Corepressor SMRT is required to maintain Hox transcriptional memory during somitogenesis

Abstract

Nuclear hormone receptors (NRs), such as retinoic acid receptors (RARs), play critical roles in vertebrate development and homeostasis by regulating target gene transcription. Their activity is controlled by ligand-dependent release of corepressors and subsequent recruitment of coactivators, but how these individual receptor modes contribute to development are unknown. Here, we show that mice carrying targeted knockin mutations in the corepressor Silencing Mediator of Retinoid and Thyroid hormone receptor (SMRT) that specifically disable SMRT function in NR signaling (SMRT^mRID), display defects in cranial neural crest cell-derived structures and posterior homeotic transformations of axial vertebrae. SMRT^mRID embryos show enhanced transcription of RAR targets including Hox loci, resulting in respecification of vertebral identities. Up-regulated histone acetylation and decreased H3K27 methylation are evident in the Hox loci whose somitic expression boundaries are rostrally shifted. Furthermore, enhanced recruitment of super elongation complex is evident in rapidly induced non-Pol II-paused targets in SMRT^mRID embryonic stem cells. These results demonstrate that SMRT-dependent repression of RAR is critical to establish and maintain the somitic Hox code and segmental identity during fetal development via epigenetic marking of target loci.

Keywords: SMRT; homeotic transformation; retinoic acid receptor; somitogenesis; transcriptional repression.

Fig. 1.

Axial homeotic transformation, its rescue by retinoid antagonist, and enhanced sensitivity to RA-induced abnormality in SMRT^mRID. (A) Schematic diagram of axial transformations in SMRT^mRID. Vertebrae are depicted as ovals. Yellow ovals with vv indicate presence of tuberculi anterior. Location of the 14th rib in SMRT^mRID is indicated in red. (B) Posterior transformation in cervical vertebrae of SMRT^mRID. Filled circles and blue arrows indicate position of tuberculum anterior (TA). Incomplete rib anlages on C7 (54% of SMRT^mRID) are indicated by black arrowheads. (C) Anterior transformation of lumbar vertebrae to thoracic. Presence of the 14th rib is indicated by arrowhead. (D) Rescue of cervical transformation in SMRT^mRID by RARα antagonist (BMS 204493) treatment at E10.5. WT and SMRT^mRID cervical columns are shown after antagonist treatment. (E) Typical cranial abnormality evident in SMRT^mRID after exogenous RA exposure at E10 with single dose of 100 mg/kg. Fusion between basioccipital (bo) and exoccipital (eo) bones in SMRT^mRID (white arrowheads). (F) Severe caudal truncations in SMRT^mRID after RA exposure as in E.

Fig. 2.

Altered Hox expression domains in SMRT^mRID. (A) Relative abundance of Hoxc5 mRNA along the body axis at E11.5. A, B, and C denote the regions of the embryo depicted in D. The data are shown as the mean ± SEM. n = 5, **P < 0.005 by Student’s t test. (B) Whole mount in situ hybridization showing anterior expansion of Hoxc5 expression boundary at E10.5. Somites are numbered from the forelimb level and arrow indicates the boundary of Hoxc5 expression. (C) Section in situ of E10.5 axial somites. Note Hoxc5 expression boundary is shifted to the seventh somite in comparison with WT embryos. (D) Anterior, A; middle, B; and caudal, C regions containing somites were used for mRNA quantitation. (E) Anterior expansion of Hoxc5 expression boundary at E11.5. (F) Section in situ showing anterior expansion of Hoxc5 expression domain in prevertebrae of E11.5 SMRT^mRID embryo. * indicates anterior prevertebrae expressing Hoxc5. (G) Relative abundance of Hoxc5 mRNA and in situ hybridization showing normalization of anterior Hoxc5 expression boundary at E11.5 by RAR antagonist (BMS 493) treatment at E10.5. The data are shown as the mean ± SEM. n = 5.

Fig. 3.

Epigenetic spatial control of Hox genes in SMRT^mRID mice. (A) Recruitment of RAR and HDAC3 to the Hoxc4 RARE in three axial somites regions (F, front; M, middle; R, rear depicted in B). The data are shown as the mean + SEM. n ≥ 3. Locations of ChIP primer pairs are indicated as arrowheads in HoxC loci diagram. (B) Epigenetic histone modification status of HoxC loci in three axial somites regions (F, front; M, middle; R, rear depicted). The data are shown as the mean + SEM. n ≥ 3. (C) Recruitment of Jmjd3 and Suz12 containing PRC2 complex to Hoxc5 exon 1 region in three axial somite regions (F, M, R, depicted in B). The data are shown as the mean + SEM. n ≥ 3. (D) Hoxc5 derepression in PC19 EC cells by SMRT-C overexpression and 50 nM HDAC inhibitor, TSA. The data are shown as the mean + SEM. n = 5. (E) siRNA-mediated knockdown of SMRT or Ezh2 expression resulted in Hoxc5 derepression in P19 EC cells. The data are shown as the mean ± SEM. n = 5. ns, not statistically significant; *P < 0.05; **P < 0.005 by Student’s t test.

Fig. 4.

Derepression of RA-degradation enzyme Cyp26a1 in SMRT^mRID and enhanced recruitment of SEC to its regulatory region. (A) Enhanced RAR-dependent β-galactosidase activity in SMRT^mRID: RAREhsplacZ at E10.5. (B) Preserved caudal boundary of RAR transcriptional activity visualized by LacZ staining (blue). Somites from the tail to the boundary of LacZ signals are numbered. (C and D) Increased Cyp26a1 expression in SMRT^mRID by in situ hybridization in C and qPCR in D. The data are shown as the mean ± SEM. n = 5. (E and F) Expression levels of Cyp26b1 at E10.5 by in situ hybridization in E and qPCR in F. Arrows indicate sites of high Cyp26 expression. The data are shown as the mean ± SEM. n = 5. (G) Embryonic tissue regions, A–C, used for mRNA quantitation by qPCR in D and F. (H) Increased mRNA RA-degradation enzyme Cyp26a1 in the livers of 10-d-old and 2-mo-old SMRT^mRID mice. The data are shown as the mean ± SEM. n = 10. (I) Increased Cyp26a1 protein levels in liver of 2-mo-old SMRT^mRID. (J) Temporal kinetics of RA-activated Cyp26a1 mRNA in established WT and SMRT^mRID ES cells. SEC-recruited, not Pol II-paused genes including Cyp26a1 are rapidly super induced by RA in SMRT^mRID ES cells. Time appears in hour scale. The data are shown as the mean ± SEM. n = 5. (K) Enhanced enrichment of SEC component AFF4 at Cyp26a1 RARE in SMRT^mRID ES cells. The data are shown as the mean + SEM. n = 5. (L) Loss of SMRT/RAR association in mRID results in decreased threshold RA concentration for RAR target gene activation. (M) Model for SMRT as a genetic antagonist to RA-transcriptional gradient. SMRT also plays a role in retinoid homeostasis by repressing the RA-degradation pathway. Derepression of RAR targets such as Hoxc5 in cervical somites results in anterior expansion of the Hoxc5 boundary, ultimately resulting in posterior homeotic transformation of cervical vertebrae. Derepression of Cyp26a1 counteracts to increased RA-transcriptional activity by degrading RA in SMRT^mRID. *P < 0.05; **P < 0.005 by Student’s t test.