Genome-wide binding of posterior HOXA/D transcription factors reveals subgrouping and association with CTCF

Homeotic genes code for key transcription factors (HOX-TFs) that pattern the animal body plan. During embryonic development, Hox genes are expressed in overlapping patterns and function in a partially redundant manner. In vitro biochemical screens probing the HOX-TF sequence specificity revealed largely overlapping sequence preferences, indicating that co-factors might modulate the biological function of HOX-TFs. However, due to their overlapping expression pattern, high protein homology, and insufficiently specific antibodies, little is known about their genome-wide binding preferences. In order to overcome this problem, we virally expressed tagged versions of limb-expressed posterior HOX genes (HOXA9-13, and HOXD9-13) in primary chicken mesenchymal limb progenitor cells (micromass). We determined the effect of each HOX-TF on cellular differentiation (chondrogenesis) and gene expression and found that groups of HOX-TFs induce distinct regulatory programs. We used ChIP-seq to determine their individual genome-wide binding profiles and identified between 12,721 and 28,572 binding sites for each of the nine HOX-TFs. Principal Component Analysis (PCA) of binding profiles revealed that the HOX-TFs are clustered in two subgroups (Group 1: HOXA/D9, HOXA/D10, HOXD12, and HOXA13 and Group 2: HOXA/D11 and HOXD13), which are characterized by differences in their sequence specificity and by the presence of cofactor motifs. Specifically, we identified CTCF binding sites in Group 1, indicating that this subgroup of HOX-proteins cooperates with CTCF. We confirmed this interaction by an independent biological assay (Proximity Ligation Assay) and demonstrated that CTCF is a novel HOX cofactor that specifically associates with Group 1 HOX-TFs, pointing towards a possible interplay between HOX-TFs and chromatin architecture

Viral expression of HOX-TFs in chicken micromass culture (chMM) modifies chondrogenic cell differentiation.

<p>(A) Individual HOX-TF expressing chMM cultures stained with Alcian blue (top) and Eosin (bottom). Alcian Blue staining of four biological replicates was quantified and compared to mock-infected chMM. Error bars indicate standard deviation from four replicates. (B) Hierarchical clustering of differentially regulated genes in the nine HOX-TF expressing cultures (all RNA-seq shown in replicates). The top 50 differentially regulated genes from each sample were selected (Criteria: p-Val ≤10e-5, base mean≥30, fold change≥2) and for each replicate, the log2-transformed fold changes relative to mock-infected cultures of these 205 genes were subjected to hierarchical clustering.</p

AP1 and CTCF binding sites are overrepresented in Group 1 HOX-TF binding sites.

<p>(A) <i>De novo</i> motif analysis of all Group 1 HOX-TF peaks (here, HOXA10 results) identifies overrepresented binding sites. A comparison of these motifs to known AP1 and CTCF motifs is shown below. (B) Centrimo analysis identifies the position of best binding site matches in all peak sequences. Blue and black lines indicate enrichment of the given HOXA10 or HOXD13 motif shown below, respectively. Yellow lines indicate enrichment for CTCF motif shown below. (C) The overlap of peaks containing a HOX (Group 1- blue, Group 2- black) or a CTCF (yellow) binding site. The red overlap indicates peaks containing a HOX and a CTCF binding site.</p

Acetylcholine Receptor Pathway Mutations Explain Various Fetal Akinesia Deformation Sequence Disorders

Impaired fetal movement causes malformations, summarized as fetal akinesia deformation sequence (FADS), and is triggered by environmental and genetic factors. Acetylcholine receptor (AChR) components are suspects because mutations in the fetally expressed γ subunit (CHRNG) of AChR were found in two FADS disorders, lethal multiple pterygium syndrome (LMPS) and Escobar syndrome. Other AChR subunits α1, β1, and δ (CHRNA1, CHRNB1, CHRND) as well as receptor-associated protein of the synapse (RAPSN) previously revealed missense or compound nonsense-missense mutations in viable congenital myasthenic syndrome; lethality of homozygous null mutations was predicted but never shown. We provide the first report to our knowledge of homozygous nonsense mutations in CHRNA1 and CHRND and show that they were lethal, whereas novel recessive missense mutations in RAPSN caused a severe but not necessarily lethal phenotype. To elucidate disease-associated malformations such as frequent abortions, fetal edema, cystic hygroma, or cardiac defects, we studied Chrna1, Chrnb1, Chrnd, Chrng, and Rapsn in mouse embryos and found expression in skeletal muscles but also in early somite development. This indicates that early developmental defects might be due to somite expression in addition to solely muscle-specific effects. We conclude that complete or severe functional disruption of fetal AChR causes lethal multiple pterygium syndrome whereas milder alterations result in fetal hypokinesia with inborn contractures or a myasthenic syndrome later in life

Genome-wide binding of posterior HOXA/D transcription factors reveals subgrouping and association with CTCF

Homeotic genes code for key transcription factors (HOX-TFs) that pattern the animal body plan. During embryonic development, Hox genes are expressed in overlapping patterns and function in a partially redundant manner. In vitro biochemical screens probing the HOX-TF sequence specificity revealed largely overlapping sequence preferences, indicating that co-factors might modulate the biological function of HOX-TFs. However, due to their overlapping expression pattern, high protein homology, and insufficiently specific antibodies, little is known about their genome-wide binding preferences. In order to overcome this problem, we virally expressed tagged versions of limb-expressed posterior HOX genes (HOXA9-13, and HOXD9-13) in primary chicken mesenchymal limb progenitor cells (micromass). We determined the effect of each HOX-TF on cellular differentiation (chondrogenesis) and gene expression and found that groups of HOX-TFs induce distinct regulatory programs. We used ChIP-seq to determine their individual genome-wide binding profiles and identified between 12,721 and 28,572 binding sites for each of the nine HOX-TFs. Principal Component Analysis (PCA) of binding profiles revealed that the HOX-TFs are clustered in two subgroups (Group 1: HOXA/D9, HOXA/D10, HOXD12, and HOXA13 and Group 2: HOXA/D11 and HOXD13), which are characterized by differences in their sequence specificity and by the presence of cofactor motifs. Specifically, we identified CTCF binding sites in Group 1, indicating that this subgroup of HOX-proteins cooperates with CTCF. We confirmed this interaction by an independent biological assay (Proximity Ligation Assay) and demonstrated that CTCF is a novel HOX cofactor that specifically associates with Group 1 HOX-TFs, pointing towards a possible interplay between HOX-TFs and chromatin architecture