Top PDF DNA Mediated Charge Transport Signaling Within the Cell

DNA Mediated Charge Transport Signaling Within the Cell

DNA Mediated Charge Transport Signaling Within the Cell

electrochemically, likely reflecting better coupling of the 4Fe-4S cluster to DNA while DinG unwinds DNA, which could have interesting biological implications. Atomic force microscopy experiments demonstrate that DinG and EndoIII cooperate at long range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Knocking out DinG in CC104 cells leads to a decrease in MutY activity that is rescued by EndoIII D138A, but not EndoIII Y82A. DinG, thus, appears to help MutY find its substrate using DNA-mediated CT, but do MutY or EndoIII aid DinG in a similar way? The InvA strain of bacteria was used to observe DinG activity, since DinG activity is required within InvA to maintain normal growth. Silencing the gene encoding EndoIII in InvA results in a significant growth defect that is rescued by the overexpression of RNAseH, a protein that dismantles the substrate of DinG, R-loops. This establishes signaling between DinG and EndoIII. Furthermore, rescue of this growth defect by the expression of EndoIII D138A, the catalytically inactive but CT-proficient mutant of EndoIII, is also observed, but expression of EndoIII Y82A, which is CT-deficient but enzymatically active, does not rescue growth. These results provide strong evidence that DinG and EndoIII utilize DNA-mediated signaling to process DNA damage. This work thus expands the scope of DNA-mediated signaling within the cell, as it indicates that DNA-mediated signaling facilitates the activities of DNA repair enzymes across the genome, even for proteins from distinct repair pathways.
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DNA-Mediated Hole and Electron Transport

DNA-Mediated Hole and Electron Transport

First and foremost, I must thank my research advisor, Professor Jacqueline K. Barton. She has always been a great mentor to me. She is the person who led me into the fascinating world of DNA-mediated charge transport. During the first year in the lab, she had great patience to educate and transform me from a theoretical chemist to a handy bioinorganic experimentalist. I am amazed by her undying unyielding enthusiasm for chemistry and science in general. Her encouragement and brilliant ideas about research kept my spirits high as I struggled through the failures in experiments. I have always remembered the exciting spirit fulfilling me after every time that I discussed my projects with her. I admire her high standards for scientific researches and from her I learned to be a better critical thinker. She is truly an inspirational leader who brings together such a wonderful group of intelligent people who have contributed to my thesis work in various ways. Lastly, and perhaps mostly importantly, I thank you for the tremendous help you have provided for my scientific career. I eagerly look forward to our future interaction.
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Spectroscopic characterization of DNA-mediated charge transfer

Spectroscopic characterization of DNA-mediated charge transfer

1.1 Schematic illustration of the DNA double helix 3 1.2 Illustration of a Rh(III) modified duplex with 5’-GG-3’ sites up to 200 Å away 4 1.3 Reaction coordinate diagram and couplings for electron transfer 7 1.4 Assemblies in which long-range charge transport was demonstrated 12 1.5 Illustration of duplex DNA self-assembled monolayers on gold 14 1.6 Assemblies highlighting the importance of stacking to charge transfer in DNA 18 1.7 Effect of protein binding on long-range guanine oxidation 20 1.8 Ethidium, guanine, and 7-deazaguanine structures and reactivity 22 1.9 Structures, reactivity, and stacking of modified bases in DNA 24 1.10 Distance dependence of electron transfer through DNA-modified electrodes 26 1.11 Incorporation of a mismatch in DNA-modified gold electrodes 27 1.12 Energetics and coupling involved in various charge transfer mechanisms 31
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DNA-mediated Charge Transport in a Biological Context: Cooperation among Metalloproteins to Find Lesions in the Genome

DNA-mediated Charge Transport in a Biological Context: Cooperation among Metalloproteins to Find Lesions in the Genome

times in Beverly Hills and beyond. It has been an amazing experience doing science alongside people who are passionate about what they do, and through this, we will always be connected to one another. Thanks to Professor Jeff Zaleski, my undergraduate research advisor at Indiana University, for encouraging me to proceed to graduate school and continue following my passion of chemistry. My friends who I met through frisbee, hip-hop, and those in other chemistry research labs have meant so much and contributed a great amount during my time here. Silva and Agnes have become special friends within the department, and I am extremely lucky to have had the interactions with them that I did. Also, I must mention the teachers and leaders who impacted my life prior to my time at Caltech. I have always felt blessed to have teachers who not only imparted so much knowledge but also were deeply involved in shaping my life-from elementary school to high school-a huge thank you to them. To my friends who have been like siblings over the years: (Brock, Mitzi, Sarah C., Alanna, Lindsay, Carolyn, Dawn, Debra, Michelle, Harmony, Sarah T., Nyssa, and Heather), who have been there without hesitation, taught me so much, and were there to make many happy and exciting memories: thank you.
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Activation of Transcription from a Distance: Investigations on the Oxidation of SoxR by DNA Mediated Charge Transport

Activation of Transcription from a Distance: Investigations on the Oxidation of SoxR by DNA Mediated Charge Transport

Here, using DNA-modified HOPG electrodes, we have demonstrated that DNA association positively shifts the redox potential of SoxR to 200 mV versus NHE. The +490 mV shift between the free and DNA-bound states of SoxR is functionally crucial, since it keeps SoxR reduced at intracellular potentials, estimated to be -260 to -280 mV in E. coli (35). For example, Figure 2.6 shows standard and free midpoint potentials for a variety of cellular redox pairs, and where DNA-bound and free SoxR are positioned along this series. While numerous redox couples, ranging from glutathione to FADH, are oxidants for soluble SoxR, they are reductants to DNA- bound SoxR. In fact, the positive shift in potential associated with DNA binding means that DNA-bound SoxR is primarily in the reduced, transcriptionally silent form in vivo. Oxidative stress serves to promote oxidation of DNA-bound SoxR, activating the numerous genes required to protect the organism. This provides a rationale for how SoxR can serve as an effective sensor of oxidative stress in E. coli.
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Regulation of Wild Type and Mutant p53 through DNA mediated Charge Transport

Regulation of Wild Type and Mutant p53 through DNA mediated Charge Transport

2.3B. We chose concentrations of DNA and p53 for near-complete binding of p53 throughout the scope of the experiment. Because oxidized p53 does not bind to this sequence of DNA, 7 the ratio of the radiolabeled bound DNA to the total signal of DNA gives the total amount of p53 binding in a lane. Oxidation data were normalized to the amount of DNA binding to p53 in the zero irradiation time points (at least 80% of total DNA present). As expected, each mutant shows dissociation from the AQ-DNA when subjected to DNA-mediated oxidation, at a total oxidation amount similar to WT at the same irradiation time point of 30 min except for C275S. The dissociation for the sample without covalent photooxidant is minimal, at most 10% after an hour of irradiation. The extent of oxidation also resembles the result seen for the MDM2 recognition element in the previous study. There is not a statistical difference between the amount of
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Expanding the Repertoire of DNA-Mediated Signaling in DNA Repair

Expanding the Repertoire of DNA-Mediated Signaling in DNA Repair

DNA is constantly assaulted by a variety of damage sources, ranging from endogenous reactive oxygen species and chemical damaging agents to external high energy UV light (1, 2). Each of these agents causes damage that, if unrepaired, leads to extended genomes, mutations, and cell death. Cells have a variety of DNA repair pathways for specific types of damage, such as base excision repair (BER), which involves the excision of a single damaged base, followed by gap filling and ligation by a polymerase (3). Nucleotide excision repair (NER), in contrast, involves larger substrates that distort the structure of DNA and are repaired by an entirely different family of enzymes (4, 5). Mismatch repair (MMR) machinery uniquely repairs mismatches that occur through DNA polymerization errors (2). While each pathway is almost entirely independent, some enzymatic overlap exists; for example, DNA polymerase ε is utilized in both NER and BER. Although the activity of each of the proteins in these pathways is understood, it is not known how proteins from each pathway target their specific lesions (6). In cells, these repair pathways work in tandem to repair the genome before
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Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways

Investigations of DNA-Mediated Redox Signaling Between E.coli DNA Repair Pathways

We have uncovered details about the effect of the protein expression levels on the efficiency of DNA-mediated redox signaling for DNA lesion detection. EndoIII is the preferred redox-signaling partner over MutY for multiple repair pathways. We demonstrated this preference in both UV-sensitivity and InvA growth assays when comparing the magnitude of the growth defect in ∆ nth and ∆ muty strains (Figures 4.4, 4.5). From our UV-sensitivity results, very low expression levels of EndoIII were enough to fully rescue growth phenotypes as all tunable expression setups rescued before addition of inducer (Figures 4.7, 4.9). Overexpression of WT EndoIII exacerbated strain growth after UV-treatment whereas overexpression of D138A, a catalytically deficient EndoII point-mutant, did not. Thus, an excess of enzymatically active EndoIII harms the cell during the dire need for proper DNA repair, likely due to non-specific enzyme activity. These results suggest that the DNA lesion scanning system, composed of 4Fe4S DNA repair proteins, is a finely tuned system that is sensitive to perturbation. Overexpression of one of the components, EndoIII, could have made scarce important cellular resources. Given that these 4Fe4S proteins requires the expression and fidelity iron-sulfur assembly pathway, a multi-protein network for the assembly and loading of FeS clusters, the metabolic tax of EndoIII overexpression was likely overwhelming for the cell [22]. Even with genome integration, addition of inducer always decreased cellular growth, further confirming the aforementioned metabolic tax (Figure 4.10).
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Charge Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA

Charge Mediated Pyrin Oligomerization Nucleates Antiviral IFI16 Sensing of Herpesvirus DNA

IFI16 oligomerization state promotes interactions with transcriptional modu- lators during HSV-1 infection. A fundamental question brought forward by our findings is how oligomerization can contribute to IFI16-mediated antiviral response. Our identification of IFI16 PYD mutants that can either retain or lose association with incoming viral genomes provided us with the tools to decipher which protein inter- actions are specifically promoted by IFI16 oligomerization. We expressed IFI16 PYD R23Q or R23K GFP-tagged constructs in HEK293Ts. Cells were infected with ICP0-RF and harvested at 6 h post-infection (hpi), when expression of cytokines during HSV-1 infection was shown to be increased (7). We conducted immunoaffinity purifications (IPs) via the GFP tag and analyzed interactions (in biological duplicates) by tandem mass spectrometry (MS) (nanoscale liquid chromatography coupled to tandem mass spectrometry [nLC-MS/MS]) on a Q Exactive HF hybrid quadrupole Orbitrap mass spectrometer (Fig. 4A). Parallel control isolations in infected cells expressing GFP alone allowed the use of the computational program “significance analysis of interactome” (SAINT) (33, 50) for assessing the specificity of interactions. To further increase the stringency of the analysis, these associations were subsequently filtered by comparison with the CRAPome database (34) to remove proteins frequently observed in control
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DNA-Mediated Charge Transfer Between [4Fe-4S] Cluster Glycosylases

DNA-Mediated Charge Transfer Between [4Fe-4S] Cluster Glycosylases

MutY homologues are found in all three domains of life and are part of a repair system that helps cells respond to oxidative stress. When excess reactive oxygen species form in vivo, these species can damage DNA, particularly at guanine residues since guanine has the lowest oxidation potential of all the nucleobases [41, 42]. When guanine is oxidized, it forms 7,8-dihydro-8- oxoguanine (8-oxo-G, Figure 1.2) [43]. Excess intracellular 8-oxo-G is removed by the enzyme MutT [11]. If it gets incorporated into DNA, it is excised by MutM [11, 12]. However, if 8-oxo-G remains misincorporated, then subsequent rounds of DNA replication will mistakenly pair an adenine molecule with it [44]. The next round of DNA replication will then place a thymine across from this adenine, resulting in a G:C  T:A transversion. MutY is the “final defense” against these transversions, as it removes adenine mispaired with 8-oxo-G [11, 44, 45]. The enzymes that repair 8-oxo-G-based lesions were first discovered by genetic mapping of cells with a strong G:C  T:A mutator phenotype. The resulting experiments identified the mutY locus as being among those responsible [45].
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Magnetic Field Effects and Biophysical Studies on DNA Charge Transport and Repair

Magnetic Field Effects and Biophysical Studies on DNA Charge Transport and Repair

Recently DinG, a DNA damage response helicase from E. coli, was shown to contain a 4Fe-4S cluster (30). DinG is part of the SOS response, which is activated by DNA damaging agents and cellular stressors. DinG shares homology with the nucleotide excision repair protein XPD as well as with a host of Superfamily 2 helicases from archaea and eukaryotes that are linked to human disease and share a conserved 4Fe-4S domain (5). DinG unwinds DNA that has single-stranded overhangs with a 5′ to 3′ polarity (31). DNA-RNA hybrid duplexes that form within a DNA bubble, termed R- loops, represent a unique substrate that DinG has been shown to unwind in vitro (32). Importantly, DinG is required to unwind R-loops in vivo in order to resolve stalled replication forks and thus to maintain the integrity of the genome (33). Here we examine the DNA-bound redox properties of DinG and explore more generally crosstalk among redox-active DNA-processing enzymes in E. coli via 4Fe-4S clusters.
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DNA-Mediated Charge Transport for Long-Range Sensing and Protein Detection

DNA-Mediated Charge Transport for Long-Range Sensing and Protein Detection

Conformational gating and charge delocalization also help to explain why such severe attenuation of DNA CT is observed when the structure of the DNA π-stack is distorted. Structural perturbations to the π-stack cause the affected bases to preferentially adopt un- stacked conformations. When it comes to the formation of delocalized domains that facilitate CT, a primarily un-stacked base functions like a rotating disc in a multi-disk combination lock that is stuck on the wrong number. Although the other disks might turn fluidly to the correct combination the disk that is stuck in the wrong orientation will still prevent the lock from opening. Likewise, the bases surrounding a lesion, mismatch, or bound protein may be free to move into CT-active conformations, but the un-stacked base disrupts the formation of a domain over which charge could delocalize, dramatically shutting off DNA CT. Charge delocalization additionally accounts for the fast CT rates that are measured electrochemically: states in which the charge is delocalized over large domains would necessarily be lower in energy than a state in which the charge is localized on an individual base, thus enabling charge injection at the applied potentials used in electrochemistry experiments.
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DNA Mediated Charge Transport Devices for Protein Detection

DNA Mediated Charge Transport Devices for Protein Detection

Given the anti-cooperative nature of TBP binding observed upon thermodynamic investigations of this protein binding to OCT-DNA monolayers, we also investigated the relative kinetics of TBP binding to these monolayers and to thiolated DNA films. Rotating disk electrode (RDE) experiments were undertaken to determine the binding kinetics of TBP on both high density thiol-DNA and low-density OCT-DNA monolayers. RDEs remove diffusion as a factor when determining kinetics of a system. 44, 48 The loss of an electrochemical DM signal upon TBP binding over time therefore reports on the kinetics of protein binding. Because the number of TBP binding sites is fixed, the solution concentration of protein is in large enough excess to be unaffected by the amount of protein bound to the surface, and the rate of TBP diffusion to the surface is removed as a factor, we can analyze the kinetics of TBP binding to the surfaces with a Langmuir kinetics model. As is evident in Figure 2.13, which shows the decrease in charge determined from the area of the reductive peak plotted as a function of time, the rate of signal decrease for both the high density and ultra low-density monolayers upon TBP binding is almost identical. As is apparent in the figure, the RDEs produce similar overall signal attenuations to stationary electrodes for both types of DMEs. When the data are fit to this Langmuir equation for protein binding kinetics, the k obs for high density
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Investigating DNA-Mediated Charge Transport by Time-Resolved Spectroscopy

Investigating DNA-Mediated Charge Transport by Time-Resolved Spectroscopy

conduit for charge, the probe should interact strongly with the DNA base stack. Such an interaction can be difficult to achieve, considering the geometry of DNA. In general, the only access a diffusing molecule has to the base stack is either at the ends of the DNA strand or within the relatively narrow major and minor grooves which run lengthwise along the sides of the DNA molecule. Probes which are too large, or which are strongly negatively charged and therefore are repelled by the phosphate backbone of DNA, do not easily interface with the DNA π-stack. Second, depending on the function of the probe, it must provide a straightforward means of either initiating or reporting on DNA CT, or both. Often, the photophysical or electrochemical properties of a molecule are utilized for these purposes. Some probes may also report CT events through chemical pathways such as degradation. Third, the probe should not degrade or interact chemically with the DNA strand or with other components of the sample unless this is by design. Not only must the probe be stable enough to persist in solution, but the excited state of the molecule must also be stable if photochemical means are used to initiate or report CT, and the various redox states of the molecule must be able to withstand the charge transfer process. Finally, the ideal probe would be synthetically versatile and easy to build or modify in order to control sensitively the parameters of the experiment. Metallointercalators, transition metal complexes which bind DNA primarily by intercalation, are one class of molecules that fulfill all of these requirements.
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Fundamental mechanisms and biological applications of DNA mediated charge transport

Fundamental mechanisms and biological applications of DNA mediated charge transport

It is well established that the π-stack of the DNA double helix can serve as an efficient medium for charge transport [1-5]. With reactions spanning distances over 200 Å, this process is acutely sensitive to the intervening bridging bases [6, 7]. Oxidative base damage resulting from DNA-mediated charge transport reaction has particular relevance in the field of aging and in many diseases including cancer and neurodegenerative disorders [8-10]. Guanine, having the lowest oxidation potential of the naturally occurring bases can effectively serve as a hole trap [11]. Upon oxidation, the neutral guanine radical, with a millisecond lifetime, can react irreversibly with water or oxygen to form permanent damage products such as 8-oxo-G, oxazolone, and imidazolone [12]. While biochemical techniques to probe guanine damage yields at long range have been auspicious in underscoring the exquisite sensitivity of charge transport to base stacking and sequence, due to the slow trapping rate of the guanine radical, these studies are inevitably convoluted by processes such as back electron transfer and hence provide information several steps removed from the initial transport event [13].
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DNA Mediated Charge Transport in DNA Repair

DNA Mediated Charge Transport in DNA Repair

Though the E. coli rnf genes have not been biochemically characterized, it has been demonstrated that inactivation of these genes has an effect on SoxR mediated soxS expression (25). The soxRS system senses oxidative stress and activates transcription of a wide variety of genes to protect against and repair oxidative damage (interestingly, one of the genes targeted is yggX (19)) (26). Activation of the soxRS regulon is mediated by SoxR, a [2Fe2S] cluster transcription factor (27-29). Upon oxidation of the cluster in SoxR from the 1+ to the 2+ state, transcription of soxS is initiated. SoxS transcription is transient; within minutes after administration of oxidants has ceased, SoxR is rereduced and soxS is no longer transcribed (29). The pathways for oxidation and rereduction of SoxR are not fully understood, though SoxR is activated within the cell by administration of paraquat (29) and it has been demonstrated in vitro that SoxR can be oxidized from a distance, in a DNA-mediated fashion, by guanine radicals or electrochemical methods (30, 31). Inactivation of the E. coli rnf genes slows the deactivation of soxS expression, indicating that the rnf gene products may be involved in the rereduction of SoxR (25).
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Exploring DNA Mediated Charge Transport with Fast Radical Traps

Exploring DNA Mediated Charge Transport with Fast Radical Traps

Molecular charge transport (CT) has been subject to extensive theoretical and experimental studies, 1-4 since nanoscale device elements provide both novel sensing platforms and the potential to extend Moore’s Law beyond the current limits of solid- state lithography. The properties of individual assemblies can be difficult to predict, however, because the mechanism of CT can change as a result of small variations in donor and bridge energies, bridge length, or environmental factors. A transition from exponential to geometric distance dependence is frequently interpreted as being due to a change in the dominant mechanism from coherent superexchange over short bridges to incoherent hopping over long bridges. In fact, it is assumed that fast, coherent CT over long distances is impossible, as a bridge low enough in potential to mediate long-range superexchange will be rapidly occupied by charge itself, and that incoherent CT will then dominate. 5 Given these conditions, it is not surprising that a variety of bridging systems have been found to transition between superexchange and hopping for increasing bridge length and decreasing separation of bridge and donor energy levels. 6-7
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Electrochemical sensors based on DNA mediated charge transport chemistry

Electrochemical sensors based on DNA mediated charge transport chemistry

To take nothing away from the science done in the Barton Labs, one of my favorite things about working for Jackie is that she attracts a wonderful group of people and I have truly enjoyed my experience in lab for that reason. I am sure I could list everyone that I have worked with over the last five years along with something for which I should thank that person, but for the sake of brevity, here is the short list. Shana Kelley and Scott Rajski helped me to settle in to lab and learn the skills I needed to get started. Donato Ceres and Greg Drummond have been excellent co-DNA electrochemists and friends. Julia Salas is a talented undergraduate student who worked with me for two years. Pratip Bhattacharya, Duncan Odom, Chris Treadway, Melanie O'Neill, Matthias Pascaly, Dave Vicic, Eva Rueba, and Jon Hart have all contributed in various ways to increase both my knowledge of science and research as well as my enjoyment of graduate school. I particularly want to thank Kim
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RIP3 promotes colitis-associated colorectal cancer by controlling tumor cell proliferation and CXCL1-induced immune suppression

RIP3 promotes colitis-associated colorectal cancer by controlling tumor cell proliferation and CXCL1-induced immune suppression

Figure 3. RIP3 promotes epithelial cell proliferation and tumor growth without affecting apoptosis. (A) The extent of intestinal epithelial cell proliferation in the colons of DSS-treated mice was determined by BrdU labeling and immunohistochemistry. The percentage of BrdU-positive cells among all crypt cells in the colons was enumerated. Original magnification, ×100. (B) Proliferation was determined by Ki-67 staining and the percentage of Ki-67-positive cells among all crypt cells in the colons of DSS-treated mice was calculated. Original magnification, ×100. (C-D) Colons of adenoma-bearing mice were stained with antibodies against PCNA and Ki-67. The percentage of PCNA- (C) and Ki-67-positive cells (D) within colonic crypts was determined. Original magnification, ×200. (E) Western blot analysis of RIP3 and p-MLKL expression in the colons of AOM/DSS-treated WT and RIP3 -/- mice with representative densitometry. (F) The number of TUNEL + cells/visual field in colon tissues from control mice and mice
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A TGFβ Smad4 Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development

A TGFβ Smad4 Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development

detected compromised myogenin transcript expression not only in tongue myogenic cells, but also in head muscles, including masseter and extraocular muscles. Although a recent study shows that distinct regulatory cascades regulate extraocular and branchiomeric muscle progenitor cell fates (Sambasivan et al., 2009), our results suggest that Smad4-mediated TGF signaling is universally required by skeletal muscle progenitor cells in the craniofacial region to induce myogenin expression, which allows lineage progression and promotes myoblast terminal differentiation. Myoblast fusion is a key cellular process that shapes the formation and repair of muscle. In vitro data suggest that myoblast fusion can be further partitioned into two phases. First, individual myoblasts undergo fusion with one another to generate nascent myotubes, which contain few nuclei. In the second phase of fusion, additional differentiated myoblasts incorporate into the forming myotube, leading to the further maturation of the nascent myofiber during which the myofiber increases in size and begins to express contractile proteins (Rochlin et al., 2010). Following the fusion of myoblasts into multinucleated myofibers, myonuclei move to a peripheral position and spread along the length of the myofiber. We found that, in Myf5-Cre;Smad4 flox/flox mice, the myoblast fusion
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