Global analysis of primary mesenchyme cell cis-regulatory modules by chromatin accessibility profiling.

BMC Genomics. 2018 Mar 20;19(1):206

Authors: Shashikant T, Khor JM, Ettensohn CA

Abstract
BACKGROUND: The developmental gene regulatory network (GRN) that underlies skeletogenesis in sea urchins and other echinoderms is a paradigm of GRN structure, function, and evolution. This transcriptional network is deployed selectively in skeleton-forming primary mesenchyme cells (PMCs) of the early embryo. To advance our understanding of this model developmental GRN, we used genome-wide chromatin accessibility profiling to identify and characterize PMC cis-regulatory modules (CRMs).
RESULTS: ATAC-seq (Assay for Transposase-Accessible Chromatin using sequencing) analysis of purified PMCs provided a global picture of chromatin accessibility in these cells. We used both ATAC-seq and DNase-seq (DNase I hypersensitive site sequencing) to identify > 3000 sites that exhibited increased accessibility in PMCs relative to other embryonic cell lineages, and provide both computational and experimental evidence that a large fraction of these sites represent bona fide skeletogenic CRMs. Putative PMC CRMs were preferentially located near genes differentially expressed by PMCs and consensus binding sites for two key transcription factors in the PMC GRN, Alx1 and Ets1, were enriched in these CRMs. Moreover, a high proportion of candidate CRMs drove reporter gene expression specifically in PMCs in transgenic embryos. Surprisingly, we found that PMC CRMs were partially open in other embryonic lineages and exhibited hyperaccessibility as early as the 128-cell stage.
CONCLUSIONS: Our work provides a comprehensive picture of chromatin accessibility in an early embryonic cell lineage. By identifying thousands of candidate PMC CRMs, we significantly enhance the utility of the sea urchin skeletogenic network as a general model of GRN architecture and evolution. Our work also shows that differential chromatin accessibility, which has been used for the high-throughput identification of enhancers in differentiated cell types, is a powerful approach for the identification of CRMs in early embryonic cells. Lastly, we conclude that in the sea urchin embryo, CRMs that control the cell type-specific expression of effector genes are hyperaccessible several hours in advance of gene activation.

PMID: 29558892 [PubMed - in process]

A conserved Shh cis-regulatory module highlights a common developmental origin of unpaired and paired fins.

Nat Genet. 2018 Mar 19;:

Authors: Letelier J, de la Calle-Mustienes E, Pieretti J, Naranjo S, Maeso I, Nakamura T, Pascual-Anaya J, Shubin NH, Schneider I, Martinez-Morales JR, Gómez-Skarmeta JL

Abstract
Despite their evolutionary, developmental and functional importance, the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka (Oryzias latipes) ZRS and enhancer assays identify the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolishes shh expression and completely truncates pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.

PMID: 29556077 [PubMed - as supplied by publisher]

Investigating Evolutionarily Conserved Molecular Mechanisms Controlling Gene Expression in the Notochord.

Adv Exp Med Biol. 2018;1029:81-99

Authors: Maguire JE, Pandey A, Wu Y, Di Gregorio A

Abstract
Ascidian embryos have been employed as model systems for studies of developmental biology for well over a century, owing to their desirable blend of experimental advantages, which include their rapid development, traceable cell lineage, and evolutionarily conserved morphogenetic movements. Two decades ago, the development of a streamlined electroporation method drastically reduced the time and cost of transgenic experiments, and, along with the elucidation of the complete genomic sequences of several ascidian species, propelled these simple chordates to the forefront of the model organisms available for studies of regulation of gene expression. Numerous ascidian sequences with tissue-specific enhancer activity were isolated and rapidly characterized through systematic in vivo experiments that would require several weeks in most other model systems. These cis-regulatory sequences include a large collection of notochord enhancers, which have been used to visualize notochord development in vivo, to generate mutant phenotypes, and to knock down genes of interest. Moreover, their detailed characterization has allowed the reconstruction of different branches of the notochord gene regulatory network. This chapter describes how the use of transgenic techniques has rendered the ascidian Ciona a competitive model organism for studies of notochord development, evolution, and gene regulation.

PMID: 29542082 [PubMed - in process]

Cooperative recruitment of Yan via a high-affinity ETS supersite organizes repression to confer specificity and robustness to cardiac cell fate specification.

Genes Dev. 2018 Mar 13;:

Authors: Boisclair Lachance JF, Webber JL, Hong L, Dinner A, Rebay I

Abstract
Cis-regulatory modules (CRMs) are defined by unique combinations of transcription factor-binding sites. Emerging evidence suggests that the number, affinity, and organization of sites play important roles in regulating enhancer output and, ultimately, gene expression. Here, we investigate how the cis-regulatory logic of a tissue-specific CRM responsible for even-skipped (eve) induction during cardiogenesis organizes the competing inputs of two E-twenty-six (ETS) members: the activator Pointed (Pnt) and the repressor Yan. Using a combination of reporter gene assays and CRISPR-Cas9 gene editing, we suggest that Yan and Pnt have distinct syntax preferences. Not only does Yan prefer high-affinity sites, but an overlapping pair of such sites is necessary and sufficient for Yan to tune Eve expression levels in newly specified cardioblasts and block ectopic Eve induction and cell fate specification in surrounding progenitors. Mechanistically, the efficient Yan recruitment promoted by this high-affinity ETS supersite not only biases Yan-Pnt competition at the specific CRM but also organizes Yan-repressive complexes in three dimensions across the eve locus. Taken together, our results uncover a novel mechanism by which differential interpretation of CRM syntax by a competing repressor-activator pair can confer both specificity and robustness to developmental transitions.

PMID: 29535190 [PubMed - as supplied by publisher]

A long range distal enhancer controls temporal fine-tuning of PAX6 expression in neuronal precursors.

Dev Biol. 2018 Feb 24;:

Authors: Lacomme M, Medevielle F, Bourbon HM, Thierion E, Kleinjan DJ, Roussat M, Pituello F, Bel-Vialar S

Abstract
Proper embryonic development relies on a tight control of spatial and temporal gene expression profiles in a highly regulated manner. One good example is the ON/OFF switching of the transcription factor PAX6 that governs important steps of neurogenesis. In the neural tube PAX6 expression is initiated in neural progenitors through the positive action of retinoic acid signaling and downregulated in neuronal precursors by the bHLH transcription factor NEUROG2. How these two regulatory inputs are integrated at the molecular level to properly fine tune temporal PAX6 expression is not known. In this study we identified and characterized a 940-bp long distal cis-regulatory module (CRM), located far away from the PAX6 transcription unit and which conveys positive input from RA signaling pathway and indirect repressive signal(s) from NEUROG2. These opposing regulatory signals are integrated through HOMZ, a 94bp core region within E940 which is evolutionarily conserved in distant organisms such as the zebrafish. We show that within HOMZ, NEUROG2 and RA exert their opposite temporal activities through a short 60bp region containing a functional RA-responsive element (RARE). We propose a model in which retinoic acid receptors (RARs) and NEUROG2 repressive target(s) compete on the same DNA motif to fine tune temporal PAX6 expression during the course of spinal neurogenesis.

PMID: 29486153 [PubMed - as supplied by publisher]

SOX2 regulates common and specific stem cell features in the CNS and endoderm derived organs.

PLoS Genet. 2018 Feb 12;14(2):e1007224

Authors: Hagey DW, Klum S, Kurtsdotter I, Zaouter C, Topcic D, Andersson O, Bergsland M, Muhr J

Abstract
Stem cells are defined by their capacities to self-renew and generate progeny of multiple lineages. The transcription factor SOX2 has key roles in the regulation of stem cell characteristics, but whether SOX2 achieves these functions through similar mechanisms in distinct stem cell populations is not known. To address this question, we performed RNA-seq and SOX2 ChIP-seq on embryonic mouse cortex, spinal cord, stomach and lung/esophagus. We demonstrate that, although SOX2 binds a similar motif in the different cell types, its target regions are primarily cell-type-specific and enriched for the distinct binding motifs of appropriately expressed interacting co-factors. Furthermore, cell-type-specific SOX2 binding in endodermal and neural cells is most often found around genes specifically expressed in the corresponding tissue. Consistent with this, we demonstrate that SOX2 target regions can act as cis-regulatory modules capable of directing reporter expression to appropriate tissues in a zebrafish reporter assay. In contrast, SOX2 binding sites found in both endodermal and neural tissues are associated with genes regulating general stem cell features, such as proliferation. Notably, we provide evidence that SOX2 regulates proliferation through conserved mechanisms and target genes in both germ layers examined. Together, these findings demonstrate how SOX2 simultaneously regulates cell-type-specific, as well as core transcriptional programs in neural and endodermal stem cells.

PMID: 29432416 [PubMed - as supplied by publisher]

Threshold-dependent transcriptional discrimination underlies stem cell homeostasis.

Proc Natl Acad Sci U S A. 2016 10 11;113(41):E6298-E6306

Authors: Perales M, Rodriguez K, Snipes S, Yadav RK, Diaz-Mendoza M, Reddy GV

Abstract
Transcriptional mechanisms that underlie the dose-dependent regulation of gene expression in animal development have been studied extensively. However, the mechanisms of dose-dependent transcriptional regulation in plant development have not been understood. In Arabidopsis shoot apical meristems, WUSCHEL (WUS), a stem cell-promoting transcription factor, accumulates at a higher level in the rib meristem and at a lower level in the central zone where it activates its own negative regulator, CLAVATA3 (CLV3). How WUS regulates CLV3 levels has not been understood. Here we show that WUS binds a group of cis-elements, cis- regulatory module, in the CLV3-regulatory region, with different affinities and conformations, consisting of monomers at lower concentration and as dimers at a higher level. By deleting cis elements, manipulating the WUS-binding affinity and the homodimerization threshold of cis elements, and manipulating WUS levels, we show that the same cis elements mediate both the activation and repression of CLV3 at lower and higher WUS levels, respectively. The concentration-dependent transcriptional discrimination provides a mechanistic framework to explain the regulation of CLV3 levels that is critical for stem cell homeostasis.

PMID: 27671653 [PubMed - indexed for MEDLINE]

FastBill: An Improved Tool for Prediction of Cis-Regulatory Modules.

J Comput Biol. 2017 Mar;24(3):193-199

Authors: Wilczynski B, Tiuryn J

Abstract
Here, we provide a new software tool, called FastBill, for prediction of evolutionarily conserved cis-regulatory modules. It improves on the previous version of our program, called Billboard, by improving the statistical significance calculation. It is also faster than the original Billboard, allowing for large-scale analyses, including multiple informant species. We illustrate the utility of FastBill by performing a large-scale computational experiment of enhancer prediction in the promoter area of more than 150 Drosophila melanogaster genes that possess annotated experimentally verified enhancers. FastBill is written in Python and is freely available for download as a standalone tool.

PMID: 27710048 [PubMed - indexed for MEDLINE]

Computational exploration of cis-regulatory modules in rhythmic expression data using the "Exploration of Distinctive CREs and CRMs" (EDCC) and "CRM Network Generator" (CNG) programs.

PLoS One. 2018;13(1):e0190421

Authors: Bekiaris PS, Tekath T, Staiger D, Danisman S

Abstract
Understanding the effect of cis-regulatory elements (CRE) and clusters of CREs, which are called cis-regulatory modules (CRM), in eukaryotic gene expression is a challenge of computational biology. We developed two programs that allow simple, fast and reliable analysis of candidate CREs and CRMs that may affect specific gene expression and that determine positional features between individual CREs within a CRM. The first program, "Exploration of Distinctive CREs and CRMs" (EDCC), correlates candidate CREs and CRMs with specific gene expression patterns. For pairs of CREs, EDCC also determines positional preferences of the single CREs in relation to each other and to the transcriptional start site. The second program, "CRM Network Generator" (CNG), prioritizes these positional preferences using a neural network and thus allows unbiased rating of the positional preferences that were determined by EDCC. We tested these programs with data from a microarray study of circadian gene expression in Arabidopsis thaliana. Analyzing more than 1.5 million pairwise CRE combinations, we found 22 candidate combinations, of which several contained known clock promoter elements together with elements that had not been identified as relevant to circadian gene expression before. CNG analysis further identified positional preferences of these CRE pairs, hinting at positional information that may be relevant for circadian gene expression. Future wet lab experiments will have to determine which of these combinations confer daytime specific circadian gene expression.

PMID: 29298348 [PubMed - in process]

MLL3/MLL4/COMPASS Family on Epigenetic Regulation of Enhancer Function and Cancer.

Cold Spring Harb Perspect Med. 2016 Nov 01;6(11):

Authors: Sze CC, Shilatifard A

Abstract
During development, precise spatiotemporal patterns of gene expression are coordinately controlled by cis-regulatory modules known as enhancers. Their crucial role in development helped spur numerous studies aiming to elucidate the functional properties of enhancers within their physiological and disease contexts. In recent years, the role of enhancer malfunction in tissue-specific tumorigenesis is increasingly investigated. Here, we direct our focus to two primary players in enhancer regulation and their role in cancer pathogenesis: MLL3 and MLL4, members of the COMPASS family of histone H3 lysine 4 (H3K4) methyltransferases, and their complex-specific subunit UTX, a histone H3 lysine 27 (H3K27) demethylase. We review the most recent evidence on the underlying roles of MLL3/MLL4 and UTX in cancer and highlight key outstanding questions to help drive future research and contribute to our fundamental understanding of cancer and facilitate identification of therapeutic opportunities.

PMID: 27638352 [PubMed - indexed for MEDLINE]

CRISPR/Cas9 and Active Genetics-based trans-species replacement of the endogenous Drosophilakni-L2 CRM reveals unexpected complexity.

Elife. 2017 Dec 23;6:

Authors: Xu XS, Gantz VM, Siomava N, Bier E

Abstract
The knirps (kni) locus encodes transcription factors required for induction of the L2 wing vein in Drosophila. Here, we employ diverse CRISPR/Cas9 genome editing tools to generate a series of targeted lesions within the endogenous cis-regulatory module (CRM) required for kni expression in the L2 vein primordium. Phenotypic analysis of these 'in locus' mutations based on both expression of Kni protein and adult wing phenotypes, reveals novel unexpected features of L2-CRM function including evidence for a chromosome pairing-dependent process that promotes transcription. We also demonstrate that self-propagating active genetic elements (CopyCat elements) can efficiently delete and replace the L2-CRM with orthologous sequences from other divergent fly species. Wing vein phenotypes resulting from these trans-species enhancer replacements parallel features of the respective donor fly species. This highly sensitive phenotypic readout of enhancer function in a native genomic context reveals novel features of CRM function undetected by traditional reporter gene analysis.

PMID: 29274230 [PubMed - as supplied by publisher]

Origins and Specification of the Drosophila Wing.

Curr Biol. 2017 Nov 30;:

Authors: Requena D, Álvarez JA, Gabilondo H, Loker R, Mann RS, Estella C

Abstract
The insect wing is a key evolutionary innovation that was essential for insect diversification. Yet despite its importance, there is still debate about its evolutionary origins. Two main hypotheses have been proposed: the paranotal hypothesis, which suggests that wings evolved as an extension of the dorsal thorax, and the gill-exite hypothesis, which proposes that wings were derived from a modification of a pre-existing branch at the dorsal base (subcoxa) of the leg. Here, we address this question by studying how wing fates are initially specified during Drosophila embryogenesis, by characterizing a cis-regulatory module (CRM) from the snail (sna) gene, sna-DP (for dorsal primordia). sna-DP specifically marks the early primordia for both the wing and haltere, collectively referred to as the DP. We found that the inputs that activate sna-DP are distinct from those that activate Distalless, a marker for leg fates. Further, in genetic backgrounds in which the leg primordia are absent, the DP are still partially specified. However, lineage-tracing experiments demonstrate that cells from the early leg primordia contribute to both ventral and dorsal appendage fates. Together, these results suggest that the wings of Drosophila have a dual developmental origin: two groups of cells, one ventral and one more dorsal, give rise to the mature wing. We suggest that the dual developmental origins of the wing may be a molecular remnant of the evolutionary history of this appendage, in which cells of the subcoxa of the leg coalesced with dorsal outgrowths to evolve a dorsal appendage with motor control.

PMID: 29225023 [PubMed - as supplied by publisher]

Gata6 restricts Isl1 to the posterior of nascent hindlimb buds through Isl1 cis-regulatory modules.

Dev Biol. 2017 Nov 29;:

Authors: Tahara N, Akiyama R, Theisen JWM, Kawakami H, Wong J, Garry DJ, Kawakami Y

Abstract
Isl1 is required for two processes during hindlimb development: initiation of the processes directing hindlimb development in the lateral plate mesoderm and configuring posterior hindlimb field in the nascent hindlimb buds. During these processes, Isl1 expression is restricted to the posterior mesenchyme of hindlimb buds. How this dynamic change in Isl1 expression is regulated remains unknown. We found that two evolutionarily conserved sequences, located 3' to the Isl1 gene, regulate LacZ transgene expression in the hindlimb-forming region in mouse embryos. Both sequences contain GATA binding motifs, and expression pattern analysis identified that Gata6 is expressed in the flank and the anterior portion of nascent hindlimb buds. Recent studies have shown that conditional inactivation of Gata6 in mice causes hindlimb-specific pre-axial polydactyly, indicating a role of Gata6 in anterior-posterior patterning of hindlimbs. We studied whether Gata6 restricts Isl1 in the nascent hindlimb bud through the cis-regulatory modules. In vitro experiments demonstrate that GATA6 binds to the conserved GATA motifs in the cis-regulatory modules. GATA6 repressed expression of a luciferase reporter that contains the cis-regulatory modules by synergizing with Zfpm2. Analyses of Gata6 mutant embryos showed that ISL1 levels are higher in the anterior of nascent hindlimb buds than in wild type. Moreover, we detected a greater number of Isl1-transcribing cells in the anterior of nascent hindlimb buds in Gata6 mutants. Our results support a model in which Gata6 contributes to repression of Isl1 expression in the anterior of nascent hindlimb buds.

PMID: 29197504 [PubMed - as supplied by publisher]

A novel method for predicting activity of cis-regulatory modules, based on a diverse training set.

Bioinformatics. 2017 Jan 01;33(1):1-7

Authors: Yang W, Sinha S

Abstract
MOTIVATION: With the rapid emergence of technologies for locating cis-regulatory modules (CRMs) genome-wide, the next pressing challenge is to assign precise functions to each CRM, i.e. to determine the spatiotemporal domains or cell-types where it drives expression. A popular approach to this task is to model the typical k-mer composition of a set of CRMs known to drive a common expression pattern, and assign that pattern to other CRMs exhibiting a similar k-mer composition. This approach does not rely on prior knowledge of transcription factors relevant to the CRM or their binding motifs, and is thus more widely applicable than motif-based methods for predicting CRM activity, but is also prone to false positive predictions.
RESULTS: We present a novel strategy to improve the above-mentioned approach: to predict if a CRM drives a specific gene expression pattern, assess not only how similar the CRM is to other CRMs with similar activity but also to CRMs with distinct activities. We use a state-of-the-art statistical method to quantify a CRM's sequence similarity to many different training sets of CRMs, and employ a classification algorithm to integrate these similarity scores into a single prediction of the CRM's activity. This strategy is shown to significantly improve CRM activity prediction over current approaches.
AVAILABILITY AND IMPLEMENTATION: Our implementation of the new method, called IMMBoost, is freely available as source code, at https://github.com/weiyangedward/IMMBoost CONTACT: sinhas@illinois.eduSupplementary information: Supplementary data are available at Bioinformatics online.

PMID: 27609510 [PubMed - indexed for MEDLINE]

Toward understanding the evolution of vertebrate gene regulatory networks: comparative genomics and epigenomic approaches.

Brief Funct Genomics. 2016 Jul;15(4):315-21

Authors: Martinez-Morales JR

Abstract
Vertebrates, as most animal phyla, originated >500 million years ago during the Cambrian explosion, and progressively radiated into the extant classes. Inferring the evolutionary history of the group requires understanding the architecture of the developmental programs that constrain the vertebrate anatomy. Here, I review recent comparative genomic and epigenomic studies, based on ChIP-seq and chromatin accessibility, which focus on the identification of functionally equivalent cis-regulatory modules among species. This pioneer work, primarily centered in the mammalian lineage, has set the groundwork for further studies in representative vertebrate and chordate species. Mapping of active regulatory regions across lineages will shed new light on the evolutionary forces stabilizing ancestral developmental programs, as well as allowing their variation to sustain morphological adaptations on the inherited vertebrate body plan.

PMID: 26293604 [PubMed - indexed for MEDLINE]

Characterization of the Autophagy related gene-8a (Atg8a) promoter in Drosophila melanogaster.

Int J Dev Biol. 2017;61(8-9):551-555

Authors: Bali A, Shravage BV

Abstract
Autophagy is an evolutionarily conserved process which is upregulated under various stress conditions, including nutrient stress and oxidative stress. Amongst autophagy related genes (Atgs), Atg8a (LC3 in mammals) is induced several-fold during nutrient limitation in Drosophila. The minimal Atg8a cis-regulatory module (CRM) which mediates transcriptional upregulation under various stress conditions is not known. Here, we describe the generation and analyses of a series of Atg8a promoter deletions which drive the expression of an mCherry-Atg8a fusion cassette. Expression studies revealed that a 200 bp region of Atg8a is sufficient to drive expression of Atg8a in nutrient rich conditions in fat body and ovaries, as well as under nutrient deficient conditions in the fat body. Furthermore, this 200 bp region can mediate Atg8a upregulation during developmental histolysis of the larval fat body and under oxidative stress conditions induced by H2O2. Finally, the expression levels of Atg8a from this promoter are sufficient to rescue the lethality of the Atg8a mutant. The 200 bp promoter-fusion reporter provides a valuable tool which can be used in genetic screens to identify transcriptional and post-transcriptional regulators of Atg8a.

PMID: 29139541 [PubMed - in process]

Organizing combinatorial transcription factor recruitment at cis-regulatory modules.

Transcription. 2017 Nov 06;:1-15

Authors: Dubois-Chevalier J, Mazrooei P, Lupien M, Staels B, Lefebvre P, Eeckhoute J

Abstract
Gene transcriptional regulation relies on cis-regulatory DNA modules (CRMs), which serve as nexus sites for integration of multiple transcription factor (TF) activities. Here, we provide evidence and discuss recent literature indicating that TF recruitment to CRMs is organized into combinations of trans-regulatory protein modules (TRMs). We propose that TRMs are functional entities composed of TFs displaying the most highly interdependent chromatin binding which are, in addition, able to modulate their recruitment to CRMs through inter-TRM effects. These findings shed light on the architectural organization of TF recruitment encoded by their recognition motifs within CRMs.

PMID: 29105538 [PubMed - as supplied by publisher]

An Emerging Regulatory Landscape for Skeletal Development.

Trends Genet. 2016 Dec;32(12):774-787

Authors: Hojo H, McMahon AP, Ohba S

Abstract
Skeletal development creates the physical framework that shapes our body and its actions. In the past two decades, genetic studies have provided important insights into the molecular processes at play, including the roles of signaling pathways and transcriptional effectors that coordinate an orderly, progressive emergence and expansion of distinct cartilage and bone cell fates in an invariant temporal and spatial pattern for any given skeletal element within that specific vertebrate species. Genome-scale studies have provided additional layers of understanding, moving from individual genes to the gene regulatory landscape, integrating regulatory information through cis-regulatory modules into cell type-specific gene regulatory programs. This review discusses our current understanding of the transcriptional control of mammalian skeletal development, focusing on recent genome-scale studies.

PMID: 27814929 [PubMed - indexed for MEDLINE]

Redundant CArG Box Cis-motif Activity Mediates SHATTERPROOF2 Transcriptional Regulation during Arabidopsis thaliana Gynoecium Development.

Front Plant Sci. 2017;8:1712

Authors: Sehra B, Franks RG

Abstract
In the Arabidopsis thaliana seed pod, pod shatter and seed dispersal properties are in part determined by the development of a longitudinally orientated dehiscence zone (DZ) that derives from cells of the gynoecial valve margin (VM). Transcriptional regulation of the MADS protein encoding transcription factors genes SHATTERPROOF1 (SHP1) and SHATTERPROOF2 (SHP2) are critical for proper VM identity specification and later on for DZ development. Current models of SHP1 and SHP2 regulation indicate that the transcription factors FRUITFULL (FUL) and REPLUMLESS (RPL) repress these SHP genes in the developing valve and replum domains, respectively. Thus the expression of the SHP genes is restricted to the VM. FUL encodes a MADS-box containing transcription factor that is predicted to act through CArG-box containing cis-regulatory motifs. Here we delimit functional modules within the SHP2 cis-regulatory region and examine the functional importance of CArG box motifs within these regulatory regions. We have characterized a 2.2kb region upstream of the SHP2 translation start site that drives early and late medial domain expression in the gynoecium, as well as expression within the VM and DZ. We identified two separable, independent cis-regulatory modules, a 1kb promoter region and a 700bp enhancer region, that are capable of giving VM and DZ expression. Our results argue for multiple independent cis-regulatory modules that support SHP2 expression during VM development and may contribute to the robustness of SHP2 expression in this tissue. Additionally, three closely positioned CArG box motifs located in the SHP2 upstream regulatory region were mutated in the context of the 2.2kb reporter construct. Mutating simultaneously all three CArG boxes caused a moderate de-repression of the SHP2 reporter that was detected within the valve domain, suggesting that these CArG boxes are involved in SHP2 repression in the valve.

PMID: 29085379 [PubMed]

Genome-wide discovery of active regulatory elements and transcription factor footprints in Caenorhabditis elegans using DNase-seq.

Genome Res. 2017 Oct 26;:

Authors: Sternberg P, Ho M, Quintero-Cadena P

Abstract
Deep sequencing of size-selected DNase I-treated chromatin (DNase-seq) allows high resolution measurement of chromatin accessibility to DNase I cleavage, permitting identification of de novo active cis-regulatory modules (CRMs) and individual transcription factor (TF) binding sites. We adapted DNase-seq to nuclei isolated from C. elegans embryos and L1 arrest larvae to generate high-resolution maps of TF binding. Over half of embryonic DNase I hypersensitive sites (DHSs) were annotated as noncoding, with 24% in intergenic, 12% in promoters and 28% in introns, with similar statistics observed in L1 arrest larvae. Noncoding DHSs are highly conserved and enriched in marks of enhancer activity and transcription. We validated noncoding DHSs against known enhancers from myo-2, myo-3, hlh-1, elt-2 and lin-26/lir-1 and recapitulated 15 of 17 known enhancers. We then mined DNase-seq data to identify putative active CRMs and TF footprints. Using DNase-seq data improved predictions of tissue-specific expression compared to motifs alone. In a pilot functional test, 10 of 15 DHSs from pha-4, icl-1 and ceh-13 drove reporter gene expression in transgenic C. elegans. Overall, we provide experimental annotation of 26,644 putative CRMs in the embryo containing 55,890 TF footprints, and 15,841 putative CRMs in the L1 arrest larvae containing 32,685 TF footprints.

PMID: 29074739 [PubMed - as supplied by publisher]