Mitochondrial Stress Engages E2F1 Apoptotic Signaling to Cause Deafness

SummaryMitochondrial dysfunction causes poorly understood tissue-specific pathology stemming from primary defects in respiration, coupled with altered reactive oxygen species (ROS), metabolic signaling, and apoptosis. The A1555G mtDNA mutation that causes maternally inherited deafness disrupts mitochondrial ribosome function, in part, via increased methylation of the mitochondrial 12S rRNA by the methyltransferase mtTFB1. In patient-derived A1555G cells, we show that 12S rRNA hypermethylation causes ROS-dependent activation of AMP kinase and the proapoptotic nuclear transcription factor E2F1. This retrograde mitochondrial-stress relay is operative in vivo, as transgenic-mtTFB1 mice exhibit enhanced 12S rRNA methylation in multiple tissues, increased E2F1 and apoptosis in the stria vascularis and spiral ganglion neurons of the inner ear, and progressive E2F1-dependent hearing loss. This mouse mitochondrial disease model provides a robust platform for deciphering the complex tissue specificity of human mitochondrial-based disorders, as well as the precise pathogenic mechanism of maternally inherited deafness and its exacerbation by environmental factors.PaperFlic

Relative abundance of the human mitochondrial transcription system and distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression

Human mitochondrial transcription requires the bacteriophage-related RNA polymerase, POLRMT, the mtDNA-binding protein, h-mtTFA/TFAM, and two transcription factors/rRNA methyltransferases, h-mtTFB1 and h-mtTFB2. Here, we determined the steady-state levels of these core transcription components and examined the consequences of purposeful elevation of h-mtTFB1 or h-mtTFB2 in HeLa cells. On a per molecule basis, we find an ∼6-fold excess of POLRMT to mtDNA and ∼3-fold more h-mtTFB2 than h-mtTFB1. We also estimate h-mtTFA at ∼50 molecules/mtDNA, a ratio predicted to support robust transcription, but not to coat mtDNA. Consistent with a role for h-mtTFB2 in transcription and transcription-primed replication, increased mitochondrial DNA and transcripts result from its over-expression. This is accompanied by increased translation rates of most, but not all mtDNA-encoded proteins. Over-expression of h-mtTFB1 did not significantly influence these parameters, but did result in increased mitochondrial biogenesis. Furthermore, h-mtTFB1 mRNA and protein are elevated in response to h-mtTFB2 over-expression, suggesting the existence of a retrograde signal to the nucleus to coordinately regulate expression of these related factors. Altogether, our results provide a framework for understanding the regulation of human mitochondrial transcription in vivo and define distinct roles for h-mtTFB1 and h-mtTFB2 in mitochondrial biogenesis and gene expression that together likely fine-tune mitochondrial function

Sarcomere function activates a p53-dependent DNA damage response that promotes polyploidization and limits in vivo cell engraftment.

Human cardiac regeneration is limited by low cardiomyocyte replicative rates and progressive polyploidization by unclear mechanisms. To study this process, we engineer a human cardiomyocyte model to track replication and polyploidization using fluorescently tagged cyclin B1 and cardiac troponin T. Using time-lapse imaging, in vitro cardiomyocyte replication patterns recapitulate the progressive mononuclear polyploidization and replicative arrest observed in vivo. Single-cell transcriptomics and chromatin state analyses reveal that polyploidization is preceded by sarcomere assembly, enhanced oxidative metabolism, a DNA damage response, and p53 activation. CRISPR knockout screening reveals p53 as a driver of cell-cycle arrest and polyploidization. Inhibiting sarcomere function, or scavenging ROS, inhibits cell-cycle arrest and polyploidization. Finally, we show that cardiomyocyte engraftment in infarcted rat hearts is enhanced 4-fold by the increased proliferation of troponin-knockout cardiomyocytes. Thus, the sarcomere inhibits cell division through a DNA damage response that can be targeted to improve cardiomyocyte replacement strategies

A GWAS in Latin Americans identifies novel face shape loci, implicating VPS13B and a Denisovan introgressed region in facial variation

To characterize the genetic basis of facial features in Latin Americans, we performed a genome-wide association study (GWAS) of more than 6000 individuals using 59 landmark-based measurements from two-dimensional profile photographs and ~9,000,000 genotyped or imputed single-nucleotide polymorphisms. We detected significant association of 32 traits with at least 1 (and up to 6) of 32 different genomic regions, more than doubling the number of robustly associated face morphology loci reported until now (from 11 to 23). These GWAS hits are strongly enriched in regulatory sequences active specifically during craniofacial development. The associated region in 1p12 includes a tract of archaic adaptive introgression, with a Denisovan haplotype common in Native Americans affecting particularly lip thickness. Among the nine previously unidentified face morphology loci we identified is the VPS13B gene region, and we show that variants in this region also affect midfacial morphology in mice

Human Embryonic Craniofacial Tissue Epigenomic Data and Chromatin State Segmentations from the Cotney Lab at UConn Health

<h4>Human Embryonic Craniofacial Tissue Epigenomic Data and Chromatin State Segmentations from the Cotney Lab at UConn Health</h4

Evolution of Human Limb Development

<p>Posters, presentations, and data from my project on the evoluton of gene regulation in human limb development.</p

Genome-wide mapping of human-specific regulatory functions during embryonic development

<p>Talk I gave at the 2013 Gordon Research Seminar for Human Genetics and Genomics.</p

The evolution of human-specific regulatory functions in the embryonic limb

<p>Poster presented at the Biology of Genomes 2013 meeting at Cold Spring Harbor Laboratory.</p

Divergent evolution of the paralogous human mitochondrial transcription factors, h-mtTFB1 and h-mtTFB2, to fulfill unique functions in mitochondrial gene expression, biogenesis, and retrograde signaling

<p>Mitochondria are essential organelles, resulting from an ancient endosymbiosis,that are found in virtually all eukaryotes. To maintain proper function, human mitochondria have maintained distinct genomes and a dedicated gene expression system. The human mitochondrial transcription system consists of three types of proteins: POLRMT, a bacteriophage-like RNA polymerase; h-mtTFA, a mitochondrial DNA binding protein; and the mtTFBs, two factors that provide a physical link between hmtTFA and POLRMT required for transcription initiation. The human mtTFBs, hmtTFB1 and h-mtTFB2, are unique transcription factors exhibiting homology with N6-adenine RNA dimethyltransferases. Phylogenetic analysis suggests a very early duplication of the endosymbiont KsgA gene as the source of these two paralogs. Consistent with this ancestry, I demonstrate that both h-mtTFB1 and h-mtTFB2 have maintained RNA methyltransferase activity. Overexpression of h-mtTFB1 in human cells increases the level of methylated 12S rRNA and induces mitochondrial mass. hmtTFB2 overexpression elevates mitochondrial DNA levels, transcripts, mass, membrane potential, and surprisingly induces a coordinate increase in h-mtTFB1 expression. This indicates that h-mtTFB1 is the major 12S methyltransferase and h-mtTFB2 is involved primarily in mitochondrial transcription. These results also suggest a major role for these factors in coordinating mitochondrial biogenesis. By combining their activities, a robust remodeling of the mitochondrial compartment occurs, increasing organelle mass and greater respiratory capacity. This response is dependent specifically upon methylation activity of h-mtTFB1 and indicates that the methylation status of 12S rRNA is a metric for mitochondrial function. In support of this idea cells harboring the mtDNA mutation A1555G, a mutation in 12S rRNA linked to non-syndromic deafness, have elevated methylated 12S rRNA and phenotypes similar to those associated with h-mtTFB1 overexpression. I propose that 12S rRNA methylation status is regularly monitored by the cell and either induces expression of h-mtTFB1 to improve overall methylation or induces mitochondrial mass in preparation for more OXPHOS complexes to be produced by fully methylated mitochondrial ribosomes. Altogether this research indicates that these factors are intimately involved in regulating mitochondrial biogenesis. Inappropriate modulation of their levels or activities might contribute to human disease by producing mitochondria deficient in transcription, translation, or respiration.</p> <p> </p

Identifying Human Craniofacial Enhancers

<p>Talk I gave 10/22/2013 at the annual meeting for the Society of Craniofacial Genetics and Developmental Biology.</p