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Spatially specific expression of Hoxb4 is dependent on the ubiquitous transcription factor NFY

Jonathan Gilthorpe^1,*,¶, Marie Vandromme^1,,¶, Tim Brend^1,, Alejandro Gutman¹, Dennis Summerbell^1,, Nick Totty^2, and Peter W. J. Rigby^1,,**

¹ Division of Eukaryotic Molecular Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Ludwig Institute for Cancer Research, 91 Riding House Street, London W1W 7BS, UK
^* Present address: Department of Developmental Neurobiology, King’s College London, Guy’s Campus, London Bridge, London SE1 1UL, UK
Present address: Laboratoire de Biologie Moleculaire et Cellulaire, UMR 5665 CNRS/ENS Lyon, 46 Allee d’Italie, 69364 Lyon Cedex 07, France
Present address: Section of Gene Function and Regulation, The Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London SW3 6JB, UK
Present address: Protein Analysis Laboratory, Cancer Research UK, PO Box123, Lincoln’s Inn Fields, London WC2A 3PX, UK
^¶ These authors contributed equally to this article and should both be considered first authors

View larger version (69K): [in a new window]   Fig. 1. (A) Expression patterns of region C constructs. (a-e) CHZ. (f,g) Hoxb4 promoter-region C construct. (h-l) CR1 deletions. (A) Lateral and dorsal views of a 10 somite (So) stage embryo, showing anterior boundaries of expression: neural tube adjacent to So 4 (yellow arrow); somitic and flank mesoderm at So 6/7 (black arrow). Typical of Hox gene expression domains, staining is weaker in So 7 (light blue triangle) than in So 8 (dark blue). (b) 9.5 dpc: somitic boundary regresses to So 13/14 (triangles); neural boundary regresses to So 6/7 (yellow arrow). (c) 10.5dpc: staining in flank mesoderm (fm). (d) 12 dpc: strong staining in spinal ganglia up to the first cervical nerve (cn1) and ventral neural tube, extending anteriorly (open triangle). (e) Transverse section (TS), forelimb level of a similar embryo (drg, dorsal root ganglia; sg, sympathetic ganglia; f, floorplate; v, ventral root of spinal nerve). (f) 11 dpc embryo (Hoxb4 promoter, construct 5) (Whiting et al., 1991): note strong anterior domains of expression in the neural tube up to the spinal cord/hindbrain boundary (yellow arrow) and in the somitic mesoderm between the anterior So 6/7 boundary and So14 (white arrows). (g) TS forelimb level of the same embryo: note strong staining in sclerotomal derivatives (open triangles) and the dorsal aorta (d). (h,i) Lateral and dorsal views of a 12 dpc embryo (CHZ559-599): boundaries of expression in the neural tube (yellow arrow) and in the somitic mesoderm (blue triangle) are indicated. (j,k) Lateral and dorsal views of a 12dpc embryo (CHZ515-607): consistent expression is restricted to a domain in the ventral neural tube (vnt). (l) TS thoracic level of the same embryo. Scale bars: 100µm. (B) Sequence alignment showing a comparison between CR1 of the mouse Hoxb4 intron and those of other paralogous group 4 Hox genes, identical bases are highlighted in black. Numbering is with respect to that of region C (bp 1 is the first base of the SalI site in exon 1, +321 of Hoxb4). The number of base pairs (bp) in each aligned sequence is shown on the right. The extents of the two deletions in constructs CHZ559-599 and CHZ515-607 are marked with blue lines and the boundaries of a possible cis-positive regulatory element (bp 515-558), identified by these deletions, are marked by red triangles. The margins of the 28bp HB-1 element (bp 574-601) are shown (black triangles). The locations of four conserved motifs are marked below (I-IV). (C) Schematic diagram of the transgenes in A. The hsp68 promoter-lacZ reporter, which is common to each construct, is not shown to scale. Exons are shaded grey. CR1 is represented by a black rectangle within the intron (white rectangle) flanked by MunI and SfiI restriction sites. CHZ515-607 carries a deletion of the entire CR1 region (Aparicio et al., 1995). An example is shown for comparison. Exp. # denotes the total number of independent transgenic F0 embryos and lines generated with each construct giving a consistent pattern of expression.

View larger version (51K): [in a new window]   Fig. 2. The localization of binding sites within CR1. (A) Sequences of probes used in EMSA experiments to localize the binding of factors within CR1. The region of interest described in Fig. 1B,C is marked by black arrows. Mutations; m1, m2, m3 and m4 are underlined and the sequences important for binding are boxed. (B) Results of EMSA experiments using probes depicted in A: E, 10.5dpc mouse embryo whole-cell extract; F9 EC, F9 EC nuclear extract; Neuro2a, Neuro2a whole-cell extract. Black arrows indicate the position of the two retarded bands. (C,D) EMSA experiments comparing HoxTF/YY1 sites from different genes, by competition (C) and direct binding (D). HoxPwt contains wild-type (wt) sequences from +143 to +169 of the Hoxb4 5'-untranslated region and myogenin comprises sequences from +9 to + 35 of the mouse myogenin promoter (Yee and Rigby, 1993; Gutman et al., 1994). MyoD contains sequences from –628 to –602 of the mouse MyoD1 gene (Zingg et al., 1991). Myogenin-mut and MyoDm contain the mutation CCA to AAC at positions +17 to +19 and –620 to –618, respectively.

View larger version (64K): [in a new window]   Fig. 3. Identification of HoxTF as NFY. (A) Protein fractionation experiments determine the approximate apparent molecular weight of HoxTF. A schematic of the method used is shown in the left. The binding potential of renatured F9 EC nuclear extract polypeptide fractions was analysed by EMSA using the MyoD probe. Molecular mass ranges of the fractions used are indicated at the top. (B) A schematic of the HoxTF purification scheme and a silver-stained SDS-PAGE gel of the purified proteins is shown on the left. Lanes A1 and A2 contain proteins eluted from the first DNA-affinity column bearing the MyoDm or the MyoD oligonucleotides, respectively. Lane B contains proteins eluted after passage of the A2 eluate over the MyoD affinity column. The purified proteins with molecular masses of 48, 45, and 36 kDa are indicated by arrows. (C) Confirmation of NFY binding to b4Cwt by EMSA. NFY* indicates the complex supershifted by the addition of anti-NFYA antibody (lane 6).

View larger version (68K): [in a new window]   Fig. 4. In vivo requirement for the NFY site in CR1. (A) EMSA competition experiments showing the ability of specific mutations to interfere with the binding of YY1 and NF-Y to b4Cwt. On the right is a schematic of the various mutations and reporter constructs. Mutated nucleotides are underlined. The binding characteristics of the probes are summarized on the right (b4Cwt, wild type; b4C-mN+Y, double mutation; b4C-mYY1, YY1 specific mutation; b4C-mNFY1 and b4C-mNFY2, NFY specific mutations; +, binding; +/–, partial binding; –, no binding). (B) Transgenic mouse embryos stained for ß-galactosidase activity showing the expression patterns derived from the mutant NF-Y/YY1 constructs. (a) Lateral and (b) dorsal views of a 12.5dpc embryo carrying construct CHZ-mN+Y. Residual staining was consistently observed in nervous system (open triangles). (c,d) Two different 9.5-10dpc embryos carrying the same construct. Weak somitic expression is visible at the level of So 13/14 (blue triangle in c) or in the most caudal somites (blue arrow in d), as is weak expression in the flank mesoderm (red arrow in d). (e) Lateral and (f) dorsal views of an 11.5dpc embryo carrying construct CHZ-mYY1. Black arrows indicate ectopic neural expression and blue arrowhead the anterior limit of somitic expression. Flank mesoderm staining is unaffected (red arrow). (g) Lateral and (h) dorsal view of a 12.5 dpc embryo carrying construct CHZ-mNFY2. (i) TS at the forelimb level of a similar embryo. (j) Lateral and (k) dorsal views of similar 12.5 dpc embryos carrying construct CHZ-mNFY1. (l) TS at the forelimb level of a similar embryo. drg, dorsal root ganglion; v, ventral root; f, floorplate; sg, sympathetic ganglia. Scale bars: 100 µm.

View larger version (34K): [in a new window]   Fig. 5. Activity of a single isolated NFY/YY1 binding element. (A-C) Lateral views of three transient F0 transgenic 12 dpc embryos stained for ß-galactosidase activity showing the expression patterns derived with the b4C-511-558-mYY1 construct. A schematic diagram of the construct is shown on the right. Consistent staining can be seen within the neural tube, cranial (black arrows in A and B) and spinal ganglia (white arrows in A and C).

View larger version (28K): [in a new window]   Fig. 6. Comparison of NFY/YY1 sites. (A) An alignment of NFY/YY1 binding sites from the Hoxb4 intron and promoter. The sequence of the forward strand is shown above aligned with the consensus for YY1 (Hyde-DeRuyscher et al., 1995). The sequence of the reverse strand is shown below aligned with the consensus for NFY (Mantovani, 1998). In each case, the core nucleotides of the site are shown in bold. Nucleotides that are unfavourable for binding based upon either consensus are represented in lower case. Variant nucleotides within the core sequences that are thought to abolish YY1 or NFY binding are shown in red. (B) The consensus sequence (forward strand) for the Hoxb4 NFY/YY1 motif. Shown below is a representation of the CR1 NFY/YY1 site. Nucleotides over which YY1 makes base-specific contacts in the major grove are highlighted on the upper strand. Similarly, nucleotides over which NFY makes base-specific contacts in the minor and major grooves are shown on the lower strand. Nucleotides that are predicted to enhance (green) or reduce (red) the affinity of either factor for its site are shown on the appropriate strand. Core nucleotides are shown in bold. (C) Predicted NFY sites from the Hoxc8 early enhancer and the Hox8/Hox7-Hox6 four cluster sequence (H8/7-6 FCS). Sites are shown in the reverse orientation with respect to transcription. The acquisition of alternative CCAAT boxes in the mouse and human HoxC cluster is underlined. Bold, lowercase and red nucleotides have equivalent meanings to those in A.