SCA17) . Previou (SCA17) . TBP dim (SCA17) . FriedmaP dimers . This inLarge scale genomic studies mainly focus on Single Nucleotide Polymorphisms (SNPs) and Copy Number Variations (CNVs) as the major sources of phenotypic variation. Here, we describe studies demonstrating how tandem repeat variation can influence phenotypes and, can be considered as a third source of genetic variation.i.e., quantitative trait loci). However, the examples described here, show that many TRs can confer quantitative changes in phenotypes through changes in one genetic locus (coding or regulatory region). In some other cases , variable TRs do function as binary switches (ON/OFF switching of cell-surface proteins in bacteria).From the evidence gleaned so far, (with the exception of repeat expansions in human diseases) TR variations do not result in deleterious changes rather in small incremental phenotypic changes . This quantitative phenotypic variation is commonly believed to depend on complex interactions between multiple genes  [The formation of TRs in genomes may be a random process (resulting from polymerase slippage) , but oncno acid) .The appearance of TRs in genes could lead to the acquisition of a novel function and their retention could signal an evolutionary relevant path. This seems to be the case for the huntingtin protein, where the acquisition of a regulatory activity controlling neural adhesion in complex mammalian nervous systems, coincides with the appearance of glutamine repeats. Evolutionary distant heterologues of huntingtin, lacking the repeat region, fail to substitute the activity of the repeat-containing mammalian protein .Taken together, tandem repeats are an underestimated, understudied source of genetic variation which, by means of their instabilities, may facilitate a rapid evolution of genes and their associated phenotypes.


Mitosis is accompanied by dramatic changes in chromatin organization and nuclear architecture. Transcription halts globally and most sequence-specific transcription factors and co-factors are ejected from mitotic chromatin. How then does the cell maintain its transcriptional identity throughout the cell division cycle? It has become clear that not all traces of active transcription and gene repression are erased within mitotic chromatin. Many histone modifications are stable or only partially diminished throughout mitosis. In addition, some sequence-specific DNA binding factors have emerged that remain bound to select sites within mitotic chromatin, raising the possibility that they function to transmit regulatory information through the transcriptionally silent mitotic phase, a concept that has been termed “mitotic bookmarking.” Here we review recent approaches to studying potential bookmarking factors with regards to their mitotic partitioning, and summarize emerging ideas concerning the in vivo functions of mitotically bound nuclear factors. Mitosis imposes dramatic and dynamic changes on nuclear organization and gene expression in eukaryotic cells. In metazoans, the nuclear envelope temporarily disintegrates, most nuclear structures are transiently dissolved or rearranged, and nuclear transcription by all three polymerases is globally arrested. Silencing of gene expression is accompanied by the separation of most transcriptional regulators from mitotic chromatin. Following chromosome segregation and re-formation of the nuclear envelope, bulk transcription resumes in the daughter cells that ultimately re-acquire gene expression patterns that are often similar to or indistinguishable from those of the mother cell. It has been widely assumed that these transitions present a problem for the cell’s maintenance of transcriptional identity, prompting investigations into mechanisms that ensure rapid and faithful restoration of gene expression upon re-entry into the G1 phase of the cell cycle. Such mechanisms fall under the category of epigenetics in the stricter sense of the word by providing a cellular memory function throughout the cell division cycle . TherefoSuch considerations led to the exploration of diverse mechanisms of gene bookmarking in mitosis to prevent potentially detrimental alterations in gene expression upon re-entry into G1 . These iA different perspective on the effects of mitosis on gene expression comes from the notion that genome-wide perturbations in transcription factor occupancy might facilitate changes in cell fate by allowing the reshaping of transcription programs. An impressive example is the observation that zygotes are capable of reprogramming somatic nuclei only after recipient cells have been arrested in mitosis , suggestIn this article instead of providing an extension of previous comprehensive reviews of known factors and histone marks that persist on mitotic chromatin, we aim to highlight recent technical and conceptual developments that approach questions of mitotic bookmarking.Here we provide a brief review of frequently used approaches to study potential mitotic memory mechanisms, since inconsistencies or controversies in the literature can be rooted in different methodologies used. Immunofluorescence (IF) microscopy is commonly employed to globally survey mitotic partitioning of nuclear factors and persistence of histone marks. An advantage of IF is that it detects endogenous proteins in their natural context, but it requires suitable antibodies for which the epitope is not occluded by chromatin compaction during mitosis, by mitosis-specific post-translational modifications, or because of fixation. These problems can be sidestepped by live cell-imaging with ectopically expressed fluorophore-tagged molecules. However, overexpression might lead to shifts in mitotic occupancy patterns. The monitoring of histone marks in living cells is more challenging, although strategies have been developed using fluorescence resonance energy transfer (FRET)-based indicator molecules that specifically interact with a given histone mark ,15. GiveTo localize nuclear factors or histone marks on specific genomic sites in mitosis, chromatin immunoprecipitation (ChIP) can be used conventionally or in combination with high throughput sequencing. Concerns about epitope recognition in mitotic cells are similar to those described for IF. The preparation of pure mitotic cells for ChIP is essential and has been aided by the recent development of effective protocols that use antibodies against phosphorylated histone H3 serine 10, a modification globally enriched during mitosis in all cell types, for fluorescence activated cell sorting (FACS) ,17. ThisThe combinatorial use of IF, live cell imaging, and ChIP does not only serve to corroborate key results, but can also uncover new concepts. For example, the hematopoietic transcription factor GATA1 globally separates from mitotic chromosomes as revealed by IF ,18. HoweAdditional strategies to assess mitotic chromatin binding involve the fractionation of mitotic chromosomes followed by western blotting  or unbiThe methods mentioned so far are useful tools for untangling the protein composition and post-translational modifications of mitotic chromosomes. However, the mere presence of a factor or histone mark on mitotic chromatin is insufficient to establish a functional role in mitotic memory. In several studies select mitotically retained factors were depleted by RNAi, leading to a delay in postmitotic target gene reactivation ,20,23-26et al. devised a strategy to deplete GATA1 selectively in mitosis [In an attempt to determine a mitotic bookmarking function for GATA1 Kadauke  mitosis . GATA1 w mitosis . It shouFinally, it is worth noting that mitotic partitioning of nuclear factors and histone marks can vary between cell types and might be influenced by whether primary or transformed cells are used . It is Finally, transcription factors both influence and are influenced by their chromatin environment. The dynamics of histone modifications during mitosis and possible roles in bookmarking are discussed elsewhere .Mechanisms that allow dispersal of nuclear factors and modulate histone modifications during mitosis have been extensively studied. Distinct kinetics of dissociation and re-association of factors during mitosis as well as varying stabilities of histone marks argue that multiple mechanisms control mitotic partitioning. Nevertheless, common themes have emerged that apply to broad classes of molecules, including dynamic phosphorylation of histones and nuclear factors. We will not discuss these mechanisms here as they have been thoughtfully covered in several recent reviews ,10,28. IAlthough global occupancy of transcription factors is sometimes reduced during mitosis ,19-21, iThe observed transcription factor occupancy patterns typically fall into distinct classes. At some sites nuclear factors are retained at normal levels while at others binding is diminished, which might be a result of fewer molecules per occupied site or fewer alleles being bound in heterogenous cell populations. In addition, new sites might be occupied by nuclear factors only in mitosis, thus signaling a shift in binding properties ,19,35. WDNA-binding factors generally occupy sites in a sequence-specific fashion, and variation in sequence can affect binding dynamics. Comparative genome-wide occupancy profiles of sequence-specific transcription factors in interphase and mitosis are now beginning to be determined ,20,21. DD. melanogaster GAGA transcription factor shifts from its interphase binding sites to centromeric alpha-satellite GA repeats [n repeats in intergenic regions remote from any annotated genes [An emerging theme of mitosis-specific occupancy involves repetitive DNA sequence elements. During mitosis the  repeats ,36. More repeats . The majed genes . Why woued genes . It is aSeveral features of chromatin such as DNase hypersensitivity, various modifications of DNA and histones, and histone variants persist at least in part through mitosis while others are dynamic throughout the cell cycle -47. It sHowever, some features of chromatin structure influence transcription factor binding profiles during mitosis. FoxA1 binding in mitosis seems to favor sites of high nucleosome density, consistent with its strong affinity for nucleosomes . It has n repeats (see previous section).The post-translational histone modifications examined so far fail to clearly distinguish interphase-only from persistent transcription factor binding sites ,20, althHox locus, even though the polycomb target mark H3K27me3 is present at this locus throughout mitosis [The polycomb protein PSC is partially maintained on chromatin in mitosis but not at the prototypic PSC target  mitosis . Notablyin vivo will be an important question for the future. It is notable that PP2A can also reverse the mitotic inactivation of the SWI/SNF chromatin remodeling complex [Dynamic phosphorylation of histones, transcription factors, and the mitotic condensation machinery control transitions through mitosis ,28, and  complex , but sinAcetylation of histones is at least partially maintained during mitosis . ReadersTranscription co-regulators are typically loaded onto chromatin via DNA binding proteins. It is therefore expected that mitotic partitioning of the former mirrors that of the latter. Correspondingly, the majority of cofactors are separated from mitotic chromosomes Table . A notabFor over half a century it has been known that transcription is globally silenced during mitosis ,115, yetTo what extent mitotically stable features of chromatin are required for the propagation of transcriptional information and maintenance is still mostly subject to speculation but has moved closer within our reach. Important related questions to be addressed include whether there is a direct role for mitosis in facilitating changes in transcription patterns to allow cell fate decisions during development, and whether alleviation of bookmarking facilitates cellular reprogramming or lineage switching. Finally, it will be important to explore whether perturbation of mitotic bookmarking accounts for developmental disorders or malignancies.ASH2L: Absent, small, or homeotic-like protein; BET: bromodomain-ET; BRD: bromodomain-containing protein; ChIP: chromatin immunoprecipitation; FACS: fluorescence-activated cell sorting; FoxA1: forkhead box A1; FOG 1: Friend of GATA 1; FRET: fluorescence resonance energy transfer; GATA1: GATA binding factor 1; HDAC1: Histone deacetylase 1; IF: immunofluorescence; P-TEFb: positive transcription elongation factor b; PSC: posterior sex combs; RbBP5: Retinoblastoma-binding protein 5; SWI/SNF: switch/sucrose nonfermentable; TAL: transactivator-like; TAL1: T-cell acute lymphocytic leukemia protein 1; TBP: TATA-binding protein; TLE: Transducin-like enhancer protein 1The authors declare that they have no competing interests.SK and GAB drafted and edited the manuscript. Both authors have read and approved the final manuscript.


Flavivirus. At the current time, there are no approved prophylactics  or specific therapeutics available to prevent or treat human infections by these pathogens. Due to their minimal genome, these viruses require many host molecules for their replication and this offers a therapeutic avenue wherein host factors can be exploited as treatment targets. Since several host factors appear to be shared by many flaviviruses the strategy may result in pan-flaviviral inhibitors and may also attenuate the rapid emergence of drug resistant mutant viruses. The scope of this strategy is greatly enhanced by the recent en masse identification of host factors impacting on WNV and DENV infection. Excellent proof-of-principle experimental demonstrations for host-targeted control of infection and infection-induced pathogenesis have been reported for both WNV and DENV. These include exploiting not only those host factors supporting infection, but also targeting host processes contributing to pathogenesis and innate immune responses. While these early studies validated the host-targeting approach, extensive future investigations spanning a range of aspects are needed for a successful deployment in humans.West Nile (WNV) and Dengue (DENV) viruses are major arboviral human pathogens belonging to the genus  Flaviviridae family [West Nile virus (WNV) and the four dengue viruses DENV1-4) are flaviviruses belonging to the -4 are fle family . Dengue e family . The gloe family . All flaviviruses have a ~11 kb positive strand RNA genome and mRNA, which is translated into a single polyprotein that is cleaved into three structural and seven nonstructural (NS) proteins necessary for viral propagation [Given their genome’s limited coding capacity and their lifecycle’s complexity, it is not surprising that flaviviruses require scores of host factors ,6. TheseIn this chapter we will provide a review on the current understanding of the host factors that impact replication of WNV and DENV focusing primarily on those factors we deem particularly druggable. As implied by this focus, our review will be emphasizing only those factors in the human  host. En masse approaches have accelerated the discovery of host factors that impact propagation of WNV and DENV by interrogating tens of thousands of gene products or interactions simultaneously. While these methods have important limitations and should be considered hypotheses generating exercises that result in lists of candidate host factors that must be tested carefully, they have generated very important data. et al. (2007) identified three pathways containing many component genes with altered expression upon DENV infection of cells in culture and DENV infected blood samples: NF-kappaB initiated immune responses, type I interferon (IFN) and the ubiquitin proteasome pathway [et al. (2013) focused on the expression of mRNA isoforms  and concluded that isoforms of genes implicated in the innate immune responses were differentially processed during infection with wild type and attenuated strains of DENV [Genome-scale analyses of transcript levels, using one of many approaches , have revealed the identity of host mRNAs that significantly vary after WNV ,8,9 and  pathway . Among s of DENV . A few s of DENV  or DENV  of DENV ,19,20,21 of DENV .The logic behind interrogating gene expression differences is the assumption that dependency or restriction factors will be overrepresented among factors with altered expression  and indeed several studies suggest that this is likely true ,19. et al. screened all 10 DENV proteins (using multiple bait constructs for each) against a human liver cDNA library to identify 105 proteins as potential interactors [et al. screened DENV NS3 and NS5 against a cDNA library derived from human liver, spleen, brain and bronchial epithelia, and identified 108 human proteins that interact with NS3 or NS5 or both [Many methods look globally for host gene products that physically interact with viral proteins or RNAs. Two-hybrid interaction screens, which identify protein-protein interactions, have been used to define the protein interactome for WNV ,24,25,26eractors . The auteractors ), the co or both . Proteinen masse. Several groups have applied this method to identify host (human and mosquito) proteins that interact with WNV and DENV gene products (both proteins and RNAs) [et al. (2011) identified scores of proteins that interact with 5' and 3' UTR DENV-2 sequences, including the DEDD-box helicase DDX6, the 3'–5' exonuclease ERI3, which was also identified using functional genomics (see below), and stress granule associated proteins G3BP1, G3BP2 and CAPRIN-1 [Affinity chromatography is another commonly used method to determine protein-protein and RNA-protein interactions nd RNAs) ,43,44,45nd RNAs) . Althougnd RNAs) . Since Wnd RNAs) . RNA chrnd RNAs) ,42,45. Mnd RNAs) ,48,49,50en masse functional analysis in human cells  involves either gain of function screens accomplished by overexpressing cDNAs, or more commonly, RNAi-mediated loss of function screens . There has been one published genome-scale screen for human host factors of WNV [et al. (2008) provided the first look at the extensive array of host factors that impacted WNV replication and tested this set of gene products for their impact on DENV [et al. (2009) adapted DENV-2 for growth in drosophila cells and screened a dsRNA library for insect host factors in insect cells. The human homologs of the identified candidate factors were tested for an effect on DENV-2 replication revealing remarkable conservation for required factors in the human and mosquito hosts [Currently the s of WNV  and one s of WNV , and sevs of WNV ,52,53,54 on DENV . Sessionet al. [There is important debate regarding the reproducibility and on-target accuracy of RNAi screens. It should be acknowledged that some genes cannot be knocked down efficiently and that some genes may have redundant functions. Careful experimentation can ensure significant reproducibility, as observed in the case of a human genome-scale RNAi screen for host factors required for replication of yellow fever virus (17D vaccine strain) (YFV-17D) ,56. For et al. . The oveet al. ). Recently, a genome-wide association study (GWAS) coupled with validation of significantly associated markers was performed to identify genes associated with dengue shock syndrome (DSS), by comparing pediatric patients with controls. This study identified association of major histocompatibility complex (MHC) class I polypeptide-related sequence B (MICB), and phospholipase C, epsilon 1 (PLCE1) with DSS , and a sAs there are no approved therapies to inhibit or prevent flaviviral infections such as those caused by WNV and dengue virus, intensive research using three general approaches have been considered to attain this goal: inhibition of viral gene products ; inhibition of host dependency factors; and modulation of host restriction factors ,65. in vitro, they caused severe toxicity in animals when administered for two weeks [Flaviviridae family) helicase has been inhibited with quinolone derivatives, which also inhibit the HCV RdRp activity [There are many compounds targeting flaviviral NS proteins, however, few exhibit pan-flaviviral activities despite significant conservation in the sequence of NS proteins across flaviviruses . The higwo weeks ,70. One wo weeks . The flawo weeks ,73, as fwo weeks . Althougwo weeks . The cycwo weeks . Recentlwo weeks  and funcwo weeks . Hepatitactivity  and someactivity . Recentlactivity . Whereas most of the aforementioned studies used small molecule inhibitors, other reports describe the use of morpholinos to inhibit DENV2 growth in tissue culture  and of RAs discussed above, flaviviruses require the activity of scores of host factors to propagate efficiently, allowing drugs that inactivate these factors to be used to combat infections. This general approach has gained recent momentum in part because of the successful use of CCR5 antagonists in HIV-1 infected individuals . While sIt has become increasingly evident that cellular metabolic pathways are heavily co-opted by flaviviruses to successfully infect host cells ,87. Thisin vitro. The drug methyl-β-cyclodextran that sequesters cholesterol, and 25-hydroxycholesterol which affects the cholesterol transcription factor functioning, were also found to reduce WNV infection in vitro [in vitro [in vitro anti-flaviviral effects of various drugs interfering with lipid metabolism, and approved use of statins in humans, no in vivo study has yet been reported evaluating their anti-flaviviral therapeutic potential in either animal models and humans.Lipid metabolism is tightly associated with flaviviral infections. For example, cholesterol is essential for the infection of cells by both WNV and DENV. It was found that treatment with lovastatin, the widely used cardiovascular drug that inhibits HMG CoA reductase , blocks both WNV (Kunjin strain)  and DENVin vitro ,127. Simin vitro ,128. Furin vitro ,86. All de novo pyrimidine biosynthesis, was shown to inhibit both WNV and DENV, in vitro [Another well-known metabolic target against flaviviruses is nucleotide metabolism. Ribavirin, ribavirin triphosphate (RTP) and ETAR are believed to be inhibitors of inosine-5'-monophosphate dehydrogenase (IMPDH) ,97. Ribain vitro . In addiin vitro . Thus, sin vitro and in vivo in mice [O-butanoyl derivative of castanospermine  has been recently tested in human as a therapy against dengue virus infection [in vitro, and reduce viremina as well as DENV infection induced disease and death in mice [In addition to the above-described well-documented role of lipid and nucleotide metabolic pathways in flaviviral infection, several aspects of post-translational modifications  have also been demonstrated as drug targets against flaviviral infections. Among these, the carbohydrate modification pathways have been extensively investigated in the context of flaviviral infections. An α-glucosidase inhibitor, castanospermine, was found to be effective in blocking dengue virus, though not WNV, both  in mice ,96. The nfection . Separatnfection . The α-g in mice ,98. Inte in mice . Collectet al. (2012) identified IU1, a small molecule inhibitor of the ubiquitin specific protease USP14, as a potent inhibitor of DENV infection [Several additional cellular processes involved in protein processing are also identified as potential host targets against flaviviral infection. One study reported that the inhibition of the cyclophilin family of peptidyl prolyl isomerases using cyclosporine interferes with the replication of both WNV and DENV, at sub-cytotoxic concentrations . Mechaninfection . An earlnfection . Similarnfection . BecauseSeveral studies have taken a drug-based discovery approach to identify host molecules that can be exploited to control flaviviral infections. Excellent studies aimed at discovering host factors that can be targeted to control flaviviral infection have in fact revealed several such druggable candidates. Yang and colleagues focused on the role of kinases in dengue infection, and identified the kinases Fyn and c-Src (using the drugs AZD0530 and Dasatinib) as druggable host proviral factors ,102. OurA major initial step in viral infection is the entry into target host cells. Therefore the cellular entry stage of viral infection has always been an attractive area of antiviral research. The dengue and West Nile viruses are no exception to this trend.N-acetyl-D-glucosamine or N-acetyl-D-galactosamine) and a uronic acid (D-glucuronic or L-iduronic acid) or D-galactose units. Studies showed that cell surface glycosaminoglycan modifications  are important requirements for the infection of dengue viruses. Particularly, highly sulfated heparan sulfate expression was shown to be essential for dengue viral entry [Cladosiphon okamuranus, potently interfered with the infection of dengue virus serotype 2, but not other dengue serotypes [Gymnogongrus griffithsiae and Cryptonemia crenulata) displayed predominant inhibitory activity against dengue virus, primarily to serotype 2 [in vitro, and found that they all were active against dengue virus with the first one being most potent, followed by the other two, in decreasing order [in vitro [Our understanding of the host cellular receptors and co-receptors of both WNV and dengue virus entry is incomplete, and no definitive receptor has been established to date for either of these viruses. More information is available for the entry requirements of dengue virus than WNV. Several proteins have been reported as putative host targets needed for the cellular entry of dengue virus . These ial entry ,142. In al entry ,144. A val entry . Severalerotypes . The antrotype 2 . Anotherng order . Howeverng order . Synthetin vitro . As summThe third approach involves modulation of the host response. Diverse arms of the immune response contributes to the control of flaviviral infections; however, the focus of this review will only be on those host immune factors and mechanisms that offer non-traditional and emerging avenues for intervention. The traditional aspects of immune control such as vaccines are reviewed elsewhere ,148,149.in vivo. In an early trial of Type II interferons (IFNs) for prophylaxis and treatment of yellow fever virus (YFV) infection, we showed that IFN-γ treatment could ameliorate the severity of YFV in Saimiri sciureus (squirrel monkeys) and Macaca mulatta (rhesus monkeys), but did not change mortality in the latter [The best-studied antiviral systems are the pattern recognition receptors and interferons . Type I e latter , a modele latter . In anote latter . The thee latter . As an ae latter . In a ree latter . AlthougInterferons exert their antiviral functions through a diverse array of interferon-stimulated genes (ISG) and mechanisms, which could also be developed as therapeutic strategies . There ain vitro WNV infection [Autophagy is another host process that can be exploited to limit flaviviral infections. Autophagy is a complex cellular process involved in both cellular component degradation and recycling, and is also integrated with a wide variety of physiological processes. Not surprisingly, autophagy is also associated with viral infections, and is known to serve as either a proviral or an antiviral immune mechanism. While autophagy did not impact nfection , successnfection ,121,160.nfection . in vivo using appropriate animal models.In summary, several excellent avenues exist to target non-traditional aspects of host antiviral immunity to mitigate viral infections. Future attention to exploit their human therapeutic potential and clinical application warrants extensive validation Diverse sets of immune and non-immune host molecules are involved in disease pathogenesis during viral infections. Targeting these molecules may help to control infection-triggered worsening of physiological homeostasis, in turn enabling the body to control viruses by itself. There are several examples for these, relevant to flaviviruses. Several host targets including cytokines have already been tested to alleviate pathogenesis of flaviviruses. The platelet-activating factor receptor (PAFR) has been documented to play a key role in dengue virus pathogenesis. A recent report describes successful use of two platelet-activating factor receptor antagonists to decrease thrombocytopenia, vascular permeability and mortality in a mouse model of dengue infection . in vitro [Several excellent studies have also revealed multiple host targets to mitigate WNV pathogenesis. In one study, targeting the cytokine macrophage migration inhibitory factor (MIF) with both a small molecule antagonist and an antibody was found to protect against WNV pathogenesis . Similarin vitro . This inin vitro [Viral infections can also cause structural damage to host organ systems, leading to pathogenesis. Therefore any drug that can prevent these infection induced “structural damages” of the host will have great clinical value. For example, a major mechanism by which dengue virus harms the human host is by inducing damage to vasculature, leading to vascular leakage. Recently, both mast cell stabilizing drugs and leukotriene receptor antagonist have been found to be effective to control vascular leakage in mice injected with dengue virus . The WNVin vitro . This obThe above-cited studies argue that targeting of both host restriction mechanisms and pathogenesis processes, perhaps simultaneously, may eventually become part of a comprehensive therapeutic regimen to manage flaviviral infection and disease of humans. However, several of these approaches are still in their development phase, and will have to endure extensive maturation before their practical clinical application can be evaluated in humans. The studies described in this review clearly demonstrated that several host molecules hold the potential to serve as therapeutic targets to treat flaviviral infections. These include both metabolic pathways and non-metabolic targets. While some of these host molecules have already been tested in animal models of infection or humans, most await detailed experimentation and validation. The host-targeting antivirals may not result in rapid formation of drug resistant mutations in the viruses.en masse approaches  need to be mined and adequately validated to identify candidates with the potential for modulation by drugs. There is also a need for newer discovery studies to identify essential host factors impacting flavivirus infections in the most appropriate cell types . These genetic study-based identifications of the flavivirus host factors should be complemented with more focused screens and validations using drugs that exclusively target known host molecules. Such an approach will provide us with a wider choice of drug targets and drugs against viruses; (ii) In vivo validations. The druggable host target genes that are already identified (as well as the to-be-discovered targets) to regulate viral infection need to be validated using appropriate gene knockout in vivo models of infections. Given the current availability of better animal models for WNV , one would anticipate faster progress in the case of WNV than for DENV. Similarly, the drugs targeting the identified host factors should also be evaluated using the most suitable animal model that mimics infection induced disease in humans; (iii) Cross-protection. Because different flaviviruses have a significant degree of conservation in their genome, some of the mechanisms involved in their interaction with host cells are also likely to be conserved. Therefore, any drug or drug target identified as useful against a particular flavivirus should be tested against a wide range of medically relevant flaviviruses of the genus Flavivirus. This will help to identify pan-flaviviral therapeutics; and (iv) Drugs boosting host innate defenses. Particular emphasis should be given to discover drugs that can augment various innate antiviral mechanisms of cells. In addition to enhancing the generic production of interferon, such drugs should ideally activate specific effector mechanisms of cells against viruses. For example, a drug that can enhance the production or antiviral activity of interferon stimulated antiviral genes may offer better protection from infection, and less toxicity to the host. A sub-category of immune response boosting drugs should also have the ability to attenuate viral interference with host immune defenses. Future attempts to develop host-targeted therapeutic approaches to successfully contain the threat of flaviviral infections should take into account at least four general areas of research: (i) Systematic identification and validation of host targets implicated in flaviviral infections. The multitude of flaviviral infection-associated host molecules  identified through various In summary, a multipronged approach integrating both host target and host-directed drug discovery will ultimately facilitate the identification of effective therapeutics against flaviviruses.


DNA damage contributes to cancer development and aging. Congenital syndromes that affect DNA repair processes are characterized by cancer susceptibility, developmental defects, and accelerated aging . DNA dam Figure 1). Cellular metabolism can produce reactive oxygen species (ROS) and alkylating agents, while cells can be exposed to ultraviolet (UV), ionizing radiation (IR), and a variety of genotoxic chemicals .Given the frequency and impact of DNA damage, highly sophisticated DNA repair systems have evolved. These systems recognize specific types of lesions and induce DNA damage signaling. Failure of DNA repair has been associated with severe disorders in humans, often associated with occurrence of cancer and/or premature aging  to DSBs is particularly well-understood. As the presence of a DSB poses a major obstacle for further cell division a sophisticated network of DDR signaling is ignited . GeneticThe initial recognition of DSBs involves binding of the trimeric Mre11–Rad50–Nbs1 (MRN) complex consisting of MRE11, NBS1, and RAD50 to the broken DNA ends . The MRNCells with defective DSB repair, like ATM mutants, are highly IR sensitive. Heritable mutations in ATM result in ataxia telangiectasia (AT), a rare recessive syndrome that is associated with progressive neurodegeneration, variable kinds of immune-deficiencies and a predisposition to lymphoid cancer . PatientWhile DNA damage checkpoint signaling halt the cell cycle, at least two distinct DSB repair machineries are activated depending on the phase of the cell cycle . In S/G2In the case of HR, both, reduced and elevated activity, have been associated with a predisposition to cancer . For exaLike DSB repair, the employment of removal mechanisms of ICLs alters depending on cell cycle stage. During G1 the excision repair cross-complementation group 1–xeroderma pigmentosum group F (ERCC1–XPF) endonuclease initiates the ICL removal . When ICThe inability to repair ICLs in FA patients leads to replication fork collapses, particularly in actively dividing cells such as the hematopoietic system. Consequently, FA is characterized by bone marrow failure due to chromosomal aberration and leukemic transformation of cells. Also FA is characterized by cancer predisposition and hypersensitivity to crosslinking agents like mitomycin C, cisplatin, or diepoxybutane .Also the Werner protein (WRN) has been linked to DNA repair when ICLs cause replication fork breakdown . WRN hasWRN underlie Werner syndrome (WS), which is a rare autosomal recessive disease. WS patients not only exhibit elevated cancer predisposition, but also develop symptoms of premature aging. The progeroid features of WS patients are particularly well recognized as a premature onset of aging as they develop typically during the third decade of life. Classic forms of progeroid syndromes are also found in patients carrying mutations in related RecQ helicases. BLS, and Rothmund–Thomson syndrome (RTS) are caused by mutations in the Bloom’s, and RECQL4 helicases, respectively , atypical WS, restrictive dermopathy (RD), and mandibuloacral dysplasia (MAD) are caused by mutations in lamin A and the ZMPSTE24 farnesyltransferase required for lamin A processing . The nucsitivity . It was sitivity . Consistsitivity . In addisitivity . TogetheTable 1). Unlike most of the progeroid syndromes discussed above, mutations that affect the two distinct branches of NER are linked either to cancer susceptibility or to premature aging. Defects in the global genome (GG-) NER branch cause the skin cancer susceptibility syndrome xeroderma pigmentosum (XP), while mutations affecting the transcription-coupled (TC-) NER branch lead to progeroid syndromes such as Cockayne syndrome (CS) that is characterized by postnatal growth retardation and accelerated aging but not cancer (Mutations in nucleotide excision repair (NER) underlie a variety of skin cancer predisposing and degenerative disorders ; Table 1t cancer . The disIn vivo experiments monitoring transcription elongation speed have revealed that RNAPII often stalls even in the absence of exogenous DNA damage. Transcription appears rather like stop-and-go traffic than a continuous process. This might have to do with spontaneous DNA damage, structural tension, histone remodeling, or regulatory events, all of which might impede elongation. During elongation, the transcription complex might also serve as control mechanism for DNA integrity, particularly in post-replicative cell types. The sensitivity of ongoing transcription to stall at DNA adducts was suggested to function as a “damage dosimeter” . The CSB protein, comprising a switch/sucrose nonfermentable (SWI/SNF)-like DNA-dependent ATPase, is associated with RNAPII and upon stalling recruits the WD40 domain protein CSA. CSB is stabilized by the recently discovered UV-stimulated scaffold protein A (UVSSA) protein that ubiquitylates the stalled RNAPII , and characteristic brittle hair and nails. Especially the brittle structure of the hair and nails has been linked to defects in completing transcription leading to deficiency of high-sulfur matrix proteins . In addition, TFIIH has been implicated in transcription that is mediated by RNAPI . Stalling of transcription complexes can also have detrimental consequences for the cell when for example a replication fork collides with a blocked RNAPII complex -tagged NER proteins in cell culture . This meFigure 2). XPC also mediates initiation of the transcription of the stem cell inducer Nanog together with Oct4/Sox2  experiments . Here, tct4/Sox2 . The XPFct4/Sox2 . Interes mutants . TAF10 cNot only factors acting in NER but also the proteins that are dysfunctional in HGPS and WS have been connected to transcription. Disruption of nuclear structure by transfection of a dominant negative lamin A mutant is followed by reduced transcription as measured by bromouridine-triphosphate (BrUTP) incorporation . FurtherTaken together several lines of evidence indicate that responses to transcription-blocking lesions are powerful tumor suppressors. Particularly, the apparent cancer protection in TC-NER defective patients suggests that cellular growth impairment together with pro-apoptotic signaling in response to RNAPII stalling could effectively limit cancer cell proliferation. Importantly, there are p53-dependent as well as -independent responses making transcription-blockage mediated tumor suppression also relevant when p53 is mutated . Indeed,Igf-1r mice are not only protected from aging but also from neurodegenerative diseases as demonstrated in mouse models for Alzheimer’s disease (The responses to transcription-blockage, namely attenuation of the somatotropic axis, might also provide targets for therapies aimed to prevent age-related diseases . An earl disease . There a disease . Althoug disease .In conclusion, it will be of pivotal importance to establish the role of transcription impairments in progeroid syndromes and during the course of DNA damage accumulation with aging. It will be particularly interesting to further explore the distinct consequences of impaired transcription elongation and initiation and to establish how cellular response mechanisms to stalled transcription complexes impact on the physiological adjustments of the aging organism.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.