Available online at www.sciencedirect.com
ScienceDirect Long range regulation of the sonic hedgehog gene Eve Anderson and Robert E Hill The regulatory architecture that controls developmental genes is often a collection of enhancers that, in combination, generate a complex spatial and temporal pattern of expression. These enhancers populate domains operating at long distances and, in the case of the sonic hedgehog (Shh) locus, this regulatory domain covers 900–1000 kb. Within this context each embryonic tissue that expresses Shh has acquired its own regulatory apparatus which may require the activity from several distinct enhancers. Expression of Shh in the developing limb bud is driven by a single enhancer that interprets a myriad of genetic information to initiate expression in the posterior margin of the limb bud, inhibits expression along the anterior margin, defines the level of expression, and sets the tissue boundary of expression. Addresses MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK
a rich source of regulatory mutations that give rise to congenital abnormalities. Chromosomal lesions and translocation which disrupt the regulatory domain are responsible for many cases of holoprosencephaly (HPE) [6]. Alternatively, well over 20 point mutations within the limb specific enhancer called the ZRS, or chromosomal duplications that increase ZRS numbers (reviewed previously [7]) cause a spectrum of limb defects, predominantly preaxial polydactyly of the hands and feet. Other regulatory mutations such as a point mutation within the brain enhancer, SBE2, which disrupts binding of Six3 has been identified within an individual with HPE [8]. Thus, to determine the basis of a number of human diseases it is important to understand the mechanisms used to regulate gene expression. In this review we will focus on the regulatory apparatus for the Shh gene to illustrate the complexity required to express a single gene during development.
Corresponding author: Hill, Robert E ([email protected])
Composition of the Shh regulatory domain Current Opinion in Genetics & Development 2014, 27:54–59 This review comes from a themed issue on Developmental mechanisms, patterning and evolution Edited by Lee A Niswander and Lori Sussel
0959-437X/# 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gde.2014.03.011
Introduction A common feature of genes that regulate development, such as transcription factors and signalling molecules, is the extensive collection of cis-regulatory information that resides in the vicinity of the protein coding sequence. These cis-acting elements lie in large ‘gene deserts’ (large genomic expanses devoid of genes) but often extend to the introns of neighbouring genes and these elements organise temporal and spatial transcriptional gene control [1,2]. The sonic hedgehog (Shh) locus is an example of long range regulation. Unlike holo-enhancers [3] and regulators of HoxD genes [4], which are composed of complexes of interacting regulators, Shh expression pattern is a summation of distinct enhancer activities. Mutations in regulatory elements have been predicted to be a major source of human disease and congenital abnormalities given that the vast majority of disease associated variants map to noncoding regions of the genome [5]. The extensive regulatory domain of Shh is Current Opinion in Genetics & Development 2014, 27:54–59
Regulation of expression of few mammalian genes has been investigated to the extent of the Shh gene. The region lying 50 of the Shh gene comprises a large genomic desert within which a number of potential cis-regulators of Shh reside or flank [9,10] (Figure 1). As a general rule, conservation of noncoding sequence elements within this domain highlights regulators of Shh expression. As is becoming more commonly recognised, enhancers do not always act independently and what is clear from the analysis of the Shh locus is that individual enhancer activities often overlap. Although there may be some sharing of enhancer information, on the whole it appears that each tissue or region possesses an intrinsic means for its own regulation and thus, the Shh spatial and temporal expression pattern is directed by an assemblage of autonomous enhancer activities. Using a transgenic reporter assay in mice, two intronic enhancers SFPE1 and SFPE2 [11], and an enhancer 35 kb upstream of Shh, SBE1, were identified (Figure 1). Both SFPE1 and SFPE2 function redundantly to direct LacZ expression within the floor plate of the spinal cord and hindbrain [9]. Furthermore, homozygous deletions of the SBE1 sequence which drives expression within the ventral midbrain and caudal region of the telencephalon were found to result in viable mice [11]. Initiation of Shh expression in the midline of the midbrain and caudal diencephalon occurred correctly at E8.5; however, this was not maintained beyond E10.0, suggesting that SBE1 function overlaps with an, as yet, unidentified enhancer which controls Shh transcription at earlier time points before E10.0 [12]. www.sciencedirect.com
Cis-regulation of the Shh gene Anderson and Hill 55
Figure 1
? Shh
PE SB 1 SF E1 PE 2
SF
G E SL
SB E SB 3 E2 SB E4
M AC M S1 FC S4 M RC S1
ZR
S
Lmbr1 Rnf32
Current Opinion in Genetics & Development
The genomic domain near the Shh gene representing the assemblage of enhancers that underlie the spatial and temporal pattern of expression. A schematic illustration of the multiple sites of Shh expression within the E11.5 embryo: expression within the epithelial lining of the pharyngeal cavity, lungs and digestive tube is shown on the left and the brain on the right, the colours used match the relevant enhancers (coloured bars) which are shown in their genomic context. MACS1 and SLGE are shown to act together to drive expression within the gut epithelium whereas both MFCS4 and MRCS1 are shown to act independently. SBE1 which drives expression within the brain has been shown to act redundantly with an unknown enhancer.
A cluster of three cis-regulatory elements are located 600– 900 kb upstream of Shh which direct the overlapping and sequential expression along the epithelial lining of the pharyngeal cavity, lungs and digestive tubes [10] (Figure 1). Thus, the action of these enhancers partitions the epithelium into three continuous domains. The enhancer MACS1 drives expression in the posterior reaches of the gut epithelium, in the tracheal tube, the lungs, intestine and urogenital tract. The MRCS1 is responsible for the expression in anterior epithelia including in the tooth, hard palate and tongue. The MFCS4 enhancer directs expression in between and overlapping the other two enhancers in the soft palate epiglottis, arytenoids and tympanic tube. Recently, an additional enhancer was identified approximately 100 kb from Shh. This 1.7 kb enhancer designated SLGE (Shh lung, gut enhancer) drives expression overlapping that of the MACS1 enhancer [13]. The SLGE enhancer is www.sciencedirect.com
suggested to extend gut expression to later stages of development (E12.5) after MACS1 has become inactive. Multiple enhancers driving similar expression patterns are well known and in Drosophila these redundant regulatory elements are referred to as shadow enhancers [14,15]. Shadow enhancer activity has recently been identified in vertebrates in the HoxB complex [16]. Shadow enhancers provide robustness to stabilise the transcriptional response to genetic or environmental variability. In the Shh regulatory locus, it is not clear if multiple enhancers controlling overlapping expression in a single tissue satisfy the criteria of a shadow enhancer. Certainly the SBE1 enhancer activity and the activity from the unknown element are overlapping but are not redundant. In this respect, multiple enhancers may serve to extend the temporal window of expression, which may be important in a varying regulatory environment which Current Opinion in Genetics & Development 2014, 27:54–59
56 Developmental mechanisms, patterning and evolution
undergoes significant changes during development. In addition, multiple enhancers in a single tissue type, such as the cluster of three enhancers controlling Shh in the endodermal derived gut epithelium, may function to divide the regulatory load among different enhancers. To further determine the apparent complementary roles of the Shh enhancers, the gold standard remains the removal of specific enhancer sequences to investigate the phenotypic outcome.
Long range regulation in a single developing tissue The most distally located enhancer in the Shh domain is an 800 bp highly conserved long-range regulatory element. In contrast to the above, this enhancer, referred to as the ZRS (also the MFCS1) [17,18], appears to be the sole regulator of expression directing SHH to a restricted domain in the posterior margin of the developing limb called the zone of polarising activity (ZPA). This regulator operates over approximately 1 Mb (in human) to regulate Shh expression (Figure 1) and a deletion of the element results in a phenocopy of the abnormal limb seen in the Shh null mutation [19]. The expression of Shh in the ZPA extends from the posterior edge of the limb bud but has no histological boundary along the anterior most limits of the domain. In the limb this restricted, focal expression of Shh is crucial for specifying digits along the posterior (side with the little finger and toe) to anterior (side with the thumb and great toe) axes. Digits form from a reiterative periodic pattern in the limb bud such that each digit forms at a specific distance from the other digits resulting in a repeating pattern of digit, and non-digit [20]. The most recent proposal to explain SHH activity is an integrated model, which in addition to morphogen activity, incorporates growth as a key function in early patterning [21]. Hence the role of the posterior Shh expression is to organise this periodic digital pattern to regulate the number of digits, perhaps by regulating the growth of the limb bud and in addition, the morphogen activity specifies individual characteristics to each digit. This posterior positioning of Shh expression is the consequence of multiple developmental events that polarise the limb. Pathways responsible have been identified that operate to both restrict Shh expression to the posterior margin of the limb bud and set the anterior boundary. Initially the limb is polarised at the earliest stages of limb bud outgrowth which is meditated by the mutually antagonistic interactions of Gli3 and Hand2 [22] (Figure 2a). Initiation of Shh expression follows and is dependent on Hand2 in the posterior of the limb bud and during these early stages the posterior genes from the HoxA and HoxD (genes 10–13) complexes are also required [23,24]. A conditional limb mutation for Hand2 results in the loss of Shh expression and a phenotype that Current Opinion in Genetics & Development 2014, 27:54–59
resembles the Shh null limb [25]. The role of HAND2 is dependent on the DNA binding capacity of its helix– loop–helix domain [26] operating at the ZRS. Hoxd10 and Hoxd13 also interact directly with the ZRS [27] and furthermore HOXD13 and HAND2 form a protein complex that transactivates the ZRS, and notably this process is interfered with by a repressor of Shh expression, GLI3R [25]. Shh is inactive, but primed for expression in the anterior margin of the limb [28] and under certain genetic circumstances can be ectopically expressed resulting in the generation of limb defects such as preaxial polydactyly [7]. Genetic analysis showed that several genes are responsible for repressing Shh expression in the anterior domain and thus restricting Shh to the ZPA (Figure 2b). Alx4 is specifically expressed in the anterior margin of the limb and when deleted anterioposterior polarity is disrupted and Shh is induced in the anterior of the limb bud [29,30]. The ETS transcription factor genes, Etv4 and Etv5, are expressed along the distal edge of the developing limb bud [31] and are maintained at a high level by the expression of FGFs in the overlying apical ectodermal ridge. Loss of both ETV genes results in the ectopic expression of Shh in the anterior of the limb bud and the production of extra anterior digits. ETV4 and ETV5 bind to the ZRS and mutation of the binding sites results in expansion of Shh expression into the anterior domain. However, ALX4 and ETV4/5 may fit into a more complex picture that includes TWIST1 [32]. This picture includes the observation that FGF expression in the AER has two functions, the first is to increase and maintain the expression of Shh in the ZPA but secondly, acting through the inducible ETV genes it inhibits the expression in the posterior domain. TWIST1 is central to these two functions. At low levels, in response to FGF signalling, TWIST1 induces the FGF receptor responsible for regulating the levels of Shh in the posterior domain, whereas in the anterior domain at higher levels induces the production ALX4 and the two ETV proteins. Two other ETS transcription factors, ETS1 and GABPa, are responsible for defining the extent and therefore boundary of Shh expression (Figure 2c) [31]. The anterior extent of expression is regulated by multiple ETS binding sites within the ZRS. Removal of a subset of these sites narrows the width of expression, whereas an extra site extends the expression domain further anteriorly. Surprisingly, some of the early developmental events involved in polarising the limb are specific for the forelimbs or the hindlimbs. For example, the Hox9 paralogs (Hoxa-d9) are responsible for Hand2 expression, which establishes the initial asymmetry [33] but only in the forelimbs (Figure 2a). Moreover, the Hox5 paralogs (particularly Hoxa5, Hoxb5 and Hoxc5) (Figure 2b) are www.sciencedirect.com
Cis-regulation of the Shh gene Anderson and Hill 57
Figure 2
Initiation of Shh
(a)
Restriction of Shh to ZPA
(b)
AER-FGF Hand2 Shh HoxA/D Hand2 Gli3
Twist
Shh
Etv4/5 FGFR1 Alx4
Gli3 HoxB/D10 + Hand2
AER-FGF
Hand2 + HoxA/D10-13 Twist (low)
Twist (high)
Hoxa9, b9,c9,d9 Forelimbs
Hand2
?
Etv4/5
FGFR1
Alx4
Hindlimbs Shh
Shh
Forelimbs Hox5 (a5,b5,c5) (c)
Formation of Shh Expression Boundary AER
ETS1/GABPα
AER ETS1 sites within the ZRS
Shh
Wild-type ZRS
Deletion of ETS1 site
Additional ETS1 site
Current Opinion in Genetics & Development
A survey of the interactions required to express Shh in a single developing tissue, the limb bud. (a) The polarity of the limb is defined early in development by the antagonist activity of GLI3 in the anterior and by HAND2 (which downregulates GLI3) in the posterior of the limb bud. Initiation of Shh expression is dependent on HAND2 expression and the posterior HoxA/D genes (Hox10-13). Polarity of the forelimbs is dependent on the Hox9 paralogues, whereas in the hindlimbs the mechanism is unknown. (b) Shh expression is repressed in the anterior margin and thus restricted to the posterior margin of the limb. The genes Alx4, Etv4 and Etv5 are involved in this restriction within both the hindlimbs and the forelimbs, whereas additionally in the forelimbs the Hox5 genes (Hoxa5, Hoxb5 and Hoxc5) are also involved. The repression of Shh may be regulated by Twist1 expression in the limb, which upregulates both the anterior repressors and the posterior FGF signalling pathway. (c) The expression boundary of Shh is determined by ETS transcription factor binding sites. Within the limb, ETS1/GABPa is expressed across the limb bud (left). In the wildtype embryo this leads to Shh production within the ZPA (right depicted in yellow). Deletion of one of the ETS1 sites causes a reduction in area of Shh expression (right depicted in mauve) whereas the addition of an ETS1 site results in an increased Shh expression area (right depicted in dark blue). Abbreviations: AER, apical ectodermal ridge; FGFs, fibroblast growth factors 4, 8, 9 and 17. www.sciencedirect.com
Current Opinion in Genetics & Development 2014, 27:54–59
58 Developmental mechanisms, patterning and evolution
necessary to maintain the expression of ZPA located Shh, but only in the forelimbs [34]. These Hox genes pose a satisfying solution as to how initial limb polarity is established but leaves no answers as to hindlimb polarity, only hinting at other Hox genes being involved.
Chromosomal conformation and topological domains 3D-FISH measurements and 3C (chromosomal conformational capture) analysis suggest that the ZRS and Shh gene interact via a chromosomal looping mechanism [28]. This occurs as a two-step process whereby firstly, looping brings the ZRS close to the Shh promoter, and Shh then moves out of its own chromosome territory which correlates with gene activation. It is proposed that the locus is being held in three states: silent, in which the gene and enhancer do not contact; poised, in which there is some contact and ultimately; and active, where contact occurs and the gene is transcribed. The Shh enhancers can operate over hundreds of kilobases but they only operate in their own genomic space, without affecting the neighbouring genes. Conclusions from Hi-C experiments (a method for identifying higher order chromatin interactions genome wide) suggest that the genome of mice and humans is organised in large chromatin interaction domains known as ‘topological domains’ or TADs (topological associated domains) [35,36]. These domains are believed to correlate with regions of the genome that constrain the spread of heterochromatin and consist of highly self-interacting regions in which tens of genes and hundreds of enhancers reside. These domains are speculated to constrain looping interactions between enhancers and promoters and set the boundaries of coordinated gene regulation, and indeed Shh and the ZRS are found to occupy the same topological domain [36].
Conclusions Genome sequencing in multiple species and the recent publication of the large ENCODE project [37] have been invaluable in gaining an elemental understanding of gene regulation. To gain an insight into the layers of regulatory information required to control spatiotemporal expression of even a single developmental gene, however, is a challenging task. Genetic manipulation and analysis of all coding regions within the genome is a current goal of mouse geneticists and these approaches have been streamlined with the advent of TALENS and CRISPR/Cas technologies [38]. Moving these technologies into the realm of manipulating regulatory domains will be crucial for unravelling the hierarchy of gene expression control. Recent studies that enable chromosomal engineering using the Sleeping Beauty transposable element is a useful tool for systematically mapping large regulatory domains [3,39,40]. Applying such genetic approaches may be the next comprehensive Current Opinion in Genetics & Development 2014, 27:54–59
strategy for understanding the coordinated regulatory events responsible for the expression of Shh and other developmental genes.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
1.
Woolfe A, Goodson M, Goode DK, Snell P, McEwen GK et al.: Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol 2005, 3:e7.
2.
Pennacchio LA, Ahituv N, Moses AM, Prabhakar S, Nobrega MA et al.: In vivo enhancer analysis of human conserved noncoding sequences. Nature 2006, 444:499-502.
3.
Marinic M, Aktas T, Ruf S, Spitz F: An integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape. Dev Cell 2013, 24:530-542. This paper shows an organisation of cis-regulators known as a holoenhancer that function as a coherent unit to generate the expression of the Fgf8 gene.
4.
Montavon T, Soshnikova N, Mascrez B, Joye E, Thevenet L et al.: A regulatory archipelago controls Hox genes transcription in digits. Cell 2011, 147:1132-1145.
5.
Makrythanasis P, Antonarakis SE: Pathogenic variants in nonprotein-coding sequences. Clin Genet 2013, 84:422-428.
6.
Roessler E, Ward DE, Gaudenz K, Belloni E, Scherer SW et al.: Cytogenetic rearrangements involving the loss of the Sonic Hedgehog gene at 7q36 cause holoprosencephaly. Hum Genet 1997, 100:172-181.
7.
Anderson E, Peluso S, Lettice LA, Hill RE: Human limb abnormalities caused by disruption of hedgehog signaling. Trends Genet 2012, 28:364-373.
8.
Jeong Y, Leskow FC, El-Jaick K, Roessler E, Muenke M et al.: Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein. Nat Genet 2008, 40:1348-1353.
9.
Jeong Y, El-Jaick K, Roessler E, Muenke M, Epstein DJ: A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers. Development 2006, 133:761-772.
10. Sagai T, Amano T, Tamura M, Mizushina Y, Sumiyama K et al.: A cluster of three long-range enhancers directs regional Shh expression in the epithelial linings. Development 2009, 136:1665-1674. 11. Epstein DJ, McMahon AP, Joyner AL: Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3dependent and -independent mechanisms. Development 1999, 126:281-292. 12. Jeong Y, Dolson DK, Waclaw RR, Matise MP, Sussel L et al.: Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity. Development 2011, 138:531-541. 13. Tsukiji N, Amano T, Shiroishi T: A novel regulatory element for Shh expression in the lung and gut of mouse embryos. Mech Dev 2013, 131:127-136. A novel, non-conserved enhancer was reported for driving expression of Shh in the mouse gut. In the gut epithelium, the expression of Shh is achieved by the seamless contribution of multiple enhancers. 14. Frankel N, Davis GK, Vargas D, Wang S, Payre F et al.: Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 2010, 466:490-493. 15. Perry MW, Boettiger AN, Bothma JP, Levine M: Shadow enhancers foster robustness of Drosophila gastrulation. Curr Biol 2010, 20:1562-1567. www.sciencedirect.com
Cis-regulation of the Shh gene Anderson and Hill 59
16. Nolte C, Jinks T, Wang X, Martinez Pastor MT, Krumlauf R: Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development. Dev Biol 2013, 383:158-173. Identifies a shadow enhancer operating at the HoxB complex in mice that participates with other local enhancer to modulate gene expression during cardiac development. 17. Lettice LA, Heaney SJ, Purdie LA, Li L, de Beer P et al.: A longrange Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet 2003, 12:1725-1735. 18. Sagai T, Masuya H, Tamura M, Shimizu K, Yada Y et al.: Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog (Shh). Mamm Genome 2004, 15:23-34. 19. Sagai T, Hosoya M, Mizushina Y, Tamura M, Shiroishi T: Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb. Development 2005, 132:797-803. 20. Sheth R, Marcon L, Bastida MF, Junco M, Quintana L et al.: Hox genes regulate digit patterning by controlling the wavelength of a Turing-type mechanism. Science 2012, 338:1476-1480. A landmark paper that defines in mathematical and genetic terms the self-organising capacity of the developing limb bud. 21. Towers M, Mahood R, Yin Y, Tickle C: Integration of growth and specification in chick wing digit-patterning. Nature 2008, 452:882-886. 22. te Welscher P, Fernandez-Teran M, Ros MA, Zeller R: Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling. Genes Dev 2002, 16:421-426. 23. Kmita M, Tarchini B, Zakany J, Logan M, Tabin CJ et al.: Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function. Nature 2005, 435:1113-1116. 24. Tarchini B, Duboule D, Kmita M: Regulatory constraints in the evolution of the tetrapod limb anterior–posterior polarity. Nature 2006, 443:985-988. 25. Galli A, Robay D, Osterwalder M, Bao X, Benazet JD et al.: Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development. PLoS Genet 2010, 6:e1000901. 26. Liu N, Barbosa AC, Chapman SL, Bezprozvannaya S, Qi X et al.: DNA binding-dependent and -independent functions of the Hand2 transcription factor during mouse embryogenesis. Development 2009, 136:933-942. 27. Capellini TD, Di Giacomo G, Salsi V, Brendolan A, Ferretti E et al.: Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression. Development 2006, 133:2263-2273. 28. Amano T, Sagai T, Tanabe H, Mizushina Y, Nakazawa H et al.: Chromosomal dynamics at the Shh locus: limb bud-specific
www.sciencedirect.com
differential regulation of competence and active transcription. Dev Cell 2009, 16:47-57. 29. Panman L, Drenth T, Tewelscher P, Zuniga A, Zeller R: Genetic interaction of Gli3 and Alx4 during limb development. Int J Dev Biol 2005, 49:443-448. 30. Kuijper S, Feitsma H, Sheth R, Korving J, Reijnen M et al.: Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh. Dev Biol 2005, 285:533-544. 31. Lettice LA, Williamson I, Wiltshire JH, Peluso S, Devenney PS et al.: Opposing functions of the ETS factor family define Shh spatial expression in limb buds and underlie polydactyly. Dev Cell 2012, 22:459-467. Demonstrates ETS transcription factor binding sites contained in the ZRS that both restricts ectopic expression of Shh and determines the posterior boundary of expression in the limb bud. 32. Krawchuk D, Weiner SJ, Chen YT, Lu BC, Costantini F et al.: Twist1 activity thresholds define multiple functions in limb development. Dev Biol 2010, 347:133-146. 33. Xu B, Wellik DM: Axial Hox9 activity establishes the posterior field in the developing forelimb. Proc Natl Acad Sci U S A 2011, 108:4888-4891. 34. Xu B, Hrycaj SM, McIntyre DC, Baker NC, Takeuchi JK et al.: Hox5 interacts with Plzf to restrict Shh expression in the developing forelimb. Proc Natl Acad Sci U S A 2013, 110:1943819443. This paper shows a previously unexpected role for the Hox5 paralogues (Hoxa5, Hoxb5 and Hoxc5) in forelimb patterning and are involved in repressing Shh in the anterior, ectopic domain, thus restricting Shh expression in the limb. 35. Dixon JR, Selvaraj S, Yue F, Kim A, Li Y et al.: Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 2012, 485:376-380. Using HiC technology at a high resolution, this paper describes the organisation of the genome into local interaction domains called topological domains. 36. Smallwood A, Ren B: Genome organization and long-range regulation of gene expression by enhancers. Curr Opin Cell Biol 2013, 25:387-394. 37. Consortium TEP: A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 2011, 9:e1001046. 38. Gaj T, Gersbach CA, Barbas CF III: ZFN, TALEN, and CRISPR/ Cas-based methods for genome engineering. Trends Biotechnol 2013, 31:397-405. 39. Kokubu C, Horie K, Abe K, Ikeda R, Mizuno S et al.: A transposonbased chromosomal engineering method to survey a large cisregulatory landscape in mice. Nat Genet 2009, 41:946-952. 40. Ruf S, Symmons O, Uslu VV, Dolle D, Hot C et al.: Large-scale analysis of the regulatory architecture of the mouse genome with a transposon-associated sensor. Nat Genet 2011, 43:379386.
Current Opinion in Genetics & Development 2014, 27:54–59
ScienceDirect Long range regulation of the sonic hedgehog gene Eve Anderson and Robert E Hill The regulatory architecture that controls developmental genes is often a collection of enhancers that, in combination, generate a complex spatial and temporal pattern of expression. These enhancers populate domains operating at long distances and, in the case of the sonic hedgehog (Shh) locus, this regulatory domain covers 900–1000 kb. Within this context each embryonic tissue that expresses Shh has acquired its own regulatory apparatus which may require the activity from several distinct enhancers. Expression of Shh in the developing limb bud is driven by a single enhancer that interprets a myriad of genetic information to initiate expression in the posterior margin of the limb bud, inhibits expression along the anterior margin, defines the level of expression, and sets the tissue boundary of expression. Addresses MRC Human Genetics Unit, MRC Institute of Genetics and Molecular Medicine, University of Edinburgh, Crewe Road, Edinburgh EH4 2XU, UK
a rich source of regulatory mutations that give rise to congenital abnormalities. Chromosomal lesions and translocation which disrupt the regulatory domain are responsible for many cases of holoprosencephaly (HPE) [6]. Alternatively, well over 20 point mutations within the limb specific enhancer called the ZRS, or chromosomal duplications that increase ZRS numbers (reviewed previously [7]) cause a spectrum of limb defects, predominantly preaxial polydactyly of the hands and feet. Other regulatory mutations such as a point mutation within the brain enhancer, SBE2, which disrupts binding of Six3 has been identified within an individual with HPE [8]. Thus, to determine the basis of a number of human diseases it is important to understand the mechanisms used to regulate gene expression. In this review we will focus on the regulatory apparatus for the Shh gene to illustrate the complexity required to express a single gene during development.
Corresponding author: Hill, Robert E ([email protected])
Composition of the Shh regulatory domain Current Opinion in Genetics & Development 2014, 27:54–59 This review comes from a themed issue on Developmental mechanisms, patterning and evolution Edited by Lee A Niswander and Lori Sussel
0959-437X/# 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.gde.2014.03.011
Introduction A common feature of genes that regulate development, such as transcription factors and signalling molecules, is the extensive collection of cis-regulatory information that resides in the vicinity of the protein coding sequence. These cis-acting elements lie in large ‘gene deserts’ (large genomic expanses devoid of genes) but often extend to the introns of neighbouring genes and these elements organise temporal and spatial transcriptional gene control [1,2]. The sonic hedgehog (Shh) locus is an example of long range regulation. Unlike holo-enhancers [3] and regulators of HoxD genes [4], which are composed of complexes of interacting regulators, Shh expression pattern is a summation of distinct enhancer activities. Mutations in regulatory elements have been predicted to be a major source of human disease and congenital abnormalities given that the vast majority of disease associated variants map to noncoding regions of the genome [5]. The extensive regulatory domain of Shh is Current Opinion in Genetics & Development 2014, 27:54–59
Regulation of expression of few mammalian genes has been investigated to the extent of the Shh gene. The region lying 50 of the Shh gene comprises a large genomic desert within which a number of potential cis-regulators of Shh reside or flank [9,10] (Figure 1). As a general rule, conservation of noncoding sequence elements within this domain highlights regulators of Shh expression. As is becoming more commonly recognised, enhancers do not always act independently and what is clear from the analysis of the Shh locus is that individual enhancer activities often overlap. Although there may be some sharing of enhancer information, on the whole it appears that each tissue or region possesses an intrinsic means for its own regulation and thus, the Shh spatial and temporal expression pattern is directed by an assemblage of autonomous enhancer activities. Using a transgenic reporter assay in mice, two intronic enhancers SFPE1 and SFPE2 [11], and an enhancer 35 kb upstream of Shh, SBE1, were identified (Figure 1). Both SFPE1 and SFPE2 function redundantly to direct LacZ expression within the floor plate of the spinal cord and hindbrain [9]. Furthermore, homozygous deletions of the SBE1 sequence which drives expression within the ventral midbrain and caudal region of the telencephalon were found to result in viable mice [11]. Initiation of Shh expression in the midline of the midbrain and caudal diencephalon occurred correctly at E8.5; however, this was not maintained beyond E10.0, suggesting that SBE1 function overlaps with an, as yet, unidentified enhancer which controls Shh transcription at earlier time points before E10.0 [12]. www.sciencedirect.com
Cis-regulation of the Shh gene Anderson and Hill 55
Figure 1
? Shh
PE SB 1 SF E1 PE 2
SF
G E SL
SB E SB 3 E2 SB E4
M AC M S1 FC S4 M RC S1
ZR
S
Lmbr1 Rnf32
Current Opinion in Genetics & Development
The genomic domain near the Shh gene representing the assemblage of enhancers that underlie the spatial and temporal pattern of expression. A schematic illustration of the multiple sites of Shh expression within the E11.5 embryo: expression within the epithelial lining of the pharyngeal cavity, lungs and digestive tube is shown on the left and the brain on the right, the colours used match the relevant enhancers (coloured bars) which are shown in their genomic context. MACS1 and SLGE are shown to act together to drive expression within the gut epithelium whereas both MFCS4 and MRCS1 are shown to act independently. SBE1 which drives expression within the brain has been shown to act redundantly with an unknown enhancer.
A cluster of three cis-regulatory elements are located 600– 900 kb upstream of Shh which direct the overlapping and sequential expression along the epithelial lining of the pharyngeal cavity, lungs and digestive tubes [10] (Figure 1). Thus, the action of these enhancers partitions the epithelium into three continuous domains. The enhancer MACS1 drives expression in the posterior reaches of the gut epithelium, in the tracheal tube, the lungs, intestine and urogenital tract. The MRCS1 is responsible for the expression in anterior epithelia including in the tooth, hard palate and tongue. The MFCS4 enhancer directs expression in between and overlapping the other two enhancers in the soft palate epiglottis, arytenoids and tympanic tube. Recently, an additional enhancer was identified approximately 100 kb from Shh. This 1.7 kb enhancer designated SLGE (Shh lung, gut enhancer) drives expression overlapping that of the MACS1 enhancer [13]. The SLGE enhancer is www.sciencedirect.com
suggested to extend gut expression to later stages of development (E12.5) after MACS1 has become inactive. Multiple enhancers driving similar expression patterns are well known and in Drosophila these redundant regulatory elements are referred to as shadow enhancers [14,15]. Shadow enhancer activity has recently been identified in vertebrates in the HoxB complex [16]. Shadow enhancers provide robustness to stabilise the transcriptional response to genetic or environmental variability. In the Shh regulatory locus, it is not clear if multiple enhancers controlling overlapping expression in a single tissue satisfy the criteria of a shadow enhancer. Certainly the SBE1 enhancer activity and the activity from the unknown element are overlapping but are not redundant. In this respect, multiple enhancers may serve to extend the temporal window of expression, which may be important in a varying regulatory environment which Current Opinion in Genetics & Development 2014, 27:54–59
56 Developmental mechanisms, patterning and evolution
undergoes significant changes during development. In addition, multiple enhancers in a single tissue type, such as the cluster of three enhancers controlling Shh in the endodermal derived gut epithelium, may function to divide the regulatory load among different enhancers. To further determine the apparent complementary roles of the Shh enhancers, the gold standard remains the removal of specific enhancer sequences to investigate the phenotypic outcome.
Long range regulation in a single developing tissue The most distally located enhancer in the Shh domain is an 800 bp highly conserved long-range regulatory element. In contrast to the above, this enhancer, referred to as the ZRS (also the MFCS1) [17,18], appears to be the sole regulator of expression directing SHH to a restricted domain in the posterior margin of the developing limb called the zone of polarising activity (ZPA). This regulator operates over approximately 1 Mb (in human) to regulate Shh expression (Figure 1) and a deletion of the element results in a phenocopy of the abnormal limb seen in the Shh null mutation [19]. The expression of Shh in the ZPA extends from the posterior edge of the limb bud but has no histological boundary along the anterior most limits of the domain. In the limb this restricted, focal expression of Shh is crucial for specifying digits along the posterior (side with the little finger and toe) to anterior (side with the thumb and great toe) axes. Digits form from a reiterative periodic pattern in the limb bud such that each digit forms at a specific distance from the other digits resulting in a repeating pattern of digit, and non-digit [20]. The most recent proposal to explain SHH activity is an integrated model, which in addition to morphogen activity, incorporates growth as a key function in early patterning [21]. Hence the role of the posterior Shh expression is to organise this periodic digital pattern to regulate the number of digits, perhaps by regulating the growth of the limb bud and in addition, the morphogen activity specifies individual characteristics to each digit. This posterior positioning of Shh expression is the consequence of multiple developmental events that polarise the limb. Pathways responsible have been identified that operate to both restrict Shh expression to the posterior margin of the limb bud and set the anterior boundary. Initially the limb is polarised at the earliest stages of limb bud outgrowth which is meditated by the mutually antagonistic interactions of Gli3 and Hand2 [22] (Figure 2a). Initiation of Shh expression follows and is dependent on Hand2 in the posterior of the limb bud and during these early stages the posterior genes from the HoxA and HoxD (genes 10–13) complexes are also required [23,24]. A conditional limb mutation for Hand2 results in the loss of Shh expression and a phenotype that Current Opinion in Genetics & Development 2014, 27:54–59
resembles the Shh null limb [25]. The role of HAND2 is dependent on the DNA binding capacity of its helix– loop–helix domain [26] operating at the ZRS. Hoxd10 and Hoxd13 also interact directly with the ZRS [27] and furthermore HOXD13 and HAND2 form a protein complex that transactivates the ZRS, and notably this process is interfered with by a repressor of Shh expression, GLI3R [25]. Shh is inactive, but primed for expression in the anterior margin of the limb [28] and under certain genetic circumstances can be ectopically expressed resulting in the generation of limb defects such as preaxial polydactyly [7]. Genetic analysis showed that several genes are responsible for repressing Shh expression in the anterior domain and thus restricting Shh to the ZPA (Figure 2b). Alx4 is specifically expressed in the anterior margin of the limb and when deleted anterioposterior polarity is disrupted and Shh is induced in the anterior of the limb bud [29,30]. The ETS transcription factor genes, Etv4 and Etv5, are expressed along the distal edge of the developing limb bud [31] and are maintained at a high level by the expression of FGFs in the overlying apical ectodermal ridge. Loss of both ETV genes results in the ectopic expression of Shh in the anterior of the limb bud and the production of extra anterior digits. ETV4 and ETV5 bind to the ZRS and mutation of the binding sites results in expansion of Shh expression into the anterior domain. However, ALX4 and ETV4/5 may fit into a more complex picture that includes TWIST1 [32]. This picture includes the observation that FGF expression in the AER has two functions, the first is to increase and maintain the expression of Shh in the ZPA but secondly, acting through the inducible ETV genes it inhibits the expression in the posterior domain. TWIST1 is central to these two functions. At low levels, in response to FGF signalling, TWIST1 induces the FGF receptor responsible for regulating the levels of Shh in the posterior domain, whereas in the anterior domain at higher levels induces the production ALX4 and the two ETV proteins. Two other ETS transcription factors, ETS1 and GABPa, are responsible for defining the extent and therefore boundary of Shh expression (Figure 2c) [31]. The anterior extent of expression is regulated by multiple ETS binding sites within the ZRS. Removal of a subset of these sites narrows the width of expression, whereas an extra site extends the expression domain further anteriorly. Surprisingly, some of the early developmental events involved in polarising the limb are specific for the forelimbs or the hindlimbs. For example, the Hox9 paralogs (Hoxa-d9) are responsible for Hand2 expression, which establishes the initial asymmetry [33] but only in the forelimbs (Figure 2a). Moreover, the Hox5 paralogs (particularly Hoxa5, Hoxb5 and Hoxc5) (Figure 2b) are www.sciencedirect.com
Cis-regulation of the Shh gene Anderson and Hill 57
Figure 2
Initiation of Shh
(a)
Restriction of Shh to ZPA
(b)
AER-FGF Hand2 Shh HoxA/D Hand2 Gli3
Twist
Shh
Etv4/5 FGFR1 Alx4
Gli3 HoxB/D10 + Hand2
AER-FGF
Hand2 + HoxA/D10-13 Twist (low)
Twist (high)
Hoxa9, b9,c9,d9 Forelimbs
Hand2
?
Etv4/5
FGFR1
Alx4
Hindlimbs Shh
Shh
Forelimbs Hox5 (a5,b5,c5) (c)
Formation of Shh Expression Boundary AER
ETS1/GABPα
AER ETS1 sites within the ZRS
Shh
Wild-type ZRS
Deletion of ETS1 site
Additional ETS1 site
Current Opinion in Genetics & Development
A survey of the interactions required to express Shh in a single developing tissue, the limb bud. (a) The polarity of the limb is defined early in development by the antagonist activity of GLI3 in the anterior and by HAND2 (which downregulates GLI3) in the posterior of the limb bud. Initiation of Shh expression is dependent on HAND2 expression and the posterior HoxA/D genes (Hox10-13). Polarity of the forelimbs is dependent on the Hox9 paralogues, whereas in the hindlimbs the mechanism is unknown. (b) Shh expression is repressed in the anterior margin and thus restricted to the posterior margin of the limb. The genes Alx4, Etv4 and Etv5 are involved in this restriction within both the hindlimbs and the forelimbs, whereas additionally in the forelimbs the Hox5 genes (Hoxa5, Hoxb5 and Hoxc5) are also involved. The repression of Shh may be regulated by Twist1 expression in the limb, which upregulates both the anterior repressors and the posterior FGF signalling pathway. (c) The expression boundary of Shh is determined by ETS transcription factor binding sites. Within the limb, ETS1/GABPa is expressed across the limb bud (left). In the wildtype embryo this leads to Shh production within the ZPA (right depicted in yellow). Deletion of one of the ETS1 sites causes a reduction in area of Shh expression (right depicted in mauve) whereas the addition of an ETS1 site results in an increased Shh expression area (right depicted in dark blue). Abbreviations: AER, apical ectodermal ridge; FGFs, fibroblast growth factors 4, 8, 9 and 17. www.sciencedirect.com
Current Opinion in Genetics & Development 2014, 27:54–59
58 Developmental mechanisms, patterning and evolution
necessary to maintain the expression of ZPA located Shh, but only in the forelimbs [34]. These Hox genes pose a satisfying solution as to how initial limb polarity is established but leaves no answers as to hindlimb polarity, only hinting at other Hox genes being involved.
Chromosomal conformation and topological domains 3D-FISH measurements and 3C (chromosomal conformational capture) analysis suggest that the ZRS and Shh gene interact via a chromosomal looping mechanism [28]. This occurs as a two-step process whereby firstly, looping brings the ZRS close to the Shh promoter, and Shh then moves out of its own chromosome territory which correlates with gene activation. It is proposed that the locus is being held in three states: silent, in which the gene and enhancer do not contact; poised, in which there is some contact and ultimately; and active, where contact occurs and the gene is transcribed. The Shh enhancers can operate over hundreds of kilobases but they only operate in their own genomic space, without affecting the neighbouring genes. Conclusions from Hi-C experiments (a method for identifying higher order chromatin interactions genome wide) suggest that the genome of mice and humans is organised in large chromatin interaction domains known as ‘topological domains’ or TADs (topological associated domains) [35,36]. These domains are believed to correlate with regions of the genome that constrain the spread of heterochromatin and consist of highly self-interacting regions in which tens of genes and hundreds of enhancers reside. These domains are speculated to constrain looping interactions between enhancers and promoters and set the boundaries of coordinated gene regulation, and indeed Shh and the ZRS are found to occupy the same topological domain [36].
Conclusions Genome sequencing in multiple species and the recent publication of the large ENCODE project [37] have been invaluable in gaining an elemental understanding of gene regulation. To gain an insight into the layers of regulatory information required to control spatiotemporal expression of even a single developmental gene, however, is a challenging task. Genetic manipulation and analysis of all coding regions within the genome is a current goal of mouse geneticists and these approaches have been streamlined with the advent of TALENS and CRISPR/Cas technologies [38]. Moving these technologies into the realm of manipulating regulatory domains will be crucial for unravelling the hierarchy of gene expression control. Recent studies that enable chromosomal engineering using the Sleeping Beauty transposable element is a useful tool for systematically mapping large regulatory domains [3,39,40]. Applying such genetic approaches may be the next comprehensive Current Opinion in Genetics & Development 2014, 27:54–59
strategy for understanding the coordinated regulatory events responsible for the expression of Shh and other developmental genes.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest
1.
Woolfe A, Goodson M, Goode DK, Snell P, McEwen GK et al.: Highly conserved non-coding sequences are associated with vertebrate development. PLoS Biol 2005, 3:e7.
2.
Pennacchio LA, Ahituv N, Moses AM, Prabhakar S, Nobrega MA et al.: In vivo enhancer analysis of human conserved noncoding sequences. Nature 2006, 444:499-502.
3.
Marinic M, Aktas T, Ruf S, Spitz F: An integrated holo-enhancer unit defines tissue and gene specificity of the Fgf8 regulatory landscape. Dev Cell 2013, 24:530-542. This paper shows an organisation of cis-regulators known as a holoenhancer that function as a coherent unit to generate the expression of the Fgf8 gene.
4.
Montavon T, Soshnikova N, Mascrez B, Joye E, Thevenet L et al.: A regulatory archipelago controls Hox genes transcription in digits. Cell 2011, 147:1132-1145.
5.
Makrythanasis P, Antonarakis SE: Pathogenic variants in nonprotein-coding sequences. Clin Genet 2013, 84:422-428.
6.
Roessler E, Ward DE, Gaudenz K, Belloni E, Scherer SW et al.: Cytogenetic rearrangements involving the loss of the Sonic Hedgehog gene at 7q36 cause holoprosencephaly. Hum Genet 1997, 100:172-181.
7.
Anderson E, Peluso S, Lettice LA, Hill RE: Human limb abnormalities caused by disruption of hedgehog signaling. Trends Genet 2012, 28:364-373.
8.
Jeong Y, Leskow FC, El-Jaick K, Roessler E, Muenke M et al.: Regulation of a remote Shh forebrain enhancer by the Six3 homeoprotein. Nat Genet 2008, 40:1348-1353.
9.
Jeong Y, El-Jaick K, Roessler E, Muenke M, Epstein DJ: A functional screen for sonic hedgehog regulatory elements across a 1 Mb interval identifies long-range ventral forebrain enhancers. Development 2006, 133:761-772.
10. Sagai T, Amano T, Tamura M, Mizushina Y, Sumiyama K et al.: A cluster of three long-range enhancers directs regional Shh expression in the epithelial linings. Development 2009, 136:1665-1674. 11. Epstein DJ, McMahon AP, Joyner AL: Regionalization of Sonic hedgehog transcription along the anteroposterior axis of the mouse central nervous system is regulated by Hnf3dependent and -independent mechanisms. Development 1999, 126:281-292. 12. Jeong Y, Dolson DK, Waclaw RR, Matise MP, Sussel L et al.: Spatial and temporal requirements for sonic hedgehog in the regulation of thalamic interneuron identity. Development 2011, 138:531-541. 13. Tsukiji N, Amano T, Shiroishi T: A novel regulatory element for Shh expression in the lung and gut of mouse embryos. Mech Dev 2013, 131:127-136. A novel, non-conserved enhancer was reported for driving expression of Shh in the mouse gut. In the gut epithelium, the expression of Shh is achieved by the seamless contribution of multiple enhancers. 14. Frankel N, Davis GK, Vargas D, Wang S, Payre F et al.: Phenotypic robustness conferred by apparently redundant transcriptional enhancers. Nature 2010, 466:490-493. 15. Perry MW, Boettiger AN, Bothma JP, Levine M: Shadow enhancers foster robustness of Drosophila gastrulation. Curr Biol 2010, 20:1562-1567. www.sciencedirect.com
Cis-regulation of the Shh gene Anderson and Hill 59
16. Nolte C, Jinks T, Wang X, Martinez Pastor MT, Krumlauf R: Shadow enhancers flanking the HoxB cluster direct dynamic Hox expression in early heart and endoderm development. Dev Biol 2013, 383:158-173. Identifies a shadow enhancer operating at the HoxB complex in mice that participates with other local enhancer to modulate gene expression during cardiac development. 17. Lettice LA, Heaney SJ, Purdie LA, Li L, de Beer P et al.: A longrange Shh enhancer regulates expression in the developing limb and fin and is associated with preaxial polydactyly. Hum Mol Genet 2003, 12:1725-1735. 18. Sagai T, Masuya H, Tamura M, Shimizu K, Yada Y et al.: Phylogenetic conservation of a limb-specific, cis-acting regulator of Sonic hedgehog (Shh). Mamm Genome 2004, 15:23-34. 19. Sagai T, Hosoya M, Mizushina Y, Tamura M, Shiroishi T: Elimination of a long-range cis-regulatory module causes complete loss of limb-specific Shh expression and truncation of the mouse limb. Development 2005, 132:797-803. 20. Sheth R, Marcon L, Bastida MF, Junco M, Quintana L et al.: Hox genes regulate digit patterning by controlling the wavelength of a Turing-type mechanism. Science 2012, 338:1476-1480. A landmark paper that defines in mathematical and genetic terms the self-organising capacity of the developing limb bud. 21. Towers M, Mahood R, Yin Y, Tickle C: Integration of growth and specification in chick wing digit-patterning. Nature 2008, 452:882-886. 22. te Welscher P, Fernandez-Teran M, Ros MA, Zeller R: Mutual genetic antagonism involving GLI3 and dHAND prepatterns the vertebrate limb bud mesenchyme prior to SHH signaling. Genes Dev 2002, 16:421-426. 23. Kmita M, Tarchini B, Zakany J, Logan M, Tabin CJ et al.: Early developmental arrest of mammalian limbs lacking HoxA/HoxD gene function. Nature 2005, 435:1113-1116. 24. Tarchini B, Duboule D, Kmita M: Regulatory constraints in the evolution of the tetrapod limb anterior–posterior polarity. Nature 2006, 443:985-988. 25. Galli A, Robay D, Osterwalder M, Bao X, Benazet JD et al.: Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development. PLoS Genet 2010, 6:e1000901. 26. Liu N, Barbosa AC, Chapman SL, Bezprozvannaya S, Qi X et al.: DNA binding-dependent and -independent functions of the Hand2 transcription factor during mouse embryogenesis. Development 2009, 136:933-942. 27. Capellini TD, Di Giacomo G, Salsi V, Brendolan A, Ferretti E et al.: Pbx1/Pbx2 requirement for distal limb patterning is mediated by the hierarchical control of Hox gene spatial distribution and Shh expression. Development 2006, 133:2263-2273. 28. Amano T, Sagai T, Tanabe H, Mizushina Y, Nakazawa H et al.: Chromosomal dynamics at the Shh locus: limb bud-specific
www.sciencedirect.com
differential regulation of competence and active transcription. Dev Cell 2009, 16:47-57. 29. Panman L, Drenth T, Tewelscher P, Zuniga A, Zeller R: Genetic interaction of Gli3 and Alx4 during limb development. Int J Dev Biol 2005, 49:443-448. 30. Kuijper S, Feitsma H, Sheth R, Korving J, Reijnen M et al.: Function and regulation of Alx4 in limb development: complex genetic interactions with Gli3 and Shh. Dev Biol 2005, 285:533-544. 31. Lettice LA, Williamson I, Wiltshire JH, Peluso S, Devenney PS et al.: Opposing functions of the ETS factor family define Shh spatial expression in limb buds and underlie polydactyly. Dev Cell 2012, 22:459-467. Demonstrates ETS transcription factor binding sites contained in the ZRS that both restricts ectopic expression of Shh and determines the posterior boundary of expression in the limb bud. 32. Krawchuk D, Weiner SJ, Chen YT, Lu BC, Costantini F et al.: Twist1 activity thresholds define multiple functions in limb development. Dev Biol 2010, 347:133-146. 33. Xu B, Wellik DM: Axial Hox9 activity establishes the posterior field in the developing forelimb. Proc Natl Acad Sci U S A 2011, 108:4888-4891. 34. Xu B, Hrycaj SM, McIntyre DC, Baker NC, Takeuchi JK et al.: Hox5 interacts with Plzf to restrict Shh expression in the developing forelimb. Proc Natl Acad Sci U S A 2013, 110:1943819443. This paper shows a previously unexpected role for the Hox5 paralogues (Hoxa5, Hoxb5 and Hoxc5) in forelimb patterning and are involved in repressing Shh in the anterior, ectopic domain, thus restricting Shh expression in the limb. 35. Dixon JR, Selvaraj S, Yue F, Kim A, Li Y et al.: Topological domains in mammalian genomes identified by analysis of chromatin interactions. Nature 2012, 485:376-380. Using HiC technology at a high resolution, this paper describes the organisation of the genome into local interaction domains called topological domains. 36. Smallwood A, Ren B: Genome organization and long-range regulation of gene expression by enhancers. Curr Opin Cell Biol 2013, 25:387-394. 37. Consortium TEP: A user’s guide to the encyclopedia of DNA elements (ENCODE). PLoS Biol 2011, 9:e1001046. 38. Gaj T, Gersbach CA, Barbas CF III: ZFN, TALEN, and CRISPR/ Cas-based methods for genome engineering. Trends Biotechnol 2013, 31:397-405. 39. Kokubu C, Horie K, Abe K, Ikeda R, Mizuno S et al.: A transposonbased chromosomal engineering method to survey a large cisregulatory landscape in mice. Nat Genet 2009, 41:946-952. 40. Ruf S, Symmons O, Uslu VV, Dolle D, Hot C et al.: Large-scale analysis of the regulatory architecture of the mouse genome with a transposon-associated sensor. Nat Genet 2011, 43:379386.
Current Opinion in Genetics & Development 2014, 27:54–59
