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First published online 25 August 2004
doi: 10.1242/dev.01344

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SMAD-mediated modulation of YY1 activity regulates the BMP response and cardiac-specific expression of a GATA4/5/6-dependent chick Nkx2.5 enhancer

¹ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Avenue Boston, MA 02115, USA
² Cardiology Department, Children's Hospital Boston, 300 Longwood Avenue, Boston, MA 02115, USA

View larger version (37K): [in a new window]   Fig. 1. BMP response and in vivo transgene expression maps to CAR3 enhancer. (A) Genomic flanking regions of chick Nkx2.5 containing CAR enhancers as shown were assayed for BMP response in P19 cells in the context of both hsp68-lacZ and Nkx2.5-luciferase reporters (Nkx2.5-lux). Only the CAR3 enhancer is BMP responsive. (B) Deletion mapping of BMP response and transgenic expression for 3 kb 3' genomic flanking region of chick Nkx2.5which contains CAR3, using hsp68-lacZ promoter/reporter. Nucleotide positions of 5' and 3' ends of deleted flanking regions are given relative to transcriptional start site of chick Nkx2.5. Extent of the genomic fragments yielding positive reporter lacZ expression in F0 transient transgenic mouse embryos are shown by black bars (C3-2, C3-4, C3-6, C3-8 and C3-10); negative reciprocal fragments are represented as gray bars (C3-1, C3-3, C3-5, C3-7 and C3-9). BMP response of these hsp-lacZ-CAR3 reporters is shown to immediate right of their representations, expressed as normalized fold induction by BMP. (C-L) Representative X-gal stained E7.5 F0 transient transgenic mice embryos injected with C3 reporters C3-1 to C3-10. (M) BMP response of 2 kb CAR3 (fragment C3-2) and 200 bp BMPRE (fragment C3-10) enhancers linked to Nkx2.5-lux reporters (Nkx2.5-lux-CAR3 and Nkx2.5-lux-BMPRE, respectively). Extents of CAR3 and BMPRE enhancers and nucleotide positions relative to transcriptional start site are shown as above. BMP response in P19 cell assay is shown to right as fold induction above basal. (N-S) Representative X-gal stained F0 transient transgenic mice embryos injected with Nkx2.5-lacZ-CAR3 (N-Q) and Nkx-lacZ-BMPRE (R,S). Embryonic stages are shown at bottom left of pictures. Frequency of transient or stable (C3-2) transgene expression in cardiac region (cardiac crescent): C3-1, 0/20 embryos; C3-2, 2/3 lines; C3-3, 0/18 embryos; C3-4, 4/5 embryos; C3-5, 1/19 embryos; C3-6, 1/1 embryos; C3-7, 1/12 embryos; C3-8, 12/12 embryos; C3-9, 0/5 embryos; C3-10, 12/16 embryos. CC, cardiac crescent; LS, lateral somite; OFT, outflow tract; AL, allantois; BA, branchial arches; St, stomach; RV, right ventricle; H, heart; LV, left ventricle.

View larger version (45K): [in a new window]   Fig. 2. SMAD consensus sites bind MH1 domain of SMAD4 and are required for BMP response of CAR3. (A) Oligonucleotides used for SMAD protein gel shifts are shown as colored horizontal lines over a sequence of 200 bp BMPRE, representing chick Nkx2.5 genomic sequences from +2150 to +2350. Nucleotide positions in BMPRE are renumbered from 1-200 for convenience. Consensus SMAD-binding sites or SBEs are shown boxed in green; numerical designations are shown in green circles. Nucleotide substitutions made for mutant oligos and reporters are displayed in red under their cognate sites, as are correspondingly altered sequences within the BMPRE (SBE1, nucleotides 35-65; SBE2, nucleotides 80-120; SBE3, nucleotides 120-160). (B) Gel shifts performed with purified GST, GST-SMAD1 MH1 or GST-SMAD4 MH1 domain proteins on labeled wild-type (lanes 1-3, 7-9 and 13-15, respectively) or mutant (mut, lanes 4-6, 10-12 and 16-18, respectively) SBE oligomers. (C) P19 cell BMP response assays for wild-type Nkx2.5-lux-CAR3 (left) or Nkx2.5-lux-BMPRE (right) reporters, or cognate reporters bearing specific SBE mutations shown in A. Nkx2.5-lux constructs are shown schematically over bar graph. (D-O) Representative X-gal stained F0 transient transgenic mice embryos injected with wild-type or SBE mutant (mut) Nkx2.5-lacZ-CAR3 reporters. Embryonic stages are in the bottom left-hand corners. Whole-mount embryo at stages E7.5 (D,G,J,M) and E10.5 (E,H,K,N) and E10.5 hearts (F,I,L,O) are shown as designated for Nkx2.5-lacZ-CAR3 WT (D-F), mut SBE1 (G-I), mut SBE2 (J-L) and mut SBE3 (M-O) as shown in A. Cardiac staining shown was representative of the following numbers of transient transgenic embryos bearing Nkx2.5-lacZ constructs driven by CAR3 reporters: wild type, 3/5 E7.5 and 4/4 E10.5 embryos displayed expression in the cardiac crescent and forming heart; CAR3mut SBE1, 2/4 E7.5 embryos displayed robust transgene expression in the cardiac crescent and 9/9 E10.5 embryos either failed to express the transgene or displayed very weak transgene expression in the outflow tract; CAR3mut SBE2, 7/7 E7.5 embryos displayed robust transgene expression in the cardiac crescent and 2/2 E10.5 embryos failed to express the transgene; CAR3mut SBE3, 7/7 E7.5 embryos and 3/3 E10.5 embryos either failed to express the transgene or displayed residual transgene expression in the outflow tract. Abbreviations are as in previous figures. RA, right atrium; LA, left atrium; LV, left ventricle.

View larger version (39K): [in a new window]   Fig. 3. Embryonic gel shift analysis of BMPRE. (A,B) Dissected regions of HH stage 6-8 and 24 (day 3) chick embryos are depicted as boxed regions of whole mount chick embryos stained by in situ hybridization for Nkx2.5 mRNA. Whole-cell extracts were made from either anterior lateral plate (ALP) or posterior primitive streaks (PPS) dissected from stage 6-8 chick embryos (A), or from hearts (Hrt) from stage 24 (day 3) chick embryos (B). (C) EMSA oligomers derived from 200 bp BMPRE enhancer sequence (see Fig. 2). Numbered and colored horizontal lines show extent of 30 or 40 bp double stranded oligomers used for gel shift assays. (D) Gel shifts obtained with anterior lateral plate (A), posterior primitive streak (P) or 3 day heart whole cell extracts. Discrete shifts were found for eight sites labeled A-E. Sites A1-A3 (nucleotides 1-30), lanes 1, 2, 21; site B (nucleotides 30-60 and 45-75), lanes 5-8, 23, 24; site C (nucleotides 60-100), lanes 9, 10, 25; site C' (nucleotides 100-140), lanes 13, 14, 27; site D (nucleotides 120-160), lanes 15, 16, 28; site E (nucleotides 160-200), lanes 19, 20, 30. Multiple shifts seen with Site A oligo in different extracts are labeled separately A1-3 to the left of the autoradiogram, and are as discussed in the text.

View larger version (42K): [in a new window]   Fig. 4. Characterization of BMPRE gel shift binding sites. (A) 200 bp BMPRE is shown with numbered horizontal lines of selected double-stranded oligonucleotides shifted by embryonic extracts (see Fig. 3 and text). Likely binding sites for embryonic gel shifts are boxed (orange for Site B, red for C and C', blue for Site D. Nucleotide changes made in mutant oligos are shown in red under their cognate site and are also highlighted in either white or red on the 200 bp BMPRE sequence. (B) Gel shifts of putative GATA-binding sites. Oligomers 3, 5 and 7, containing Sites B, C and C', respectively, were used in EMSA experiments with nuclear extracts prepared from control COS cells, or COS cells expressing GATA4, GATA5 and GATA6. All three oligomers bind all cardiac GATAs (lanes 2-4, site B; lanes 10-12, site C; lanes 18-20, site C') and binding is greatly reduced or eliminated upon mutation of consensus GATA-binding sites within the oligomers (lanes 6-8, mutant site B; lanes 14-16, mutant site C; lanes 22-24, mutant site C'). (C) Gel shifts and antibody supershifts of either wild-type (WT) (lanes 1-3, 7-9, 13-15 and 19) or mutant (mut) (lanes 4-6, 10-12, 16-18 and 20) site B, C, C' and D oligomers with day 3 heart whole cell extracts. Supershifts for sites B (lanes 1-6), C (lanes 7-12) and C' (lanes 13-18) were performed with either control mouse Ig (lanes 1, 4, 7, 10, 13 and 16), anti-GATA4 (lanes 2, 5, 8, 11, 14 and 17) or anti-GATA-6 (lanes 3, 6, 9, 12, 15 and 18) rabbit polyclonal antibodies as indicated. (D) BMP responsiveness of either wild-type Nkx2.5-lux-CAR3 reporter or similar reporters bearing mutations in either sites B, C/C' or D (as diagrammed in A). (E-L) Representative X-gal stained F0 transient transgenic mice embryos containing either wild type or mutant Nkx2.5-lacZ-CAR3 reporters. Embryonic stages are shown in the bottom left-hand corner. Results shown are representative of the following numbers of transgenic embryos: mut Site B, 11/11 embryos; mut site C/C', 14/15 embryos (1/15 E8.5 embryos displayed residual heart staining); mut Site D, 7/7 embryos. Abbreviations are as in previous figures.

View larger version (19K): [in a new window]   Fig. 5. SMAD-dependent BMP response is associated with site D activity. (A) BMPRE sequence is displayed. Colored horizontal lines show extent of double-stranded oligonucleotides used to create multimerized enhancers encompassing SBE1 plus GATA6 and GATA4/5 binding sites (SMAD region 1 or SR1, orange line), SBE2 plus GATA4-binding sites (SMAD region 2 or SR2, red line) and SBE3 plus binding site D (SMAD region 3 or SR3, blue line). Site D and SBE3 mutations are displayed below sequence of BMPRE in red, and corresponding altered nucleotides are displayed as white. (B) BMP response of Nkx2.5-lux driven by either the 2 kb CAR3, the 200 bp BMPRE, or 5x-multimerized repeats of SR1, 2 or 3. Reporters are shown on the left, with colored arrowheads representing each repeat and its orientation. Cognate BMP response is shown to the immediate right of the construct schematic, and is expressed as fold activation over basal activity. (C) BMP induction of SR3 requires both binding site D and SBE3. BMP response of Nkx2.5-lux driven by either 4x-multimerized repeats of SR3-WT, or multimers of SR3 bearing mutations in either binding site D or associated SMAD-binding element 3 (SBE3) (shown in A). (D) SMAD4 dependence of SR3-mediated BMP response. BMP4 response of Nkx2.5-lux-4xSR3 was assayed in the SMAD4-deficient MDA-MB468 cell line in the absence or presence of co-transfected SMAD1 and SMAD4 expression vehicles. Activation is seen only in the presence of both co-expressed SMAD proteins and BMP4 (bottom lanes).

View larger version (26K): [in a new window]   Fig. 6. BMP responsiveness of SR3/CAR3 and cardiac expression of a CAR3-driven transgene requires intact binding sites for transcription factor YY1. (A) Sequence of SR3 region encompassing site D (blue box) and SBE3 (green box). Sense and antisense YY1 consensus binding sites are shown above putative YY1-binding sites within SR3. Point mutations in putative YY1 binding sites A and B are shown in red below their cognate nucleotides. (B) YY1 binds to SR3. EMSA with oligos encoding either WT SR3 (lanes 1 and 5) or SR3 with mutations in putative YY1-binding site A (lane 2), site B (lane 3) or sites A+B (lane 4) with either P19 cell nuclear extract (top panel), whole cell extracts from day3 (HH Stage 24) chick hearts (middle panel) or purified HA-tagged YY1 protein (lower panel). Point mutation of the YY1 consensus binding site B in the SR3 oligomer significantly decreased or abolished DNA-binding activities in all samples (lane 3), as did mutations of both sites A and B (lane 4). Anti-YY1 polyclonal antibody added to the EMSA disrupted the interaction of factors in both P19 cells and day 3 chick hearts with the SR3 oligomer (lane 5). (C) Point mutation of the YY1 consensus site B in the context of an Nkx2.5-lux-CAR3 reporter (schematized at top) results in loss of BMP response in P19 cells. Results are shown in duplicate for wild-type Nkx2.5-lux-CAR3 (left) and Nkx2.5-lux-CAR3 mutB (right). (D-G) YY1-binding sites are required for CAR3-driven transgene expression in the heart. Representative X-gal staining patterns are shown for transgenic mice embryos containing Nkx2.5-lacZ-CAR3 mut B (shown in D). lacZ expression is abrogated at all stages assayed, indicating a requirement for YY1-binding sites in CAR3 to drive transgene expression in the heart (compare with the robust cardiac expression of the parental Nkx2.5-lacZ-CAR3 construct in Fig. 2D-F). Embryonic stages are shown in the lower left-hand corner. Results are representative of 3/3 E7.5 and 5/5 E10.5 transgenic embryos. Abbreviations are as in previous figures.

View larger version (24K): [in a new window]   Fig. 7. BMP SMADs associate with the N terminus of YY1: (A) Co-immunoprecipitation of YY1 with the SMAD1/4 complex. Whole-cell extracts from either BMP-stimulated COS cells transfected with expression vehicles encoding either human YY1 (lanes 1-4), Myc-tagged (MT) SMAD1 (lanes 2 and 4) or myc/Flag-tagged (MT/F)-SMAD4 (lanes 3 and 4). SMAD-associated proteins were immunoprecipitated from cell extracts with an anti-myc monoclonal antibody, subjected to polyacrylamide gel eletrophoresis (PAGE) and detected following western blot with either anti-myc or anti-YY1 polyclonal antibodies. Levels of YY1 present in the input extract used for immunoprecipitation are shown in bottom panel. Bands corresponding to YY1, SMAD1 and SMAD4 are indicated by arrowheads on the right, as are molecular mobility markers. (B) Mapping the SMAD1/4 interaction domain of YY1 by co-immunoprecipitation. COS cells were transfected with expression vehicles encoding either wild-type (WT) Flag-tagged (F)-YY1(1-414) or various Flag-tagged deletion mutants of YY1, plus expression vehicles encoding MT/F-SMAD4 and MT-SMAD1, as indicated. SMAD-associated proteins were immunoprecipitated from cell extracts with an anti-Myc monoclonal antibody, subjected to polyacrylamide gel eletrophoresis (PAGE) and detected following western blot with anti-Flag monoclonal antibody (upper panel). Anti-Flag western blot of 2.5% of input used in co-immunoprecipitation confirms substantial expression of all tagged deletion mutants of YY1 and SMAD4 in the various cell extracts (lower panel); in addition, equivalent levels of MT-SMAD1 expression was detected in the various cell extracts (data not shown). Deletion mutant used in each sample is shown above the lanes; band representing Flag/MT SMAD 4 (Sm4) is shown by arrowheads on the right, as are those of YY1 isoforms, bracketed on the right. Results shown are representative of three independent co-immunoprecipitation experiments. (C) The SMAD1/4 interaction domain of YY1 maps between residues 1-170. Schematic representation of YY1 structural regions is shown at top [adapted, with permission, from Thomas and Seto (Thomas and Seto, 1999)]; solid bars showing extent of various YY deletion mutants assayed in B. Gray box identifies SMAD1/4-interacting region of YY1 based upon amino acids shared by all YY1 deletion mutants that associate with SMAD1/4. Relative strength of SMAD1/4 association of various YY1 deletion mutants is indicated on extreme left by either +++ (strong association), ++ (weak but detectable association), no mark (no detectable association). (D) Co-expression of YY1(261-414), which lacks the SMAD1/4 interaction domain, inhibits BMP induction of a CAR3-driven reporter. P19 cells were transfected with Nkx2.5-lux-CAR3, Tk-renilla-luciferase (for normalization) and expression vehicles encoding either SMADs1 and 4 and p21E1b (to inhibit apoptosis due to loss of YY1 activity), plus increasing amounts (0-100 ng/well) of expression vectors encoding either wild-type YY1(1-414) (right lanes) or N-terminally truncated YY1(261-414) (left lanes). YY1(261-414) is able to bind to Site D in SR3 (data not shown) but is incapable of SMAD1/4 interaction (see above), and blocks the ability of BMP signals to induce the expression of a CAR3-driven reporter.

View larger version (18K): [in a new window]   Fig. 8. CAR3-mediated cardiac expression requires the collaboration of multiple factors. Diagram outlines three arms of BMP signaling that synergistically activate CAR3-driven cardiac expression: SMAD nuclear localization and direct interaction with SBE sites located in CAR3; induction of cardiac GATA proteins; and SMAD-mediated modulation of the activity of the ubiquitously expressed YY1 repressor/activator.