Discovery of genes required for body axis and limb formation by global identification of retinoic acid regulated epigenetic marks

Abstract

Identification of target genes that mediate required functions downstream of transcription factors is hampered by the large number of genes whose expression changes when the factor is removed from a specific tissue and the numerous binding sites for the factor in the genome. Retinoic acid (RA) regulates transcription via RA receptors bound to RA response elements (RAREs) of which there are thousands in vertebrate genomes. Here, we combined ChIP-seq for epigenetic marks and RNA-seq on trunk tissue from wild-type and Aldh1a2-/-embryos lacking RA synthesis that exhibit body axis and forelimb defects. We identified a relatively small number of genes with altered expression when RA is missing that also have nearby RA-regulated deposition of H3K27ac (gene activation mark) or H3K27me3 (gene repression mark) associated with conserved RAREs, suggesting they have important downstream functions. RA-regulated epigenetic marks were identified near RA target genes already known to be required for body axis and limb formation, thus validating our approach, plus many other candidate RA target genes were found. Nr2f1, Nr2f2, Meis1, and Meis2 gene family members were identified by our approach, and double knockouts of each family demonstrated previously unknown requirements for body axis and/or limb formation. These findings demonstrate that our method for identifying RA-regulated epigenetic marks can be used to discover genes important for development.

Identification of downstream effectors of retinoic acid specifying the zebrafish pancreas by integrative genomics

“…Interestingly, 24 RAR sites were found in extremely conserved non-coding regions and sequences could be aligned from zebrafish to human. This is the case for RAR sites located in some hox clusters (Nolte, De Kumar, and Krumlauf 2019), near meis2 (Berenguer et al 2020), nrip1a, ncoa3 and in the skap1 gene (this study). Such high conservation suggests that the level of expression of these RA target genes must be precisely controlled.…”

“…This gene set includes many genes previously shown to be regulated by RA in other germ layers like hox, nr2f, cyp26a, dhrs3 or hnf1b genes (Feng et al 2010b; C. E. Love and Prince 2012; Berenguer et al 2020; Hernandez et al 2004; Nolte, De Kumar, and Krumlauf 2019) indicating that the regulatory network triggered by RA is shared, at least partially, between the three germ layers. Analysis of the zebrafish RAR cistrome at the end of gastrulation confirms hox genes as direct RAR targets (Nolte, De Kumar, and Krumlauf 2019), acting on the anterio-posterior patterning of the endoderm, but the data also pinpoint 4 direct RAR target genes (i.e.…”

Axial skeleton anterior-posterior patterning is regulated through feedback regulation between Meis transcription factors and retinoic acid

“…Given that retinoic acid has been shown to regulate Meis expression in different settings (Berenguer et al, 2020;Mercader et al, 2000;Oulad-Abdelghani et al, 1997;Yashiro et al, 2004), we studied whether the elimination Raldh2-mediated RA synthesis affects axial Meis expression. We studied Meis1 and Meis2 mRNA and protein expression in Sox2 Cre ; Raldh2 flox/flox embryos and found that both genes presented a reduction of transcripts along the trunk region of E8 embryos ( Figure 6U-X).…”

“…The active form of vitamin-A, retinoic acid (RA), regulates gene expression during embryonic development by binding to nuclear receptors RARα, RARβ or RARγ (Rhinn and Dolle, 2012). Meis genes have been identified in screens for RA targets (Berenguer et al, 2020;Oulad-Abdelghani et al, 1997) and respond to RA fluctuations in vivo (Mercader et al, 2000;Yashiro et al, 2004). RA excess produces axial skeleton alterations and modifies the Hox AP expression domains (Kessel and Gruss, 1991) and mutations in RA-receptor genes result in homeotic transformations, however, the mechanism by which this takes place is not clear.…”

Identification of Downstream Effectors of Retinoic Acid Specifying the Zebrafish Pancreas by Integrative Genomics.

“…Although several regulatory genes have been identi ed as important downstream targets of RA, such as Hox or Hnf1b genes 8, [15][16][17][18] , the regulatory network triggered by RA in endoderm during gastrulation is still unclear. Many transcriptomic studies have identi ed RA-regulated genes in murine, xenopus or zebra sh embryos [19][20][21][22][23][24][25] ; however these reports were often done on whole embryos and not done speci cally on endodermal tissues. Still two studies performed on xenopus endoderm have nevertheless reported the involvement of Ndrg1, Fzd4 and Hnf1b genes as crucial mediators of RA signalling for pancreas development 18,26 ; however, it is not yet known if the role of these regulatory genes is conserved among vertebrates.…”