Tracing the evolution of lineage-specific transcription factor binding sites in a birth-death framework

Changes in cis-regulatory element composition that result in novel patterns of gene expression are thought to be a major contributor to the evolution of lineage-specific traits. Although transcription factor binding events show substantial variation across species, most computational approaches to study regulatory elements focus primarily upon highly conserved sites, and rely heavily upon multiple sequence alignments. However, sequence conservation based approaches have limited ability to detect lineage-specific elements that could contribute to species-specific traits. In this thesis, we describe a novel framework that utilizes a birth-death model to trace the evolution of lineage-specific binding sites without relying on detailed base-by-base cross-species alignments. Our model was applied to analyze the evolution of binding sites based on the ChIP-seq data for six transcription factors (GATA1, SOX2, CTCF, MYC, MAX, ETS1) along the lineage toward human after human-mouse common ancestor. We estimate that a substantial fraction of binding sites (∼58-79% for each factor) in humans have origins since the divergence with mouse. Over 15% of all binding sites are unique to hominids. Such elements are often enriched near genes associated with neural-related functions and pathways, and harbor more common SNPs than older binding sites in the human genome. These results support the ability of our method to identify lineage-specific regulatory elements and help understand their roles in shaping variation in gene regulation across species

The mammary cellular hierarchy and breast cancer.

Advances in the study of hematopoietic cell maturation have paved the way to a deeper understanding the stem and progenitor cellular hierarchy in the mammary gland. The mammary epithelium, unlike the hematopoietic cellular hierarchy, sits in a complex niche where communication between epithelial cells and signals from the systemic hormonal milieu, as well as from extra-cellular matrix, influence cell fate decisions and contribute to tissue homeostasis. We review the discovery, definition and regulation of the mammary cellular hierarchy and we describe the development of the concepts that have guided our investigations. We outline recent advances in in vivo lineage tracing that is now challenging many of our assumptions regarding the behavior of mammary stem cells, and we show how understanding these cellular lineages has altered our view of breast cancer

IR Laser-Induced Gene Expression for Tracking Development of Single Embryonic Neurons and Glia in C. Elegans

The assembly of neural circuits requires a complex choreography of developmental events: neurons must be generated, extend neurites at the correct time and location, and then integrate extracellular information, like long-range guidance cues or cellular contacts, with an internal developmental program to make correct wiring decisions. Visualizing neural-circuit assembly in vivo can provide insight into how these events are coordinated. The C. elegans embryo, which contains only 222 neurons and 56 glia, is an attractive setting to study nervous system development comprehensively in an intact, living organism. However, methods to label and track optically-resolvable neurites or manipulate single neurons through gene expression do not exist, as most embryonic reporters are broadly expressed. Here, I present a method for expressing fluorescent reporters or any gene of interest in specific C. elegans embryonic neurons, glia, or other cell types, without cell specific drivers. Our method is based on a previous setup (Kamei et al., 2009), and uses an infrared (IR) laser to localize heat to the volume of a single precursor cell in the embryo. This induces gene expression in the progeny of that cell (1-4 cells/embryo) through heat-shock-response regulatory elements. I perform significant optimizations to adapt this strategy to cells in the C. elegans embryo, which are highly sensitive to heat toxicity. Direct temperature measurements of IR heating in the embryo reveal that cells are heated to physiological temperatures (320C) for 5 minute durations using our modified irradiation protocol. These conditions lead to high rates of gene induction (\u3e60%) with no signs of damage. First, I use our system to label and track single neurons during early nervous system assembly. These studies reveal a retrograde extension mechanism for axon growth in specific interneurons. I also study the etiology of axon-guidance defects in sax-3/Robo and vab-1/EphR mutants; these studies suggest that a timing/competence mechanism controls axon-outgrowth dynamics in the nerve ring. Next, I demonstrate the versatility of IR irradiation by performing cell-specific rescues, determining DAF-6/Patched-related site of action during sensory-organ development. Finally, I demonstrate that IR cell irradiation can be used to perform simultaneous ablation and labeling of cells in the same embryo. I use this system to uncover a role for the amphid sheath glia in dendrite extension. As IR induction can be used for targeted labeling, gene expression, and ablation without the need for cell-specific drivers, this tool opens to door to high resolution systematic analyses of C. elegans morphogenesis

Epigenomes in Cardiovascular Disease.

If unifying principles could be revealed for how the same genome encodes different eukaryotic cells and for how genetic variability and environmental input are integrated to impact cardiovascular health, grand challenges in basic cell biology and translational medicine may succumb to experimental dissection. A rich body of work in model systems has implicated chromatin-modifying enzymes, DNA methylation, noncoding RNAs, and other transcriptome-shaping factors in adult health and in the development, progression, and mitigation of cardiovascular disease. Meanwhile, deployment of epigenomic tools, powered by next-generation sequencing technologies in cardiovascular models and human populations, has enabled description of epigenomic landscapes underpinning cellular function in the cardiovascular system. This essay aims to unpack the conceptual framework in which epigenomes are studied and to stimulate discussion on how principles of chromatin function may inform investigations of cardiovascular disease and the development of new therapies

Functional cis-regulatory modules encoded by mouse-specific endogenous retrovirus

Cis-regulatory modules contain multiple transcription factor (TF)-binding sites and integrate the effects of each TF to control gene expression in specific cellular contexts. Transposable elements (TEs) are uniquely equipped to deposit their regulatory sequences across a genome, which could also contain cis-regulatory modules that coordinate the control of multiple genes with the same regulatory logic. We provide the first evidence of mouse-specific TEs that encode a module of TF-binding sites in mouse embryonic stem cells (ESCs). The majority (77%) of the individual TEs tested exhibited enhancer activity in mouse ESCs. By mutating individual TF-binding sites within the TE, we identified a module of TF-binding motifs that cooperatively enhanced gene expression. Interestingly, we also observed the same motif module in the in silico constructed ancestral TE that also acted cooperatively to enhance gene expression. Our results suggest that ancestral TE insertions might have brought in cis-regulatory modules into the mouse genome

Colorectal Cancer Through Simulation and Experiment

Colorectal cancer has continued to generate a huge amount of research interest over several decades, forming a canonical example of tumourigenesis since its use in Fearon and Vogelstein’s linear model of genetic mutation. Over time, the field has witnessed a transition from solely experimental work to the inclusion of mathematical biology and computer-based modelling. The fusion of these disciplines has the potential to provide valuable insights into oncologic processes, but also presents the challenge of uniting many diverse perspectives. Furthermore, the cancer cell phenotype defined by the ‘Hallmarks of Cancer’ has been extended in recent times and provides an excellent basis for future research. We present a timely summary of the literature relating to colorectal cancer, addressing the traditional experimental findings, summarising the key mathematical and computational approaches, and emphasising the role of the Hallmarks in current and future developments. We conclude with a discussion of interdisciplinary work, outlining areas of experimental interest which would benefit from the insight that mathematical and computational modelling can provide

Sox10 regulates enteric neural crest cell migration in the developing gut

Concurrent Sessions 1: 1.3 - Organs to organisms: Models of Human Diseases: abstract no. 1417th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, VII Latin American Society of Developmental Biology Meeting and XI Congreso de la Sociedad Mexicana de Biologia del Desarrollo. The Conference's web site is located at http://www.inb.unam.mx/isdb/Sox10 is a HMG-domain containing transcription factor which plays important roles in neural crest cell survival and differentiation. Mutations of Sox10 have been identified in patients with Waardenburg-Hirschsprung syndrome, who suffer from deafness, pigmentation defects and intestinal aganglionosis. Enteric neural crest cells (ENCCs) with Sox10 mutation undergo premature differentiation and fail to colonize the distal hindgut. It is unclear, however, whether Sox10 plays a role in the migration of ENCCs. To visualize the migration behaviour of mutant ENCCs, we generated a Sox10NGFP mouse model where EGFP is fused to the N-terminal domain of Sox10. Using time-lapse imaging, we found that ENCCs in Sox10NGFP/+ mutants displays lower migration speed and altered trajectories compared to normal controls. This behaviour was cell-autonomous, as shown by organotypic grafting of Sox10NGFP/+ gut segments onto control guts and vice versa. ENCCs encounter different extracellular matrix (ECM) molecules along the developing gut. We performed gut explant culture on various ECM and found that Sox10NGFP/+ ENCCs tend to form aggregates, particularly on fibronectin. Time-lapse imaging of single cells in gut explant culture indicated that the tightly-packed Sox10 mutant cells failed to exhibit contact inhibition of locomotion. We determined the expression of adhesion molecule families by qPCR analysis, and found integrin expression unaffected while L1-cam and selected cadherins were altered, suggesting that Sox10 mutation affects cell adhesion properties of ENCCs. Our findings identify a de novo role of Sox10 in regulating the migration behaviour of ENCCs, which has important implications for the treatment of Hirschsprung disease.postprin

Evolution of gene regulation in ruminants differs between evolutionary breakpoint regions and homologous synteny blocks

The role of chromosome rearrangements in driving evolution has been a long-standing question of evolutionary biology. Here we focused on ruminants as a model to assess how rearrangements may have contributed to the evolution of gene regulation. Using reconstructed ancestral karyotypes of Cetartiodactyls, Ruminants, Pecorans, and Bovids, we traced patterns of gross chromosome changes. We found that the lineage leading to the ruminant ancestor after the split from other cetartiodactyls was characterized by mostly intrachromosomal changes, whereas the lineage leading to the pecoran ancestor (including all livestock ruminants) included multiple interchromosomal changes. We observed that the liver cell putative enhancers in the ruminant evolutionary breakpoint regions are highly enriched for DNA sequences under selective constraint acting on lineage-specific transposable elements (TEs) and a set of 25 specific transcription factor (TF) binding motifs associated with recently active TEs. Coupled with gene expression data, we found that genes near ruminant breakpoint regions exhibit more divergent expression profiles among species, particularly in cattle, which is consistent with the phylogenetic origin of these breakpoint regions. This divergence was significantly greater in genes with enhancers that contain at least one of the 25 specific TF binding motifs and located near bovidae-to-cattle lineage breakpoint regions. Taken together, by combining ancestral karyotype reconstructions with analysis of cis regulatory element and gene expression evolution, our work demonstrated that lineage-specific regulatory elements colocalized with gross chromosome rearrangements may have provided valuable functional modifications that helped to shape ruminant evolution

Cortical interneuron specification and diversification in the era of big data

Inhibition in the mammalian cerebral cortex is mediated by a small population of highly diverse GABAergic interneurons. These largely local neurons are interspersed among excitatory projection neurons and exert pivotal regulation on the formation and function of cortical circuits. We are beginning to understand the extent of GABAergic neuron diversity and how this is generated and shaped during brain development in mice and humans. In this review, we summarise recent findings and discuss how new technologies are being used to further advance our knowledge. Understanding how inhibitory neurons are generated in the embryo is an essential pre-requisite of stem cell therapy, an evolving area of research, aimed at correcting human disorders that result in inhibitory dysfunction

Analysis of craniofacial defects in Six1/Eya1-associated Branchio-Oto-Renal Syndrome

Poster Session I - Morphogenesis: 205/B10117th ISDB 2013 cum 72nd Annual Meeting of the Society for Developmental Biology, 7th Latin American Society of Developmental Biology Meeting and 11th Congreso de la Sociedad Mexicana de Biologia del Desarrollo.Branchio-Oto-Renal (BOR) syndrome patients exhibit craniofacial and renal anomalies as well as deafness. BOR syndrome is caused by mutations in Six1 or Eya1, both of which regulate cell proliferation and differentiation. The molecular mechanism underlying the craniofacial and branchial arch (BA) defects in BOR syndrome is unclear. We have found that Hoxb3 is up-regulated in the second branchial arch (BA2) of Six1-/- mutants. Moreover, Hoxb3 over-expression in transgenic mice leads to BA abnormalities which are similar to the BA defects in Six1-/- or Eya1-/- mutants, suggesting a regulatory relationship among Six1, Eya1 and Hoxb3 genes. The aim of this study is to investigate the molecular mechanism underlying abnormal BA development in BOR syndrome using Six1 and Eya1 mutant mice. Two potential Six1 binding sites were identified on the Hoxb3 gene. In vitro and in vivo Chromatin IP assays showed that Six1 could directly bind to one of the sites specifically. Furthermore, using a chick in ovo luciferase assay we showed that Six1 could suppress gene expression through one of the specific binding sites. On the other hand, in Six1-/- mutants, we found that the Notch ligand Jag1 was up-regulated in BA2. Similarly, in Hoxb3 transgenic mice, ectopic expression of Jag1 could be also detected in BA2. To investigate the activation of Notch signaling pathway, we found that Notch intracellular domain (NICD), a direct indicator of Notch pathway activation, was up-regulated in BAs of Six1-/-; Eya1-/- double mutants. Our results indicate that Hoxb3 and Notch signaling pathway are involved in mediating the craniofacial defects of Six1/Eya1-associated Branchio-Oto-Renal Syndrome.postprin