List of Top 25 Articles of Bioinformatics

Jan Mar 2014

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Sr. No Title

1 Generation of Gene-Modified Cynomolgus Monkey via Cas9/RNA-Mediated Gene Targeting in One-Cell Embryos
Summary
Monkeys serve as important model species for studying human diseases and developing therapeutic strategies, yet the application of
monkeys in biomedical researches has been significantly hindered by the difficulties in producing animals genetically modified at the
desired target sites. Here, we first applied the CRISPR/Cas9 system, a versatile tool for editing the genes of different organisms, to target
monkey genomes. By coinjection of Cas9 mRNA and sgRNAs into one-cell-stage embryos, we successfully achieve precise gene targeting in
cynomolgus monkeys. We also show that this system enables simultaneous disruption of two target genes (Ppar- and Rag1) in one step,
and no off-target mutagenesis was detected by comprehensive analysis. Thus, coinjection of one-cell-stage embryos with Cas9 mRNA and
sgRNAs is an efficient and reliable approach for gene-modified cynomolgus monkey generation.


2 One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering

Summary
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-
consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting
technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the
simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty - 8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection
of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes
with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations
simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple
genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.

3 Hallmarks of Cancer: The Next Generation

Summary:

The hallmarks of cancer comprise six biological capabilities acquired during the multistep development of human tumors. The hallmarks
constitute an organizing principle for rationalizing the complexities of neoplastic disease. They include sustaining proliferative signaling,
evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and
metastasis. Underlying these hallmarks are genome instability, which generates the genetic diversity that expedites their acquisition, and
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inflammation, which fosters multiple hallmark functions. Conceptual progress in the last decade has added two emerging hallmarks of
potential generality to this listreprogramming of energy metabolism and evading immune destruction. In addition to cancer cells, tumors
exhibit another dimension of complexity: they contain a repertoire of recruited, ostensibly normal cells that contribute to the acquisition
of hallmark traits by creating the tumor microenvironment. Recognition of the widespread applicability of these concepts will
increasingly affect the development of new means to treat human cancer.

4 Hydrolysis of lignocellulosic materials for ethanol production: a review
Abstract
Lignocellulosic biomass can be utilized to produce ethanol, a promising alternative energy source for the limited crude oil. There are
mainly two processes involved in the conversion: hydrolysis of cellulose in the lignocellulosic biomass to produce reducing sugars, and
fermentation of the sugars to ethanol. The cost of ethanol production from lignocellulosic materials is relatively high based on current
technologies, and the main challenges are the low yield and high cost of the hydrolysis process. Considerable research efforts have been
made to improve the hydrolysis of lignocellulosic materials. Pretreatment of lignocellulosic materials to remove lignin and hemicellulose
can significantly enhance the hydrolysis of cellulose. Optimization of the cellulase enzymes and the enzyme loading can also improve the
hydrolysis. Simultaneous saccharification and fermentation effectively removes glucose, which is an inhibitor to cellulase activity, thus
increasing the yield and rate of cellulose hydrolysis.

5 Crystal Structure of Cas9 in Complex with Guide RNA and Target DNA
Summary
The CRISPR-associated endonuclease Cas9 can be targeted to specific genomic loci by single guide RNAs (sgRNAs). Here, we report the
crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 resolution. The structure revealed a
bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively
charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the
HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and noncomplementary strands of
the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the
protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular
mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing
technologies.

6 Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR/Cas System
Summary
The spatiotemporal organization and dynamics of chromatin play critical roles in regulating genome function. However, visualizing
specific, endogenous genomic loci remains challenging in living cells. Here, we demonstrate such an imaging technique by repurposing the
bacterial CRISPR/Cas system. Using an EGFP-tagged endonuclease-deficient Cas9 protein and a structurally optimized small guide (sg)
RNA, we show robust imaging of repetitive elements in telomeres and coding genes in living cells. Furthermore, an array of sgRNAs tiling
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along the target locus enables the visualization of nonrepetitive genomic sequences. Using this method, we have studied telomere
dynamics during elongation or disruption, the subnuclear localization of the MUC4 loci, the cohesion of replicated MUC4 loci on sister
chromatids, and their dynamic behaviors during mitosis. This CRISPR imaging tool has potential to significantly improve the capacity to
study the conformation and dynamics of native chromosomes in living human cells.

7 Declining NAD^+ Induces a Pseudohypoxic State Disrupting Nuclear-Mitochondrial Communication during Aging
Summary
Ever since eukaryotes subsumed the bacterial ancestor of mitochondria, the nuclear and mitochondrial genomes have had to closely
coordinate their activities, as each encode different subunits of the oxidative phosphorylation (OXPHOS) system. Mitochondrial
dysfunction is a hallmark of aging, but its causes are debated. We show that, during aging, there is a specific loss of mitochondrial, but not
nuclear, encoded OXPHOS subunits. We trace the cause to an alternate PGC-1/-independent pathway of nuclear-mitochondrial
communication that is induced by a decline in nuclear NAD+ and the accumulation of HIF-1 under normoxic conditions, with parallels to
Warburg reprogramming. Deleting SIRT1 accelerates this process, whereas raising NAD+ levels in old mice restores mitochondrial function
to that of a young mouse in a SIRT1-dependent manner. Thus, a pseudohypoxic state that disrupts PGC-1/-independent nuclear-
mitochondrial communication contributes to the decline in mitochondrial function with age, a process that is apparently reversible.

8 Microbiota Modulate Behavioral and Physiological Abnormalities Associated with Neurodevelopmental Disorders
Summary
Neurodevelopmental disorders, including autism spectrum disorder (ASD), are defined by core behavioral impairments; however, subsets
of individuals display a spectrum of gastrointestinal (GI) abnormalities. We demonstrate GI barrier defects and microbiota alterations in
the maternal immune activation (MIA) mouse model that is known to display features of ASD. Oral treatment of MIA offspring with the
human commensalBacteroides fragilis corrects gut permeability, alters microbial composition, and ameliorates defects in communicative,
stereotypic, anxiety-like and sensorimotor behaviors. MIA offspring display an altered serum metabolomic profile,
and B. fragilis modulates levels of several metabolites. Treating naive mice with a metabolite that is increased by MIA and restored
by B. fragilis causes certain behavioral abnormalities, suggesting that gut bacterial effects on the host metabolome impact behavior.
Taken together, these findings support a gut-microbiome-brain connection in a mouse model of ASD and identify a potential probiotic
therapy for GI and particular behavioral symptoms in human neurodevelopmental disorders.

9 Reversing DNA Methylation: Mechanisms, Genomics, and Biological Functions
Methylation of cytosines in the mammalian genome represents a key epigenetic modification and is dynamically regulated during
development. Compelling evidence now suggests that dynamic regulation of DNA methylation is mainly achieved through a cyclic
enzymatic cascade comprised of cytosine methylation, iterative oxidation of methyl group by TET dioxygenases, and restoration of
unmodified cytosines by either replication-dependent dilution or DNA glycosylase-initiated base excision repair. In this review, we discuss
the mechanism and function of DNA demethylation in mammalian genomes, focusing particularly on how developmental modulation of
the cytosine-modifying pathway is coupled to active reversal of DNA methylation in diverse biological processes.
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10 One-Step Generation of Mice Carrying Reporter and Conditional Alleles by CRISPR/Cas-Mediated Genome Engineering
Summary
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we
created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as
DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in
the Nanog, theSox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites
in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of
five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.

11 Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity
Summary
Targeted genome editing technologies have enabled a broad range of research and medical applications. The Cas9 nuclease from the
microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the
DNA target and thereby promote undesired off-target mutagenesis. Here, we describe an approach that combines a Cas9 nickase mutant
with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity,
simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically
recognized bases for target cleavage. We demonstrate that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell
lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a
wide variety of genome editing applications that require high specificity.

12 Premature Termination of Reprogramming In Vivo Leads to Cancer Development through Altered Epigenetic Regulation
Summary
Cancer is believed to arise primarily through accumulation of genetic mutations. Although induced pluripotent stem cell (iPSC) generation
does not require changes in genomic sequence, iPSCs acquire unlimited growth potential, a characteristic shared with cancer cells. Here,
we describe a murine system in which reprogramming factor expression in vivo can be controlled temporally with doxycycline (Dox).
Notably, transient expression of reprogramming factors in vivo results in tumor development in various tissues consisting of
undifferentiated dysplastic cells exhibiting global changes in DNA methylation patterns. The Dox-withdrawn tumors arising in the kidney
share a number of characteristics with Wilms tumor, a common pediatric kidney cancer. We also demonstrate that iPSCs derived from
Dox-withdrawn kidney tumor cells give rise to nonneoplastic kidney cells in mice, proving that they have not undergone irreversible
genetic transformation. These findings suggest that epigenetic regulation associated with iPSC derivation may drive development of
particular types of cancer.




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13 Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors

Summary
Differentiated cells can be reprogrammed to an embryonic-like state by transfer of nuclear contents into oocytes or by fusion with
embryonic stem (ES) cells. Little is known about factors that induce this reprogramming. Here, we demonstrate induction of pluripotent
stem cells from mouse embryonic or adult fibroblasts by introducing four factors, Oct3/4, Sox2, c-Myc, and Klf4, under ES cell culture
conditions. Unexpectedly, Nanog was dispensable. These cells, which we designated iPS (induced pluripotent stem) cells, exhibit the
morphology and growth properties of ES cells and express ES cell marker genes. Subcutaneous transplantation of iPS cells into nude mice
resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to
mouse embryonic development. These data demonstrate that pluripotent stem cells can be directly generated from fibroblast cultures by
the addition of only a few defined factors.
14 Microbiota-Generated Metabolites Promote Metabolic Benefits via Gut-Brain Neural Circuits
Summary
Soluble dietary fibers promote metabolic benefits on body weight and glucose control, but underlying mechanisms are poorly understood.
Recent evidence indicates that intestinal gluconeogenesis (IGN) has beneficial effects on glucose and energy homeostasis. Here, we show
that the short-chain fatty acids (SCFAs) propionate and butyrate, which are generated by fermentation of soluble fiber by the gut
microbiota, activate IGN via complementary mechanisms. Butyrate activates IGN gene expression through a cAMP-dependent
mechanism, while propionate, itself a substrate of IGN, activates IGN gene expression via a gut-brain neural circuit involving the fatty acid
receptor FFAR3. The metabolic benefits on body weight and glucose control induced by SCFAs or dietary fiber in normal mice are absent in
mice deficient for IGN, despite similar modifications in gut microbiota composition. Thus, the regulation of IGN is necessary for the
metabolic benefits associated with SCFAs and soluble fiber.

15 Mechanism of surfactant effect in enzymatic hydrolysis of lignocellulose
Abstract
Lignocellulose is a potential substrate for ethanol production. However, high cellulose conversion requires high enzyme loading, which
makes the process less economically feasible. Addition of surfactants to enzymatic hydrolysis of lignocellulose increases the conversion of
cellulose into soluble sugars. The mechanism is not known for the increase of lignocellulose hydrolysis by surfactant addition, therefore,
experiments were designed to explore mechanisms of surfactant effects. A number of surfactants were screened for their ability to
improve enzymatic hydrolysis of steam-pretreated spruce (SPS). Non-ionic surfactants were found to be the most effective. Studies of
adsorption of the dominating cellulase ofTrichoderma reesei, Cel7A (CBHI), during hydrolysis showed that the anionic and non-ionic
surfactants reduced enzyme adsorption to the lignocellulose substrate. The approximate reduction of enzyme adsorption was from 90%
adsorbed enzyme to 80% with surfactant addition. Cellulase stability in the presence of surfactants was studied by activity and
fluorescence measurements. Surfactants were shown to have only a weak effect on cellulase temperature stability. Our conclusions from
studies of lignocellulose and delignified substrates are that the improved conversion of lignocellulose with surfactant can be explained by
the reduction of the unproductive enzyme adsorption to the lignin part of the substrate. This is due to hydrophobic interaction of
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surfactant with lignin on the lignocellulose surface, which releases unspecifically bound enzyme. A new approach with mixed charged and
non-ionic surfactants has been introduced to further improve the positive effect of the surfactant addition.

16 Epigenetic Priming of Memory Updating during Reconsolidation to Attenuate Remote Fear Memories
Summary
Traumatic events generate some of the most enduring forms of memories. Despite the elevated lifetime prevalence of anxiety disorders,
effective strategies to attenuate long-term traumatic memories are scarce. The most efficacious treatments to diminish recent (i.e., day-
old) traumata capitalize on memory updating mechanisms during reconsolidation that are initiated upon memory recall. Here, we show
that, in mice, successful reconsolidation-updating paradigms for recent memories fail to attenuate remote (i.e., month-old) ones. We find
that, whereas recent memory recall induces a limited period of hippocampal neuroplasticity mediated, in part, by S-nitrosylation of
HDAC2 and histone acetylation, such plasticity is absent for remote memories. However, by using an HDAC2-targeting inhibitor (HDACi)
during reconsolidation, even remote memories can be persistently attenuated. This intervention epigenetically primes the expression of
neuroplasticity-related genes, which is accompanied by higher metabolic, synaptic, and structural plasticity. Thus, applying HDACis during
memory reconsolidation might constitute a treatment option for remote traumata.

17 Serpins Promote Cancer Cell Survival and Vascular Co-Option in Brain Metastasis
Summary
Brain metastasis is an ominous complication of cancer, yet most cancer cells that infiltrate the brain die of unknown causes. Here, we
identify plasmin from the reactive brain stroma as a defense against metastatic invasion, and plasminogen activator (PA) inhibitory serpins
in cancer cells as a shield against this defense. Plasmin suppresses brain metastasis in two ways: by converting membrane-bound
astrocytic FasL into a paracrine death signal for cancer cells, and by inactivating the axon pathfinding molecule L1CAM, which metastatic
cells express for spreading along brain capillaries and for metastatic outgrowth. Brain metastatic cells from lung cancer and breast cancer
express high levels of anti-PA serpins, including neuroserpin and serpin B2, to prevent plasmin generation and its metastasis-suppressive
effects. By protecting cancer cells from death signals and fostering vascular co-option, anti-PA serpins provide a unifying mechanism for
the initiation of brain metastasis in lung and breast cancers.

18 Hippo Signaling Regulates Microprocessor and Links Cell-Density-Dependent miRNA Biogenesis to Cancer
Summary
Global downregulation of microRNAs (miRNAs) is commonly observed in human cancers and can have a causative role in tumorigenesis.
The mechanisms responsible for this phenomenon remain poorly understood. Here, we show that YAP, the downstream target of the
tumor-suppressive Hippo-signaling pathway regulates miRNA biogenesis in a cell-density-dependent manner. At low cell density, nuclear
YAP binds and sequesters p72 (DDX17), a regulatory component of the miRNA-processing machinery. At high cell density, Hippo-mediated
cytoplasmic retention of YAP facilitates p72 association with Microprocessor and binding to a specific sequence motif in pri-miRNAs.
Inactivation of the Hippo pathway or expression of constitutively active YAP causes widespread miRNA suppression in cells and tumors
and a corresponding posttranscriptional induction of MYC expression. Thus, the Hippo pathway links contact-inhibition regulation to
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miRNA biogenesis and may be responsible for the widespread miRNA repression observed in cancer.

19 ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering

Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) comprise a powerful class of tools that are
redefining the boundaries of biological research. These chimeric nucleases are composed of programmable, sequence-specific DNA-
binding modules linked to a nonspecific DNA cleavage domain. ZFNs and TALENs enable a broad range of genetic modifications by
inducing DNA double-strand breaks that stimulate error-prone nonhomologous end joining or homology-directed repair at specific
genomic locations. Here, we review achievements made possible by site-specific nuclease technologies and discuss applications of these
reagents for genetic analysis and manipulation. In addition, we highlight the therapeutic potential of ZFNs and TALENs and discuss future
prospects for the field, including the emergence of clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas-based RNA-
guided DNA endonucleases.
20 Neurogenesis in the Striatum of the Adult Human Brain
Summary
In most mammals, neurons are added throughout life in the hippocampus and olfactory bulb. One area where neuroblasts that give rise to
adult-born neurons are generated is the lateral ventricle wall of the brain. We show, using histological and carbon-14 dating approaches,
that in adult humans new neurons integrate in the striatum, which is adjacent to this neurogenic niche. The neuronal turnover in the
striatum appears restricted to interneurons, and postnatally generated striatal neurons are preferentially depleted in patients with
Huntingtons disease. Our findings demonstrate a unique pattern of neurogenesis in the adult human brain.

21 Cannabidiol protects liver from binge alcohol-induced steatosis by mechanisms including inhibition of oxidative stress and increase in
autophagy
Abstract
Acute alcohol drinking induces steatosis, and effective prevention of steatosis can protect liver from progressive damage caused by
alcohol. Increased oxidative stress has been reported as one mechanism underlying alcohol-induced steatosis. We evaluated whether
cannabidiol, which has been reported to function as an antioxidant, can protect the liver from alcohol-generated oxidative stress-induced
steatosis. Cannabidiol can prevent acute alcohol-induced liver steatosis in mice, possibly by preventing the increase in oxidative stress and
the activation of the JNK MAPK pathway. Cannabidiol per se can increase autophagy both in CYP2E1-expressing HepG2 cells and in mouse
liver. Importantly, cannabidiol can prevent the decrease in autophagy induced by alcohol. In conclusion, these results show that
cannabidiol protects mouse liver from acute alcohol-induced steatosis through multiple mechanisms including attenuation of alcohol-
mediated oxidative stress, prevention of JNK MAPK activation, and increasing autophagy.




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22 The Bacterial Cytoplasm Has Glass-like Properties and Is Fluidized by Metabolic Activity
Summary
The physical nature of the bacterial cytoplasm is poorly understood even though it determines cytoplasmic dynamics and hence cellular
physiology and behavior. Through single-particle tracking of protein filaments, plasmids, storage granules, and foreign particles of
different sizes, we find that the bacterial cytoplasm displays properties that are characteristic of glass-forming liquids and changes from
liquid-like to solid-like in a component size-dependent fashion. As a result, the motion of cytoplasmic components becomes
disproportionally constrained with increasing size. Remarkably, cellular metabolism fluidizes the cytoplasm, allowing larger components to
escape their local environment and explore larger regions of the cytoplasm. Consequently, cytoplasmic fluidity and dynamics dramatically
change as cells shift between metabolically active and dormant states in response to fluctuating environments. Our findings provide
insight into bacterial dormancy and have broad implications to our understanding of bacterial physiology, as the glassy behavior of the
cytoplasm impacts all intracellular processes involving large components.

23 The Hallmarks of Cancer

After a quarter century of rapid advances, cancer research has generated a rich and complex body of knowledge, revealing cancer to be a
disease involving dynamic changes in the genome. The foundation has been set in the discovery of mutations that produce oncogenes
with dominant gain of function and tumor suppressor genes with recessive loss of function; both classes of cancer genes have been
identified through their alteration in human and animal cancer cells and by their elicitation of cancer phenotypes in experimental models
(Bishop and Weinberg 1996).
Some would argue that the search for the origin and treatment of this disease will continue over the next quarter century in much the
same manner as it has in the recent past, by adding further layers of complexity to a scientific literature that is already complex almost
beyond measure. But we anticipate otherwise: those researching the cancer problem will be practicing a dramatically different type of
science than we have experienced over the past 25 years. Surely much of this change will be apparent at the technical level. But
ultimately, the more fundamental change will be conceptual.

24 Collective Invasion in Breast Cancer Requires a Conserved Basal Epithelial Program
Summary
Carcinomas typically invade as a cohesive multicellular unit, a process termed collective invasion. It remains unclear how different
subpopulations of cancer cells contribute to this process. We developed three-dimensional (3D) organoid assays to identify the most
invasive cancer cells in primary breast tumors. Collective invasion was led by specialized cancer cells that were defined by their expression
of basal epithelial genes, such as cytokeratin-14 (K14) and p63. Furthermore, K14+ cells led collective invasion in the major human breast
cancer subtypes. Importantly, luminal cancer cells were observed to convert phenotypically to invasive leaders following induction of
basal epithelial genes. Although only a minority of cells within luminal tumors expressed basal epithelial genes, knockdown of either K14
or p63 was sufficient to block collective invasion. Our data reveal that heterotypic interactions between epithelial subpopulations are
critical to collective invasion. We suggest that targeting the basal invasive program could limit metastatic progression.
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25 Interactome Maps of Mouse Gene Regulatory Domains Reveal Basic Principles of Transcriptional RegulationSummary
A key finding of the ENCODE project is that the enhancer landscape of mammalian cells undergoes marked alterations during ontogeny.
However, the nature and extent of these changes are unclear. As part of the NIH Mouse Regulome Project, we here combined DNaseI
hypersensitivity, ChIP-seq, and ChIA-PET technologies to map the promoter-enhancer interactomes of pluripotent ES cells and
differentiated B lymphocytes. We confirm that enhancer usage varies widely across tissues. Unexpectedly, we find that this feature
extends to broadly transcribed genes, including Myc and Pim1 cell-cycle regulators, which associate with an entirely different set of
enhancers in ES and B cells. By means of high-resolution CpG methylomes, genome editing, and digital footprinting, we show that these
enhancers recruit lineage-determining factors. Furthermore, we demonstrate that the turning on and off of enhancers during
development correlates with promoter activity. We propose that organisms rely on a dynamic enhancer landscape to control basic cellular
functions in a tissue-specific manner.