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A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2 [ATAC-seq]

ABSTRACT: During development cell fates are specified by tightly controlled gene expression programs. PBX TALE transcription factors control gene regulatory networks (GRN) that direct vertebrate tissue patterning and organ morphogenesis. How PBX1/2 proteins achieve context-specific functions, despite widespread embryonic Pbx expression, remains elusive. In mouse limbs, mesenchymal-specific loss of PBX1/2 or of the limb regulator HAND2 results in strikingly similar phenotypes, suggesting that PBX1/2- and HAND2-dependent programs converge to control limb development. To investigate this scenario using the murine hindlimb model, we combined tissue-specific and temporally controlled mutagenesis to multi-omics approaches on dissected hindlimb buds. This resulted in the reconstruction of a GRN that is collaboratively directed by PBX1/2-HAND2, demonstrating that Pbx1-Hand2 genetically interact in vivo during pentadactylous hindlimb patterning, with PBX1 concomitantly acting as an upstream regulator of Hand2. At organismal-level resolution the GRN is active within restricted subsets of posterior-proximal hindlimb mesenchymal cells, wherein Pbx1/2 and Hand2 are co-expressed with their target genes. Profiling the binding of Pbx1 and Hand2 genome-wide across multiple tissues revealed that HAND2 can act selectively on a subset of PBX-bound regions to impart limb patterning functionality. Our research elucidates mechanisms on the limb-specific activities of PBX1/2, while informing general principles by which promiscuous transcription factors cooperate with select cofactors to instruct distinct developmental programs.

ORGANISM(S): Mus musculus

PROVIDER: GSE197855 | GEO | 2023/04/26

REPOSITORIES: GEO

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Project description:ChIP-Sequencing on Shox2-HA E12.5 and E13.5 Limb and Palate, as well as Pbx on E12.5 limb . Abstract: Vertebrate appendage patterning is programmed by Hox-TALE factors-bound regulatory elements. However, it remains enigmatic which cell lineages are commissioned by Hox-TALE factors to generate regional specific pattern and whether other Hox-TALE co-factors exist. In this study, we investigated the transcriptional mechanisms controlled by the Shox2 transcriptional regulator in limb patterning. Harnessing an osteogenic lineage-specific Shox2 inactivation approach we show that despite widespread Shox2 expression in multiple cell lineages, lack of the stylopod observed upon Shox2 deficiency is a specific result of Shox2 loss of function in the osteogenic lineage. ChIP-Seq revealed robust interaction of Shox2 with cis-regulatory enhancers clustering around skeletogenic genes that are also bound by Hox-TALE factors, supporting a lineage autonomous function of Shox2 in osteogenic lineage fate determination and skeleton patterning. Pbx ChIP-Seq further allowed the genome-wide identification of cis-regulatory modules exhibiting co-occupancy of Pbx, Meis, and Shox2 transcriptional regulators. Integrative analysis of ChIP-Seq and RNA-Seq data and transgenic enhancer assays indicate that Shox2 patterns the stylopod as a repressor via interaction with enhancers active in the proximal limb mesenchyme and antagonizes the repressive function of TALE factors in osteogenesis. Shox2/TALE For ChIP-Seq, the list of libraries below, including controls, were generated [listed in the format of (antibody)-target-tissue-stage]: (Î±-HA)-Shox2-Limb-E12.5, (Î±-HA)-Shox2-Limb-E13.5, (Î±-HA)-Shox2-Palate-E12.5, (Î±-HA)-Shox2-Limb/Palate-E12.5, (Î±-Pbx)-Pbx-Limb-E12.5, Input (control), (Î±-HA)-Mixed Limb/Palate from Shox2+/+ mice-E12.5 (control). *The attached signal tracks(*.bigwig) were generated by âbdgcmp (MACS2) to filter out background signal(by filtering against the signal track obtained from (Î±-HA)-Mixed Limb/Palate from Shox2+/+ mice-E12.5 (control)) and subsequently convert to bigwig for analysis and visualization.

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A spatio-temporally constrained gene regulatory network directed by PBX1/2 acquires limb patterning specificity via HAND2 [ATAC-seq]

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