Cooperative DNA binding is a key feature of transcriptional regulation. Here we examined the role of cooperativity in Notch signaling by CRISPR-mediated engineering of mice in which neither Notch1 ...nor Notch2 can homo- or heterodimerize, essential for cooperative binding to sequence-paired sites (SPS) located near many Notch-regulated genes. Although most known Notch-dependent phenotypes were unaffected in Notch1/2 dimer-deficient mice, a subset of tissues proved highly sensitive to loss of cooperativity. These phenotypes include heart development, compromised viability in combination with low gene dose, and the gut, developing ulcerative colitis in response to 1% dextran sulfate sodium (DSS). The most striking phenotypes-gender imbalance and splenic marginal zone B-cell lymphoma-emerged in combination with gene dose reduction or when challenged by chronic fur mite infestation. This study highlights the role of the environment in malignancy and colitis and is consistent with Notch-dependent anti-parasite immune responses being compromised in Notch dimer-deficient animals.
The lipogenic enzyme stearoyl CoA desaturase (SCD) plays a key role in tumor lipid metabolism and membrane architecture. SCD is often up-regulated and a therapeutic target in cancer. Here, we report ...the unexpected finding that median expression of SCD is low in glioblastoma relative to normal brain due to hypermethylation and unintentional monoallelic co-deletion with phosphatase and tensin homolog (PTEN) in a subset of patients. Cell lines from this subset expressed undetectable SCD, yet retained residual SCD enzymatic activity. Unexpectedly, these lines evolved to survive independent of SCD through unknown mechanisms. Cell lines that escaped such genetic and epigenetic alterations expressed higher levels of SCD and were highly dependent on SCD for survival. Last, we identify that SCD-dependent lines acquire resistance through a previously unknown FBJ murine osteosarcoma viral oncogene homolog B (FOSB)-mediated mechanism. Accordingly, FOSB inhibition blunted acquired resistance and extended survival of tumor-bearing mice treated with SCD inhibitor.
Despite a common understanding that Gli TFs are utilized to convey a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. Using the mouse ...model (
), we demonstrated that rather than being driven by a Hh threshold, robust Gli3 transcriptional activity during skeletal and glossal development required interaction with the basic helix-loop-helix TF Hand2. Not only did genetic and expression data support a co-factorial relationship, but genomic analysis revealed that Gli3 and Hand2 were enriched at regulatory elements for genes essential for mandibular patterning and development. Interestingly, motif analysis at sites co-occupied by Gli3 and Hand2 uncovered mandibular-specific, low-affinity, 'divergent' Gli-binding motifs (
GBMs). Functional validation revealed these
GBMs conveyed synergistic activation of Gli targets essential for mandibular patterning and development. In summary, this work elucidates a novel, sequence-dependent mechanism for Gli transcriptional activity within the craniofacial complex that is independent of a graded Hh signal.
How complex networks of activators and repressors lead to exquisitely specific cell-type determination during development is poorly understood. In the
Drosophila eye, expression patterns of ...Rhodopsins define at least eight functionally distinct though related subtypes of photoreceptors. Here, we describe a role for the transcription factor gene
defective proventriculus (
dve) as a critical node in the network regulating Rhodopsin expression.
dve is a shared component of two opposing, interlocked feedforward loops (FFLs). Orthodenticle and Dve interact in an incoherent FFL to repress Rhodopsin expression throughout the eye. In R7 and R8 photoreceptors, a coherent FFL relieves repression by Dve while activating Rhodopsin expression. Therefore, this network uses repression to restrict and combinatorial activation to induce cell-type-specific expression. Furthermore, Dve levels are finely tuned to yield cell-type- and region-specific repression or activation outcomes. This interlocked FFL motif may be a general mechanism to control terminal cell-fate specification.
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► Dve is a key transcription factor in the network regulating Rhodopsins in the fly eye ► Dve is a shared component of two opposing, interlocked feedforward loops (FFLs) ► An incoherent FFL represses expression of Rhodopsins in outer photoreceptors ► A coherent FFL relieves Dve repression and activates Rhodopsins in inner photoreceptors
In Drosophila, the development of the compound eye depends on the movement of a morphogenetic furrow (MF) from the posterior (P) to the anterior (A) of the eye imaginal disc. We define several ...subdomains along the A-P axis of the eye disc that express distinct combinations of transcription factors. One subdomain, anterior to the MF, expresses two homeobox genes, eyeless (ey) and homothorax (hth), and the zinc-finger gene teashirt (tsh). We provide evidence that this combination of transcription factors may function as a complex and that it plays at least two roles in eye development: it blocks the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. A key step in the transition from an immature proliferative state to a committed state in eye development is the repression of hth by the BMP-4 homolog Decapentaplegic (Dpp).
During Drosophila embryogenesis, segments, each with an anterior and posterior compartment, are generated by the segmentation genes while the Hox genes provide each segment with a unique identity. ...These two processes have been thought to occur independently. Here we show that abdominal Hox proteins work directly with two different segmentation proteins, Sloppy paired and Engrailed, to repress the Hox target gene Distalless in anterior and posterior compartments, respectively. These results suggest that segmentation proteins can function as Hox cofactors and reveal a previously unanticipated use of compartments for gene regulation by Hox proteins. Our results suggest that these two classes of proteins may collaborate to directly control gene expression at many downstream target genes.
A major question in development is how different specialized cell types arise from a common progenitor. In the adult Drosophila compound eye, color discrimination is achieved by UV-, blue- and ...green-sensitive photoreceptors (PRs). These different PR subsets arise from neuronal precursors called R7 and R8 cells. Recent studies have demonstrated that R7-based UV-sensitive PRs require the repression of R8-based blue/green-sensitive PR characteristics to properly develop. This repression is mediated by the transcription factor Prospero (Pros). Here, we report that Senseless (Sens), a Drosophila ortholog of the vertebrate Gfi1 transcription factor, plays an opposing role to Pros by both negatively regulating R7-based features and positively enforcing R8-based features during terminal differentiation. In addition, we demonstrate that Pros and Sens function together with the transcription factor Orthodenticle (Otd) to oppositely regulate R7 and R8 PR Rhodopsin gene expression in vitro. These data show that sens, previously shown to be essential for neuronal specification, also controls differentiation of specific neuronal subtypes in the retina. Interestingly, Pros has recently been shown to function as a tumor suppressor, whereas Gfi1 is a well-characterized oncogene. Thus, we propose that sens/pros antagonism is important for regulating many biological processes.
Notch pathway haploinsufficiency can cause severe developmental syndromes with highly variable penetrance. Currently, we have a limited mechanistic understanding of phenotype variability due to gene ...dosage. Here, we unexpectedly found that inserting an enhancer containing pioneer transcription factor sites coupled to Notch dimer sites can induce a subset of
haploinsufficiency phenotypes in
with wild type
gene dose. Using
genetics, we show that this enhancer induces Notch phenotypes in a Cdk8-dependent, transcription-independent manner. We further combined mathematical modeling with quantitative trait and expression analysis to build a model that describes how changes in Notch signal production versus degradation differentially impact cellular outcomes that require long versus short signal duration. Altogether, these findings support a 'bind and discard' mechanism in which enhancers with specific binding sites promote rapid Cdk8-dependent Notch turnover, and thereby reduce Notch-dependent transcription at other loci and sensitize tissues to gene dose based upon signal duration.
Hox transcription factors specify numerous cell fates along the anterior–posterior axis by regulating the expression of downstream target genes. While expression analysis has uncovered large numbers ...of de-regulated genes in cells with altered Hox activity, determining which are direct versus indirect targets has remained a significant challenge. Here, we characterize the DNA binding activity of Hox transcription factor complexes on eight experimentally verified cis-regulatory elements. Hox factors regulate the activity of each element by forming protein complexes with two cofactor proteins, Extradenticle (Exd) and Homothorax (Hth). Using comparative DNA binding assays, we found that a number of flexible arrangements of Hox, Exd, and Hth binding sites mediate cooperative transcription factor complexes. Moreover, analysis of a Distal-less regulatory element (DMXR) that is repressed by abdominal Hox factors revealed that suboptimal binding sites can be combined to form high affinity transcription complexes. Lastly, we determined that the anterior Hox factors are more dependent upon Exd and Hth for complex formation than posterior Hox factors. Based upon these findings, we suggest a general set of guidelines to serve as a basis for designing bioinformatics algorithms aimed at identifying Hox regulatory elements using the wealth of recently sequenced genomes.
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