Jonathon T. Hill

MMAPPR: Mutation Mapping Analysis Pipeline for Pooled RNA-seq

Forward genetic screens in model organisms are vital for identifying novel genes essential for developmental or disease processes. One drawback of these screens is the labor-intensive and sometimes inconclusive process of mapping the causative mutation. To leverage high-throughput techniques to improve this mapping process, we have developed a Mutation Mapping Analysis Pipeline for Pooled RNA-seq (MMAPPR) that works without parental strain information or requiring a preexisting SNP map of the organism, and adapts to differential recombination frequencies across the genome. MMAPPR accommodates the considerable amount of noise in RNA-seq data sets, calculates allelic frequency by Euclidean distance followed by Loess regression analysis, identifies the region where the mutation lies, and generates a list of putative coding region mutations in the linked genomic segment. MMAPPR can exploit RNA-seq data sets from isolated tissues or whole organisms that are used for gene expression and transcriptome analysis in novel mutants. We tested MMAPPR on two known mutant lines in zebrafish, nkx2.5 and tbx1, and used it to map two novel ENU-induced cardiovascular mutants, with mutations found in the ctr9 and cds2 genes. MMAPPR can be directly applied to other model organisms, such as Drosophila and Caenorhabditis elegans, that are amenable to both forward genetic screens and pooled RNA-seq experiments. Thus, MMAPPR is a rapid, cost-efficient, and highly automated pipeline, available to perform mutant mapping in any organism with a well-assembled genome.

Ghrelin expression in the mouse pancreas defines a unique multipotent progenitor population.

Pancreatic islet cells provide the major source of counteractive endocrine hormones required for maintaining glucose homeostasis; severe health problems result when these cell types are insufficiently active or reduced in number. Therefore, the process of islet endocrine cell lineage allocation is critical to ensure there is a correct balance of islet cell types. There are four endocrine cell types within the adult islet, including the glucagon-producing alpha cells, insulin-producing beta cells, somatostatin-producing delta cells and pancreatic polypeptide-producing PP cells. A fifth islet cell type, the ghrelin-producing epsilon cells, is primarily found during gestational development. Although hormone expression is generally assumed to mark the final entry to a determined cell state, we demonstrate in this study that ghrelin-expressing epsilon cells within the mouse pancreas do not represent a terminally differentiated endocrine population. Ghrelin cells give rise to significant numbers of alpha and PP cells and rare beta cells in the adult islet. Furthermore, pancreatic ghrelin-producing cells are maintained in pancreata lacking the essential endocrine lineage regulator Neurogenin3, and retain the ability to contribute to cells within the pancreatic ductal and exocrine lineages. These results demonstrate that the islet ghrelin-expressing epsilon cells represent a multi-potent progenitor cell population that delineates a major subgrouping of the islet endocrine cell populations. These studies also provide evidence that many of hormone-producing cells within the adult islet represent heterogeneous populations based on their ontogeny, which could have broader implications on the regulation of islet cell ratios and their ability to effectively respond to fluctuations in the metabolic environment during development.

Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products

Background: Genome editing techniques, including ZFN, TALEN, and CRISPR, have created a need to rapidly screen many F1 individuals to identify carriers of indels and determine the sequences of the mutations. Current techniques require multiple clones of the targeted region to be sequenced for each individual, which is inefficient when many individuals must be analyzed. Direct Sanger sequencing of a polymerase chain reaction (PCR) amplified region surrounding the target site is efficient, but Sanger sequencing genomes heterozygous for an indel results in a string of “double peaks” due to the mismatched region. Results: To facilitate indel identification, we developed an online tool called Poly Peak Parser (available at http://yost.genetics.utah.edu/software.php) that is able to separate chromatogram data containing ambiguous base calls into wild‐type and mutant allele sequences. This tool allows the nature of the indel to be determined from a single sequencing run per individual performed directly on a PCR product spanning the targeted site, without cloning. Conclusions: The method and algorithm described here facilitate rapid identification and sequence characterization of heterozygous mutant carriers generated by genome editing. Although designed for screening F1 individuals, this tool can also be used to identify heterozygous indels in many contexts. Developmental Dynamics 243:1632–1636, 2014.

Genome-Wide Analysis Reveals the Unique Stem Cell Identity of Human Amniocytes

Human amniotic fluid contains cells that potentially have important stem cell characteristics, yet the programs controlling their developmental potency are unclear. Here, we provide evidence that amniocytes derived from multiple patients are marked by heterogeneity and variability in expression levels of pluripotency markers. Clonal analysis from multiple patients indicates that amniocytes have large pools of self-renewing cells that have an inherent property to give rise to a distinct amniocyte phenotype with a heterogeneity of pluripotent markers. Significant to their therapeutic potential, genome-wide profiles are distinct at different gestational ages and times in culture, but do not differ between genders. Based on hierarchical clustering and differential expression analyses of the entire transcriptome, amniocytes express canonical regulators associated with pluripotency and stem cell repression. Their profiles are distinct from human embryonic stem cells (ESCs), induced-pluripotent stem cells (iPSCs), and newborn foreskin fibroblasts. Amniocytes have a complex molecular signature, coexpressing trophoblastic, ectodermal, mesodermal, and endodermal cell-type-specific regulators. In contrast to the current view of the ground state of stem cells, ESCs and iPSCs also express high levels of a wide range of cell-type-specific regulators. The coexpression of multilineage differentiation markers combined with the strong expression of a subset of ES cell repressors in amniocytes suggests that these cells have a distinct phenotype that is unlike any other known cell-type or lineage.

Ghrelin is dispensable for embryonic pancreatic islet development and differentiation

Ghrelin is a peptide hormone that has been implicated in the regulation of food intake and energy homeostasis. Ghrelin is predominantly produced in the stomach, but is also expressed in many other tissues where its functions are not well characterized. In the rodent and human pancreas, ghrelin levels peak at late gestation and gradually decline postnatally. Several studies have suggested that ghrelin regulates beta cell function during embryonic development and in the adult. In addition, in a number of mouse models, ghrelin cells appear to replace insulin- and glucagon-producing cells in the islet. In this analysis, we investigated whether the absence or overexpression of ghrelin influenced the development and differentiation of the pancreatic islet during embryonic development. These studies revealed that ghrelin is dispensable for normal pancreas development during gestation. Conversely, we demonstrated that elevated ghrelin in the Nkx2.2 null islets is not responsible for the absence of insulin- and glucagon-producing cells. Finally, we have also determined that in the absence of insulin, ghrelin cells form in their normal numbers and ghrelin is expressed at wild type levels.

Nkx2.2 Activates the Ghrelin Promoter in Pancreatic Islet Cells

Nkx2.2 is an essential regulator of pancreatic endocrine differentiation. Nkx2.2-null mice are completely devoid of beta-ells and have a large reduction of alpha- and PP cells. In the place of these islet populations, there is a corresponding increase in the ghrelin-positive epsilon-cells. Molecular studies have indicated that Nkx2.2 functions as an activator and repressor to regulate islet cell fate decisions. To determine whether Nkx2.2 is solely important for islet cell fate decisions or also has the capability to control ghrelin at the promoter level, we studied the transcriptional regulation of the ghrelin promoter within the pancreas, in vitro and in vivo. These studies demonstrate that both of the previously identified transcriptional start sites in the ghrelin promoter are active within the embryonic pancreas; however, the long transcript is preferentially up-regulated in the Nkx2.2-null pancreas. We also show that the promoter region between -619 and -488 bp upstream of the translational start site is necessary for repression of ghrelin in alphaTC1 and betaTC6 cells. Surprisingly, we also show that Nkx2.2 is able to bind to and activate the ghrelin promoter in several cell lines that do or do not express endogenous ghrelin. Together, these results suggest that the up-regulation of ghrelin expression in the Nkx2.2-null mice is not due to loss of repression of the ghrelin promoter in the nonghrelin islet populations. Furthermore, Nkx2.2 may contribute to the activation of ghrelin in mature islet epsilon-cells.

Novel computational analysis of protein binding array data identifies direct targets of Nkx2.2 in the pancreas

BackgroundThe creation of a complete genome-wide map of transcription factor binding sites is essential for understanding gene regulatory networks in vivo. However, current prediction methods generally rely on statistical models that imperfectly model transcription factor binding. Generation of new prediction methods that are based on protein binding data, but do not rely on these models may improve prediction sensitivity and specificity.ResultsWe propose a method for predicting transcription factor binding sites in the genome by directly mapping data generated from protein binding microarrays (PBM) to the genome and calculating a moving average of several overlapping octamers. Using this unique algorithm, we predicted binding sites for the essential pancreatic islet transcription factor Nkx2.2 in the mouse genome and confirmed >90% of the tested sites by EMSA and ChIP. Scores generated from this method more accurately predicted relative binding affinity than PWM based methods. We have also identified an alternative core sequence recognized by the Nkx2.2 homeodomain. Furthermore, we have shown that this method correctly identified binding sites in the promoters of two critical pancreatic islet β-cell genes, NeuroD1 and insulin2, that were not predicted by traditional methods. Finally, we show evidence that the algorithm can also be applied to predict binding sites for the nuclear receptor Hnf4α.ConclusionsPBM-mapping is an accurate method for predicting Nkx2.2 binding sites and may be widely applicable for the creation of genome-wide maps of transcription factor binding sites.

Poly Peak Parser: Method and software for identification of unknown indels using Sanger Sequencing of PCR products

Background: Genome editing techniques, including ZFN, TALEN, and CRISPR, have created a need to rapidly screen many F1 individuals to identify carriers of indels and determine the sequences of the mutations. Current techniques require multiple clones of the targeted region to be sequenced for each individual, which is inefficient when many individuals must be analyzed. Direct Sanger sequencing of a polymerase chain reaction (PCR) amplified region surrounding the target site is efficient, but Sanger sequencing genomes heterozygous for an indel results in a string of “double peaks” due to the mismatched region. Results: To facilitate indel identification, we developed an online tool called Poly Peak Parser (available at http://yost.genetics.utah.edu/software.php) that is able to separate chromatogram data containing ambiguous base calls into wild‐type and mutant allele sequences. This tool allows the nature of the indel to be determined from a single sequencing run per individual performed directly on a PCR product spanning the targeted site, without cloning. Conclusions: The method and algorithm described here facilitate rapid identification and sequence characterization of heterozygous mutant carriers generated by genome editing. Although designed for screening F1 individuals, this tool can also be used to identify heterozygous indels in many contexts. Developmental Dynamics 243:1632–1636, 2014.

Poly peak parser: Method and software for identification of unknown indels using sanger sequencing of polymerase chain reaction products: POLY PEAK PARSER

Background: Genome editing techniques, including ZFN, TALEN, and CRISPR, have created a need to rapidly screen many F1 individuals to identify carriers of indels and determine the sequences of the mutations. Current techniques require multiple clones of the targeted region to be sequenced for each individual, which is inefficient when many individuals must be analyzed. Direct Sanger sequencing of a polymerase chain reaction (PCR) amplified region surrounding the target site is efficient, but Sanger sequencing genomes heterozygous for an indel results in a string of “double peaks” due to the mismatched region. Results: To facilitate indel identification, we developed an online tool called Poly Peak Parser (available at http://yost.genetics.utah.edu/software.php) that is able to separate chromatogram data containing ambiguous base calls into wild‐type and mutant allele sequences. This tool allows the nature of the indel to be determined from a single sequencing run per individual performed directly on a PCR product spanning the targeted site, without cloning. Conclusions: The method and algorithm described here facilitate rapid identification and sequence characterization of heterozygous mutant carriers generated by genome editing. Although designed for screening F1 individuals, this tool can also be used to identify heterozygous indels in many contexts. Developmental Dynamics 243:1632–1636, 2014.