Project description:Analysis of the expression profile of adult mice heterozygous for Pitx2 isoform C. Total RNA obtained from isolated left and right atria from 5 Pitx2c heterozygous mice and 5 wild-type littermates.
Project description:Pitx2c, a homeodomain transcription factor, is classically known for its left-right patterning role. However, an early wave of pitx2 expression occurs at the onset of gastrulation in several species, indicating a possible earlier role that remains relatively unexplored. Here, we show that in zebrafish, maternal-zygotic (MZ) pitx2c mutants exhibit a shortened body axis indicative of convergence and extension (CE) defects. Live imaging reveals that MZpitx2c mutants display less persistent mesendodermal migration during later stages of gastrulation. Transplant experiments indicate that Pitx2c functions cell non-autonomously to regulate this cell behavior by modulating cell shape and protrusive activity. Using transcriptomic analyses and candidate gene approaches, we identify transcriptional changes in components of the chemokine-ECM-integrin dependent mesendodermal migration network. Together, our results define pathways downstream of Pitx2 that are required during early embryogenesis and reveal novel functions for Pitx2 as a regulator of morphogenesis.
Project description:Abnormalities provoked in human cells heterozygous for clinically relevant truncating mutations in BRCA2 by their exposure to naturally occurring aldehydes define a mechanism promoting carcinogenesis in mutation carriers. The ubiquitous metabolite and environmental toxin, formaldehyde, triggers replication fork instability and structural chromosomal aberrations in BRCA2 heterozygous cells. These abnormalities arise from a previously unrecognized effect of formaldehyde to selectively induce the proteasomal degradation of BRCA2, inducing functional haploinsufficiency only in cells where BRCA2 expression is already compromised by a heterozygous mutation. Similar effects are observed with acetaldehyde, a toxic product of alcohol catabolism. Replication fork instability and chromosomal aberrations in aldehyde-exposed cells arise from the unscheduled accumulation of RNA-DNA hybrids, revealing a mechanism driving genomic instability in BRCA2 heterozygous cells. We propose a model for cancer pathogenesis in which aldehyde exposure unmasks the carcinogenic potential of heterozygous BRCA2 mutations, with public health implications in mutation carriers.
Project description:Genome-wide association studies (GWAS) have found that increased risk for atrial fibrillation (AF), the most common type of arrhythmia in humans, is associated with non-coding sequence variants located in proximity to the PITX2 homeobox gene. Using cardiomyocyte-specific epigenomic and comparative genomic analyses, we identified two AF-associated enhancers neighboring PITX2 with varying degrees of conservation in mice. Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9 mediated deletion of a 20 kb long topologically engaged enhancer lead to reduced Pitx2c transcription and AF predisposition. Allele-specific ChIP-seq and CUT&RUN experiments showed that long-range interaction of this AF-associated region with the Pitx2c promoter was required for maintenance of the Pitx2c promoter chromatin state. Moreover, long-range looping was mediated by CTCF, as the genetic disruption of an intronic CTCF binding site caused decreased Pitx2c cardiac expression, AF predisposition, and reduced active chromatin marks on Pitx2. Our findings reveal that AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2
Project description:Genome-wide association studies (GWAS) have found that increased risk for atrial fibrillation (AF), the most common type of arrhythmia in humans, is associated with non-coding sequence variants located in proximity to the PITX2 homeobox gene. Using cardiomyocyte-specific epigenomic and comparative genomic analyses, we identified two AF-associated enhancers neighboring PITX2 with varying degrees of conservation in mice. Pitx2c promoter directly contacted the AF-associated enhancer regions. CRISPR/Cas9 mediated deletion of a 20 kb long topologically engaged enhancer lead to reduced Pitx2c transcription and AF predisposition. Allele-specific ChIP-seq and CUT&RUN experiments showed that long-range interaction of this AF-associated region with the Pitx2c promoter was required for maintenance of the Pitx2c promoter chromatin state. Moreover, long-range looping was mediated by CTCF, as the genetic disruption of an intronic CTCF binding site caused decreased Pitx2c cardiac expression, AF predisposition, and reduced active chromatin marks on Pitx2. Our findings reveal that AF risk variants located at 4q25 reside in genomic regions possessing long-range transcriptional regulatory functions directed at PITX2
Project description:Single cell transcriptome analyses of whole kidney marrow cells isolated from wild type and rad21 heterozygous mutant adult zebrafish.
Project description:Heterozygous CHMs have been believed as synonymous with dispermic moles (two sperm origin). The possibility of diploid sperm origin has not been considered. We assessed whether heterozygous CHMs would be of dispermic or diploid sperm origin. In all the cases, centromeric zygosity was random, i.e., mixed status. Theoretically, if the heterozygous CHMs were of diploid sperm origin, the centromeric status will be all homozygous or all heterozygous. Thus, all the analyzed heterozygous CHMs were considered to be of dispermic origin. Three cases showed the trisomy while we did not notice them with STR analysis. Diploid sperm fertilization can be rare during the development of heterozygous androgenetic CHM.