The Isl1/Ldb1 complex orchestrates heart-specific chromatin organization and transcriptional regulation

Cardiac stem/progenitor cells hold great potential for regenerative therapies however the mechanisms regulating their expansion and differentiation remain insufficiently defined. Here we show that the multi-adaptor protein Ldb1 is a central regulator of cardiac progenitor cell differentiation and second heart field (SHF) development. Mechanistically, we demonstrate that Ldb1 binds to the key regulator of SHF progenitors Isl1 and protects it from proteasomal degradation. Furthermore, the Isl1/Ldb1 complex promotes long-range promoter-enhancer interactions at the loci of the core cardiac transcription factors Mef2c and Hand2. Chromosome conformation capture followed by sequencing identified surprisingly specific, Ldb1-mediated interactions of the Isl1/Ldb1 responsive Mef2c anterior heart field enhancer with genes which play key roles in cardiac progenitor cell function and cardiovascular development. Importantly, the expression of these genes was downregulated upon Ldb1 depletion and Isl1/Ldb1 haplodeficiency. In conclusion, the Isl1/Ldb1 complex orchestrates a network for heart-specific transcriptional regulation and coordination in three-dimensional space during cardiogenesis

The <it>SLEEPER</it> genes: a transposase-derived angiosperm-specific gene family

<p>Abstract</p> <p>Background</p> <p><it>DAYSLEEPER</it> encodes a domesticated transposase from the hAT-superfamily, which is essential for development in <it>Arabidopsis thaliana</it>. Little is known about the presence of <it>DAYSLEEPER</it> orthologs in other species, or how and when it was domesticated. We studied the presence of <it>DAYSLEEPER</it> orthologs in plants and propose a model for the domestication of the ancestral <it>DAYSLEEPER</it> gene in angiosperms.</p> <p>Results</p> <p>Using specific BLAST searches in genomic and EST libraries, we found that <it>DAYSLEEPER</it>-like genes (hereafter called <it>SLEEPER</it> genes) are unique to angiosperms. Basal angiosperms as well as grasses (Poaceae) and dicotyledonous plants possess such putative orthologous genes, but <it>SLEEPER</it>-family genes were not found in gymnosperms, mosses and algae. Most species contain more than one <it>SLEEPER</it> gene. All <it>SLEEPER</it>s contain a C₂H₂ type BED-zinc finger domain and a hATC dimerization domain. We designated 3 motifs, partly overlapping the BED-zinc finger and dimerization domain, which are hallmark features in the <it>SLEEPER</it> family. Although <it>SLEEPER</it> genes are structurally conserved between species, constructs with <it>SLEEPER</it> genes from grapevine and rice did not complement the <it>daysleeper</it> phenotype in Arabidopsis, when expressed under control of the <it>DAYSLEEPER</it> promoter. However these constructs did cause a dominant phenotype when expressed in Arabidopsis. Rice plant lines with an insertion in the <it>RICESLEEPER</it>1 or 2 locus displayed phenotypic abnormalities, indicating that these genes are functional and important for normal development in rice. We suggest a model in which we hypothesize that an ancestral hAT transposase was retrocopied and stably integrated in the genome during early angiosperm evolution. Evidence is also presented for more recent retroposition events of <it>SLEEPER</it> genes, such as an event in the rice genome, which gave rise to the <it>RICESLEEPER</it>1 and 2 genes.</p> <p>Conclusions</p> <p>We propose the ancestral <it>SLEEPER</it> gene was formed after a process of retro-transposition during the evolution of the first angiosperms. It may have acquired an important function early on, as mutation of two <it>SLEEPER</it> genes in rice, like the <it>daysleeper</it> mutant in <it>A. thaliana</it> gave a developmental phenotype indicative of their importance for normal plant development.</p

Epigenetic Alterations at Genomic Loci Modified by Gene Targeting in Arabidopsis thaliana

Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It is considered an effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, and is widely used in mice, with the potential of becoming routine in many species. Nevertheless, the epigenetic status of the targeted allele remains largely unexplored. Using GT-modified lines of the model plant Arabidopsis thaliana, we show that the DNA methylation profile of the targeted locus is changed following GT. This effect is non-directional as methylation can be either completely lost, maintained with minor alterations or show instability in the generations subsequent to GT. As DNA methylation is known to be involved in several cellular processes, GT-related alterations may result in unexpected or even unnoticed perturbations. Our analysis shows that GT may be used as a new tool for generating epialleles, for example, to study the role of gene body methylation. In addition, the analysis of DNA methylation at the targeted locus may be utilized to investigate the mechanism of GT, many aspects of which are still unknown

Mechanistic models for modification of methylation status of targeted alleles by GT.

<p>Methylated cytosines are indicated by grey lollipops. The GT vector is denoted by a red line, with a red asterisk illustrating the genetic perturbation introduced by the vector. If gap enlargement occurs following DSB induction (pathway on the left), the methylation template is lost and the targeted allele loses its methylation. However, if GT occurs via strand assimilation (pathway on the right) or via DSB induction but without gap enlargement (pathway in the middle), the methylation pattern of the WT can be restored upon maintenance methylation.</p

Methylation changes at the targeted <i>PPOX</i> locus in lines TGT-2 and TGT-3.

<p>(<b>A</b>) The methylation landscape in TGT-2 and TGT-3 lines (upper and lower graphs, respectively). Blue circles, triangles and squares correspond to WT, T2 and T3 generations, respectively. Dotted lines mark the CG positions at which the methylation level had changed relative to WT. (<b>B</b>) CG positions that lost methylation stability in the two targeted lines. CG position is denoted beneath each bar. The numbers above each bar denote the level of methylation as fraction.</p