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1. Cis-regulatory modes of Ultrabithorax inactivation in butterfly forewings

   https://doi.org/10.7554/eLife.90846.3  

   Tendolkar, Amruta; Mazo-Vargas, Anyi; Livraghi, Luca; Hanly, Joseph J; Van_Horne, Kelsey C; Gilbert, Lawrence E; Martin, Arnaud (January 2024, eLife)

   Hoxgene clusters encode transcription factors that drive regional specialization during animal development: for example the Hox factor Ubx is expressed in the insect metathoracic (T3) wing appendages and differentiates them from T2 mesothoracic identities.Hoxtranscriptional regulation requires silencing activities that prevent spurious activation and regulatory crosstalks in the wrong tissues, but this has seldom been studied in insects other thanDrosophila, which shows a derivedHoxdislocation into two genomic clusters that disjoinedAntennapedia(Antp) andUltrabithorax(Ubx). Here, we investigated howUbxis restricted to the hindwing in butterflies, amidst a contiguousHoxcluster. By analysing Hi-C and ATAC-seq data in the butterflyJunonia coenia, we show that a Topologically Associated Domain (TAD) maintains a hindwing-enriched profile of chromatin opening aroundUbx. This TAD is bordered by a Boundary Element (BE) that separates it from a region of joined wing activity around theAntplocus. CRISPR mutational perturbation of this BE releases ectopicUbxexpression in forewings, inducing homeotic clones with hindwing identities. Further mutational interrogation of two non-coding RNA encoding regions and one putativecis-regulatory module within theUbxTAD cause rare homeotic transformations in both directions, indicating the presence of both activating and repressing chromatin features. We also describe a series of spontaneous forewing homeotic phenotypes obtained inHeliconiusbutterflies, and discuss their possible mutational basis. By leveraging the extensive wing specialization found in butterflies, our initial exploration ofUbxregulation demonstrates the existence of silencing and insulating sequences that prevent its spurious expression in forewings. 

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2. Ultrabithorax Is a Micromanager of Hindwing Identity in Butterflies and Moths

   https://doi.org/10.3389/fevo.2021.643661  

   Tendolkar, Amruta; Pomerantz, Aaron F.; Heryanto, Christa; Shirk, Paul D.; Patel, Nipam H.; Martin, Arnaud (March 2021, Frontiers in Ecology and Evolution)

   null (Ed.)

   The forewings and hindwings of butterflies and moths (Lepidoptera) are differentiated from each other, with segment-specific morphologies and color patterns that mediate a wide range of functions in flight, signaling, and protection. The Hox gene Ultrabithorax ( Ubx ) is a master selector gene that differentiates metathoracic from mesothoracic identities across winged insects, and previous work has shown this role extends to at least some of the color patterns from the butterfly hindwing. Here we used CRISPR targeted mutagenesis to generate Ubx loss-of-function somatic mutations in two nymphalid butterflies ( Junonia coenia , Vanessa cardui ) and a pyralid moth ( Plodia interpunctella ). The resulting mosaic clones yielded hindwing-to-forewing transformations, showing Ubx is necessary for specifying many aspects of hindwing-specific identities, including scale morphologies, color patterns, and wing venation and structure. These homeotic phenotypes showed cell-autonomous, sharp transitions between mutant and non-mutant scales, except for clones that encroached into the border ocelli (eyespots) and resulted in composite and non-autonomous effects on eyespot ring determination. In the pyralid moth, homeotic clones converted the folding and depigmented hindwing into rigid and pigmented composites, affected the wing-coupling frenulum, and induced ectopic scent-scales in male androconia. These data confirm Ubx is a master selector of lepidopteran hindwing identity and suggest it acts on many gene regulatory networks involved in wing development and patterning. 

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3. Hox proteins interact to pattern neuronal subtypes in Caenorhabditis elegans males

   https://doi.org/10.1093/genetics/iyac010  

   Kalis, Andrea K; Sterrett, Maria C; Armstrong, Cecily; Ballmer, Amarantha; Burkstrand, Kylie; Chilson, Elizabeth; Emlen, Estee; Ferrer, Emma; Loeb, Seanna; Olin, Taylor; et al (February 2022, Genetics)

   Hobert, O (Ed.)

   Abstract Hox transcription factors are conserved regulators of neuronal subtype specification on the anteroposterior axis in animals, with disruption of Hox gene expression leading to homeotic transformations of neuronal identities. We have taken advantage of an unusual mutation in the Caenorhabditis elegans Hox gene lin-39, lin-39(ccc16), which transforms neuronal fates in the C. elegans male ventral nerve cord in a manner that depends on a second Hox gene, mab-5. We have performed a genetic analysis centered around this homeotic allele of lin-39 in conjunction with reporters for neuronal target genes and protein interaction assays to explore how LIN-39 and MAB-5 exert both flexibility and specificity in target regulation. We identify cis-regulatory modules in neuronal reporters that are both region-specific and Hox-responsive. Using these reporters of neuronal subtype, we also find that the lin-39(ccc16) mutation disrupts neuronal fates specifically in the region where lin-39 and mab-5 are coexpressed, and that the protein encoded by lin-39(ccc16) is active only in the absence of mab-5. Moreover, the fates of neurons typical to the region of lin-39-mab-5 coexpression depend on both Hox genes. Our genetic analysis, along with evidence from Bimolecular Fluorescence Complementation protein interaction assays, supports a model in which LIN-39 and MAB-5 act at an array of cis-regulatory modules to cooperatively activate and to individually activate or repress neuronal gene expression, resulting in regionally specific neuronal fates. 

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4. Plant polymerase IV sensitizes chromatin through histone modifications to preclude spread of silencing into protein-coding domains

   https://doi.org/10.1101/gr.277353.122  

   Hari Sundar G, Vivek; Swetha, Chenna; Basu, Debjani; Pachamuthu, Kannan; Raju, Steffi; Chakraborty, Tania; Mosher, Rebecca A.; Shivaprasad, P.V. (May 2023, Genome Research)

   Across eukaryotes, gene regulation is manifested via chromatin states roughly distinguished as heterochromatin and euchromatin. The establishment, maintenance, and modulation of the chromatin states is mediated using several factors including chromatin modifiers. However, factors that avoid the intrusion of silencing signals into protein-coding genes are poorly understood. Here we show that a plant specific paralog of RNA polymerase (Pol) II, named Pol IV, is involved in avoidance of facultative heterochromatic marks in protein-coding genes, in addition to its well-established functions in silencing repeats and transposons. In its absence, H3K27 trimethylation (me3) mark intruded the protein-coding genes, more profoundly in genes embedded with repeats. In a subset of genes, spurious transcriptional activity resulted in small(s) RNA production, leading to post-transcriptional gene silencing. We show that such effects are significantly pronounced in rice, a plant with a larger genome with distributed heterochromatin compared withArabidopsis. Our results indicate the division of labor among plant-specific polymerases, not just in establishing effective silencing via sRNAs and DNA methylation but also in influencing chromatin boundaries. 

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5. Parallel evolution of ancient, pleiotropic enhancers underlies butterfly wing pattern mimicry

   https://doi.org/10.1073/pnas.1907068116  

   Lewis, James J.; Geltman, Rachel C.; Pollak, Patrick C.; Rondem, Kathleen E.; Van Belleghem, Steven M.; Hubisz, Melissa J.; Munn, Paul R.; Zhang, Linlin; Benson, Caleb; Mazo-Vargas, Anyi; et al (November 2019, Proceedings of the National Academy of Sciences)

   Color pattern mimicry in Heliconius butterflies is a classic case study of complex trait adaptation via selection on a few large effect genes. Association studies have linked color pattern variation to a handful of noncoding regions, yet the presumptive cis-regulatory elements (CREs) that control color patterning remain unknown. Here we combine chromatin assays, DNA sequence associations, and genome editing to functionally characterize 5 cis-regulatory elements of the color pattern gene optix . We were surprised to find that the cis-regulatory architecture of optix is characterized by pleiotropy and regulatory fragility, where deletion of individual cis-regulatory elements has broad effects on both color pattern and wing vein development. Remarkably, we found orthologous cis-regulatory elements associate with wing pattern convergence of distantly related comimics, suggesting that parallel coevolution of ancestral elements facilitated pattern mimicry. Our results support a model of color pattern evolution in Heliconius where changes to ancient, multifunctional cis-regulatory elements underlie adaptive radiation. 

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