The Control Of Hox Specificity And Activity

admin — Sat, 19 May 2012 12:19:49 +0000

The Hox genes encode a conserved family of homeodomain-containing transcription factors that specify tissue and cellular identities throughout the animal kingdom. As is typical for homeodomain proteins, they tend to bind a wide range of degenerate, TAAT-containing binding sites, raising the fundamental question of how these factors achieve specificity in vivo. We use a variety of approaches, including whole genome binding studies, in vitro DNA binding assays, X-ray crystallography, in vivo functional tests, and high-throughput DNA specificity measurements, to address this question. Our most recent work (Slattery et al., 2011) reveals that Hox cofactors reveal “latent specificity” that is present in the Hox homeodomain, but cannot be used in the absence of the cofactors. In one specific case (Scr; Joshi et al., 2007) we found that latent specificity allows normally unstructured residues in the Hox protein Scr to read a DNA shape: the width of the minor groove.

Current lab members working on this project: Matt Slattery, Vikram Ranade, Namiko Abe, Rich Allan, Marios Agelopoulos (collaborations with Barry Honig and Harmen Bussemaker)

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Illustrated above are the relative affinities for all of the Drosophila Hox-Exd heterodimers bound to the top ten binding sites identified by our SELEX-seq experiments. See Slattery et al., 2011 for details.

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Shown is a space-filling model of the Hox protein Scr (blue) bound with it’s cofactor Exd (gray) to a specific binding site called fkh250 (magenta). In the presence of Exd, two basic Scr side chains, an Arg and a His (green) are positioned in such a manner that they can insert into the minor groove. This region of the minor groove is narrow, making it a poor binding site for most Hox or Hox-Exd heterodimers. See Joshi et al., 2007 for details.

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Closeup side view of a narrow minor groove in the fkh250 binding site in which two basic side chains from Scr are inserting. See Mann, 2008 for details.

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In collaboration with Barry Honig’s lab at Columbia, we generalized the concept of DNA shape recognition. Sequence-dependent differences in DNA structure, such as the examples illustrated here, can contribute to the recognition of specific DNA sequences by many DNA binding proteins. See Rohs et al., 2009.

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Another interest of the lab related to Hox proteins is how transcription factors modify chromatin structures, perhaps in a tissue specific manner, to regulate gene expression. To address these questions, we recently described a novel method to analyze the local chromatin structure in specific cell types during embryogenesis (cgChIP). This figure illustrates a summary from this work, which shows that the local chromatin structure at the Dll gene differs in thoracic expressing cells compared to abdominal non-expressing cells, due to the action of the abdominal Hox proteins. See Agelopoulos et al., 2012, for details.

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Representative publications:

Slattery et al., Cofactor Binding Evokes Latent Differences in DNA binding Specificity between Hox Proteins. Cell (2011) 147: 1270–1282. PDF

Agelopoulos et al., Developmental Regulation of Chromatin Conformation by Hox Proteins in Drosophila. Cell Reports (2012) 1: 1-10. PDF

Joshi, R. Passner, J., Rohs, R., Jain, R. Sosinsky, A., Crickmore, M.A., Jacob, V., Aggarwal, A.K., Honig, B. and Mann, RS. Functional specificity of a Hox protein mediated by the recognition of minor groove structure. Cell. (2007) 131(3):530-43.

PDF: joshi_etal

Affolter, M., Slattery, M., and Mann, R.S. A lexicon for homeodomain-DNA recognition. Cell (2008) 133:1133-1135.

Rohs, R., West, S., Sosinsky, A., Liu, P. Mann, RS, and Honig, B. The role of DNA shape in protein-DNA recognition. Nature (article) (2009) 461(7268):1248-53.

PDF: Rohs-et-al_Nature

Mann RS, Lelli KM, Joshi R. Hox specificity: unique roles for cofactors and collaborators. Curr Top Dev Biol. 2009;88:63-101

West SM, Rohs R, Mann RS, Honig B. Electrostatic interactions between arginines and the minor groove in the nucleosome. J Biomol Struct Dyn. 2010 Jun;27(6):861-6.

Rohs, R., Jin, X., West, S. Joshi, R., Honig, B. and Mann, RS. Origins of Specificity in Protein-DNA Recognition. Annual Review of Biochemistry (2010), Volume 79:233-269.

PDF:Rohs-et-al.-AnnRev

Joshi, R., Sun, L. and Mann, RS. Dissecting the functional specificities of two Hox proteins. Genes Dev. 2010:24(14); 1533-1545.

PDF:Joshi et al G&D

Slattery, M. Négre, N. Ma, L., White, KP, and Mann, RS. Genome-wide tissue-specific occupancy of the Hox protein Ultrabithorax and Hox cofactor Homothorax in Drosophila.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0014686.

PDF: Slattery-et-al-PLoSONE

Motor Neuron Development And Circuitry In The Adult Leg

admin — Wed, 16 May 2012 15:53:31 +0000

Adult fruit flies walk using six legs that move in a highly coordinated and stereotyped manner. The motor neurons that command these movements are located in the central nervous system (CNS) and send axons to the legs that synapse onto specific muscles. We are characterizing the molecular signatures that give each of these motor neurons their specific identities, focusing on the lineages that they are derived from and the transcription factors they express. In addition, we are developing assays that allow us to monitor fly walking at high resolution, with the goal of carrying out genetic screens to find genes and neurons that are required for motor coordination in the adult.

Current lab members working on this project: Cesar Mendes, Jonathan Enriquez, Samantha Emmert, Alex Plana

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High speed video slowed down to reveal the walking pattern of a fruit fly. Flies walk most typically using the so-called tripod gait, where three legs are in stance phase (touching the surface) and three legs are in swing phase (swinging forward) at any one time.

Using our new method based on frustrated Total Internal Reflection (fTIR) we now visualize and quantify footsteps as shown in the video WT_fly_processed. See Mendes et al. for details.

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~50 Motor neuron axons and some sensory neuron cell bodies labeled in the adult leg by vGlut-Gal4, UAS-CD8-GFP.

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7 of the ~50 adult leg motor neurons, labeled here, all derive from a single neuroblast, Lin B, as visualized by a MARCM clone experiment.

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A single coxa motor neuron cell body and its dendrites is labeled in the adult CNS by a MARCM experiment. See Baek and Mann 2009 for details.

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A single coxa motor neuron axon synapsing onto a muscle in the adult leg. See Baek and Mann 2009 for details.

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An adult CNS with all adult motor neurons labeled via the vglut-Gal4 driver line. See Baek and Mann 2009 for details.

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The sensory and motor neuron circuitry relevant to adult walking in the fruit fly.

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Representative publications:

Baek M, Mann RS. Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila. J Neurosci. (2009) May 27;29(21):6904-16.

PDF: Baek and Mann JNeuro

César S Mendes, Imre Bartos, Turgay Akay, Szabolcs Márka, Richard S Mann. Quantification of gait parameters in freely walking wild type and sensory deprived Drosophila melanogaster. eLife 2013 10.7554/eLife.00231

Proximal Distal Axis Formation In The Appendages

admin — Wed, 16 May 2012 15:03:17 +0000

Overview of PD axis formation in the fly leg

Unlike the anterior-posterior (AP) and dorsal-ventral (DV) axes, the proximal-distal (PD) axis must be generated de novo, each time an appendage develops. For the leg, this axis extends from the distal-most tip (the tarsal claw) to the body (proximal). The axis is first formed during embryogenesis and is elaborated during larval development. Key molecular markers for this axis are the homeodomain-encoding gene Distalless (Dll), expressed in the distal-most portion of the leg, the gene dachshund (dac), expressed in medial positions, and homothorax (hth), expressed in the most proximal regions of the appendage and body wall. Our goal is to define the molecular pathways that specify this axis, and our general approach is to identify and characterize the cis-regulatory elements that control the expression of these, and other, PD genes during development. This “bottom-up” approach has revealed a transcription factor cascade that is initially triggered by the joint activities of two secreted morphogens, Wingless (Wg) and Decapentaplegic (Dpp), but is then elaborated by feed-forward loops and autoregulation.

Current lab members working on this project: Aurélie Jory, Rich Allan, Roumen Voutev, Matt Slattery, Suzie Tozier

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Artist: Carlos Estella

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A mid-stage embryo stained for Dll (red) and Hth (blue). The three round expression domains mark the appendage primordia in the three thoracic segments.

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A stage 14 embyro stained for Wg (blue), pMad (green, a readout of Dpp signaling), and the Dll (LT)-lacZ reporter gene (red). Expression of LT-lacZ marks the future Dll+dac domains and is triggered by Wg+Dpp signaling. Expression in the abdominal segments is repressed by the abdominal Hox genes.

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Six snapshots of leg development, from left to right: embryogenesis, 1st instar larva, 2nd instar larva, 3rd instar larva, pupariation, adult. Images are stained for Hth (blue), Dac (green), and Dll (red).

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Evolving relationship between Dpp and Wg during leg development. From left to right: embryogenesis, 1st instar, 2nd instar, early 3rd instar, late 3rd instar. Images are stained for Wg (red) and a reporter for Dpp (green).

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Shown is a 3rd instar leg disc stained for Dll (blue), Dac (red and far right panel), and expression from a dac reporter construct, dacHI-lacZ (green and center panel). See Giorgianni and Mann, 2011 for more details.

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Representative publications:

Estella, C., Voutev, R. and Richard S. Mann, A Dynamic Network of Morphogens and Transcription Factors Patterns the Fly Leg. In Serge Plaza and François Payre, editors: Current Topics in Developmental Biology, Vol. 98, Burlington: Academic Press, 2012, pp. 173-198. PDF

Estella, C. and Mann, R.S. Non-redundant selector and growth-promoting functions of two sister genes, buttonhead and Sp1, in Drosophila leg development. PLoS Genet. 2010 Jun 24;6(6):e1001001. PDF

Giorgianni, M. and Mann, RS. Establishment of medial fates along the proximodistal axis of the Drosophila leg through direct activation of dachshund by Distalless. Developmental Cell 2011: 19;20(4):455-68.. PDF

McKay, D., Estella, C. and Mann R.S. The origins of the Drosophila leg as revealed by the cis-regulatory architecture of the Distalless gene. Development. 2009 Jan;136(1):61-71. PDF

Estella, C., McKay, D., and Mann, R.S. Direct integrations of Wingless, Decapentaplegic, and autoregulatory inputs into Distalless during Drosophila leg development. Dev. Cell, (2008) 14:86-96.

Estella, C. and Mann, R.S. Logic of Wg and Dpp induction of distal and medial fates in the Drosophila leg. Development, (2008), 135:627-636. PDF

The Developmental Control Of Tissue Growth And Organ Size

admin — Wed, 16 May 2012 14:12:20 +0000

The genetic control of organ size and cell proliferation is poorly understood. Our work addresses these questions from two different directions. In one, we ask how the Hox gene Ubx is able to reduce the number of cells in the haltere compared to the homologous wing. One important answer to this question is that Ubx appears to be modifying many different parameters of the Dpp pathway, including how far the Dpp morphogen can diffuse from its site of production. See Crickmore et al. 2006, 2007. In a second project we are asking how the Hippo tumor suppressor pathway, which is a core component of the cell proliferation machinery in nearly all tissues, is being used in a tissue-specific manner. We found that while the Hippo pathway in wing cells use the transcription factor Scalloped (Sd), in eye progenitor cells they use the homeodomain protein Hth. Thus, we hypothesize that the choice of transcription factor can provide tissue-specificity to this otherwise generic proliferation-regulating pathway. See Peng et al., 2009 for more details.

Current lab members working on this project: Roumen Voutev, Matt Slattery, Vikram Ranade, Rich Allan

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Haltere (left) and wing (right) imaginal discs have the same overall morphology but differ in size. They also differ in that all haltere cells, but not wing cells, express the Hox protein Ubx, stained here in green.

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Clones of cells (labeled in green) that mis-express Hth and the Zn-finger protein Teashirt (Tsh) overproliferate by inhibiting the Hippo pathway. See Bessa et al., 2002 and Peng et al., 2009.

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An eye imaginal disc stained for CycB and a marker of the morphogenetic furrow. CycB is expressed strongly ahead (anterior) of the furrow.

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A series of dorsal appendages that vary in size. The wild type wing is on top and the wild type haltere is on the bottom. See Crickmore 2006 for details.

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Differences in Dpp expression in the wild type haltere (left) compared to the wild type wing (right). See Crickmore, 2006 for details.

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Representative publications:

Bessa J, B. Gebelein, F. Pichaud, F. Casares, and R.S. Mann (2002) Combinatorial control of Drosophila eye development by Eyeless, Homothorax, and Teashirt. Genes Dev. 16:2415-2427.

Crickmore, MA and R.S. Mann (2006) Hox Control of Organ Size by Regulation of Morphogen Production and Mobility. Science (Article), 313(5783):63-8.

PDF: Crickmore+Mann_text+SOM

Crickmore MA, Mann RS. (2006) Hox control of morphogen mobility and organ development through regulation of glypican expression. Development. 2007 Jan;134(2):327-34.

PDF: Crickmore+MannDev

Peng, W., Slattery, M. and Mann RS. Transcription factor choice in the Hippo signaling pathway: homothorax and yorkie regulation of the microRNA bantam in the progenitor domain of the Drosophila eye imaginal disc. Genes Dev (2009) 23(19):2307-19.

PDF: Peng et al_G&D

Computational And High-Throughput Approaches For Analyzing Cis-Regulatory Elements

admin — Wed, 16 May 2012 13:43:02 +0000

With the increasing number of eukaryotic genomes available, high-throughput automated tools for identification of regulatory DNA sequences are becoming increasingly feasible. We are developing both a number of computational tools to discover transcriptional regulatory regions, as well as novel high-throughput methods to analyze DNA binding specificities on a global scale. We also continue to investigate the role of sequence-dependent DNA shape in protein-DNA interactions.

Current lab members working on this project: Matt Slattery, Namiko Abe, Andy Sterling (collaborations with Barry Honig and Harmen Bussemaker)

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Examples of DNA structures taken from protein-DNA crystal structures in the Protein Data Base (PDB) showing a wide variety of DNA shapes involved in DNA recognition by proteins (see Rohs et al., 2010).

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Shown is an example of the output of the EDGI tool (http://luna.bioc.columbia.edu/alona/EDGI/). Motifs shared among a set of related Drosophila species are shown by the vertical lines. Clusters of conserved motifs are indicated by the graph in the lower panel. See Sosinsky et. al., (2007) for details.

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Relevant links:

EDGI: http://luna.bioc.columbia.edu/alona/EDGI/

TargetExplorer: http://trantor.bioc.columbia.edu/Target_Explorer/

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Representative publications:

Sosinsky A., Bonin K., Mann R.S., Honig B. (2003) Target Explorer: an automated tool for the identification of new target genes for a specified set of transcription factors. NAR, 31:3589-92.

Sosinsky, A., Honig, B., Mann, R.S., and Califano, A. Discovering transcriptional regulatory regions in Drosophila by a nonalignment method for phylogenetic footprinting. Proc Natl Acad Sci U S A, 2007. 104(15): p. 6305-10.

PDF: EDGI_PNAS

Rohs, R., West, S., Sosinsky, A., Liu, P. Mann, RS, and Honig, B. The role of DNA shape in protein-DNA recognition. Nature (article) (2009) 461(7268):1248-53.

West SM, Rohs R, Mann RS, Honig B.Electrostatic interactions between arginines and the minor groove in the nucleosome. J Biomol Struct Dyn. 2010 Jun;27(6):861-6.

Rohs, R., Jin, X., West, S. Joshi, R., Honig, B. and Mann, RS. Origins of Specificity in Protein-DNA Recognition. Annual Review of Biochemistry (2010), Volume 79:233-269.

Enhancer Silencing In Response To Gene Dosage And Natural Variation

admin — Wed, 16 May 2012 13:23:21 +0000

We discovered that reporter genes inserted into the Ubx gene, which report subsets of the complex transcriptional inputs into this gene, are silenced in response to increasing the Ubx dose or in response to natural genetic variation. For the second phenomenon, we showed that these reporter genes are turned off in patches of cells in the haltere imaginal disc when they are crossed into strains of Drosophila melanogaster that come from various places around the world. These observations argue that the same cis-regulatory elements have distinct activities depending on the genetic background. See Crickmore et al., 2009

Current lab members working on this project: Vikram Ranade

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Artist: Vikram Ranade

Shown above is a haltere imaginal disc with patches of cells that no longer express a Ubx enhancer trap (GFP+) due to epigenetic silencing. The image was modified to represent continents on the globe, to make the point that crosses with wild populations throughout the world can trigger this silencing. See Crickmore et al., 2009 for more details.

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An example of a haltere imaginal disc stained for Ubx (red) and a Ubx enhancer trap (green) from an F1 animal in which one parent came from our lab stock and the other parent came from North Carolina. Patches of non-expressing cells where the enhancer trap was silenced (lack of GFP+) can be observed. See Crickmore et al., 2009 for more details.

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Representative publications:

Crickmore, MA, Ranade, V. and Mann RS. Regulation of Ubx expression by epigentic silencing in response to Ubx levels and genetic variation. PLoS Genetics (2009) 5: e1000633.

PDF: Crickmore etalPG